Polymeric chromophores, compositions containing same, and methods of preparing and using same - Patents.com

Abstract

Described herein are polymeric chromophores comprising a dye, a polymer, and, optionally, a bioconjugate group. The polymeric chromophores can have a structure represented by the following: ABC, or CAB, where A is a dye, B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C, if present, comprises a bioconjugate group. Also described herein are compositions comprising the polymeric chromophores, and methods of preparing and using the polymeric chromophores.

Description

[Technical field]

[0001] Government support statement This invention was made with Government support under Grant No. DE-SC0001035 awarded by the Department of Energy. The Government has certain rights in this invention.

[0002] The present invention relates generally to polymeric chromophores comprising a dye, a polymer segment, and, optionally, a bioconjugate group. The present invention also relates to compositions comprising the polymeric chromophores, as well as methods of preparing and using the same. [Background technology]

[0003] Many applications of chromophores are carried out in aqueous solutions, yet most organic chromophores are hydrophobic or slightly polar. There are many approaches to encapsulating chromophores, but none yet meet the criteria of synthetic simplicity, lack of fluorophore-fluorophore quenching, and the presence of a single bioconjugatable group. Summary of the Invention [Means for solving the problem]

[0004] A first aspect of the present invention is directed to a compound having a structure represented by: A,B,C, or CAB Where: A is a dye (e.g., a fluorophore), optionally wherein the dye has a molecular weight ranging from about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, optionally wherein the polymer has a molecular weight ranging from about 1,000 Da, 5,000 Da, or 10,000 Da to about 175,000 Da; and C is optional, where C comprises a bioconjugate group.

[0005] Another aspect of the invention is directed to a composition comprising a compound of the invention and, optionally, water. Also, an aspect of the invention is directed to a composition comprising a particle (e.g., a particle comprising a core and a shell), wherein the particle comprises a compound having a structure represented by: A,B,C, or CAB Where: A is a dye (e.g., a fluorophore); B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; C is optional, where C comprises a bioconjugate group; and water.

[0006] A further aspect of the present invention is directed to a method of preparing a compound, comprising polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer, attaching a dye to a first portion (e.g., a terminus or terminal portion) of the copolymer, thereby providing the compound, and optionally attaching a bioconjugate group to a second portion (e.g., another terminus or terminal portion) of the copolymer and / or optionally crosslinking the compound. Polymerizing the hydrophobic monomer and the hydrophilic monomer may comprise polymerizing via living radical polymerization in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst, to provide the copolymer.

[0007] Another aspect of the invention is directed to compounds prepared according to the methods of the invention.

[0008] Also provided according to embodiments of the invention are methods of using compounds of the invention and / or methods of using compositions of the invention, such as in flow cytometry.

[0009] A further aspect of the invention is directed to a method of detecting cells and / or particles using flow cytometry, the method comprising labeling cells and / or particles with a compound of the invention and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0010] Another aspect of the invention is directed to a method of detecting tissues and / or agents (e.g., cells, infecting agents, etc.), the method comprising administering a compound of the invention or a composition of the invention to a subject, where optionally the compound associates with the tissues and / or agents, and detecting the compound in the subject, thereby detecting the cells and / or particles.

[0011] A further aspect of the present invention is directed to biomolecules (eg, cells, antibodies, etc.) comprising one or more (eg, 1, 2, 3, 4, 5, 6 or more) compounds of the present invention.

[0012] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated in a different embodiment even if not specifically described in that context. That is, all aspects and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to modify the claims as originally filed and / or to submit new claims accordingly, including the right to amend the claims as originally filed to rely on and / or incorporate features of other claims or claims not originally so claimed. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Further features, advantages and details of the invention will be apparent to those skilled in the art from reading the drawings and the detailed description of the preferred embodiments below. And, such description is merely illustrative of the invention. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a schematic diagram of an exemplary polymeric chromophore according to an embodiment of the present invention. [Diagram 2] Figure 2 shows the SEC elution traces of copolymer 7 (solid line) and chlorine-loaded copolymer F2 (dashed line). Samples were eluted with THF and detected with a refractive index detector. [Diagram 3] Figure 3 shows three different absorption spectra. Panel (A) shows the absorption spectrum of D1 in CH2Cl2 (solid line) and the absorption (dashed) and emission (dotted) spectra of F1 in water at μM concentrations. Panel (B) shows the absorption spectrum of D2 in CH2Cl2 (solid line) and the absorption (dashed) and emission (dotted) spectra of F2 in water at μM concentrations. Panel (C) shows the absorption spectrum of D3 in toluene (solid line) and the absorption (dashed) and emission (dotted) spectra of F3 in water at μM concentrations. All spectra were measured at room temperature. [Figure 4] FIG. 4 shows dynamic light scattering (DLS) size-dependence data for F-2 at 10 mg / mL (A), 5 mg / mL (B), and 1.0 mg / mL (C). [Diagram 5] FIG. 5 shows the absorption spectra of F-2 in 1.0 M NaCl solution (top) and in water (bottom). [Figure 6] FIG. 6 shows the emission spectra in 1.0 M NaCl solution (top) and in water (bottom). [Figure 7] FIG. 7 shows DLS data for two batches of F-Ph at various concentrations in 1.0 M NaCl aqueous solution. [Figure 8] FIG. 8 shows the absorption (left) and emission (right) spectra of Pod-Rhodamine in water in the presence of various cations. [Figure 9] FIG. 9 shows the fluorescence titration spectrum of Au(III) (top graph) and Hg(II) (bottom graph). [Figure 10] FIG. 10 shows the DLS data of F-Ph in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH=7.35) at room temperature. [Figure 11] FIG. 11 shows the DLS data of F-Ph in NaCl solutions of various concentrations. [Figure 12] FIG. 12 shows a graph of the percentage of unimers (by DLS) in NaCl solutions of various concentrations and in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH=7.35) at room temperature. [Figure 13] FIG. 13 shows DLS data of P-S5-CD1 (28 kDa) in NaCl solutions of various concentrations at room temperature. [Figure 14] FIG. 14 shows the DLS data of P-S5-CD1 (28 kDa) in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. [Figure 15] FIG. 15 shows a graph of the percentage of unimers (by DLS) in NaCl solutions of various concentrations and in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. [Figure 16] FIG. 16 shows the DLS data of F-Ph and F-PMI in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. [Figure 17] FIG. 17 shows DLS data of P-S5-CD1(28 kDa) and P-S5-CD1(28 kDa)-PMI in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. [Figure 18] FIG. 18 shows the DLS data of P-S5-CD1-PMI conjugates with different molecular weights in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. [Figure 19]FIG. 19 shows DLS data of three polymer-PMI conjugates with the same molecular weight but different ratios of pendants in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0015] The terms used in the detailed description of the invention herein are for the purpose of describing particular embodiments only, and are not intended to be limiting of the invention. As used in the detailed description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to also include the plural forms unless the context clearly dictates otherwise.

[0016] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, words, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an ideal or overly formal sense unless expressly defined herein. The terms used in the detailed description of the invention herein are intended only to describe specific embodiments and are not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict in terms, the present specification shall control.

[0017] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0018] It is specifically contemplated that the various features of the invention described herein can be used in any combination unless the context dictates otherwise. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For purposes of illustration, when the present specification states that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, can be omitted and discarded.

[0019] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to include those materials or steps described that "do not materially affect one or more of the basic and novel characteristics" of the claimed invention. In Herz , 537 F.2d 549,551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, as used herein, the word "consisting essentially of" should not be construed as equivalent to "comprising."

[0020] As used herein, the words "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example and / or variant embodiments discussed herein, and are not intended to indicate a preference for one or more of the embodiments discussed herein over one or more other embodiments.

[0021] When used herein when referring to a measurable value, such as an amount or concentration, the term "about" is meant to include a variation of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified value as well as the specified value. For example, if X is a measurable value, "about X" is meant to include X as well as a variation of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of X. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0022] As used herein in reference to a chemical molecule, a "derivative" refers to a chemical molecule that has one or more atoms (e.g., hydrogen atoms), functional groups, and / or bonds that have been modified (e.g., removed, replaced, etc.) compared to the parent molecular entity. For example, a derivative of a dye can refer to a parent dye compound that has one or more atoms (e.g., hydrogen atoms) and / or functional groups that have been modified (e.g., removed) to facilitate covalent attachment to another group or residue (e.g., to facilitate covalent attachment to a polymer). In some embodiments, a derivative can include functional groups (e.g., substituents and / or auxochromes) that change the absorption spectrum of the parent molecular entity.

[0023] As used herein alone or as part of another group, "alkyl" refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms, which may be referred to as C1-C20 alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. As used herein, "lower alkyl" is a subset of alkyl, and in some embodiments, refers to a straight or branched chain hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.The term "alkyl" or "lower alkyl", unless otherwise indicated, is intended to encompass both substituted and unsubstituted alkyl or lower alkyl, and these groups may be substituted with groups selected from the following: halogen atoms, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby producing a polyalkoxy, e.g., polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl- S(O)m, haloalkyl-S(O)m, alkenyl-S(O)m, alkynyl-S(O)m, cycloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocyclo-S(O)m, heterocycloalkyl-S(O)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, where m=0, 1, 2 or 3.

[0024] As used herein alone or as part of another group, "alkenyl" refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkenyl) which may contain 1 to 8 double bonds in the straight chain, and may be referred to as C1-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term "alkenyl" or the term "lower alkenyl" is intended to include both substituted and unsubstituted alkenyl or lower alkenyl, unless otherwise indicated, and these groups may be substituted with groups as described in connection with alkyl and lower alkyl above.

[0025] As used herein alone or as part of another group, "alkynyl" refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkynyl) containing one triple bond in the straight chain, and can be referred to as C1-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term "alkynyl" or the term "lower alkynyl" is intended to include both substituted and unsubstituted alkynyl or lower alkynyl, unless otherwise indicated, and these groups can be substituted with the same groups described in connection with alkyl and lower alkyl above.

[0026] As used herein, "halogen atom" refers to any suitable halogen atom, including, for example, -F, -Cl, -Br and -I.

[0027] As used herein, "mercapto" refers to a --SH group.

[0028] As used herein, "azido" refers to the group --N3.

[0029] As used herein, "cyano" refers to the group --CN.

[0030] As used herein, "hydroxyl" refers to an --OH group.

[0031] As used herein, "nitro" refers to the group --NO.sub.2.

[0032] As used herein alone or as part of another group, "alkoxy" refers to an alkyl or lower alkyl group, as defined herein, appended to the parent molecular residue through an oxygen atom radical, -O-, (thus including substituted versions, e.g., polyalkoxy). Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0033] As used herein alone or as part of another group, "acyl" refers to a -C(O)R group, where R is any suitable substituent, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituents described herein.

[0034] As used herein alone or as part of another group, "haloalkyl" refers to at least one halogen atom, as defined herein, appended to the parent molecular residue through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.

[0035] "Alkylthio" as used herein alone or as part of another group refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a thio moiety, as defined herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0036] As used herein alone or as part of another group, "cycloalkyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 1 to 20 carbon atoms, optionally with the carbon atom substituted in a heterocyclic group as discussed below. Cycloalkyl groups may contain 0, 1, 2, or more double or triple bonds. Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may be optionally substituted with additional substituents as described herein, such as halogen atoms or lower alkyl. The term "cycloalkyl" is generic and, unless otherwise specified, is intended to include the heterocyclic groups as discussed below.

[0037] As used herein alone or as part of another group, a "heterocyclic group" or "heterocyclo" refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic or bicyclic system. Monocyclic systems are exemplified by any 5- or 6-membered ring that contains 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. The 5-membered ring has 0-2 double bonds, and the 6-membered ring has 0-3 double bonds. Representative examples of monocyclic systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic systems are exemplified by any of the above monocyclic systems fused to an aryl group, as defined herein, a cycloalkyl group, as defined herein, or another monocyclic system, as defined herein.Representative examples of bicyclic systems include, but are not limited to, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxin, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings, including their quaternized derivatives, can be optionally substituted with groups selected from the following: halogen atoms, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(O). m , haloalkyl-S(O) m , alkenyl-S(O) m , alkynyl-S(O) m , cycloalkyl-S(O) m , cycloalkylalkyl-S(O) m , aryl-S(O) m , arylalkyl-S(O) m , heterocyclo-S(O) m , heterocycloalkyl-S(O) m , amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, where m=0, 1, 2 or 3.

[0038] As used herein alone or as part of another group, "aryl" refers to a monocyclic, carbocyclic or bicyclic, carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term "aryl", unless otherwise indicated, is intended to include both substituted and unsubstituted aryl, and these groups may be substituted with the same groups described above in connection with alkyl and lower alkyl.

[0039] As used herein alone or as part of another group, "arylalkyl" refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

[0040] As used herein, "amino" refers to the group --NH.

[0041] As used herein alone or as part of another group, "alkylamino" refers to the group --NHR, where R is an alkyl group.

[0042] As used herein alone or as part of another group, an "ester" refers to a -C(O)OR group, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0043] As used herein, "formyl" refers to the group --C(O)H.

[0044] As used herein, "carboxylic acid" refers to a -C(O)OH group.

[0045] As used herein, "sulfoxyl" refers to a compound of formula -S(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0046] As used herein, "sulfonyl" refers to a compound of the formula -S(O)(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0047] As used herein, "sulfonate" refers to a salt of sulfonic acid (e.g., a sodium (Na) salt) and / or a compound of the formula -S(O)(O)OR, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0048] As used herein, "sulfonic acid" refers to a compound of the formula -S(O)(O)OH.

[0049] As used herein alone or as part of another group, "amide" refers to -C(O)NR a R b where R a and R b is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0050] As used herein alone or as part of another group, "sulfonamide" refers to -S(O)NR a R b where R a and R b is any suitable substituent, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0051] The compounds of the present invention have a polymeric fluorophore. The compounds of the present invention have a single (i.e., one) polymer attached to a single (i.e., one) dye, and optionally have a single (i.e., one) bioconjugate group, which may have a single binding site for a biomolecule. Exemplary compounds are shown in FIG. 1. In some embodiments, the single polymer is attached to both the dye and the bioconjugate group (if present). In some embodiments, the single dye is attached to both the polymer and the bioconjugate group (if present). In some embodiments, the compositions of the present invention include the compounds of the present invention in solution, such as water, aqueous solutions and / or hydrophobic solvents.

[0052] While the compounds of the invention are attached to a single biomolecule via a bioconjugate group, the biomolecule may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compounds of the invention. Thus, in some embodiments, a biomolecule and / or portion thereof may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compounds of the invention.

[0053] In some embodiments, the compound of the invention has a structure represented by: A,B,C, or CAB Where: A is a dye (e.g., a fluorophore); B is a polymer containing one or more hydrophobic units and one or more hydrophilic units; and C is optional, where C, when present, comprises a bioconjugate group.

[0054] The terms "dye" and "chromophore" are used interchangeably herein and refer to luminophores (e.g., fluorescent and / or phosphorescent molecular entities) and / or non-luminescent molecular entities (e.g., non-fluorescent and / or non-phosphorescent molecular entities). As used herein, the term "non-luminescent molecular entity" refers to a molecular entity that has no or negligible luminescence. In some embodiments, a non-luminescent molecular entity does not form an excited state of any significant lifetime and / or relaxes rapidly and essentially quantitatively to the ground state. In some embodiments, a non-luminescent molecular entity has an excited state lifetime of less than about 100 picoseconds, less than about 75 picoseconds, less than about 50 picoseconds, less than about 25 picoseconds, less than about 10 picoseconds, less than about 5 picoseconds, less than about 1 picoseconds, less than about 0.5 picoseconds, or less than about 0.1 picoseconds. In some embodiments, the non-emissive molecular entity has a quantum yield of internal conversion of greater than about 0.8, greater than about 0.85, greater than about 0.9, greater than about 0.95, greater than about 0.9, greater than about 0.999, greater than about 0.9999, or greater than about 0.99999, where a quantum yield of 1.0 corresponds to 100%. In some embodiments, the non-emissive molecular entity has a luminescence quantum yield of less than about 0.2, less than about 0.15, less than about 0.1, less than about 0.05, less than about 0.01, less than about 0.001, less than about 0.0001, or less than about 0.00001, where a quantum yield of 1.0 corresponds to 100%. It is known that the luminescence quantum yield is obtained from the competing process of radioactive decay against the sum of all processes for depolarizing the excited state manifold. Although such compounds are often referred to as "non-emissive", sensitive detection techniques are often able to detect small amounts of residual luminescence as would be expected with such low luminescence quantum yields. Although the maximum possible conversion of light input to heat output is desired, a small amount of luminescence may not adversely affect some applications, such as photoacoustic imaging. Thus, the term "non-luminescent" is used herein to refer to a molecular entity that has no luminescence or negligible luminescence. In some embodiments, the compound of the present invention comprises a dye, which is a non-luminescent molecular entity (e.g., a non-fluorescent molecular entity and / or a non-phosphorescent molecular entity). In some embodiments, the compound of the present invention comprises a dye, which is a luminophore (e.g., a fluorescent molecular entity and / or a phosphorescent molecular entity)."Fluorescent molecular entity" and "fluorophore" are used interchangeably herein and refer to a molecular entity that emits fluorescence.

[0055] The dyes of the invention can have certain spectroscopic characteristics and / or properties, such as spectroscopic characteristics and / or properties that make them suitable for use in the methods of the invention. In some embodiments, the dyes have a molecular weight ranging from about 150 Daltons (Da) to about 3,000 Da, from about 400 Da to about 1,100 Da, or from about 300 Da to about 1,000 Da. In some embodiments, the dye has a molecular weight of about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or about 3000 Da. Exemplary dyes include, but are not limited to, tetrapyrroles; rylenes, such as perylene, terylene, and quatarylene; fluoresceins, such as TET (tetramethylfluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX), and 5-carboxyfluorescein (5-FAM); phycoerythrin; resorufin dyes; coumarin dyes; rhodamine dyes, such as 6-carboxy-X-rhodamine (ROX), Texas Red, and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); cyanine dyes; phthalocyanines; boron-dipyrromethene (BODIPY) dyes; quinolines; pyrenes; acridines; stilbenes; and derivatives thereof. In some embodiments, the pigment is a tetrapyrrole, which includes porphyrins, chlorins and bacteriochlorins and their derivatives.Exemplary tetrapyrroles include, but are not limited to, those described in U.S. Pat. Nos. 6,272,038; 6,451,942; 6,420,648; 6,559,374; 6,765,092; 6,407,330; 6,642,376; and 6,946,552. No. 6,603,070; No. 6,849,730; No. 7,005,237; No. 6,916,982; No. 6,944,047; No. 6,944,047; Specification No. 7,884,280; Specification No. 7,332,599; Specification No. 7,148,361; Specification No. 7,022,862; Specification No. 6,924,375; Specification No. 7,501,5 Specification No. 07; Specification No. 7,323,561; Specification No. 7,153,975; Specification No. 7,317,108; Specification No. 7,501,508; Specification No. 7,378,520; Specification No. 7,534,807; Specification No. 7,919,770; Specification No. 7,799,910; Specification No. 7,582,751; Specification No. 8,097,609; Specification No. 8,187 Nos. 8,207,329, 7,633,007, 7,745,618, 7,994,312, 8,278,340, 9,303,165, and 9,365,722; and International Patent Application Nos. PCT / US17 / 47266 and PCT / US17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments, the dye is attached and / or bound to a monomer that is polymerized with one or more different monomers (e.g., polymerized with a hydrophobic monomer and / or a hydrophilic monomer). In some embodiments, the dye is a luminophore (i.e., a material and / or compound that is capable of emitting light and does not specify the nature of the original state (e.g., singlet state, triplet state, and / or other state). Exemplary luminophores include, but are not limited to, phosphorescent and / or fluorophores that provide phosphorescence and / or fluorescence, respectively.

[0056] In some embodiments, the compounds of the present invention include a recognition motif. In some embodiments, the dyes of the present invention may include a recognition motif, and the linker that connects the dyes of the present invention to the polymers of the present invention may include a recognition motif. The recognition motif may be attached to the dye and / or the linker. As used herein, a "recognition motif" refers to a molecular entity that can bind to a binding entity, and such binding changes the absorption spectrum of the dye and / or turns on the fluorescence of the dye. Recognition motifs and binding entities known to those skilled in the art may be used in the compounds of the present invention. Exemplary recognition motifs include, but are not limited to, crown ethers, cryptands, pincers, and / or chelating motifs. Examples of binding entities are metal ions (e.g., Hg, Cr, Li, etc.). The mechanism for altering the absorption spectrum of the dye and / or turning on the fluorescence of the dye can be accomplished by various means, such as: (i) metal ion binding promotes ring opening to generate a conjugated chromophore; or (ii) metal ion binding to an electron-rich group, which when unbound causes quenching of fluorescence, thereby quenching is stopped upon binding. In some embodiments, the compounds of the present invention act and / or function as colorimetric and / or fluorescent sensors. In some embodiments, the compounds of the present invention provide and / or enable metal ion sensing in water, optionally without the addition and / or presence of organic solvents. In some embodiments, the compounds of the present invention are used in sensing applications and / or in sensors. For example, in some embodiments, the compounds of the present invention are present in (e.g., embedded in) and / or on a sensor. The sensor can be an in vivo sensor and / or for in vivo sensing applications and / or for environmental sensing applications. The recognition motif may be at least partially solvent accessible and / or available to allow binding of a binding entity.In some embodiments, the compounds of the invention contain a recognition motif and can be used in aqueous solution, optionally for sensing applications and / or in photoacoustic imaging. In some embodiments, when the compounds of the invention are used in photoacoustic imaging and the compounds contain a recognition motif, the recognition motif can cause a shift in the absorption spectrum of the dye upon binding to a binding entity.

[0057] The polymer of the compound of the present invention may comprise one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic units and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic units. The polymer may be prepared from one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic monomers and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic monomers using any type of polymerization to provide a polymer comprising the one or more hydrophobic units and the one or more hydrophilic units. In some embodiments, the polymer may be prepared from two or more (e.g., 2, 3, 4, 5 or more) hydrophobic monomers different from each other and / or two or more (e.g., 2, 3, 4, 5 or more) hydrophilic monomers different from each other. For example, in some embodiments, a polymer of the compounds of the present invention can be prepared from at least one hydrophobic monomer, at least one first hydrophilic monomer, and at least one second hydrophilic monomer, where the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0058] As used herein, a "hydrophilic monomer" refers to a monomer that includes a hydrophilic (e.g., ionic and / or polar functional group (e.g., hydrophilic pendant functional group), optionally, the hydrophilic functional group is at the residue and / or terminal portion of the monomer. As one of ordinary skill in the art will appreciate, portions of a hydrophilic monomer may be hydrophobic, such as portions that form a polymer backbone when polymerized with other monomers and / or portions of the functional group that include ionic residues but are still referred to as hydrophilic monomers when they include hydrophilic functional groups (e.g., hydrocarbon chains). As used herein, a "hydrophilic unit" refers to a section or unit of a polymer prepared from an individual hydrophilic monomer. As used herein, a "hydrophobic monomer" refers to a monomer that includes a hydrophobic functional group (e.g., hydrophobic pendant functional group), optionally, the hydrophobic functional group is at the residue and / or terminal portion of the monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon residue (e.g., alkyl). As used herein, a "hydrophobic unit" refers to a section or unit of a polymer prepared from an individual hydrophobic monomer.

[0059] In some embodiments, the polymer of the compound of the present invention may also be referred to as a polymer segment of the compound of the present invention. The one or more hydrophobic units and the one or more hydrophilic units may be randomly distributed in the polymer. In some embodiments, the polymer is a random copolymer. The polymer may be an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. The one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio (hydrophobic units:hydrophilic units) of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, where the ratio of hydrophobic units to hydrophilic units is about 1:4 to about 1:6. The length of the polymer may be varied and / or controlled. In some embodiments, the polymer has a molecular weight in the range of about 1,000 Da to about 175,000 Da, about 5,000 Da to about 175,000 Da, about 10,000 Da to about 175,000 Da, about 20,000 Da to about 175,000 Da, about 28,000 Da to about 175,000 Da, about 28,000 Da to about 35,000 Da, about 28,000 Da to about 50,000 Da, about 100,000 Da to about 150,000 Da, about 50,000 Da to about 130,000 Da, or about 10,000 Da to about 100,000 Da. In some embodiments, the polymer has a molecular weight of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, or about 170 kilodaltons (kDa). In some embodiments, the polymer has a molecular weight of more than 28 kDa. In some embodiments, the polymer has a molecular weight of about 28 kDa to about 175 kDa. In some embodiments, the polymer has a molecular weight of about 28 kDa to about 35 kDa or about 50 kDa.

[0060] The hydrophobic and / or hydrophilic units of the polymer may have pendant functional groups. A "pendant functional group" may be a functional group that is directly attached to the polymer backbone or a functional group that is attached to a residue that is attached to the polymer backbone. The pendant functional group may be part of the hydrophobic and / or monomer and / or hydrophilic unit and / or monomer during polymerization, or may be added to the hydrophobic and / or hydrophilic unit after polymerization. In some embodiments, the pendant functional group may be attached to the hydrophobic and / or hydrophilic unit after polymerization (e.g., post-polymerization functionalization). In some embodiments, the pendant functional group comprises a charged group. In some embodiments, the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., an active ester, such as a pentafluorophenyl ester, a succinimide ester, a 2,4-dinitrophenyl ester, etc.), an azido group, a pentafluorophenyl group, a succinimide group, a fluorophenyl group, a maleimide group, an isocyanate group, or an isothiocyanate group. In some embodiments, the pendant functional group is a hydrophilic group having a terminal cationic (e.g., ammonium), anionic (e.g., sulfonate, phosphate, carboxylate), or amphoteric (e.g., a choline or choline-like group (e.g., a derivative of choline) group and, optionally, a poly(ethylene glycol) residue and / or unit. In some embodiments, the hydrophilic group is attached to the poly(ethylene glycol) residue and / or unit, and is optionally attached to the terminal portion of the poly(ethylene glycol) residue and / or unit.

[0061] In some embodiments, the hydrophobic unit has a pendant functional group comprising an alkyl (e.g., dodecyl), and / or the hydrophilic unit comprises a pendant functional group comprising a glycol (e.g., poly(ethylene glycol)), sulfone, and / or sulfonate. In some embodiments, the hydrophobic unit is prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer, and / or the hydrophilic unit is prepared from a glycol acrylate (e.g., PEG acrylate) monomer. In some embodiments, the compounds of the present invention comprise at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and at least two different hydrophilic units, the hydrophilic units comprising a first hydrophilic unit prepared from a glycol acrylate (e.g., PEG acrylate) monomer and a second hydrophilic unit prepared from a sulfonic acid acrylamide monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate acrylate monomer.

[0062] In some embodiments, one or more of the hydrophobic units and / or one or more of the hydrophilic units may comprise a charge (e.g., a positive charge or a negative charge) and / or a charged group (e.g., a cationic group or an anionic group), and the charge may inhibit non-specific binding to the compound or a portion thereof (e.g., a portion of a polymer).

[0063] In some embodiments, a hydrophobic monomer, which may be used to provide the hydrophobic units of the polymers described herein, may have a structure represented by Formula I below:

[0064] [ka]

[0065] Where: R is a hydrogen atom or a C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R1 is absent or is -O-, -NH- or -CH2-, R' is a C1-C20 alkyl, a C2-C20 alkenyl, a C2-C20 alkynyl, or a C3-C20 cycloalkyl; and R 2 is a hydrogen atom or a halogen atom, a hydroxyl, a carboxyl, an amino, a formyl, or an ester (e.g., succinimide ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.) group. 2 is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group. 2is a hydrogen atom. In some embodiments, R' in the compound of formula I is C2-C4 alkyl, C2-C6 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C16 alkyl, C8-C18 alkyl, C10-C14 alkyl, or C10-C12 alkyl. In some embodiments, R' in the compound of formula I is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R' in the compound of formula I is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl. In some embodiments, R' in the compound of formula I is C3-C5 cycloalkyl, C3-C6 cycloalkyl, C4-C20 cycloalkyl, C6-C20 cycloalkyl, C8-C16 cycloalkyl, C8-C18 cycloalkyl, C10-C14 cycloalkyl, or C10-C12 cycloalkyl. In some embodiments, R' in the compound of formula I is a C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 cycloalkyl. One of skill in the art will appreciate that in some embodiments, the monomer is, for example, an acrylate (e.g., R 1 is an oxygen atom), acrylamide (e.g., R 1 is NH) or vinyl ketones (e.g., R 1 is CH2), but it will be readily appreciated that other compounds are possible.

[0066] In some embodiments, a hydrophilic monomer, which may be used to provide the hydrophilic units of the polymers described herein, may have a structure represented by Formula II below.

[0067] [ka]

[0068] Where: R is a hydrogen atom or a C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R 3 But -(CH2CH2R 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3-, or salts thereof, wherein R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; and R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester).

[0069] In some embodiments, R in the compound of formula II 4 is arbitrarily R 3 -(CH2CH2R 5 ) n When R is -, -C1-C6 alkyl, or -C1-C6 alkyl-O-, it is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group. 3 is -C1-C6 alkyl-O- or -(CH2CH2R 5 ) n -, where R 5 is -O-, R 4can be a hydrogen atom, an alkyl (e.g., a methyl or ethyl group), a phosphono (e.g., a dihydroxyphosphoryl), a sulfono (e.g., a hydroxysulfonyl), a phosphatidylcholine, or a phosphoryl group. In some embodiments, R in the compound of formula II 3 is -C1-C6 alkyl, R 4 can be a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group, or a sulfono group. 3 is -C1-C6 alkyl-SO3- or a salt thereof, R 4 is a hydrogen atom or is absent. In some embodiments, R in the compound of formula II 3 is a salt of -C1-C6 alkyl-SO3- (e.g., a sodium salt), and R 4 is absent. In some embodiments, R in the compound of formula II 3 -(CH2CH2R 5 ) n -It is.

[0070] In some embodiments, the hydrophobic unit can have a structure represented by Formula III below:

[0071] [ka]

[0072] Where: R is a hydrogen atom or a C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R' is a C1-C20 alkyl, a C2-C20 alkenyl, a C2-C20 alkynyl, or a C3-C20 cycloalkyl; R 2is a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., a pentafluorophenyl ester, a succinimide ester, a fluorophenyl ester, or a 2,4-dinitrophenyl ester), an azide group, a maleimide group, an isocyanate group, or an isothiocyanate group, and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

[0073] In some embodiments, R in the compound of formula III 2 is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group. 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group, which may optionally be added and / or provided by pre-polymerization and / or post-polymerization functionalization. 2is a hydrogen atom. In some embodiments, R' in the compound of formula III is C2-C4 alkyl, C2-C6 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C16 alkyl, C8-C18 alkyl, C10-C14 alkyl, or C10-C12 alkyl. In some embodiments, R' in the compound of formula III is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R' in the compound of formula III is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl. In some embodiments, R' in the compound of formula III is C3-C5 cycloalkyl, C3-C6 cycloalkyl, C4-C20 cycloalkyl, C6-C20 cycloalkyl, C8-C16 cycloalkyl, C8-C18 cycloalkyl, C10-C14 cycloalkyl, or C10-C12 cycloalkyl. In some embodiments, R' in the compound of formula III is C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 cycloalkyl.

[0074] In some embodiments, the hydrophilic unit can have a structure represented by Formula IV below.

[0075] [ka]

[0076] Where: R is a hydrogen atom or a C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R 3 But -(CH2CH2R 5) n -, -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3-, or salts thereof, wherein R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

[0077] In some embodiments, R in the compound of formula IV 4 is arbitrarily R 3 -(CH2CH2R 5 ) n When R is -, -C1-C6 alkyl, or -C1-C6 alkyl-O-, it is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group. 3 -C1-C6 alkyl-O- or -(CH2CH2R 5 ) n - and R 5 -O-, R 4 can be a hydrogen atom, an alkyl (e.g., a methyl or ethyl group), a phosphono (e.g., a dihydroxyphosphoryl), a sulfono (e.g., a hydroxysulfonyl), a phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or a phosphoryl group. In some embodiments, R in the compound of formula IV3 is -C1-C6 alkyl or -(CH2CH2R 5 ) n - and R 5 When is -CH2-, R 4 can be a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group, or a sulfono group. 4 is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group. 4 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group, which may optionally be added and / or provided by pre-polymerization and / or post-polymerization functionalization. 3 is -C1-C6 alkyl-SO3- or a salt thereof, R 4 is a hydrogen atom or is absent. In some embodiments, R in the compound of formula IV 3 is a salt of -C1-C6 alkyl-SO3- (e.g., a sodium salt), and R 4 is absent. In some embodiments, R in the compound of formula IV 3 -(CH2CH2R 5 ) n -It is.

[0078] In some embodiments, the compound of the present invention may comprise and / or be a telechelic polymer, which is a polymer or prepolymer that can enter into further polymerization or other reactions through one or more of its reactive end groups.In some embodiments, the compound of the present invention may comprise and / or be a heterotelechelic polymer, which is a polymer or prepolymer that can enter into further polymerization or other reactions through a reactive end group at each end of the polymer or prepolymer, and the two reactive end groups are not the same as each other.In some embodiments, the compound of the present invention may comprise and / or be a homotelechelic polymer, which is a polymer or prepolymer that can enter into further polymerization or other reactions through a reactive end group at each end of the polymer or prepolymer, and the two reactive end groups are the same as each other.In some embodiments, the compound of the present invention may comprise and / or be a semitelechelic polymer, which is a polymer or prepolymer that can enter into further polymerization or other reactions through a reactive end group at each end of the polymer or prepolymer.

[0079] Bioconjugate groups may optionally be present in the compounds of the invention. A "bioconjugable group", "bioconjugable moiety" or "bioconjugate group" and grammatical variations thereof refer to a residue and / or functional group that is attached or conjugated to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Thus, a "bioconjugable group", "bioconjugable moiety" or "bioconjugate group" and grammatical variations thereof do not include a biomolecule. However, in some embodiments, a bioconjugate group is used to conjugate a biomolecule or a bioconjugate group or a derivative thereof is conjugated to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Exemplary bioconjugable groups include, but are not limited to, amines (including amine derivatives), such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, and the like; acids or acid derivatives, such as N-hydroxysuccinimide esters (more commonly, active esters derived from carboxylic acids, such as p-nitrophenyl esters), acid hydrazides, and the like; and other linking groups, such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyls, halogen groups, biotin, 2-iminobiotin, and the like. Linking groups, such as those described above, are known and are described in U.S. Patent Nos. 6,728,129, 6,657,884, 6,212,093, and 6,208,553. For example, a compound of the invention may have a bioconjugate group that includes a carboxylic acid, and the carboxylic acid may be used for bioconjugation to a biomolecule (e.g., via carbodiimide activation and coupling with an amino-substituted biomolecule).

[0080] In some embodiments, the biomolecule may include and / or be a protein (e.g., an antibody and / or a carrier protein), a peptide, DNA, RNA, etc. In some embodiments, the biomolecule may include a residue (e.g., a polymer) that may optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) binding sites for the compounds of the invention. In some embodiments, the biomolecule may be a member of a specific binding pair. "Specific binding pair" and "ligand-receptor binding pair" are used interchangeably herein and refer to two different molecules, where one of the molecules has an area on the surface or within a cavity of the molecule that specifically attracts or binds to a particular spatial or polar organization of the other molecule, such that both molecules have affinity for each other. Members of a specific binding pair may be referred to as a ligand and a receptor (antiligand). The term "ligand and receptor" is intended to include the entire ligand or the entire receptor, or a sufficient portion thereof for binding to occur between the ligand and the receptor. Ligand-receptor binding pairs include, but are not limited to: hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor-receptor, and interferons and interferon receptors; avidin and biotin or antibiotin; antibody-antigen pairs; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; interleukins and interleukin receptors; stimulatory factors and their receptors, such as granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor) and GMCSF receptor and macrophage colony stimulating factor (MCSF) and MCSF receptor.

[0081] The compounds of the present invention may include a dye (e.g., tetrapyrrole) covalently attached to a portion of the polymer described herein. In some embodiments, the dye may be covalently attached to a terminal portion of the polymer. If present, a bioconjugate group may also be covalently attached to a portion of the polymer, such as the terminal portion of the polymer. In some embodiments, the bioconjugate group is covalently attached to a first terminal portion (e.g., a first end) of the polymer, and the dye is covalently attached to the opposite terminal portion (e.g., the opposite end) of the polymer.

[0082] The compounds of the invention may include a dye (e.g., a tetrapyrrole) covalently attached to a portion of the polymer, and a bioconjugate group may be covalently attached to the portion of the dye. In some embodiments, the bioconjugate group is covalently attached to a first portion of the dye (e.g., a first end) and the polymer is covalently attached to a second portion of the dye (e.g., an opposite end).

[0083] In some embodiments, the compounds of the present invention or portions thereof have a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or conformational flexibility. The conformational flexibility of a molecular chain can be described and quantified by the "persistence length" of the compound or portions thereof (e.g., a polymer portion). In some embodiments, the persistence length of the compounds of the present invention can be on the order of the length of a given carbon-carbon bond.

[0084] The compounds of the present invention may be self-folding, such as self-folding in water and / or aqueous solution. As used herein, "self-folding" refers to a compound that transitions from a partially or completely extended or unfolded structure to a folded structure in response to contact with a solution (e.g., aqueous solution) or compound, where the folding is innate because the folding occurs spontaneously (i.e., without external control or force) in response to contact with a solution. In some embodiments, the compounds of the present invention self-fold in response to contact with water and / or aqueous solution. The compounds of the present invention may optionally self-fold in a unimeric micelle structure in response to contact with water and / or aqueous solution. The aqueous solution in which the compounds of the present invention fold may be a buffer solution, such as a phosphate buffer solution (e.g., phosphate buffered saline). In some embodiments, the aqueous solution (e.g., aqueous buffer) in which the compound of the present invention is folded may have a low ionic strength; for example, the aqueous solution may have a mu value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM. In some embodiments, the aqueous solution in which the compound of the present invention is folded may have a mu value of less than about 100 mM. In some embodiments, the aqueous solution in which the compound of the present invention is folded may contain 1 M NaCl. In some embodiments, the aqueous solution in which the compound of the present invention is folded may contain less than 1 M NaCl, for example less than about 0.75 M, less than about 0.5 M, or less than about 0.25 M NaCl. In some embodiments, the aqueous solution in which the compounds of the invention are folded contains 10 mM NaH2PO4 and 150 mM NaCl and has a pH of about 7.35.

[0085] In some embodiments, the compound of the present invention can be in the form of particles.The compound of the present invention can form particles, such as when contacting with a solution (e.g., an aqueous solution).In some embodiments, a single (i.e., one) compound can form particles.Therefore, the compound and the particle are present in about 1:1 ratio (i.e., there is one compound per particle).

[0086] The compound of the present invention may comprise one or more hydrophobic units in the core or inner region of the particle, and / or one or more hydrophilic units in the peripheral or outer region (e.g., shell) of the particle. In some embodiments, the particle has a micellar structure (e.g., unimer micelle structure). The compound of the present invention may comprise a dye, which may be bound to the polymer of the present invention, and when the compound is in a folded structure and / or in the form of a particle (e.g., unimer micelle structure), the dye may be encapsulated by a part of the compound (e.g., a part of the polymer). In some embodiments, the dye or a part thereof and one or more hydrophobic units may be present in the core or inner region of the particle, and one or more hydrophilic units may surround the dye and / or one or more hydrophobic units.

[0087] In some embodiments, the hydrophobic units present in the polymer of the present invention can be one or more hydrophobic units of formula III. In some embodiments, the one or more hydrophobic units have an alkyl (e.g., dodecyl) pendant functional group and / or are formed from a compound of formula I and / or an alkyl acrylate (e.g., dodecyl acrylate) monomer. In some embodiments, the hydrophilic units present in the polymer of the present invention can be one or more hydrophilic units of formula IV and / or are formed from a compound of formula II. In some embodiments, the one or more hydrophilic units include a non-ionic (i.e., neutral / uncharged) pendant functional group (e.g., PEG) and / or are formed from a non-ionic monomer (e.g., PEG acrylate (PEGA)). In some embodiments, the one or more hydrophilic units include an ionic (e.g., anionic, charged) pendant functional group (e.g., sulfonic acid and / or sulfonate) and / or are formed from an ionic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the hydrophilic unit is formed from at least two different monomers, such as a non-ionic (i.e., neutral / uncharged) hydrophilic monomer (e.g., PEG acrylate (PEGA)) and an ionic (e.g., anionic, charged) hydrophilic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). Those skilled in the art will appreciate that a monomer containing an acid, such as a sulfonic acid, can be present in its acid form and / or in its ionic form. In some embodiments, the acid-containing monomer is predominantly in its ionic form (i.e., greater than 50%). In some embodiments, the ionic hydrophilic monomer is an acid in a deprotonated form (e.g., deprotonated sulfonic acid acrylate) and / or in a salt form, such as a sodium sulfonate acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid as the sodium salt).

[0088] In some embodiments, when two or more different hydrophilic units are present in the polymer of the invention, the ratio of the two or more different hydrophilic units can vary, such as from about 10:1 to about 1:10. For example, in some embodiments, the polymer comprises nonionic (i.e., neutral / uncharged) hydrophilic units (e.g., formed from PEGylated methyl acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic units (e.g., formed from sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)) in a ratio of about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 (nonionic units:ionic units). In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units present in the backbone of the polymer of the present invention can vary. In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units present in the backbone of the polymer is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 (hydrophobic units:hydrophilic units).

[0089] In some embodiments, the polymer of the present invention comprises from about 1% to about 40% hydrophobic units based on the total molar amount of monomers used to prepare the polymer, and from about 60% to about 99% hydrophilic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer of the present invention comprises from about 1%, about 5%, about 10%, about 15%, or about 20% to about 25%, about 30%, about 35%, or about 40% hydrophobic units based on the total molar amount of monomers used to prepare the polymer, and from about 60%, about 65%, about 70%, about 75%, or about 80%, to about 85%, about 90%, about 95%, or about 99% hydrophilic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% hydrophobic units relative to the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%) hydrophobic units relative to the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98, or about 99% hydrophilic units relative to the total molar amount of monomers used to prepare the polymer.In some embodiments, the polymer comprises greater than about 70% (e.g., greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%) hydrophilic units relative to the total molar amount of monomers used to prepare the polymer.

[0090] The polymers of the present invention may have a weight fraction of hydrophobic units of from about 1%, about 5%, about 10%, about 15% or 20% to about 25%, about 30%, about 35% or about 40% based on the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophobic units of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% relative to the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophobic units of less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than 5%) relative to the total weight of the polymer.

[0091] The polymers of the present invention may have a weight fraction of hydrophilic units of about 60%, about 65%, about 70%, about 75% or about 80% to about 85%, about 90%, about 95% or about 99% based on the total weight of the polymer. In some embodiments, the polymers of the present invention can have a weight fraction of hydrophilic units of about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98, or about 99% based on the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophilic units of greater than about 70% (e.g., greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%) relative to the total weight of the polymer.

[0092] In some embodiments, the amount of unimeric micellar structures formed upon contact with the solution is about 50% to about 100%, about 75% to about 100%, about 85% to about 100%, or about 95% to about 100%, optionally as measured using a sizing method (e.g., dynamic light scattering (DLS) spectroscopy). In some embodiments, the amount of unimeric micellar structures formed upon contact with the solution is about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 109%, about 200%, about 201%, about 202%, about 203%, about 204%, about 205%, about 206%, about 207%, about 208%, about 209%, about 210%, about 211%, about 212 7%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. The solution in which the unimer is present can be an aqueous solution, such as an aqueous buffer, as described herein. In some embodiments, the aqueous solution in which the unimer is present can be a phosphate buffer (e.g., phosphate buffered saline). In some embodiments, the aqueous solution in which the unimer is present (e.g., an aqueous buffer) has a low ionic strength (e.g., may have a mu value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM). In some embodiments, the aqueous solution in which the unimer is present comprises 10 mM NaH2PO4 and 150 mM NaCl, and has a pH of about 7.35.

[0093] In some embodiments, dilution of a solution containing a compound of the invention in the form of a unimer micelle structure results in no loss or a loss of less than about 20% of the unimer micelle structure present in the solution compared to the amount of unimer micelle structure present in the solution prior to dilution. In some embodiments, the amount of unimer micelle structure present in the solution does not change upon dilution or changes by less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1% compared to the amount of unimer micelle structure present in the solution prior to dilution.

[0094] In some embodiments, a solution containing a compound of the invention in the form of a unimeric micellar structure contains less than about 50% aggregates (e.g., less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%). less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%. Thus, at least 50% or more of the compound may not aggregate and be in the form of a unimer micelle structure. In some embodiments, dilution of a solution containing a compound of the present invention in the form of a unimer micelle structure results in no or minimal additional aggregate formation compared to the amount of aggregates present in the solution before dilution. In some embodiments, the amount of aggregates present in a solution comprising a compound of the invention does not change upon dilution or changes by less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1% compared to the amount of aggregates present in the solution prior to dilution.In some embodiments, the diluted solution contains less than about 50% aggregates (e.g., less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%.

[0095] The compounds of the invention may have diameters in water and / or aqueous solutions ranging from about 1 nm to about 50 nm, or from about 3 nm to about 30 nm (eg, when folded into a unimeric micellar structure). In some embodiments, the compounds may have a diameter (e.g., when folded into a unimeric micellar structure) of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nm in water and / or aqueous solutions. In some embodiments, the compounds of the present invention may have a particulate form (ie, an at least partially folded structure).

[0096] In some embodiments, the compounds of the present invention are crosslinked, where optionally, the compounds are crosslinked when the compounds are in a folded structure. In some embodiments, the compounds of the present invention can be in solution (e.g., aqueous solution) and / or crosslinked with a crosslinking agent. Crosslinking the compounds of the present invention can include linking together two or more residues and / or functional groups (e.g., pendant functional groups) of one or more hydrophobic units and / or one or more hydrophilic units. Crosslinking can provide the compounds in a folded structure that cannot be unfolded without breaking one or more bonds formed by crosslinking. The degree or amount of crosslinking can be controlled, modified and / or adjusted, for example, by the amount of crosslinking agent reacted with the compound. In some embodiments, crosslinking the compounds can include the reactions and / or reactive entities (e.g., functional groups) listed in Table 1.

[0097] [Table 1]

[0098] In the compounds of the present invention, the fluorescence quantum yield of the dye when the compound is present in water and / or aqueous solution may be reduced by about 10% or less (e.g., about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less) compared to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent (e.g., in toluene). Depending on the bioconjugation reaction of the compound of the present invention to a biomolecule (e.g., protein), the fluorescence quantum yield of the dye may be the same or substantially the same (e.g., within ±20%) as the fluorescence quantum yield of the dye in water and / or hydrophobic solvent. In some embodiments, when the fluorescence quantum yield of the dye is 1.00 (theoretical maximum), a decrease of 10-fold or less (e.g., about 10-fold or less, about 9-fold or less, about 8-fold or less, about 7-fold or less, about 6-fold or less, about 5-fold or less, about 4-fold or less, about 3-fold or less, about 2-fold or less) may be acceptable.

[0099] In some embodiments, the compound of the present invention is water-soluble.The compound may have a water solubility in the range of about 1 mg / mL to about 10 mg / mL at room temperature.In some embodiments, the compound has a water solubility of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL or about 10 mg / mL at room temperature.

[0100] In some embodiments, the compounds and / or particles of the present invention are resistant to dilution. As used herein, "resistant to dilution" refers to compounds and / or particles that retain their structure and / or properties. In some embodiments, resistant to dilution refers to compounds and / or particles that retain a folded structure (e.g., unimeric micelle structure), which can be determined by measuring the diameter of the particle before and after dilution, and the diameter after dilution can remain within ±50% or less, ±40% or less, ±30% or less, ±20% or less, ±10% or less of the diameter before dilution. In some embodiments, resistant to dilution refers to compounds and / or particles that retain the fluorescence quantum yield of the dye after dilution within ±50% or less, ±40% or less, ±30% or less, ±20% or less, ±10% or less of the fluorescence quantum yield of the dye before dilution. In some embodiments, the compounds and / or particles of the invention remain in a folded structure when diluted up to 25-fold, 50-fold, 75-fold, or 100-fold, or when diluted to submicromolar concentrations.

[0101] According to some embodiments of the present invention, a method for preparing the compounds and / or compositions of the present invention is provided. In some embodiments, the method for preparing the compounds of the present invention includes polymerizing a hydrophilic monomer with a hydrophobic monomer to prepare a copolymer, attaching a dye to a first portion (e.g., a terminal portion or end portion) of the copolymer, and optionally attaching a bioconjugate group (e.g., a group capable of bioconjugation) to a second portion (e.g., another terminal portion or end portion) of the copolymer, thereby preparing the compound. The hydrophobic and hydrophilic monomers can be polymerized using any method known to those skilled in the art, such as, but not limited to, via condensation reaction (e.g., reaction with a diol and a diacid) and / or living radical polymerization (e.g., atom-transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, the polymerization of the hydrophobic monomer and the hydrophilic monomer is carried out in a manner that provides a copolymer with one or both groups of the copolymer being reactive (i.e., one or both end groups of the copolymer can enter into further polymerization or reaction), and the two end groups can be the same or different.In some embodiments, the polymerization of the hydrophobic monomer and the hydrophilic monomer is via living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., bromide initiator), a catalyst (e.g., ruthenium catalyst), and optionally, a cocatalyst, to prepare a copolymer.In some embodiments, the polymerization of the hydrophobic monomer and the hydrophilic monomer is via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., a thiocarbonylthio compound).

[0102] In some embodiments, the step of attaching the dye to the first part of the copolymer may include reacting the monomer comprising the dye with a hydrophobic monomer and / or a hydrophobic unit and / or a hydrophilic monomer and / or a hydrophilic unit. Thus, in some embodiments, the step of attaching the dye to the copolymer may occur during or after the polymerization step. In some embodiments, the method includes reacting the monomer comprising the dye with one or more (e.g., two or three) hydrophobic monomers and / or hydrophobic units and / or one or more (e.g., two or three) hydrophilic monomers and / or hydrophilic units during the step of polymerizing the hydrophobic monomer and the hydrophilic monomer. In some embodiments, the polymerization of one or more hydrophobic monomers and one or more hydrophilic monomers occurs via living radical polymerization (e.g., ATRP) in the presence of an initiator, and the initiator comprises the dye. In some embodiments, polymerization of one or more hydrophobic monomers and one or more hydrophilic monomers occurs via living radical polymerization (e.g., RAFT) in the presence of a radical initiator and a RAFT agent, where, optionally, the RAFT agent includes the dye.

[0103] Exemplary terminal functional groups on the copolymer where the copolymer is available for immediate dye attachment or bioconjugation reactions include, but are not limited to, the terminal functional groups set forth in Table 2. These terminal functional groups are not pendant functional groups, but may be present at either end of the copolymer.

[0104] [Table 2]

[0105] Some functional groups may be unstable under certain polymerization conditions. Thus, in some embodiments, functional groups may be introduced in protected form. As a result, these functional groups may be available for dye attachment or bioconjugation reactions upon deprotection. Exemplary protected forms of certain functional groups include, but are not limited to, those listed in Table 3.

[0106] [Table 3]

[0107] In some embodiments, a portion of the copolymer (e.g., the terminal or end portion) may contain a halogen group (e.g., Cl, Br, I). The halide portion of the copolymer may be derivatized with a nucleophile or end-capping reagent to generate functional groups for dye attachment or bioconjugation reactions. In some embodiments, a portion of the copolymer (e.g., the terminal or end portion) may have a thiol group, which may be derivatized with a reagent containing a thiol-reactive group to generate functional groups for dye attachment or bioconjugation reactions. Examples of thiol-reactive groups include, but are not limited to, halide (e.g., bromine, chlorine, iodine), alkyne, aldehyde, vinyl ketone, and / or maleimide functional groups. All functional groups listed in Tables 2 and 3 are compatible with these strategies, and additional exemplary functional groups include, but are not limited to, those listed in Table 4.

[0108] [Table 4]

[0109] Polymerizing the hydrophobic monomer and the hydrophilic monomer (optionally via ATRP or RAFT) can include polymerizing the hydrophobic monomer and the hydrophilic monomer in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 (one or more hydrophobic monomers: one or more hydrophilic monomers). In some embodiments, the ratio can be from about 1:1 to about 1:3 or about 1:6. In some embodiments, the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl acrylate) and / or the hydrophilic monomer is a glycol acrylate (e.g., PEG acrylate). In some embodiments, one or more hydrophobic monomers are polymerized (optionally via RAFT or ATRP) with two or more different hydrophilic monomers in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 (one or more hydrophobic monomers:one or more hydrophilic monomers). For example, in some embodiments, a first hydrophilic monomer can be ionic (e.g., a sulfonic acid acrylate monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate monomer), and a second hydrophilic monomer can be non-ionic (e.g., a glycol acrylate (e.g., a PEGylated methyl acrylate)). The ratio of the first hydrophilic monomer to the second hydrophilic monomer can vary (e.g., the ratio of the first hydrophilic monomer:the second hydrophilic monomer can be about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or about 1:6).

[0110] Exemplary catalysts that can be used in the method of the present invention include, but are not limited to, ruthenium complexes, iron complexes, copper complexes, nickel complexes, palladium complexes, rhodium complexes and rhenium complexes.Exemplary ruthenium complexes include, but are not limited to, dichlorotris(triphenylphosphine)ruthenium(II) [RuCl2(PPh3)3], pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride [RuCp*Cl(PPh3)2], chloro(cyclopentadienyl)bis(triphenylphosphine)ruthenium [RuCpCl(PPh3)2], dihydridotetrakis(triphenylphosphine)ruthenium(II) [RuH2(PPh3)4] and dichloro(p-cymene)ruthenium(II) dimer. Exemplary iron complexes include, but are not limited to, dichlorobis(triphenylphosphine)iron(II) [FeCl2(PPh3)2], bromo(cyclopentadienyl)dicarbonyliron(II) [FeCpBr(CO)2], and cyclopentadienyliron dicarbonyl dimer. In some embodiments, copper complexes generated in-situ with copper salts and ligands may be used, and exemplary copper salts include, but are not limited to, copper chloride, copper bromide, copper triflate, copper hexafluorophosphate, copper acetate, and the like. Exemplary nitrogen-based ligands include, but are not limited to, 2,2'-bipyridine and its derivatives, 1,10-phenanthroline and its derivatives, sparteine ​​and other diamines, and terpyridine and its derivatives. Exemplary nickel complexes include, but are not limited to, dibromobis(triphenylphosphine)nickel(II) [NiBr2(PPh3)2] and tetrakis(triphenylphosphine)nickel [Ni(PPh3)4]. An exemplary palladium complex is tetrakis(triphenylphosphine)palladium [Pd(PPh3)4]. An exemplary rhodium complex is tris(triphenylphosphine)rhodium bromide. An exemplary rhenium complex is dioxobis(triphenylphosphine)rhenium iodide. In some embodiments, the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

[0111] A co-catalyst may optionally be present in the methods of the invention, such as the step of polymerizing the hydrophobic monomer and the hydrophilic monomer. In some embodiments, a co-catalyst may be present and may be 4-(dimethylamino)-1-butanol.

[0112] In some embodiments, the method of the present invention includes hydrolyzing the copolymer, optionally in the presence of trifluoroacetic acid and water, to provide a formyl group at a first portion (e.g., a first end) of the copolymer. The method may include reacting the dye with the formyl group of the copolymer, optionally forming a hydrazone bond between the dye and the copolymer via aldehyde-hydrazide chemistry, thereby attaching the dye to the first portion of the copolymer. In some embodiments, a biomolecule may be attached by reacting the formyl group with an amine group in a bioconjugate group via reductive amination.

[0113] In some embodiments, the method of the present invention includes reacting the copolymer with mercaptoacetic acid and triethylamine to provide a carboxymethyl thioether group at a second portion (e.g., a second terminus) of the copolymer. The carboxymethyl thioether group can be derivatized to provide an N-hydroxysuccinimide ester at the second portion of the copolymer. A biomolecule (e.g., avidin) can be attached to the N-hydroxysuccinimide ester at the second portion of the copolymer.

[0114] In some embodiments, the methods of the present invention include reacting the copolymer with sodium azide to provide an azide group, and optionally attaching a dye to the azide group via copper-catalyzed azide-alkyne chemistry.

[0115] In some embodiments, the method of the present invention includes RAFT polymerization. In some embodiments, RAFT polymerization occurs in the presence of a radical initiator (e.g., AIBN) and a RAFT agent, such as a thiocarbonylthio compound. Additional examples of RAFT agents include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates, and / or xanthates.

[0116] In some embodiments, the method of the present invention comprises cleaving the thiocarbonylthio functionality present at the terminal end of the copolymer obtained using RAFT polymerization. Such cleavage can occur using any common method known in the art. For example, in some embodiments, the thiocarbonylthio functionality is cleaved via aminolysis, for example in the presence of ethanolamine, to give a free thiol. In some embodiments, the free thiol can be attached to a dye that includes maleimide functionality, thereby attaching the dye to the first portion (e.g., the terminal end) of the copolymer. In some embodiments, a biomolecule can be attached to the free thiol group of the first portion (e.g., the terminal end). In some embodiments, a biomolecule can be attached to the opposite terminal end of the polymer.

[0117] According to some embodiments, the compounds and / or compositions of the present invention can be used in flow cytometry.Flow cytometry is known and described, for example, in U.S. Patent Nos. 5,167, 5,915,925, 6,248,590, 6,589,792 and 6,890,487.In some embodiments, the particle to be detected, for example, cells, is labeled with a luminescent compound, for example, the compound of the present invention, for detection.Labeling can be performed by any suitable technique, for example, by binding the luminescent compound (for example, the compound of the present invention) to the particle or cell, for example, via an antibody that specifically binds to the particle or cell, by uptake or internalization of the luminescent compound into the cell or particle, by non-specific absorption of the luminescent compound into the cell or particle, etc. As such luminescent compounds, the compounds described herein may be useful in flow cytometry, which techniques (fluorescent activated cell sorting, or FACS) may be performed according to known techniques or modifications thereof that will be apparent to those of skill in the art based on the present disclosure.

[0118] In some embodiments, a method of detecting cells and / or particles using flow cytometry is provided, the method comprising labeling cells and / or particles with a compound of the invention and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0119] In some embodiments, a method of detecting tissues and / or factors (e.g., cells, infectious agents, etc.) in a subject is provided, the method comprising administering a compound and / or composition of the invention to a subject, where optionally the compound associates with the tissues and / or factors, and detecting the compound in the subject, thereby detecting the tissues and / or factors.

[0120] In some embodiments, methods of using the compounds of the present invention in photodynamic therapy (PDT) and / or photodynamic inactivation (PDI) are provided. Photodynamic therapy (PDT) is a form of phototherapy that includes light and photosensitizing chemicals used in combination with molecular oxygen to induce cell death (phototoxicity). PDT can be used to kill microbial cells, including bacteria, fungi, and viruses. PDT can also be used to treat cancer. When light energy is administered in photodynamic therapy (PDT) to destroy tumors, various forms of energy are within the scope of the present invention as will be understood by those skilled in the art. Such forms of energy include, but are not limited to, heat, sound, ultrasound, chemicals, light, microwaves, ionization (e.g., X-rays and gamma rays), mechanical, and / or electrical. For example, sonodynamically induced or activated agents include, but are not limited to, gallium-porphyrin complexes (see Yumita et al., Cancer Letters 112:79-86 (1997)), other porphyrin complexes such as protoporphyrin and hematoporphyrin, (see Umemura et al., Ultrasonics Sonochemistry 3:S187-S191 (1996)); other anti-cancer drugs such as daunorubicin and adriamycin used in the presence of ultrasound therapy (see Yumita et al., Japan J. Hyperthermic Oncology 3(2):175-182 (1987)).

[0121] Examples of treatment areas for PDT and / or PDI include, but are not limited to, the following:

[0122] (i) Treatment of opportunistic infections. The compounds, compositions and / or methods of the present invention may be useful for PDT of opportunistic infections, particularly soft tissue. For antibacterial treatment (via PDT) of infections, particularly wound infections, the infectious organisms may include (as non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa and / or Escherichia coli. In hospital-acquired infections, Pseudomonas aeruginosa is responsible for 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the subject is an immunocompromised subject, for example, a subject suffering from AIDS and / or a subject undergoing treatment with an immunosuppressant.

[0123] (ii) Treatment of burns. Infections caused by Staphylococcus aureus and gram-positive bacteria in general are particularly prevalent in burns (Lambrechts, 2005). Multidrug resistance of Staphylococcus aureus presents a significant medical challenge. In this regard, the compounds, compositions and / or methods of the present invention may be useful for the treatment of opportunistic infections in burns.

[0124] (iii) Sepsis. The compounds, compositions and / or methods of the invention may be useful for PDT treatment of subjects suffering from opportunistic infection with Vibrio vulnificus. Vibrio vulnificus, a gram-negative bacterium, causes primary sepsis, wound infections and / or gastrointestinal disease in humans.

[0125] (iv) Ulcers. The compounds, compositions and / or methods of the invention may be useful for PDT treatment of bacteria causing ulcers (Helicobacter pylori). In the clinic, treatment may be performed in any suitable manner, for example by inserting a fiber optic cable (similar to an endoscope but equipped for the delivery of red or near infrared light) into the stomach and / or into the diseased area.

[0126] (v) Periodontal Disease. The compounds, compositions and / or methods of the present invention may be useful in PDT for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by the overgrowth of bacteria, such as the gram-negative anaerobic bacterium Porphyromonas gingivalis. As with many PDT treatments, a targeting or solubilizing entity combined with the photoactive species is essential to properly deliver the photoactive species to the desired cells. Oral pathogens that may be targeted include, but are not limited to, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. Polymorphum, Actinomyces viscosus, and streptococci. For such uses, the compounds and / or compositions of the present invention may be applied topically (e.g., as a mouthwash or mouthrinse) and then photoadministered with an external device, an oral appliance, or a combination thereof.

[0127] (vi) Atherosclerosis. The compounds, compositions and / or methods of the present invention may be useful in PDT to treat vulnerable atherosclerotic plaque. Without wishing to be bound by a particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade thin layers of collagen in coronary arteries, resulting in thrombosis, which is often fatal (Demidova and amblin, 2004). Such bacteriochlorins that target inflammatory macrophages may be useful for PDT of vulnerable plaque.

[0128] (vii) Cosmetic and dermatological applications. The compounds, compositions and / or methods of the present invention may be useful in PDT to treat a wide range of cosmetic dermatological problems, such as hair removal, treatment of psoriasis and / or removal of skin discoloration. Ruby lasers are currently used for hair removal. In many laser treatments, melanin is the photosensitized chromophore. Such treatments work reasonably well for fair-skinned people with black hair. The compounds, compositions and / or methods of the present invention may be used as near-infrared sensitizers for hair removal, which allows targeting chromophores with more specific and / or sharper absorption bands.

[0129] (viii) Acne. The compounds, compositions and / or methods of the present invention may be useful in PDT to treat acne. Acne vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands. Approximately 80% of young people are affected. Again, growing bacterial resistance to antibiotic treatments has led to a surge in acne that is difficult to treat. Current PDT treatments for acne typically rely on the addition of aminolevulinic acid, which is converted to free base porphyrin in the hair follicle or sebaceous gland. The compounds and / or compositions of the present invention may be administered to a subject topically or parenterally (e.g., by subcutaneous injection), depending on the particular condition.

[0130] (ix) Infectious diseases. The compounds, compositions and / or methods of the present invention may be useful in PDT to treat infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which occur widely in the Mediterranean and Middle Eastern regions, are currently treated with arsenic-containing compounds. PDT has recently been used to reasonable effect in human subjects in at least one case. The use of the compounds and / or compositions of the present invention is similarly useful and potentially offers advantages, such as ease of synthesis and better spectral absorption properties.

[0131] (x) Tissue sealants. The compounds, compositions and / or methods of the present invention may be useful in PDT as tissue sealants in subjects in need thereof. Light-activated tissue sealants are attractive for sealing wounds, joining tissues and / or closing defects in tissues. There are many applications where sutures and / or staples are undesirable, and the use of such mechanical sealing methods often results in infection and / or scarring.

[0132] (xi) Neoplastic diseases. The compounds, compositions and / or methods of the invention may be useful in PDT to treat neoplastic diseases and / or cancers, including, for example, skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque stage cutaneous T-cell lymphoma and / or Kaposi's sarcoma.

[0133] During photodynamic therapy, the compound of the present invention is administered to a subject in need thereof (e.g., a subject having any of the diseases described above). The administered compound can bind to diseased tissue present within the subject, and exposure of the subject to a light source that emits appropriate light at an appropriate wavelength and intensity can activate the compound within the diseased tissue (e.g., release reactive oxygen species (ROS)), thereby treating the diseased tissue, optionally without affecting healthy tissue. For example, in some embodiments, the diseased tissue is a hyperproliferative tissue (e.g., tumor).

[0134] In some embodiments, a method of using the compounds of the present invention in photoacoustic imaging is provided. According to some embodiments, the method of the present invention includes a method of performing photoacoustic imaging. Photoacoustic imaging (PAI) is attractive in that it does not rely on optical emission for detection (Haisch, C., Quantitative analysis in medicine using photoacoustic tomography. Anal. Bioanal. Chem. 2009, 393, 473-479; Cox, B.; Laufer, JG; Arridge, SR; Beard, PCQuantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Optical emission can be affected by light scattering. In PAI, laser irradiation (for example, optionally performed using non-ionizing laser pulses) is followed by thermoelastic expansion and ultrasonic pressure waves. Detection of the ultrasonic pressure waves can be achieved via conventional ultrasound detectors. Essentially, ultrasound imaging can be performed with laser input. It is noteworthy that, in contrast to X-ray imaging methods, PAI does not rely on ionizing radiation.

[0135] Methods of the invention may include administering a compound and / or composition of the invention to a subject, where optionally, the compound associates with tissue and / or cells in the subject; irradiating at least a part or portion of the subject with a laser, where optionally, said part or portion of the subject comprises a compound of the invention; and imaging at least said part or portion of the subject, where optionally, said imaging comprises ultrasound imaging.

[0136] PAI can be performed without utilizing any exogenous contrast agent or chemical probe. In such a case, the distinct absorption of endogenous chromophores in native tissue produces a distinct signal. Absorption by hemoglobin, for example, facilitates the depiction of the presence of blood vessels. However, the molar extinction coefficient of hemoglobin is low and may be insufficient for the distinct depiction of deep tissue. In such a case, the use of contrast agents is very attractive. In some embodiments, the compounds of the present invention are used as contrast agents in PAI and / or include dyes that can be used as contrast agents in PAI.

[0137] Various substances have been investigated for use as contrast agents in PAI. Exemplary dyes for use in PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconducting polymers, and naphthalocyanines (Chitgupi, U.; Lovell, J. F. Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging. Biomed. Eng. Lett. 2018, 8, 215-221; de la Zerda, A., et al., Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 2011, 6, 346-369). In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention have the following photophysical properties: following absorption of light, the dye / compound instantly and quantitatively relaxes to the ground state without emission of light or formation of any significant lifetime metastable state. In other words, the yield of internal conversion (i.e., radiationless decay) must be quantitative, and ideally, the rate of internal conversion must be very fast, with the excited state lifetime less than 1 picosecond. This description essentially represents the "optical-to-acoustic conversion efficiency" (Cheng, K.; Cheng, Z. Near infrared receptor-targeted nanoprobes for early diagnosis of cancers. Curr. Med. Chem. 2012, 19, 4767-4785) from the perspective of molecular photophysics. The attraction of such rapid and quantitative internal conversion is that it converts all absorbed light into heat, i.e., thermal expansion, which generates ultrasound.Some research groups refer to such contrast agents as "sonochromes" (Duffy, MJ, et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49-61), to distinguish them from the more commonly known lumichromes or fluorochromes or luminophores, all of which refer to the emission of light following the absorption of incident light. In some embodiments, the compounds of the present invention are and / or include sonochromes.

[0138] In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb light in the red or near-infrared region (NIR). For example, in some embodiments, the compounds of the present invention can be used to image deep tissues, where absorption in the red or near-infrared region (NIR) is desired, since this region presents an optical window that allows the transmission of light. At shorter wavelengths, absorption by endogenous chromophores (e.g., hemoglobin, melanin) can occur. At longer wavelengths, scattering of light by overtone vibrational bands of water can be observed. In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb in the red or NIR, and the molar extinction coefficient is as large as possible, providing excellent sensitivity, e.g., 1,000 M -1 cm -1 , 10,000M -1 cm -1 , 100,000M -1 cm -1 or greater. In some embodiments, the chlorin produces a molar extinction coefficient value of about 10,000 M -1 cm -1 ~About 100,000M -1 cm -1 Q in the range y The molar extinction coefficients of the bands are shown. In some embodiments, the bacteriochlorin has a molar extinction coefficient of about 50,000 M -1 cm-1 ~About 200,000M -1 cm -1 Q in the range y The band molar extinction coefficients are shown.

[0139] In some embodiments, the method of the present invention provides multi-wavelength multiplexing. Multi-wavelength multiplexing can be achieved by using two or more absorbers as PAI contrast agents, all of which exhibit quantitative (or nearly quantitative) internal conversion, where the two or more absorbers are two or more different compounds of the present invention. The two or more different compounds of the present invention can have largely non-overlapping absorption bands. Multiplexing can be achieved by sweeping an incident light source (e.g., a laser) across the NIR and red spectral regions, with the detection of the resulting ultrasound in response to the sequential absorption of each spectrally distinct contrast agent. Alternatively, a set of multiple lasers can be used, with each laser dedicated to a different PAI contrast agent.

[0140] In some embodiments, the dye present in the compound of the invention comprises a chlorin or bacteriochlorin, where optionally the compound is used in the method of the invention for PAI. Chlorins and / or bacteriochlorins have strong and sharp long wavelength (Q y Given their long range absorption bands, chlorins and / or bacteriochlorins may be ideal for photoacoustic imaging. The chlorins and / or bacteriochlorins may be modified to produce high yields of internal conversion and / or packaged in a manner that achieves solubilization in aqueous media.

[0141] For example, tetrapyrrole macrocycles that are fluorescent in free base form can be rendered non-fluorescent by metallation with a suitable metal. Tetrapyrroles include porphyrins and hydroporphyrins, the latter including chlorins and bacteriochlorins. Given the extensive research into the preparation and study of metallotetrapyrroles over nearly a century, a true "periodic table of metallotetrapyrroles" exists. Metals that give non-luminescent tetrapyrrole chelates are well known (see, for example, Gouterman, M. Optical spectra and electronic structure. In The Porphyrins; Dolphin, D. (Ed.), Vol. III, Academic Press: New York, 1978, pp1-165). Examples of metals that can give non-luminescent tetrapyrrole chelates (valences not shown for clarity) include, but are not limited to, Fe, Co, Ni, Cu, Zr, Ru, and lanthanides. In some embodiments, the dye present in the compounds of the present invention is an iron-containing tetrapyrrole macrocycle. Iron may be particularly attractive given its presence as a natural component in human metabolism, the vast amount of research that has been devoted to iron tetrapyrroles (given the fact that heme is an iron chelate of protoporphyrin IX), and the very short excited state lifetime of iron porphyrins. In some embodiments, the compounds of the present invention comprise an iron chlorin or iron bacteriochlorin. In some embodiments, the methods of the present invention comprise administering to a subject a compound of the present invention comprising an iron chlorin or iron bacteriochlorin as a PAI contrast agent and performing photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocycle that comprises copper (e.g., Cu(II)). In some embodiments, the dye present in the compounds of the present invention comprises copper (e.g., Cu(II)) and is optionally used for photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention comprises iron (e.g., Fe(II)) and can optionally be used for oxygen sensing.

[0142] In some embodiments, the compounds of the present invention include Fe(II) tetrapyrroles that are sterically hindered and / or do not form mu-oxo dimers of Fe(III) tetrapyrroles. In some embodiments, the compounds of the present invention include Fe(III) tetrapyrroles. It should be noted that Fe(II) tetrapyrroles can coordinate to molecular oxygen and, in the absence of steric hindrance, can undergo chemical reactions to produce mu-oxo dimers of Fe(III) tetrapyrroles. In contrast, Fe(III) tetrapyrroles do not coordinate to molecular oxygen and do not undergo mu-oxo dimer formation. Fe(III) tetrapyrroles are the preferred oxidation state of iron tetrapyrroles, upon formation under aerobic conditions. A variety of long-established methods are available for the formation of Fe(III) tetrapyrroles and for the conversion of Fe(II) tetrapyrroles to Fe(III) tetrapyrroles.

[0143] The free base tetrapyrrole can provide some fluorescence (e.g., up to about 10% quantum yield), some triplet state formation (e.g., up to about 70% quantum yield), and the rest is internal conversion (e.g., up to about 20% quantum yield). As mentioned above, a convenient way to achieve about 100% quantum yield for internal conversion (i.e., radiationless decay) is to metallize the tetrapyrrole with a metal that rapidly and essentially quantitatively relaxes the excited state to the ground state by one or more mechanisms. An alternative approach to promote internal conversion versus radioactive decay (i.e., fluorescence) and intersystem crossing (i.e., triplet state formation) is to attach appropriate substituents to the tetrapyrrole. Typical substituents are those that produce spin-orbit coupling, such as heavier halogen atoms, including bromine, iodine, and astatine. Thus, in some embodiments, the introduction of one or more halogen atoms in the dyes and / or compounds of the present invention can be used alone or together with metals that provide limited emission by themselves, thereby providing rapid and essentially quantitative relaxation to ground state. Such metals include many of the metals in the periodic table. The method of metallation of tetrapyrroles is well known (Buchler, JWStatic coordination chemistry of metalloporphyrins.In Porphyrins and Metalloporphyrins; Smith, KM(Ed.),1975,Elsevier Scientific Publishing Co.:Amsterdam,pp157-231;Sanders, JKM,et al.,Axial coordination chemistry of metalloporphyrins.In The Porphyrin Handbook;Kadish, KM;Smith, KM;Guilard, R.(Eds.),Vol.3,2000,Academic Press:San Diego,pp1-48). A wide variety of heavy atom substituted arenes are excellent candidates for use as PAIs according to the methods of the invention, since heavy atoms attached to arenes are well known to result in rapid relaxation of excited states.In some embodiments, the compounds of the present invention include tetrapyrroles (e.g., tetrapyrroles with heavy atom substituents on the periphery of a macrocycle and / or a centrally chelated metal that renders them non-emissive). Such tetrapyrroles (e.g., chlorins or bacteriochlorins) can provide many possible narrow band absorptions across the red and NIR spectral regions.

[0144] While various mechanisms are described that allow the excited state of a compound to rapidly and essentially quantitatively return to the ground state, the invention is not limited thereto, and other mechanisms known in the art may be used. For example, such mechanisms may result from: (1) a high rate of internal conversion relative to the rate of radioactive decay and intersystem crossing; (2) a high rate of intersystem crossing relative to the rate of radioactive decay and internal conversion, followed by immediate and non-radiative decay of the excited multiplet state to the ground state; and / or (3) a high rate of charge transfer relative to all other rates of depopulation of the excited state, followed by charge recombination quantitatively bringing it to the ground state. Another example is conformationally distorting a macrocycle from its intrinsic planarity. Other mechanisms are known to those skilled in the art. Regardless of the mechanism, tetrapyrroles can be created that exhibit excited states with very short lifetimes and essentially quantitative relaxation to the ground state using established methods known to those skilled in the art. Rapid and nearly quantitative relaxation to the ground state can provide what is referred to herein as a "non-emissive" molecular entity that can be used in PAIs. Another example is conformationally distorting a macrocycle from its inherent planarity. Other mechanisms are known to those skilled in the art. Regardless of the mechanism, established methods known to those skilled in the art can be used to create tetrapyrroles that exhibit excited states with very short lifetimes and essentially quantitative relaxation to the ground state. The rapid and nearly quantitative relaxation to the ground state can provide what is referred to herein as a "non-emissive" molecular entity that can be used in PAIs.

[0145] The compounds of the present invention may optionally package a metallotetrapyrrole for use in PAIs. The metallotetrapyrrole may have a bioconjugable group that can be used to attach the metallotetrapyrrole to the polymers described herein to prepare the compounds of the present invention. Thus, the compounds of the present invention may include a single metallotetrapyrrole. In some embodiments, the compounds of the present invention may optionally maintain the inherent spectral characteristics of the dye (e.g., absorption spectrum, fluorescence spectrum, fluorescence quantum yield, etc.) without modification due to interactions with external entities, such as other dyes and / or biological materials (e.g., cellular components, proteins, etc.), by packaging the dye within a portion of the compound (e.g., within a polymer portion). The inclusion of a single dye (e.g., Fe(III) tetrapyrrole) within the compounds of the present invention may preserve the inherent absorption spectrum of the dye.

[0146] According to some embodiments, the dye present in the compound of the present invention may be a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity), where optionally the compound is used in a PAI. The dye may have a rapid optical to acoustic conversion. In some embodiments, the dye is a non-luminescent molecular entity and has a short excited state lifetime, where optionally the excited state lifetime is in the sub-picosecond range. Upon irradiation, the excited state may quickly return to the ground state and release heat. The heat generates a "sound wave" that can be detected by a microphone. The structure of the compound of the present invention may protect the dye from the physiological environment and / or be suitable for use in a method of performing a PAI.

[0147] In some embodiments, the compounds of the present invention provide a means for packaging hydrophobic chromophores, which can allow high solubility in water to be achieved, and / or a means for preventing the chromophores from aggregating, which can alter the appearance of the absorption band, including wavelength position, molar extinction coefficient, and band width.

[0148] The invention is described in more detail in the following non-limiting examples.

[0149] Working Example

[0150] Example 1 - Single polymer encapsulation of a single hydrophobic chromophore Random copolymers with pendant PEGylated chromophores and polymerized micelles containing hydrophobic fluorophores were studied. Finally, a design was identified with heterotelechelic, amphiphilic, random copolymers derived from two acrylate monomers (hydrophilic (pendant PEG-6) and hydrophobic (dodecyl) monomers in a 3:1 ratio) via living radical polymerization (via RuCp*Cl(PPh3)2, 4-(dimethylamino)-1-butanol and acetal-substituted initiators in ethanol at 40 °C). Hydrolysis of the acetal and subsequent reaction with a hydrophobic chlorin-hydrazide afforded polymers with a single chlorin-hydrazone (i.e., foldamers or single-chain nanoparticles, abbreviated as scNp). Examination of the chlorin-polymers in aqueous solution revealed sharp absorption / fluorescence bands and undiminished fluorescence quantum yields compared to chlorin in toluene. The approach separates the chromophore selection and the aqueous solubilization strategy into separate areas, the latter of which is currently very simple to implement.

[0151] Three hydrophobic dye-labeled amphiphilic copolymers with self-folding properties, F1-F3, were synthesized and spectroscopically characterized. The conformational features of the hydrophobic dyes and polymer backbone are shown in Scheme 1 below. The amphiphilic copolymer is composed of a 3:1 ratio of hydrophilic segments (PEG segments) and hydrophobic segments (dodecyl segments) with a molecular weight of about 120 kDa. As a random block copolymer, the copolymer in water can self-fold to form a hydrophobic core and encapsulate the hydrophobic dye, thereby protecting it from aggregation. Three hydrophobic dyes (i.e., BODIPY, chlorine, and phthalocyanine, differing in molecular size and absorption wavelength (540, 640, and 700 nm, respectively)) were loaded onto the same polymer backbone and subjected to spectroscopic measurements. Without wishing to be bound by any particular theory, the resulting distinct fluorescent properties of dye-loaded copolymers in water suggest that the effectiveness of dye encapsulation may depend on the molecular size of the dye and the length of the copolymer backbone.

[0152] [ka]

[0153] Synthesis of hydrophobic fluorophores. Generally, the dye-hydrazides used here for dye attachment are prepared from the corresponding carboxylic acid esters via amide formation. Treatment of the activated carboxyl species, BODIPY-NHS ester 1, with hydrazine hydrate afforded the desired BODIPY-hydrazide D1 in 40% yield (Scheme 2 below).

[0154] [ka]

[0155] Iodochlorin 2 was quantitatively converted to methyl ester 3 via carbonyl insertion in the presence of Pd(PPh3)4, methanol and carbon monoxide (Scheme 3 below). The methyl ester 3 was then treated with hydrazine hydrate under reflux conditions to produce the desired chlorin-hydrazide D2 in 83% yield. It was noted that the reaction needed to be carried out at concentrations below 50 mM, as more concentrated solutions would result in the reduction of D2 to the corresponding bacteriochlorin.

[0156] [ka]

[0157] The preparation of phthalocyanine-hydrazide D3 was more laborious due to the limited solubility of the macrocycle. Ethynylphthalocyanine 4 was coupled with methyl 3-(4-bromophenyl)propanoate in the presence of Pd(OAc)2 / P(o-tol)3 to give the methyl ester 5 in 13% yield (Scheme 4 below). Again, the low solubility of the macrocycle in the reaction system explains the low yield of the Sonogashira coupling reaction. The methyl ester 5 was then treated with hydrazine in a mixture of toluene and methanol to produce the desired hydrazide D3.

[0158] [ka]

[0159] Synthesis of copolymers. Living radical polymerization of monomers PEGA and LA was carried out with reported initiator 6 in the presence of RuCp*Cl(PPh3)2 and 4-dimethylaminobutanol in a 3:1 ratio (Scheme 5 below). The resulting copolymer 7 is heterotelechelic with an acetal at one end and a bromine atom at the other end. The two functional groups were derivatized for dye attachment and introduction of bioconjugable handles, respectively. The bromine atom in 7 was replaced with mercaptoacetic acid to provide a carboxyl group at the end of the copolymer open for bioconjugation. Hydrolysis of the acetal end under acidic conditions resulted in formyl copolymer 8. This copolymer 8 served as a platform for dye conjugation to generate copolymers F1-F3 loaded with the desired dyes via treatment with hydrazides D1-D3, respectively.

[0160] [ka]

[0161] SEC analysis. Taking F2 as an example, analytical SEC was used to monitor the process of the dye-binding reaction. The SEC trace shown in Figure 2 shows that the size increases upon the binding of chlorin onto the copolymer. Also, the molecular weight of copolymer 7 was found to be 1.2 × 10 based on the SEC analysis. 5 Estimated in g / mol.

[0162] Measurement of absorption and emission spectra. The copolymers F1-F3 loaded with the dyes of interest were then subjected to investigation of their spectroscopic properties in both organic solvents and aqueous solutions. The spectra are shown in FIG. 3. For both F1 (BODIPY loaded, FIG. 3, panel A) and F2 (chlorin loaded, FIG. 3, panel B), the absorption spectra of the samples in aqueous solutions at μM concentrations are comparable to those in organic solvents. Conjugation to the 120 kDa amphiphilic copolymer dramatically increases the water solubility of BODIPY D1 and chlorin D2 without strongly perturbing the spectroscopic properties. The emission bands for F1 and F2 in water remain the same as those measured in organic solvents, indicating minimal dye-dye interactions involved in aqueous solutions of F1 and F2 at μM concentrations. Nevertheless, as the molecular size of the dyes is the largest, the phthalocyanine-doped copolymer F3 exhibits an absorption spectrum in water that is completely different from that in toluene (Figure 3, panel C) and has completely quenched fluorescence. This negative result may be due to the inappropriate size of the copolymer backbone. Larger polymers may be required to encapsulate large hydrophobic chromophores such as phthalocyanine D3.

[0163] Fluorescence quantum yield. Fluorescence quantum yield values ​​were also measured for F1-F3 in water at room temperature. The data, along with other spectroscopic data, are summarized in Table 5. Taking the chlorine-linked copolymer F2 as an example, the dye-copolymer conjugate exhibited a fluorescence quantum yield of 0.18 at μM concentration (entry 6), which is similar to the value from the dye D2 alone in CH2Cl2 solution (0.19, entry 4). Similar results were observed for the BODIPY-labeled copolymer F1 (Φf=0.058.8, entry 3) and the BODIPY dye D1 (0.065, entry 1). These comparisons show the absence of dye-dye quenching due to aggregation for F1 and F2 in μM aqueous solutions. The results indicate amphiphilic copolymers as a successful platform for encapsulating hydrophobic chromophores in water when the polymer chain length is appropriate. However, as mentioned above, the phthalocyanine-labeled copolymer has a completely quenched fluorescence. Longer polymer chains may be more effective for encapsulating larger chromophores such as D3, and smaller phthalocyanine skeletons (e.g., with methyl instead of heptyl as the peripheral group) have been successfully encapsulated in the current length copolymers.

[0164] [Table 5]

[0165] Experimental Department

[0166] General methods. All commercially obtained chemicals were used as received unless otherwise stated. Reagent grade solvents (CH2Cl2, THF, methanol) and HPLC grade water were used as received. NMR data were measured in CDCl3 solutions unless otherwise stated. Compounds 1, 2, and 4, which are not commercially available, were prepared according to literature procedures. Analytical SEC experiments were performed using a Plgel 10000 Å SEC column and eluted with ACS grade THF (stabilized with 400 ppm BHT) at 35 °C at a flow rate of 1 mL / min. Samples were detected using an Agilent 1260 infinity refractive index detector. Absorption spectra were measured on an Agilent 8453 and Shimadzu UV1800 instruments using dilute (μmolar) solutions of compounds in UV-transparent (e.g., quartz) cuvettes and solvent blanks at room temperature.

[0167] 2-[6-(N-aminocarbamoyl)hex-1-yn-1-yl]-8-mesityl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (D1). A solution of 1 (9.0 mg) in THF (500 μL) was treated with hydrazine hydrate (5.3 μL) at room temperature for 30 min. The solution was then concentrated and chromatographed (silica gel, CHOH / acetic acid=9:1) to give a red solid (3.0 mg, 39%): 1 H NMR(DMSO-d6,300MHz) δ 8.85(br,1H),7.99(s,1H),7.94(s,1H),6.95(s,2H),6.76(d,J=4.2Hz,1H),6.72(s,1H),6.53(d,J=4. MALDI-MS Observed value 449.1[(M+H) + ],429.2[(MF) + ], calculated value 448.2 (M = C 25 H 27 BF2N4O).

[0168] 10-Mesityl-5-(4-methoxycarbonyl)phenyl-18,18-dimethylchlorin (3). Toluene and methanol were degassed by bubbling with argon for 1 h. (20 mg, 0.030 mmol, 1.0 equiv.) and Pd(PPh3)4 (3.5 mg, 3.0 μmol, 0.10 equiv.) were placed in a conical vial with a rubber septum and then evacuated under high vacuum. The vial was then backfilled with argon. This evacuation-purging process was repeated three times. Degassed toluene (0.50 mL) and methanol (0.50 mL) and triethylamine (21 μL, 0.15 mmol, 5.0 equiv.) were added to the vial under argon. The solution was degassed again by three freeze-pump-thaw cycles. The vial was evacuated under high vacuum at 77 K and then backfilled with carbon monoxide. A CO-filled balloon was also connected to the vial to provide extra pressure. The solution was stirred at 65° C. for 23 h, concentrated, and chromatographed (silica gel, hexane / CHCl=1:1) to give a green solid (18 mg, 100%): TLC (silica, hexane / CHCl=1:1) R f = 0.28; 1 H NMR(300MHz) δ 8.92(s,1H),8.87(s,1H),8.82(d,J=4.8Hz,1H),8.73(d,J=4.7Hz,1H),8.69(d,J=4.7Hz,1H),8.61(d,J=4.7Hz,1H),8.38(d,J=8.1Hz,2H),8. 37(s,1H),8.36(s,1H),8.22(d,J=8.3Hz,2H),7.22(s,2H),4.57(s,2H) ,4.08(s,3H),2.58(s,3H),2.03(s,6H),1.84(s,6H),-1.87(br,s,2H); 13C NMR(100MHz)δ 175.2,167.6,163.6,152.4,151.5,147.2,140.9,140.4,139.2,138.3,137.7,134.7,134.4,134.1,132.1,131.1,129. 5,128.1,128.0,127.8,123.7,123.6,120.59,120.57,96.81,94.99,52.49,51.86,46.63,31.31,21.57,21.45;ESI-MS Observation value 592.2851 [(M+H] + ], calculated value 592.2838 (M = C 39 H 36 N4O2);Lab values ​​(CH2Cl2) 415, 509, 533, 590, 641 nm.

[0169] 5-[4-(N-aminocarbamoyl)phenyl]-10-mesityl-18,18-dimethylchlorin (D2). A solution of chlorin 3 (44 mg, 75 μmol, 1.0 equiv.) in THF (1.0 mL) was treated with methanol (1.0 mL) and hydrazine hydrate (0.21 mL, 3.8 mmol, 50 equiv.) at 50° C. for 24 h. [Note: If the concentration is greater than 50 mM, reduction of the chlorin-hydrazine to the corresponding bacteriochlorin-hydrazine will occur. The bacteriochlorin can be oxidized back to the desired chlorin by treatment with DDQ (1.0 equiv.) in CHCl at room temperature for 30 min. ]The solution was then diluted with ethyl acetate, washed with water, dried over sodium sulfate, concentrated, and chromatographed (silica gel, hexane / EtOAc 1:2 to CH2Cl2 / CH3OH 9:1) to give a green solid (37 mg, 84%): 1H NMR(400MHz) δ 8.96(s,1H),8.88(s,1H),8.76(d,J=4.5Hz,1H),8.75(d,J=4.5Hz,1H),8 .62(d,J=4.7Hz,1H),8.56(d,J=4.7Hz,1H),8.44(d,J=8.1Hz,2H),8.39( s,1H),8.38(s,1H),8.30(d,J=8.0Hz,2H),7.68~7.64(m,2H),5.02(br,2) H),4.62(s,2H),2.60(s,3H),2.06(s,6H),1.85(s,6H),-1.85(br,s,2H); 13 C NMR(100MHz) δ 165.7,164.8,163.5,153.44,153.38,144.3,140.8,139.0,138.0,134.3,132.1,132.0,128.9,128.6,128.5, 127.7,126.8,123.7,88.75,82.21,53.77,42.04,31.14,30.29,29.65,21.27,18.40,17.37,12.06 Observation value 593.1 [(M+H) + ], calculated value 592.3 (M = C 38 H 36 NO).

[0170] 2-[4-(2-Methoxy-2-oxoethyl)phenyl]ethynyl-9,10,16,17,23,24-hexaheptylphthalocyanine (5). Following the standard Sonogashira coupling procedure, a solution of 4 (20 mg, 18 μmol), methyl 3-(4-bromophenyl)propanoate (4.8 mg, 20 μmol), Pd(OAc)2 (1.1 mg, 13 μmol) and P(o-tol)3 (5.5 mg, 18 μmol) in degassed toluene (6.0 mL) was degassed by three freeze-pump-thaw cycles. The mixture was stirred at 60 °C for 18 h. The resulting reaction mixture was concentrated and column chromatographed using a three column strategy [(1) silica, CH2Cl2, (2) SEC, toluene, (3) silica, CH2Cl2] to give a green solid (3.0 mg, 13%). MALDI-MS: Observed 1289.4 [(M+H) +], calculated value 1288.9 (M = C 86 H 112 N8O2).

[0171] 2-[4-(N-aminocarbamoyl)methylphenylethynyl]-9,10,16,17,23,24-hexaheptylphthalocyanine (D3). A solution of 5 (3.0 mg, 2.3 μmol) in toluene (140 μL) was treated with 6.5 μL of hydrazine hydrate (55 wt%) and methanol (10 μL). The resulting mixture was stirred at 50° C. for 16 h, after which ethyl acetate and water were added to the mixture. The organic extract was washed with brine, dried (Na2SO4), and concentrated to give a green solid, which was used directly in the next step of the synthesis.

[0172] Example 2 A general approach for polymer preparation and derivatization according to some embodiments of the present invention is shown in Scheme 6 below.

[0173] [ka]

[0174] In the approach shown in Scheme 6, the initiator is QX, where X is a halohalogen atom (e.g., Cl, Br, I) or a sulfonate (e.g., triflate), and Q can bear a dye or bear a functional group and remains intact throughout the course of the polymerization.

[0175] In case of further derivatization, the necessary groups for dye attachment can be incorporated (in the Q unit) before polymerization and used directly. Alternatively, derivatization of Q in the synthetic polymer I after polymerization can give a modified Q (denoted Q') in polymer II for dye attachment.

[0176] Provision for attachment to a biomolecule (the ω-terminus of the polymer) exists in some cases by direct use of the X-substituent in polymer I. Alternatively, the X-group can be substituted to provide a functional group W in polymer II for attachment of a biomolecule. Examples of W include azide, isocyanate, isothiocyanate, active ester (e.g., pentafluorophenyl ester, succinimide ester, 2,4-dinitrophenyl ester), maleimide, vinyl, mercapto, amino, and carboxylic acid. Derivatization at the ω-terminus in polymer I is accomplished by a single step or multiple steps (e.g., nucleophilic substitution and / or deprotection) to provide the desired functional group W in polymer II. For the prepolymerization method, the functional group is first installed in the initiator (Q unit of QX, Scheme 6) and remains intact throughout the course of polymerization.

[0177] Some examples of Q and QX are shown in Scheme 7. As shown in Scheme 7, Q is a hydroxy group. 1,2 , carboxy group 3 , amino group 4 , formyl group 4 , vinyl group 5,6 , epoxy group 7 , anhydride 8 , haloaryl group 7 , ester group 3 or an oxazoline group 8 Vinyl or allyl groups can be attached via the initiator and remain intact during polymerization without causing additional problems during crosslinking. 1,5,6 This can be achieved mainly by selecting appropriate ligands in the presence of copper(I) catalyst. However, some functional groups commonly used for dye attachment (e.g., azide group) or bioconjugation reactions cannot be introduced by the prepolymerization method (shown in Table 6).

[0178] [ka]

[0179] JPEG0007655493000017.jpg97170

[0180] It should be noted that the examples discussed herein describe attachment of the dye to the α-terminus of the polymer and the biomolecule to the ω-terminus of the polymer. However, if desired, the use of the two termini can be reversed, such that the biomolecule is attached to the α-terminus of the polymer and the dye is attached to the ω-terminus of the polymer.

[0181] References JPEG0007655493000018.jpg147170

[0182] Example 3 - Illustrative Reactions An exemplary reaction for preparing compounds of the invention containing a bridge is provided below in Scheme 8.

[0183] [ka] JPEG0007655493000020.jpg122170

[0184] Exemplary reactions for preparing compounds of the invention, including sulfonation and crosslinking, are provided below in Scheme 9.

[0185] [ka] JPEG0007655493000022.jpg255150JPEG0007655493000023.jpg229170

[0186] Example 4 An exemplary approach for polymer preparation and derivatization according to some embodiments of the present invention is shown in Scheme 10 below.

[0187] [ka]

[0188] In the approach shown in Scheme 10, Z in the RAFT agent can be aryl, alkyl, or thioalkyl, and Q can bear a functional group that remains intact during the polymerization.

[0189] In the case of further derivatization, the functional groups required for conjugation to dyes or biomolecules can be incorporated (in the Q unit) prior to polymerization and used directly. Such functional groups can be initially introduced into the Q unit of the RAFT agent and remain intact throughout the course of polymerization. Alternatively, derivatization of Q in a synthetic polymer after polymerization can provide a modified Q for conjugation of dyes or biomolecules.

[0190] Some examples of Z and Q in RAFT agents are shown below in Chart 1. Examples of Z in the RAFT agent include, but are not limited to, phenyl (optionally substituted) and / or thioalkyl groups (including branched and / or unbranched C1-C25 thioalkyl groups).

[0191] Examples of Q in the RAFT agent include, but are not limited to, carboxylate, azido, hydroxy, N-succinimidyl, vinyl, phthalimido, and / or biotinyl.

[0192] [ka]

[0193] Prior to conjugating a dye or biomolecule at the terminal end of a polymer containing a thiocarbonylthio group, the thiol group can be liberated by cleaving the thiocarbonylthio group using methods known in the art. The free thiol group can either be directly conjugated to a dye or biomolecule or can be further modified with an agent LW to provide a capped thiol (e.g., a thioether) having a suitable functional group W for coupling to the dye or biomolecule. The agent LW contains a thiol-reactive group L, which reacts with the free thiol group and also serves as a linker L' between the thiol and functional group W in the capped product.

[0194] Some examples of L and W in LW are shown below in Chart 2. Examples of L groups in LW factors include, but are not limited to, substituted halides (e.g., substituted benzyl bromides and / or alpha acids), substituted alkynes (e.g., substituted benzyl alkynes), substituted vinyl esters (e.g., alpha-vinyl esters), and / or substituted succinimides (e.g., ethylamine succinimide, ethanol succinimide).

[0195] Examples of functional groups W are carboxylic acids (e.g., -COOH, -CH2CH2COOH), amino (e.g., -NH2, -CH2CH2NH2, optionally with a protecting group: NHBoc, -CH2CH2NHBoc), aldehydes, alcohols (e.g., -CH2CH2OH) and / or alkylated alcohols (e.g., -OCH2CH2OH, -OCH2CH2NHBoc, -OCH2CH2N3, -OCH2CH2NHBoc). ≡ -CH, -OCHCH=CH2).

[0196] Derivatization of the free thiol group can be accomplished via a single step or multiple steps (eg, nucleophilic substitution and / or deprotection) to provide the desired functional group W.

[0197] [ka]

[0198] A further example of RAFT polymerization is shown in Scheme 11. The hydrophobic monomer dodecyl acrylate (LA) is polymerized in the presence of the hydrophilic monomers 2-acrylamido-2-methylpropanesulfonic acid as the sodium salt (AMPS) and PEG acrylate (PEGA) to produce a polymer. In some embodiments, one or more functional groups are present (e.g., pre-introduced) on the RAFT agent prior to polymerization. An example of such one or more functional groups is shown in Scheme 11. After polymerization, the one or more pre-introduced functional groups are placed at one terminal end of the polymer and can be used for coupling to a biomolecule or dye.

[0199] [ka]

[0200] References JPEG0007655493000028.jpg219170

[0201] Example 5 Synthesis of amphiphilic random copolymers via reversible addition-fragmentation chain transfer (RAFT) A model study of the synthesis of sulfonated amphiphilic random copolymers is shown in Scheme 12 below. Three monomers were used, one of which was hydrophobic (dodecyl acrylate (LA)) and two of which were hydrophilic (2-acrylamido-2-methylpropanesulfonic acid and PEG acrylate (PEGA) as the sodium salt (AMPS). AMPS can be prepared by basifying commercially available 2-acrylamido-2-methylpropanesulfonic acid with sodium hydroxide and / or basifying the sodium salt of commercially available 2-acrylamido-2-methylpropanesulfonic acid, which has a small amount of free acid present as a small amount of contaminant in the commercially available AMPS material. RAFT chain transfer agent 1 was used as it was available in the lab. Polymerizations with various monomer ratios were carried out in DMF (80° C.) with AIBN as the radical initiator and mesitylene as an internal standard. After polymerization, the crude product was poured into a large excess of ethyl ether to precipitate the polymer. The precipitate was then dialyzed against water to give the purified polymer.

[0202] [ka]

[0203] Dynamic Light Scattering (DLS) size analysis of amphiphilic-polymers. Each polymer was dissolved in 1.0 M NaCl aqueous solution and passed through a 200 μm membrane filter. The filtrate was examined by DLS to determine the size of the nanoparticles. The DLS size data of the various polymers are summarized in Table 7. According to the data, for the polymers without PEG groups, the best results were obtained with the use of sulfonate and lauryl groups in a ratio of 6:1, which gave 65% unimers in aqueous solution. Upon introduction of PEG groups, the percentage of unimers increased when the ratio of PEG and sulfonic acid groups was decreased from 1:1 to 1:5. At a ratio of AMPS:PEGA:LA=5:1:1, the unimers appeared to be the major species in the aqueous solution.

[0204] [Table 7]

[0205] Synthesis of polymer-chromophore conjugates via RAFT polymerization. Living radical polymerization of the monomers PEGA, LA, and AMPS was carried out with the RAFT agent 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid 2 in the presence of the radical initiator 2,2'-azobis(2-methylpropionitrile) (AIBN) in a 1:1:5 ratio (i.e., hydrophilic / hydrophobic ratio = 6:1) (Scheme 13). The resulting polymer 3 is a heterotelechelic containing a carboxyl group at one end and a thiocarbonylthio group at the other end. Aminolysis of polymer 3 with ethanolamine cleaved the thiocarbonyl group and revealed the free thiol group. In situ coupling of the latter with a hydrophobic maleimide-substituted bacteriochlorin D1 afforded the desired polymer-chromophore conjugate F-2.

[0206] [ka]

[0207] Dynamic Light Scattering (DLS) size analysis of polymer-chromophore conjugates. The polymer-chromophore samples were dissolved in 1.0 M NaCl aqueous solution and passed through a 200 μm membrane filter. The filtered solutions were examined by DLS to determine the size of the nanoparticles. The DLS size data of the various polymers are summarized in Table 8. The polymer-chromophore sample F-2 exhibited unimer form at various concentrations (Figure 4).

[0208] [Table 8]

[0209] Measurement of absorption and emission spectra and fluorescence quantum yield of F-2. The absorption and emission spectra of the target polymer-chromophore conjugate F-2 were measured at room temperature in both water and aqueous buffer (Figures 5 and 6). The spectroscopic data and fluorescence quantum yield data are summarized in Table 9 below.

[0210] The absorption and emission spectra of F-2 in aqueous solution are comparable to those of D1 in toluene, with minimal broadening and loss of Qy absorbance. The fluorescence yield of F-2 in aqueous media is 93% (in buffer) and 80% (in water) of that of D1 in toluene. These data are consistent with little chromophore aggregation in aqueous media. A single chromophore is encapsulated in an amphiphilic polymer and maintains its intrinsic fluorescence upon immersion in an aqueous environment.

[0211] [Table 9]

[0212] Example 6 Developing novel methods of molecular fluorescence or luminescence for chemical sensing using organic chromophores as recognition units is of particular interest, especially in chemistry, biology, environmental science, clinical and medical sciences (Refs. 1-3). Detection is based on (1) a shift in the absorption or emission wavelength of the fluorophore, or (2) a change in the intensity of the absorption or emission. Structural features that control the change in the wavelength or intensity of the absorption or fluorescence include, but are not limited to, twisting of double bonds, changes in conjugation patterns, "heavy" atoms, weak bonds, and opportunities for photoinduced electron transfer (PET) or electronic energy transfer (EET) (Refs. 4-10). Advantages of such detection via optical signals include: high sensitivity; the possibility of "on and off" switching; qualitative or quantitative analysis; and detection by the naked eye (Refs. 11-15).

[0213] Heavy metal ions have a detrimental effect on the environment and human health, and are therefore of great concern among chemists, biologists, environmental scientists, and medical scientists (Reference 16). The demand for sensitive and selective fluorophore sensors targeting toxic heavy metal ions is continually increasing as the challenge is significant. In 1997, Czarnik and co-workers reported the fluorescence-inducing spirolactam ring-opening of rhodamine-B hydrazide for Cu(II) detection in aqueous solutions (Reference 17). As shown in Scheme 14, non-fluorescent rhodamine-hydrazide undergoes hydrolytic ring-opening catalyzed by metal cations to give the conjugated and fluorescent rhodamine structure. The ring opening depends on the nature of the cation. The cations tested in this study included Ag(I), Al(III), Ca(II), Cd(II), Co(II), Cr(III), Cu(II), Eu(III), Fe(III), Ga(III), Gd(III), Hg(II), In(III), K(I), Li(I), Mg(II), Mn(II), Na(I), Ni(II), Pb(II), Rb(I), Sn(IV), Sr(II), U(IV), Yb(III), Zn(II), Cu(II), and Hg(II). Of these, only Cu(II) and Hg(II) caused significant changes in the absorption or fluorescence spectra. Selective detection of Cu(II) was achieved within 10 min. -7 At a concentration of 100 M, the assay was sensitive and quantitative with Cu(II).

[0214] [ka]

[0215] Several rhodamine-hydrazide analogues have been synthesized and analyzed to detect metal ions, such as Pb(II) (18), Cd(II), Fe(III), Hg(II) (19), and Sn(II) (20). However, their application in aqueous solutions requires the incorporation of organic solvents, such as acetonitrile and methanol, due to the hydrophobic nature of rhodamine-hydrazides. In this regard, we designed and synthesized Pod-Rhodamine to study metal ion sensing in pure water without the addition of organic solvents.

[0216] Pod-rhodamine was synthesized by first preparing an amphiphilic random copolymer. The synthesis of the target sulfonated amphiphilic random copolymer is shown in Scheme 15.

[0217] [ka]

[0218] The polymerization is carried out as described herein to give F-Ph, where the ratio of m:n:p is based on the reaction stoichiometry and on the synthesis of the polymer. 1 The ratio of the polymer to the total polymer is 1.0:1.0:5.0 by H NMR spectroscopy. The size of the target amphiphilic random copolymer F-Ph was also measured in aqueous solutions at various concentrations of the polymer using dynamic light scattering (DLS) spectroscopy (Figure 7). The data show that the polymer exhibits exclusively unimeric behavior in aqueous solutions, with a size distribution peak at 10 nm and no detectable aggregation.

[0219] The dithioester of F-Ph was removed by reaction with hydrazine hydrate in DMF to give the polymer F-SH containing free thiol end groups. The thiol group of F-SH was further derivatized to F-CHO bearing a formyl group by reaction with p-bromomethylbenzaldehyde in DMF. 1Examination of F-CHO by H NMR spectroscopy (in DO) gave m, n, and p of 22, 21, and 104, respectively. The m, n, and p values ​​are obtained based on a single carboxaldehyde proton. The data are then consistent with the m:n:p ratio of 1.0:1.0:5.0 expected from the initial monomer stoichiometry. 1 Note that the calculated molecular weight of F-CHO given by the m, n and p values ​​from the H NMR measurements is 39.6 kDa, which compares with the estimated molecular weight of F-Ph of 41.4 kDa estimated from the HPLC analysis (the two polymers have molecular weights that differ by only 2 Da in mass). This comparison is excellent for the HPLC and NMR measurements.

[0220] Pod-rhodamine was prepared by reaction of F-CHO with rhodamine-hydrazide I in N,N-dimethylformamide for 15 h at 40° C. Subsequent removal of unreacted dye by dialysis afforded the desired pod-rhodamine in 91% yield.

[0221] [ka]

[0222] Pod-Rhodamine was subjected to the absorption and release tests in water in the presence of various metal ions. In a vial, 1.0 mg of pod-Rhodamine was treated with a solution of metal salt in water (1.0 mL, 2 mM, 100 molar equivalents of pod-Rhodamine). The final concentration of pod-Rhodamine was 20 μM. The resulting solution was stirred at room temperature for 1 hour, after which the solution was measured by absorption and emission spectroscopy. In this study, the cations tested were: Au(III), Al(III), Ce(III), Cd(II), Co(II), Cr(II), Cu(II), Fe(III), Ga(III), Hg(II), In(III), Mg(II), Mn(II), Ni(II), Pb(II), Yb(III) and Zn(II).

[0223] The absorption and emission spectra of the various solutions are shown in FIG. 8. For absorption analysis, Au(III), Cr(II), Cu(II), Fe(III), Hg(II) and In(III) show changes in absorption. For fluorescence analysis, Au(III), Ga(III), Hg(II) and In(III) show increased fluorescence intensity compared to the blank control. The loss of fluorescence in the Cu(II) and Fe(III) samples could be attributed to the heavy atom effect. Photographs of the various reaction solutions were obtained with and without illumination. For Cr(II), a precipitate was observed during the reaction, therefore, the absorption at Cr(II) was measured using the supernatant.

[0224] Fluorescence titrations were performed with Au(III) and Hg(II) using 10 μM pod-rhodamine and 0-1.0 μM cations (excitation at 510 nm). Figure 9 shows the titration fluorescence spectra.

[0225] In summary, the key point of this work is that the rhodamine sensor remains active upon conjugation with the heterotelechelic polymer and can be used in pure water for ion sensing purposes. In contrast, literature data indicates that the use of the rhodamine sensor alone requires the use of a mixture of organic and aqueous media. Without wishing to be bound by a particular theory, this suggests that the polymer provides an organic solubilizing function for the conjugated rhodamine sensor.

[0226] References: JPEG0007655493000037.jpg204170

[0227] Example 7 An embodiment of the present invention relates to heterotelechelic polymers with a single bioconjugable group and a single chromophore for use in aqueous solutions, which is counterintuitive. The counterintuitive nature stems from the widespread belief in the field, as shown in numerous papers over the past 50 years, that the polymer or other structure needs to be loaded with as many chromophores as possible to achieve an adequate signal (e.g., brightness). One aspect of our strategy is to site-isolate the desired chromophore as a single cargo item at one end of each heterotelechelic polymer, achieve a high degree of unimeric self-assembly in aqueous solutions, and utilize the other end of the polymer for bioconjugation reactions.

[0228] One approach to solubilize hydrophobic fluorophores in aqueous solution involves conjugation to amphiphilic polymers, which self-assemble with the hydrophobic fluorophore anchored in the hydrophobic interior. A single polymer-single cargo strategy has been previously reported by our group. 9 , where amphiphilic polymers containing reactive end groups and pendant hydrophobic and hydrophilic groups were used to package hydrophobic fluorophores in aqueous solutions. One polymer in particular that was studied contained acrylate and acrylamide units in the backbone with pendant hydrophobic (dodecyl), nonionic hydrophilic (PEG9) and ionic hydrophilic (sulfonate-terminated) groups. The polymer was heterotelechelic with benzothioate and carboxylic acid end groups. The polymer, termed F-Ph, exhibited approximately 40 kDa and contained lauryl, PEG9 and sulfonate as pendant groups in a 1:1:5 ratio. The construct was freely soluble in aqueous media. Studies showed that the polymer F-Ph quantitatively formed unimers in 1 M NaCl solution at room temperature, as assessed by dynamic light scattering (DLS) spectroscopy. Conjugation of hydrophobic fluorophores (drawn from approximately eight classes of fluorophores) was performed at the termini of F-Ph polymers (i.e., one fluorophore per polymer). The resulting polymer-fluorophores retained the inherent brightness observed with the fluorophores alone in organic solvents.

[0229] Most antibodies or cells cannot survive in aqueous solutions containing such high concentrations of NaCl. We recently decided to examine F-Ph and fluorophore-containing derivatives of F-Ph in aqueous media of the type used in biology. In the PBS buffer commonly used in biology, F-Ph is 68% unimers (D at about 13 nm). h Peak) and 32% aggregated particles with dimensions greater than 100 nm (Figure 10). PBS buffer contains 0.15 M NaCl and 0.01 M phosphate. These results suggest the importance of ionic strength in the self-assembly of amphiphilic polymers.

[0230] Since a high percentage of unimers (versus aggregates) at low ionic strength is believed to be important for many biological applications, the inventors undertook a study to investigate the many factors believed to affect the assembly process of unimers. The application of most interest for amphiphilic polymers is flow cytometry. The study involved the examination of F-Ph as a function of ionic strength, as well as the preparation and examination of new polymers that differ in the nature of the pendant groups, the ratio of pendant groups, molecular weight, and the presence or absence of hydrophobic fluorophores. The laser in the DLS instrument illuminates the samples at 632 nm, which eliminates the possibility of using chromophores that absorb in this region. The fluorophore chosen for the study described here is perylene-monoimide. The perylene-monoimides, which do not absorb at 632 nm, are large hydrophobic arenes and are therefore considered viable surrogates for a variety of tetrapyrrole macrocycles (e.g., chlorins and bacteriochlorins).

[0231] 1. Polymer synthesis. A new series of amphiphilic polymers containing cyclododecyl and sulfonate pendant groups were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization. The cyclododecyl group was introduced via cyclododecyl acrylate. The sulfonate was introduced at the terminus of an N-alkyl substituted acrylamide. Cyclododecyl (CD) was previously shown to have a much stronger tendency for intramolecular self-association than lauryl groups. 2 In F-Ph, lauryl was replaced with cyclododecyl; and PEG was also deleted. Thus, the resulting polymer contained CD and sulfonate pendant groups, no PEG groups, and retained benzothioate and carboxylic acid termini. For the studies described below, polymers were prepared containing different ratios of the two types of pendant groups and / or variations in overall molecular weight. Treatment of such polymers with ethanolamine resulted in cleavage of the thiobenzoyl groups and free thiols at the termini. The resulting free thiols were reacted with perylene-monoimidemaleimide (PMI-mal) to give the corresponding polymer-fluorophore conjugates (Scheme 17).

[0232] [ka]

[0233] Seven PS-CD polymers were prepared using various initial ratios of cyclododecyl acrylate (CDA, hydrophobic monomer), sulfonate-terminated acrylamide (AMPS, hydrophilic monomer / ionic monomer), and chain-transfer reagent (CTR). Subsequent derivatization afforded the corresponding polymer-PMI conjugates (Table 10). The molecular weight of each PS-CD polymer was estimated from HPLC analysis. Here, we investigated the percentage of unimers observed as a function of changes in the following parameters: (1) the ionic strength of the solution, (2) the ratio of the two pendant groups, (3) the molecular weight of the polymer alone (lacking a fluorophore), and (4) the presence of a fluorophore - in other words, the effect of the presence and absence of a fluorophore attached to the polymer.

[0234] [Table 10]

[0235] 2. Study of the influence on unimer assembly. A. Ionic strength. DLS studies of the previous F-Ph and the new polymer P-S5-CD1 (28 kDa) (item 5, Table 10) were carried out in six media: pure water, PBS buffer and aqueous solutions with different concentrations of NaCl (0.25 M, 0.50 M, 0.75 M and 1.0 M). The raw DLS data are shown in Figure 11. In pure water, F-Ph showed three different sizes according to DLS examination. The peak at about 1 nm may be due to unfolded polymer, while some of the polymer may aggregate, resulting in a size distribution that reaches a peak at about 400 nm. The ionic strength of the PBS buffer also did not result in a complete unimer behavior of F-Ph (see Figure 10). However, in 0.25 M NaCl solution and solutions with higher ionic strength, F-Ph quantitatively provided unimers (Figure 11). The DLS data from FIG. 11 with respect to the percentage of unimers is plotted graphically in FIG.

[0236] We then turned to a similar study of the new polymer P-S5-CD1 (28 kDa). The raw DLS results in pure water and in aqueous solutions with various NaCl concentrations are shown in Figure 13, while the results in PBS buffer are shown in Figure 14. These results are plotted graphically in Figure 15. The graph shows that the polymer P-S5-CD1 (28 kDa) self-assembles to produce unimers in a quantitative manner in aqueous media containing 0.50 M NaCl or higher. Apparently, the polymer tends to assemble into particles of uniform size in aqueous media of high ionic strength.

[0237] 2.B. Effect of pendant and hydrophobic fluorophore presence. Samples of F-Ph or F-PMI were dissolved in PBS buffer and examined by DLS spectroscopy. The percentage of unimers for F-PMI conjugates was higher than that of F-Ph in PBS buffer (Figure 16). Thus, the presence of hydrophobic fluorophore cargo appears to drive the assembly of the polymer in PBS buffer.

[0238] A similar comparison was performed for P-S5-CD1(28kDa) and P-S5-CD1(28kDa)-PMI (Figure 17). The results show that P-S5-CD1(28kDa)-PMI quantitatively assembled into unimer particles in PBS buffer, whereas polymers lacking the PMI unit did not quantitatively assemble into unimers. Again, the presence of hydrophobic cargo appears to drive the assembly of the overall structure into a unimer architecture.

[0239] 2.C. Molecular Weight. DLS was measured for a series of cyclododecyl / sulfonate polymer-PMI conjugates containing the same ratio of pendants (5:1) but with different degrees of polymerization. The molecular weights, which directly reflect the degree of polymerization given a constant ratio of reacting monomers, ranged from 10 to 35 kDa. Raw DLS data for polymer-PMI conjugates in PBS buffer are shown in Figure 18. For P-S5-CD1(28 kDa)-PMI and P-S5-CD1(35 kDa)-PMI, the percentage of unimers was 100%, whereas constructs derived from lower molecular weight polymers, i.e., P-S5-CD1(10 kDa)-PMI and P-S5-CD1(18 kDa)-PMI, were not 100%. Based on this data set, a 28 kDa polymer is a sufficient size to provide 100% unimer formation for constructs containing PMI (although not completely free of PMI, as noted above).

[0240] 2.D. Pendant ratio. The effect of the pendant ratio was also studied. Three polymer-PMI conjugates were prepared, where the polymers were of approximately the same molecular weight (about 28 kDa) but exhibited different ratios of pendants. The ratio of pendant groups was 4:1, 5:1, and 6:1 for sulfonate to cyclodecyl. The raw DLS data of each construct in PBS is shown in FIG. 19. Each construct quantitatively assembled into unimer particles, and in each case was of similar size and polydispersity index (PDI). Therefore, slight changes in the pendant ratio do not result in aggregation in PBS buffer, at least for this study of the about 28 kDa polymer.

[0241] The results of the above experiments are summarized in Table 11 below. For the seven PS-CD polymers we prepared, all polymers (lacking hydrophobic fluorophores) quantitatively formed unimers in 1 M NaCl solution, but not in PBS buffer. However, conjugation to hydrophobic fluorophores allowed the polymers to have sufficient molecular weight to assemble unimers, regardless of the pendant ratio.

[0242] [Table 11]

[0243] 3. Experimental section General method for preparing polymers. A solution flask of CDA (715 mg, 3.0 mmol), AMPS (12 mmol-18 mmol), chain transfer reagent (0.06 mmol-0.27 mmol) and mesitylene (360 mg, 3.0 mmol) in DMF (27 mL) in a Schlenk tube was degassed by three freeze-pump-thaw cycles. Then, AIBN (4.9 mg, 0.030 mmol) was added to the flask. The resulting mixture was stirred at 80° C. for 24 h. Mesitylene was dissolved in 100 mL of ... 1 H was included as an internal standard to assess the conversion of each monomer via H NMR spectroscopy. The mixture was cooled to room temperature and then poured into 200 mL of diethyl ether. The precipitate was washed three times with diethyl ether. The crude polymer was then dissolved in deionized water and placed in a dialysis membrane tube equipped with two closures that allow compounds with molecular weights less than 3.5 kDa to pass through. The solution was dialyzed against deionized water, and the reservoir volume was replaced with fresh deionized water four times over a period of approximately 24 h. The dialyzed solution was lyophilized under high vacuum to give a pale pink solid (1.2-1.4 g).

[0244] General method for preparing polymer-PMI conjugates. Taking F-S5-CD1(28kDa)-PMI as a representative example, a solution of PMI-maleimide (1.0mg, 0.97μmol) and F-S5-CD1(28kDa) (18.1mg, 0.65μmol) in DMF (1mL) was treated with ethanolamine (1 drop). The mixture was stirred at 35°C for 16 hours. The mixture was then transferred into a dialysis membrane tube with two closures. The solution was then dialyzed in DMF to remove excess fluorophore. The dialysis reservoir volume was replaced with fresh DMF four times over a period of about 24 hours. The resulting solution was dried under high vacuum at 30°C. The resulting solid was dissolved in deionized water and then lyophilized to give a pink solid (17mg).

[0245] 4. Discussion A set of amphiphilic polymers was prepared with various ratios of pendant groups and various degrees of polymerization to investigate factors related to the extent of unimer formation in PBS buffer. PBS buffer is a widely accepted medium for biological assays and many clinical studies. Substitution of lauryl and PEG groups (previously used 9 ) with cyclododecyl groups was easily achieved. Comparison of polymers containing lauryl and PEG with those containing cyclododecyl showed that the latter promoted intramolecular assembly. One surprising result was that the presence of a single hydrophobic fluorophore induced self-folding of the amphiphilic polymer in aqueous solution. A polymer with a high molecular weight (approximately 28 kDa) was sufficient to completely package the hydrophobic fluorophore in PBS buffer; i.e., unimers were quantitatively formed. The ratio of sulfonate groups to cyclododecyl pendant groups could be varied in the range of 4–6:1 as long as the molecular weight was above 28 kDa, and unimers were quantitatively formed in PBS buffer accordingly. Without wishing to be bound by any particular theory, it is believed that an interplay of three factors (molecular weight, ratio of pendant groups, and the presence of hydrophobic cargo) influences the assembly of the polymer, which was clearly manifested in PBS buffer but was likely obscured in the high ionic strength conditions of 1 M NaCl, a medium that has been used extensively (if not nearly universally) by others and by the present inventors in the study of foldamer formation. In summary, the heterotelechelic PS-CD polymers described here provide a simple and accessible platform for use with hydrophobic fluorophores in potential applications in biochemistry.

[0246] 5.References JPEG0007655493000041.jpg221170

[0247] The foregoing is illustrative of the invention and should not be construed as limiting thereof. The invention is defined by the following claims, including equivalents thereto. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

1. A compound having a structure represented by A,B,C, or CAB Where: A is a dye, optionally wherein the dye has a molecular weight in the range of 150 Daltons (Da) to 3,000 Da; B is a polymer having a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units, wherein the polymer backbone comprises a first end and a second end, the first end and the second end being opposite ends of the polymer backbone, optionally, the polymer has a molecular weight in the range of 5,000 Da to 100,000 Da; and C is optional, where C comprises a bioconjugate group; wherein the dye is covalently attached only to the first end or only to the second end of the polymer backbone, and wherein the dye comprises a recognition motif selected from the group consisting of crown ethers, cryptands, pincers, chelating motifs, and any combination thereof; The hydrophobic unit has a structure represented by Formula III: 【Chemistry 1】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is absent, -O-, -NH- or -CH 2 -; R' is a C1-C20 alkyl, a C2-C20 alkenyl, a C2-C20 alkynyl, or a C3-C20 cycloalkyl; R 2 is a hydrogen atom, a halogen atom, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group, or an isothiocyanate group; and p is an integer from 1 to 100,000.

2. The compound of claim 1 , wherein the recognition motif is attached to the dye.

3. The compound of claim 1 , further comprising a linker that attaches the dye to the polymer.

4. The compound according to any one of claims 1 to 3, wherein the dye is a non-luminescent molecular entity.

5. The compound according to any one of claims 1 to 4, wherein the dye is a metal-containing tetrapyrrolic macrocycle.

6. 6. The compound of claim 1, wherein the compound comprises a bioconjugate group and the bioconjugate group is attached to the first end and the dye is covalently attached to the second end.

7. The compound according to any one of claims 1 to 6, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer.

8. 8. The compound according to any one of claims 1 to 7, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of 1:1 to 1:10, optionally wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of 1:6 (hydrophobic units:hydrophilic units).

9. The compound according to any one of claims 1 to 8, wherein the compound has conformational flexibility.

10. The compound according to any one of claims 1 to 9, wherein the compound self-folds in aqueous solution, optionally in a unimeric micellar structure.

11. The compound according to any one of claims 1 to 10, wherein the polymer is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer.

12. 12. The compound according to any one of claims 1 to 11, wherein the compound is crosslinked, optionally wherein the compound is crosslinked when the compound is in a folded conformation.

13. 13. The compound of any one of claims 1 to 12, wherein the compound folds to provide a particle, optionally wherein the particle has a diameter in the range of 1 nm to 40 nm.

14. 14. The compound according to claim 13, wherein at least a portion of said one or more hydrophobic units is present in the core of the particle and / or at least a portion of said one or more hydrophilic units is present at the periphery of the particle.

15. The compound of any one of claims 1 to 14, wherein the dye is encapsulated by a portion of the compound when the compound is in a folded conformation.

16. The compound according to any one of claims 1 to 5 or 7 to 15, wherein the polymer is a telechelic polymer or a heterotelechelic polymer.

17. The compound according to any one of claims 1 to 16, wherein the dye is hydrophobic.

18. 18. The compound according to any one of claims 1 to 17, wherein the compound is water soluble, optionally wherein the compound has a solubility in water in the range of 1 mg / mL to 10 mg / mL at room temperature.

19. 19. The compound according to any one of claims 1 to 18, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group, optionally wherein the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group, an isothiocyanate group, a phosphono group, a sulfono group, an ammonio group or a phosphatidylcholine group, and / or wherein the pendant functional group is a hydrophilic group having a terminal cation, an anion or amphoteric group, and optionally a poly(ethylene glycol) residue.

20. 20. The compound of claim 1, wherein at least one of the one or more hydrophobic units comprises an alkyl pendant group and / or at least one of the one or more hydrophilic units comprises a glycol pendant group.

21. The compound according to any one of claims 1 to 20, wherein the polymer is attached to a dye and, optionally, to a bioconjugate group.

22. R in the hydrophobic unit 2 The compound according to any one of claims 1 to 21, wherein is a hydrogen atom, or a formyl group or an ester group.

23. R in the hydrophobic unit 2 The compound according to any one of claims 1 to 21, wherein is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group.

24. The compound according to any one of claims 1 to 23, wherein the hydrophilic unit has a structure represented by formula IV: 【Chemistry 2】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is not present or is -O-, -NH- or -CH 2 - and R 3 is -(CH 2 CH 2 R 5 ) n -, -C 1 ~C 6 Alkyl, -C 1 ~C 6 Alkyl-O- and -C 1 ~C 6 Alkyl-SO 3 - or salts thereof, wherein R 5 is -O- or -CH 2 and n is an integer from 1 to 10,000. R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group, and p is an integer from 1 to 100,000.

25. R in the hydrophilic unit 4 The compound according to claim 24, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group.

26. R in the hydrophilic unit 4 The compound of claim 24, wherein is alkyl.

27. R 3 -C 1 ~C 6 Alkyl-O- or -(CH 2 CH 2 R 5 ) n - where R 5 is -O-, and R in the hydrophilic unit 4 The compound according to claim 24 or 25, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group or a phosphoryl group.

28. R 3 But, C 1 ~C 6 Alkyl or -(CH 2 CH 2 R 5 ) n - where R 5 Ga-CH 2 and R in the hydrophilic unit 4 26. The compound according to claim 24 or 25, wherein is a hydroxyl, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group or a sulfono group.

29. R 3 But -C 1 ~C 6 Alkyl-SO 3 26. The compound according to claim 24 or 25, which is - or a salt thereof.

30. 30. The compound according to any one of claims 1 to 29, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

31. The compound according to any one of claims 1 to 30, wherein said compound is and / or functions as a sensor, optionally without the addition and / or presence of an organic solvent.

32. A dye, optionally having a molecular weight in the range of 150 Daltons (Da) to 3,000 Da; a polymer having a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units, wherein the polymer backbone comprises a first end and a second end, the first end and the second end being opposite ends of the polymer backbone, optionally wherein the polymer has a molecular weight in the range of 5,000 Da to 100,000 Da; a recognition motif selected from the group consisting of crown ethers, cryptands, pincers, chelating motifs, and any combination thereof, attached to a portion of the compound; and Optionally a bioconjugate group; A compound comprising: wherein the dye is covalently attached only to the first end or only to the second end of the polymer backbone; wherein the dye comprises the recognition motif and wherein the compound comprises at least one unit having a structure represented by formula III: 【Chemistry 3】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is not present or is -O-, -NH- or -CH 2 - and R' is a C3-C20 cycloalkyl; R 2 is a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group, or an isothiocyanate group, and p is an integer from 1 to 100,000.

33. 33. The compound of claim 32, wherein the recognition motif is attached to the dye.

34. 33. The compound of claim 32, further comprising a linker attaching the dye to the polymer.

35. The compound according to any one of claims 32 to 34, wherein the dye is a non-luminescent molecular entity.

36. The compound of any one of claims 32 to 35, wherein the dye is a metal-containing tetrapyrrolic macrocycle.

37. 37. The compound of any one of claims 32 to 36, wherein the compound comprises the bioconjugate group and the dye is covalently attached to one of the first end or the second end and the bioconjugate group is attached to the other of the first end or the second end.

38. The compound according to any one of claims 32 to 37, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer.

39. 39. The compound according to any one of claims 32 to 38, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of 1:1 to 1:10, optionally wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of 1:6 (hydrophobic units:hydrophilic units).

40. 40. The compound of any one of claims 32 to 39, wherein the compound has conformational flexibility.

41. The compound according to any one of claims 32 to 40, wherein the compound self-folds in aqueous solution, optionally in a unimeric micellar structure.

42. The compound according to any one of claims 32 to 41, wherein the polymer is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer.

43. 43. The compound according to any one of claims 32 to 42, wherein the compound is crosslinked, optionally wherein the compound is crosslinked when the compound is in a folded conformation.

44. 44. The compound of any one of claims 32 to 43, wherein the compound folds to provide a particle, optionally wherein the particle has a diameter in the range of 1 nm to 40 nm.

45. 45. The compound of claim 44, wherein at least a portion of said one or more hydrophobic units is present in the core of the particle and / or at least a portion of said one or more hydrophilic units is present at the periphery of the particle.

46. 46. ​​The compound of any one of claims 32 to 45, wherein the dye is encapsulated by a portion of the compound when the compound is in a folded conformation.

47. The compound according to any one of claims 32 to 36 and 38 to 46, wherein the polymer is a telechelic polymer or a heterotelechelic polymer.

48. The compound according to any one of claims 32 to 47, wherein the dye is hydrophobic.

49. 49. The compound of any one of claims 32 to 48, wherein the compound is water soluble, optionally wherein the compound has a solubility in water in the range of 1 mg / mL to 10 mg / mL at room temperature.

50. 50. The compound according to any one of claims 32 to 49, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group, optionally wherein the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group, an isothiocyanate group, a phosphono group, a sulfono group, an ammonio group or a phosphatidylcholine group, and / or wherein the pendant functional group is a hydrophilic group having a terminal cation, an anion or amphoteric group, and optionally a poly(ethylene glycol) residue.

51. 51. The compound of any one of claims 32 to 50, wherein at least one of the one or more hydrophobic units comprises an alkyl pendant group and / or at least one of the one or more hydrophilic units comprises a glycol pendant group.

52. The compound according to any one of claims 32 to 51, wherein the polymer is attached to a dye and, optionally, to a bioconjugate group.

53. The compound of any one of claims 32 to 52, wherein R' is a C8-C12 cycloalkyl.

54. R in the hydrophobic unit 2 The compound according to any one of claims 32 to 53, wherein is a hydrogen atom, a formyl group, or an ester group.

55. R in Formula III 2 The compound according to any one of claims 32 to 54, wherein is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group.

56. The compound according to any one of claims 32 to 55, wherein the hydrophilic unit has a structure represented by formula IV: 【Chemistry 4】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is not present or is -O-, -NH- or -CH 2 - and R 3 is -(CH 2 CH 2 R 5 ) n -, -C 1 ~C 6 Alkyl, -C 1 ~C 6 Alkyl-O- and -C 1 ~C 6 Alkyl-SO 3 - or salts thereof, wherein R 5 is -O- or -CH 2 and n is an integer from 1 to 10,000. R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group, and p is an integer from 1 to 100,000.

57. R in the hydrophilic unit 4 The compound according to claim 56, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group.

58. R in Formula III 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group.

59. R 3 But -C 1 ~C 6 Alkyl-O- or -(CH 2 CH 2 R 5 ) n - where R 5 is -O-, and R in the hydrophilic unit 4 The compound according to claim 56 or 57, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, or a phosphoryl group.

60. R 3 But, C 1 ~C 6 Alkyl or -(CH 2 CH 2 R 5 ) n - where R 5 Ga-CH 2 and R in the hydrophilic unit 4 58. The compound according to claim 56 or 57, wherein is a hydroxyl, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group or a sulfono group.

61. R 3 But -C 1 ~C 6 Alkyl-SO 3 - or a salt thereof.

62. A compound according to claims 32 to 61, wherein the recognition motif is bound to a dye.

63. The compound of claim 62, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

64. 64. The compound according to any one of claims 32 to 63, which is and / or functions as a sensor, optionally without the addition and / or presence of an organic solvent.

65. A composition comprising a compound according to any one of claims 1 to 64.

66. 66. The composition of claim 65, wherein the compound forms a particle.

67. 67. The composition of claim 66, wherein a single compound is present in the particle.

68. 68. The composition of claim 66 or 67, wherein at least a portion of the one or more hydrophobic units are present in the core of the particle.

69. 69. The composition of any one of claims 66 to 68, wherein at least a portion of said one or more hydrophilic units are present in the shell of said particle.

70. 70. The composition of any one of claims 66 to 69, wherein the particles are resistant to dilution, optionally wherein the particles remain in a folded structure when the composition is diluted up to 100-fold or to sub-micromolar concentrations.

71. A composition according to any one of claims 66 to 70, wherein the dye is present in the core of the particle and / or is encapsulated by at least a portion of the polymer.

72. The composition of any one of claims 65 to 71, wherein the composition is free of organic solvents.

73. A method for preparing a compound according to any one of claims 1 to 64, comprising the steps of: polymerizing hydrophobic monomers and hydrophilic monomers to provide said polymer backbone comprising said one or more hydrophobic units and said one or more hydrophilic units; attaching said dye to said first end or said second end of said polymer backbone, thereby providing said compound, wherein said dye optionally comprises said recognition motif; The method comprising:

74. 74. The method of claim 73, wherein polymerizing the hydrophobic monomers and the hydrophilic monomers comprises polymerizing the hydrophobic monomers and the hydrophilic monomers via living radical polymerization in the presence of an initiator, a catalyst, and optionally a co-catalyst to provide the polymer.

75. 74. The method of claim 73, wherein polymerizing the hydrophobic monomers and the hydrophilic monomers comprises polymerizing the hydrophobic monomers and the hydrophilic monomers via living radical polymerization in the presence of an initiator and a RAFT agent to provide the polymer.

76. 76. The method of any one of claims 73 to 75, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer in a ratio of 1:1 to 1:

10.

77. The method of any one of claims 73 to 76, wherein the hydrophobic monomer has a structure represented by Formula I: 【Chemistry 5】 Where: R is a hydrogen atom or a C1-C8 alkyl; R 1 is not present or is -O-, -NH- or -CH 2 - and R' is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, and R 2 is a hydrogen atom, a halogen atom, a formyl group or an ester group.

78. R in the hydrophobic monomer 2 The method of claim 77, wherein is a hydrogen atom, or a formyl group or an ester group.

79. The method of any one of claims 73 to 78, wherein the hydrophilic monomer has a structure represented by Formula II: 【Chemistry 6】 Where: R is a hydrogen atom or a C1-C8 alkyl; R 1 is not present or is -O-, -NH- or -CH 2 - and R 3 But -(CH 2 CH 2 R 5 ) n -, -C 1 ~C 6 Alkyl, -C 1 ~C 6 Alkyl-O- and -C 1 ~C 6 Alkyl-SO 3 - or salts thereof, wherein R 5 is -O- or -CH 2 and n is an integer from 1 to 10,000; and R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group.

80. R in the hydrophilic monomer 4 The method of claim 79, wherein is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group.

81. R 3 Ga-C 1 ~C 6 alkyl-O-, and R in the hydrophilic monomer 4 The method of claim 79, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, or a phosphoryl group.

82. R 3 Ga-C 1 ~C 6 Alkyl or -(CH 2 CH 2 R 5 ) n - and R 5 is -O-, and R in the hydrophilic monomer 4 The method of claim 79, wherein is a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group, or a sulfono group.

83. 83. The method of any one of claims 73 to 82, further comprising attaching said recognition motif to said compound, optionally to said dye or to a portion of said polymer.

84. 73. A method of using a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72 in flow cytometry, wherein said compound comprises said bioconjugate group.

85. A method for detecting cells and / or particles using flow cytometry, comprising labeling the cells and / or particles with a compound according to any one of claims 1 to 64 or a compound prepared according to the method of any one of claims 73 to 83, wherein said compound comprises said bioconjugate group; and detecting said compounds by flow cytometry, thereby detecting said cells and / or particles.

86. An agent for use in detecting tissues and / or factors in a subject, comprising: The agent comprises a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72, wherein the compound comprises the bioconjugate group; The detection may include: administering said agent to a subject, optionally wherein said compound associates with said tissue and / or factor; and Detecting said compound in said subject, thereby detecting said tissue and / or factor. The agent comprising:

87. 1. An agent for use in treating cells and / or tissues in a subject in need thereof, comprising: The agent comprises a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72, The treatment comprises: administering said agent to a subject, where optionally said compound associates with said cells and / or tissue; and irradiating the subject or a portion of the subject with light of a wavelength and intensity sufficient to treat the cells and / or tissue, where, optionally, the light activates the compound or a portion of the compound; The agent comprising:

88. The method of claim 87, wherein the cells and / or tissues are hyperproliferative tissues.

89. 1. An agent for use in a photodynamic therapy method for treating hyperproliferative tissue in a subject in need thereof, comprising: The agent comprises a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72, wherein the compound comprises the bioconjugate group; The photodynamic therapy method comprises: administering said agent to a subject, optionally wherein said compound associates with said hyperproliferative tissue; and irradiating said hyperproliferative tissue with light of a wavelength and intensity sufficient to activate said compound or a portion of said compound, thereby treating said hyperproliferative tissue. The agent comprising:

90. An agent comprising a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72.

91. The agent according to claim 90, comprising two or more compounds according to any one of claims 1 to 64 or two or more compositions according to any one of claims 65 to 72.

92. 13. An agent for use in a photodynamic therapy method, comprising a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72, wherein said compound comprises said bioconjugate group.

93. An agent for use in photoacoustic imaging, comprising: The agent comprising a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72.

94. 1. An agent used to image tissues and / or factors in a subject, comprising: The agent comprising a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72.

95. 95. The method of claim 94, wherein detecting the compound in the subject comprises irradiating the subject or a portion of the subject with light of a wavelength and intensity sufficient to generate ultrasound, optionally wherein the irradiation is performed using a laser and / or by exposing the subject to one or more non-ionizing laser pulses.

96. 96. The method of claim 94 or 95, wherein detecting the compound in the subject comprises detecting ultrasound, optionally with an ultrasound detector.

97. The method according to any one of claims 94 to 96, wherein imaging of tissues and / or factors in the subject comprises photoacoustic imaging of the tissues and / or factors.

98. 1. An agent for use as a chromogenic, fluorescent, in vivo, oxygen and / or metal ion sensor, optionally without the addition and / or presence of an organic solvent, comprising The agent comprises a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72. The agent.

99. 1. A method for sensing metal ions, comprising: Providing a compound according to any one of claims 1 to 4, 6 to 35, and 37 to 64 or a composition according to any one of claims 65 to 72; contacting said metal ion with said compound, thereby sensing said metal ion; The method comprising:

100. 100. The method of claim 99, wherein the compound and / or metal ion is present in water, optionally without organic solvent.

101. 73. A method of using a compound according to any one of claims 1 to 64 or a composition according to any one of claims 65 to 72 as an oxygen sensor and / or oxygen binding material, wherein said compound comprises said bioconjugate group.

102. 99. The method of claim 98, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

103. 102. The method of any one of claims 99 to 101, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

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