Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers for solubilizers, dyes, and biopharmaceuticals

Abstract

Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers are provided. In some cases, the PEOZ polymers have repeating units of the structure -CHN(C(O)R)CHCHOCHCH-. The PEOZ copolymers can be used in a variety of applications, for example, to enhance the solubility of various compounds, such as drugs, dyes, and biopharmaceuticals. Also provided are methods for making the PEOZ copolymers, which can optionally produce monodisperse compositions of the PEOZ copolymers.

Description

[Background technology]

[0001] Polyethylene glycol (PEG) groups have been attached to small molecules, nucleotides, peptides, proteins, liposomes, and nanoparticles to improve solubility, stability, and pharmacokinetic properties (Chenet et al., ACS Nano, 2021, 15, 14022). In some cases, polymer-drug conjugates have been formed using PEG, such as PEG groups with molecular weights ranging from 0.3 to 60 kDa per polymer (Konget et al., Frontiers in Bioengineering and Biotechnology, 2022, 10, 879988). PEG has also been used in bioconjugation reactions and nanomedicine to extend blood circulation time and enhance drug efficacy (Thi et al., Polymers, 2020, 12, 298). Summary of the Invention [Problem to be solved by the invention]

[0002] However, some individuals who have not received the medication may have pre-existing antibodies that can bind to PEG and cause an immune response (Chenet et al., supra). At least 25 different pharmaceuticals or compositions contain PEG groups, including the mRNA-1273 vaccine for COVID-19 manufactured by Moderna (supra). In addition, treatment of subjects with drugs containing PEG groups has been observed to result in the development of anti-PEG antibodies, which may be undesirable (Thi et al., supra).

[0003] Furthermore, many common methods for synthesizing PEG and other polymers produce polydisperse compositions with broad molecular weight distributions, which can adversely limit control over polymer properties. [Means for solving the problem]

[0004] Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers are provided. In some cases, the PEOZ polymers have repeating units of the structure -CHN(C(O)R)CHCHOCHCH-. The PEOZ copolymers can be used in a variety of applications, for example, to enhance the solubility of various compounds, such as drugs, dyes, and biopharmaceuticals. Also provided are methods for making the PEOZ copolymers, which in some cases can produce monodisperse compositions of the PEOZ copolymers. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 shows the general structures of polyethylene glycol (PEG), polyoxazoline (POZ), and poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer. [Figure 2] FIG. 1 shows an exemplary solution phase synthesis of PEOZ copolymers. [Figure 3-1] FIG. 1 shows an exemplary solid-supported synthesis of PEOZ copolymers. [Figure 3-2] FIG. 1 shows an exemplary solid-supported synthesis of PEOZ copolymers. [Figure 4] FIG. 1 shows the chemical synthesis of ethylene glycol-oxazoline monomers. [Figure 5-1] FIG. 1 shows a second solution phase synthesis of PEOZ copolymers. [Figure 5-2] FIG. 1 shows a second solution phase synthesis of PEOZ copolymers. [Figure 6] FIG. 1 shows PEOZ copolymers with various side groups and end groups. [Figure 7] FIG. 1 shows further examples of PEOZ copolymers with various side groups and end groups. [Figure 8] FIG. 1 shows PEOZ copolymers with PEG or PEOZ side chains. DETAILED DESCRIPTION OF THE INVENTION

[0006] definition "Alkyl" refers to a monoradical, branched or straight-chain acyclic saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, the alkyl group has 1 to 24 carbon atoms, e.g., 1 to 12, 1 to 6, or 1 to 3 carbon atoms.

[0007] "Alkenyl" refers to a monoradical, branched or straight-chain acyclic hydrocarbonyl group having a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl.

[0008] "Alkynyl" refers to a monoradical, branched or straight-chain acyclic hydrocarbonyl group having a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n-propynyl.

[0009] "Cycloalkyl" refers to a monoradical cyclic saturated hydrocarbon group. Similarly, "cycloalkenyl" refers to a monoradical cyclic group having a carbon-carbon double bond, while "cycloalkynyl" refers to a monoradical cyclic group having a carbon-carbon triple bond.

[0010] "Heterocyclyl" refers to a monoradical cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and is not aromatic (i.e., which distinguishes heterocyclyl groups from heteroaryl groups). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0011] "Aryl" refers to an aromatic group containing at least one aromatic ring, wherein each atom of the aromatic ring is a carbon atom, i.e., none of the ring atoms is a heteroatom (e.g., O, S, N). In some cases, the aryl group has a second aromatic ring, e.g., fused to the first aromatic ring. Exemplary aryl groups are phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, and benzophenone.

[0012] "Heteroaryl" refers to an aromatic group containing at least one aromatic ring, wherein at least one of the atoms in the aromatic ring is a heteroatom (e.g., O, S, N). Exemplary heteroaryl groups include those obtained by removing a hydrogen atom from pyridine, pyrimidine, furan, thiophene, or benzothiophene.

[0013] The term "substitution" refers to the removal of one or more hydrogen atoms from an atom (e.g., a C atom or an N atom) and their replacement with another group. For example, a hydrogen atom from a phenyl (-CH) group can be replaced with a methyl group to form a -CHCHCH group. Thus, the -CHCHCH group can be considered a substituted aryl group. As another example, two hydrogen atoms from the second carbon of a propyl (-CHCHCH) group can be replaced with oxygen atoms to form a -CHC(O)CH group, which can be considered a substituted alkyl group. However, replacing a hydrogen atom from a propyl (-CHCHCH) group with a methyl group (e.g., to give -CHCH(CH)CH) is not considered a "substitution" as used herein because both the starting and ending groups are alkyl groups. However, if a propyl group is replaced with a methoxy group to give a -CHCH(OCH)CH group, the entire group cannot be considered an "alkyl" but rather a "substituted alkyl." Thus, to be considered a substituent, the replacing group must be of a different type than the original group. In addition, a group is assumed to be unsubstituted unless it is described as substituted. For example, the terms "alkyl" and "unsubstituted alkyl" are used interchangeably herein.

[0014] Exemplary substituents include alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halogen, hydroxy, nitro, sulfonate, and substituted versions thereof.

[0015] In some cases, the substituent itself may be further substituted with one or more groups. For example, a -CHCHCH group may be considered a substituted aryl, i.e., an aryl group substituted with the alkyl group ethyl. Furthermore, the ethyl group itself may be substituted with a pyridyl group to form -CHCHCHCHC5H5N, which may further be considered a substituted aryl group as used herein. In some cases, the substituent is not substituted with any other groups.

[0016] Diradical groups are further described herein, in contrast to the monoradical groups such as alkyl and aryl described above. The term "alkylene" refers to the diradical version of an alkyl group; i.e., an alkylene group is a diradical, branched or straight-chain, cyclic or acyclic saturated hydrocarbon group. Exemplary alkylene groups include diylmethane (also known as a methylene group -CH2-), 1,2-diylethane (-CH2CH2-), and 1,1-diylethane (i.e., a CHCH3 fragment in which the first atom has two single bonds to two other different groups). The term "arylene" refers to the diradical version of an aryl group; for example, 1,4-diylbenzene refers to a CH4 fragment in which two hydrogen atoms located para to each other have been removed and replaced with single bonds to other groups. The terms "alkenylene," "alkynylene," "heteroarylene," and "heterocyclene" are further used herein.

[0017] "Acyl" refers to a group of the formula -C(O)R, where R is alkyl, alkenyl, alkynyl, or substituted versions thereof. For example, an acetyl group has the formula -C(O)CH. "Carbonyl" refers to a diradical group of the formula -C(O)-.

[0018] "Alkoxy" refers to a group of the formula -O(alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl, and other groups.

[0019] "Amino" is the group -NR X R Y refers to R X and R Y are each independently H or a non-hydrogen substituent. Exemplary non-hydrogen substituents include alkyl groups (e.g., methyl, ethyl, and isopropyl).

[0020] "Carbonyl" refers to a diradical group of formula -C(O)-.

[0021] "Carboxy" is used interchangeably with carboxyl and carboxylate and refers to the -CO2H group and its salts.

[0022] "Ether" refers to a diradical group of formula -O-. For example, when an ether group is attached to an alkyl group, the entire group is an alkoxy group (e.g., -OCH or methoxy). When an ether group is attached to a carbonyl group, the entire group is an ester group of formula -OC(O)-.

[0023] "Halo" and "halogen" refer to chloro, bromo, fluoro, and iodo groups.

[0024] "Nitro" refers to a group of the formula -NO2.

[0025] Unless otherwise specified, a reference to an atom is meant to include all isotopes of that atom. For example, a reference to H is 1 H, 2 H (i.e., D, i.e., deuterium) and 3H (i.e. tritium), and references to C 12 C and all other isotopes of carbon (e.g. 13 C) and C). Unless otherwise specified, the radicals include all possible stereoisomers.

[0026] The terms "reactive moiety," "chemoselective functional group," "chemoselective tag," and "conjugated tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, optionally after activation of one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their activated esters, and groups that can react with each other via click chemistry, such as azides and alkynes (e.g., cyclooctyne groups), tetrazines, transcyclooctenes, dienes and dienophiles, and azides, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, as well as hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, phosphines, epoxides, succinimides, and the like.

[0027] As used herein, the term "sample" refers to a material or mixture of materials, optionally in liquid form, containing one or more analytes of interest. In some embodiments, the term used in its broadest sense refers to any plant, animal, or bacterial substance containing cells or producing cellular metabolites, such as tissues or fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or in vitro cell culture components, as well as samples from the environment. The term "sample" may also refer to a "biological sample." As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., bodily fluids, including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). A "biological sample" can also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or components, or a fraction or portion thereof, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, external sections of skin, respiratory tract, intestinal tract, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. In some embodiments, the sample is derived from an animal or plant. A biological sample may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of living organisms, both eukaryotic and prokaryotic, having at least a nucleus and a cell membrane. In some embodiments, cells include prokaryotic cells, e.g., from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from a biological sample from an animal, plant, or fungus.

[0028] The terms "carrier-bound" and "linked to a carrier" are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently linked to a carrier of interest. Covalent bonding may involve the chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between two moieties of interest (e.g., a carrier and a specific binding member). In some cases, non-covalent bonding may involve specific binding between two moieties of interest (e.g., two affinity moieties, such as a hapten and an antibody, or a biotin moiety and streptavidin). In some cases, non-covalent bonding may involve absorption to a substrate.

[0029] The term "polypeptide" refers to polymeric forms of amino acids of any length, including peptides ranging from 2 to 50 amino acids in length and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones in which the conventional backbone is replaced with a non-naturally occurring or synthetic backbone. Polypeptides may be of any convenient length, such as 2 or more amino acids, 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, for example, up to 500 or 1000 or more amino acids. A "peptide" may be 2 or more amino acids, for example, 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, for example, up to 50 amino acids. In some embodiments, the peptide is in the range of 5 to 30 amino acids in length.

[0030] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and refer to a compound of the formula --(CH2----O--) n- or derivatives thereof. In some embodiments, "n" is 5000 or less, e.g., 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, e.g., 3 to 15, or 10 to 15. It is understood that the PEG polymer group may be of any convenient length and may contain a variety of end groups and / or further substituents, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido end groups and / or substituents. PEG groups are further described by S. Zalipsky in "Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates", Bioconjugate Chemistry 1995, 6 (2), 150-165; "Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy", Chem. Rev., 2012, 112 (8), pp 4687-4735; "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", J.M. Harris, Ed., Plenum Press, New York, NY (1992); and "Poly(ethylene glycol) Chemistry and Biological Applications", J.M. Harris and S. Zalipsky, Eds., ACS (1997) and further described by Zhu et al. in WO 90 / 13540; WO 92 / 00748; WO 92 / 16555; WO 94 / 04193; WO 94 / 14758; WO 94 / 17039; WO 94 / 18247; WO 94 / 28937; WO 95 / 11924; WO 96 / 00080;WO 96 / 23794; WO 98 / 07713; WO 98 / 41562; WO 98 / 48837; WO 99 / 30727; WO 99 / 32134; WO 99 / 33483; WO 99 / 53951; WO 01 / 26692; WO 95 / 13312; WO 96 / 21469; WO 97 / 03106 Brochure; WO 99 / 45964; U.S. Patent No. 4,179,337; U.S. Patent No. 5,075,046; U.S. Patent No. 5,089,261; U.S. Patent No. 5,100,992; U.S. Patent No. 5,134,192; U.S. Patent No. 5,166,309; U.S. Patent No. 5,171,264; U.S. Patent No. 5,213,891; U.S. Patent No. 5,219,564; U.S. Patent No. 5,275,838; U.S. Patent No. 5,281,698; U.S. Patent No. 5,298,643; U.S. Patent No. 5,312,808 ;U.S. Patent No. 5,321,095;U.S. Patent No. 5,324,844;U.S. Patent No. 5,349,001;U.S. Patent No. 5,352,756;U.S. Patent No. 5,405,877;U.S. Patent No. 5,455,027;U.S. Patent No. 5,446,090;U.S. Patent No. 5,470,829;U.S. Patent No. 5,478,805;U.S. Patent No. 5,567,422;U.S. Patent No. 5,605,976;U.S. Patent No. 5,612,460;U.S. Patent No. 5,614,549;U.S. Patent No. 5,618,528;U.S. Patent No. 5,67 2662; U.S. Patent No. 5,637,749; U.S. Patent No. 5,643,575; U.S. Patent No. 5,650,388; U.S. Patent No. 5,681,567; U.S. Patent No. 5,686,110; U.S. Patent No. 5,730,990; U.S. Patent No. 5,739,208; U.S. Patent No. 5,756,593; U.S. Patent No. 5,808,096; U.S. Patent No. 5,824,778; U.S. Patent No. 5,824,784; U.S. Patent No. 5,840,900; U.S. Patent No. 5,874,500; U.S. Patent No. 5,880,131;Further described in U.S. Patent Nos. 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; and 6,214,966.

[0031] As used herein, the term "isolated" refers to a moiety that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, or even at least 99% free from other components with which the moiety is associated prior to purification.

[0032] As used herein, the terms "evaluating," "determining," "measuring," "assessing," and "analyzing" are used interchangeably and include both quantitative and qualitative determinations.

[0033] The term "separate," as used herein, refers to the physical separation of two elements (e.g., by size or affinity) and the degradation of one element while leaving the other intact.

[0034] The term "linker" or "linkage" refers to a linking moiety that connects two groups and has a backbone that is 100 atoms or less in length. The linker or linkage can be a covalent bond connecting two groups, or a chain that is 1 to 100 atoms in length, e.g., a chain that is 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms in length, and the linker can be linear, branched, cyclic, or a single atom. In some cases, the linker is a branched linker, which refers to a linking moiety that connects three or more groups. In some cases, one, two, three, four, or five or more carbon atoms of the linker backbone can be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker backbone has a linking functional group such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between backbone atoms may be saturated or unsaturated, and in some cases, there may be one, two, or three or fewer unsaturated bonds in the linker backbone. The linker may have one or more substituents, including, for example, an alkyl group, an aryl group, or an alkenyl group. The linker may include, but is not limited to, polyethylene glycol; an ether, which may be linear or branched, a thioether, a tertiary amine, an alkyl, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, where two or more atoms of the cyclic group, for example, 2, 3, or 4 atoms, are included in the backbone. The linker may be cleavable or non-cleavable.

[0035] As used herein, the terms "water-solubilizing group," "water-soluble group," and "WSG" are used interchangeably and refer to a group or substituent that is sufficiently solvated in an aqueous environment, e.g., under physiological conditions, and that results in improved water solubility of the molecule to which it is attached. A WSG may increase the solubility of a tandem dye or its component, e.g., a donor fluorophore or acceptor fluorophore, primarily in aqueous solution, compared to a control tandem dye or its component lacking the WSG. In some cases, a WSG may increase the solubility of a compound (e.g., a dye, tandem dye, or labeled specific binding member) compared to a control compound in which the WSG is replaced with a hydrogen atom. In some cases, a WSG increases solubility in aqueous media, e.g., distilled water, by 1% or more, e.g., 10% or more, 25% or more, 50% or more, 100% or more, or 500% or more. A water-solubilizing group may be any convenient hydrophilic group that is sufficiently solvated in an aqueous environment.

[0036] A water-soluble group (WSG) can impart a water solubility of greater than 10 mg / mL to a subject dye or polymeric tandem dye, such as greater than 20 mg / mL, greater than 30 mg / mL, greater than 40 mg / mL, greater than 50 mg / mL, greater than 60 mg / mL, greater than 70 mg / mL, greater than 80 mg / mL, greater than 90 mg / mL, or greater than 100 mg / mL. In some cases, a branched nonionic water-soluble group (WSG) can impart a water solubility of greater than 20 mg / mL to a subject dye or polymeric tandem dye, such as greater than 30 mg / mL, greater than 40 mg / mL, greater than 50 mg / mL, greater than 60 mg / mL, greater than 70 mg / mL, greater than 80 mg / mL, greater than 90 mg / mL, greater than 100 mg / mL, or more. It is understood that water-soluble dipyrromethene dyes can, under certain conditions, form discrete water-solvated nanoparticles in aqueous systems. In some cases, the water-solvated nanoparticles are less likely to aggregate and are useful in various biological assays.

[0037] The term "specific binding" refers to a direct bond between two molecules, for example, through covalent, electrostatic, hydrophobic, ionic and / or hydrogen-bonding interactions (including interactions such as salt bridges and water bridges). A specific binding member refers to a member of a pair of molecules that have binding specificity for one another. Members of a specific binding pair may be naturally occurring or wholly or partially synthetically produced. One member of the pair of molecules has a region or cavity on its surface that specifically binds to, and is therefore complementary to, a particular spatial polar organization of the other member of the pair. Thus, the members of the pair have the property of specifically binding to one another. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. Specific binding members of a binding pair exhibit high affinity and binding specificity for binding to one another. Typically, the affinity between a pair of specific binding members is in the range of 10 -6 M or less, e.g. 10 -7 M or less, e.g. 10 -8 M or less, e.g. 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, e.g. 10 -15 K below M d The affinity is characterized by a dissociation constant (KD). "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a decrease in KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity between one molecule and another is determined, for example, by measuring the on-rate of the interaction at 25°C. "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a decrease in KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity between one molecule and another is determined, for example, by measuring the on-rate of the interaction at 25°C.

[0038] A specific binding member can be proteinaceous. As used herein, the term "proteinaceous" refers to a moiety composed of amino acid residues. A proteinaceous moiety can be a polypeptide. In some cases, a proteinaceous specific binding member is an antibody. In some embodiments, a proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. For example, in humans, the recognized immunoglobulin genes include the kappa (k), lambda (l), and heavy chain loci, which contain numerous variable region genes as well as the constant region genes μ (u), δ (d), γ (g), σ (e), and α (a), which encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., USDapartment of Health and Human Services, (1991)). All antibody amino acid sequences described herein are numbered according to the Kabat system. The sequences of the framework regions of various light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope on an antigen. The term antibody is meant to include full-length antibodies and may refer to natural antibodies from any organism, artificial antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes, as further defined below.

[0039] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, ScFv, or other antigen-binding subsequences of antibodies produced by modification of whole antibodies or synthesized de novo using recombinant DNA technology. The antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonist, agonist, neutralizing antibody, inhibitory antibody, or stimulatory antibody). It is understood that the antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen-binding function or other antibody functions.

[0040] In some embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, an scFv, a diabody, or a triabody. In some embodiments, the specific binding member is an antibody. Optionally, the specific binding member is a murine antibody or binding fragment thereof. Optionally, the specific binding member is a recombinant antibody or binding fragment thereof.

[0041] "Active pharmaceutical ingredient" (API), "active agent," "pharmacologically active agent," and "drug" are used interchangeably herein and refer to a chemical compound or compound that, when administered to a living organism (e.g., a human or non-human animal), produces a desired pharmacological and / or physiological effect through local and / or systemic action.

[0042] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" mean generally safe, non-toxic, biologically or otherwise acceptable excipients, diluents, carriers, and adjuvants that are useful in the preparation of pharmaceutical compositions, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0043] A "plurality" includes at least two members. In some cases, a plurality may have 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0044] Numeric ranges are inclusive of the numbers defining the range.

[0045] Detailed Description Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers are provided. In some cases, the PEOZ polymers have repeating units of the structure -CHN(C(O)R)CHCHOCHCH-. The PEOZ copolymers can be used in a variety of applications, for example, to enhance the solubility of various compounds, such as drugs, dyes, and biopharmaceuticals. In some cases, methods for producing PEOZ copolymers are also provided, which can produce monodisperse compositions of PEOZ copolymers.

[0046] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. It is to be further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0047] When a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0048] In this specification, a range is presented with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately the number may be a number that, in the context in which the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described.

[0050] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0051] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0052] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from or combined with any of the features of the other multiple embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0053] Although the apparatus and methods have been or will be described for grammatical fluidity with functional descriptions, it should be clearly understood that the claims, unless expressly recited under 35 U.S.C. 112, should not be construed as necessarily limited in any way by limitations of "means" or "step" construction, but should be accorded the full scope of the meaning and equivalents of the definition given by the claims under the judicial theory of equivalents, and that if a claim is expressly recited under 35 U.S.C. 112, it should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0054] PEOZ Copolymer As summarized above, poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers are provided. The chemical structures of ethylene glycol and oxazoline are shown below. The PEOZ copolymers include monomer units of ethylene glycol and oxazoline. In some cases, the PEOZ copolymers are copolymers having monomer units derived from ethylene glycol and oxazoline or their analogs. The structures of ethylene glycol and oxazoline are provided below.

[0055] [ka]

[0056] For example, Figure 1 shows the chemical structure of a PEOZ copolymer according to embodiments of the present invention, along with a polyethylene glycol (PEG) polymer and a polyoxazoline (POZ) polymer. In some embodiments, the PEOZ copolymer has the structure of formula (I):

[0057] [ka]

[0058] In the formula, m is an integer ranging from 1 to 20, n is an integer in the range of 2 to 10,000; R is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azido, carboxy, cyano, ether, halogen, hydroxy, nitro, thiol, thioether, thioketo, borate, -SO2, -SO3-, a reactive moiety, and a reactive moiety protecting group; Each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxy, a reactive moiety, and a reactive moiety protecting group.

[0059] As noted above, m can vary and in some cases is an integer ranging from 1 to 20, such as 1 to 15, 1 to 10, 1 to 6, or 1 to 4. In some cases, m is 1 or 2. In some cases, m is 1, i.e., the PEOZ copolymer has the structure of formula (II):

[0060] [ka]

[0061] The variable m can vary and is optionally an integer in the range of 2 to 10,000, e.g., 2 to 5,000, 2 to 1,000, 2 to 100, 2 to 25, or 2 to 10. In some cases, m is an integer in the range of 3 to 10,000, e.g., 5 to 10,000, 10 to 10,000, or 25 to 10,000.

[0062] R can vary, and in some embodiments, R is selected from the group consisting of H, alkyl, and substituted alkyl. In some cases, R is alkyl, such as methyl.

[0063] In some cases, R comprises one or more water-solubilizing groups (e.g., as shown in Figures 6-8), e.g., to enhance the solubility of the copolymer. In some embodiments, R is a substituted alkyl group comprising a polyethylene glycol group or another PEOZ copolymer. In some embodiments, R is a substituted alkyl group comprising a sulfonic acid group (i.e., -SO3 group), which may provide increased solubility due to its charge.

[0064] In some cases, R comprises a reactive moiety or a reactive moiety protecting group (e.g., as shown in Figures 6-8). In some embodiments, the reactive moiety is configured to form a covalent bond with another group via a chemoselective reaction, such as a click chemistry reaction. Exemplary reactive moieties include thiols and maleimides or iodoacetamides, amines and carboxylic acids or their activated esters, and groups that can react with each other via click chemistry, such as azides and alkynes (e.g., cyclooctyne groups), tetrazines, trans-cyclooctenes, dienes and dienophiles, and azides, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, phosphines, epoxides, and succinimides.

[0065] The R group can further comprise a specific binding moiety or lipid for incorporation into a liposomal or cell membrane. In some cases, the R group is a specific binding member, such as an antibody, a fragment of an antibody (e.g., a Fab fragment, a F(ab')2 fragment, an ScFv, a bispecific antibody, or a trispecific antibody), an antigen, a biotin group, or an avidin group.

[0066] In some embodiments, each Y group is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, ether, hydroxy, and a reactive moiety protecting group. In some embodiments, the Y group can comprise a water-solubilizing group, a reactive moiety, a reactive moiety protecting group, a specific binding moiety, or a combination thereof.

[0067] pigment Further provided is a dye comprising a fluorophore and a PEOZ copolymer having a water-solubilizing group. The terms "fluorophore" and "chromophore" are used interchangeably herein.

[0068] In some cases, the dye comprises an organic dye. The organic dyes can vary and include, but are not limited to, cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.Organic dyes of interest include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, and Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPYFL, BODIPYFL-Br.sub.2, BODIPY 530 / 550, BODIPY558 / 568, BODIPY 564 / 570, BODIPY576 / 589, BODIPY 581 / 591, BODIPY630 / 650, BODIPY Examples of suitable water-soluble dyes include, but are not limited to, 650 / 665, BODIPYR6G, BODIPYTMR, BODIPYTR, Dyonomics dyes (e.g., DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831), dipyrromethene boron difluoride (BODIPY), biotin CF555, diethylaminocoumarin, and derivatives thereof. In some cases, the water-solubilizing group of the PEOZ copolymer is attached to an organic dye.

[0069] In some embodiments, the dye further comprises a non-conjugated polymer backbone having non-conjugated repeat units. By "non-conjugated," we mean that at least a portion of the repeat units comprise saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds) that prevent π-conjugation or extended delocalized electronic structures along the polymer backbone from one repeat unit to the next. Even if a repeat unit is not conjugated to adjacent repeat units, it is understood that such a repeat unit may have one or more isolated unsaturated groups, including unsaturated bonds (e.g., those of an alkenylene or alkynylene group) and / or aryl or heteroaryl groups, which may be part of the backbone. In some cases, each repeat unit of the polymer backbone comprises a side chain having an attached pendant group or a chemoselective tag for linking to a pendant group.

[0070] In some embodiments, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptoid. In some cases, the polymer is a hydrocarbon polymer. In other cases, the non-conjugated polymer is a PEG polymer. Further details regarding non-conjugated polymer backbones that may be used in embodiments of the present invention can be found in International Application No. PCT / US2019 / 024662, published as WO 2019 / 191482, and International Application No. PCT / US2020 / 019510, published as WO 2020 / 222894, the disclosures of which are incorporated herein by reference.

[0071] In some cases, the non-conjugated polymer backbone is a peptide of 2 to 100 amino acids, e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 amino acids. In some cases, the linear peptide backbone comprises 2 or more amino acids, e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to 100 amino acids. In some cases, the tandem dye comprises a linear peptide backbone of 5 to 30 amino acids, e.g., 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

[0072] The non-conjugated repeat units may have any convenient configuration, such as a linear, branched, or dendrimeric configuration. The polymer backbone may be a linear polymer. The polymer backbone may be branched. In some cases, the dye has multiple pendant chromophore groups, each independently linked to a non-conjugated repeat unit of the polymer backbone. The configuration of the pendant groups may be introduced during or after the synthesis of the non-conjugated polymer backbone. The pendant groups may be introduced in a random, block, or sequence-specific manner by stepwise synthesis, depending on the particular method of synthesis used.

[0073] In some cases, the non-conjugated repeat unit comprises multiple amino acid residues. In some cases, the dye comprises an organic dye attached to the non-conjugated polymer backbone. The water-solubilizing groups of the PEOZ copolymer can be attached to the non-conjugated polymer backbone and / or the organic dye. In some cases, the dye is a polymeric dye (e.g., a fluorescent polymeric dye). The fluorescent polymeric dyes used in the subject methods and systems vary. In some cases, the polymeric dye comprises a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure, including a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds), where π electrons can move from one bond to another. Thus, the conjugated backbone may impart an extended, linear structure to the polymeric dye, with restricted bond angles between the repeat units of the polymer. For example, proteins and nucleic acids are also polymers, but in some cases, they do not form extended rod structures but rather fold into higher-order, three-dimensional shapes. Additionally, CPs may form a "rigid rod" polymer backbone, exhibiting limited twist (e.g., torsion) angles between the monomeric repeat units along the polymer backbone chain. In some cases, polymeric dyes include CPs with rigid rod-like structures. The structural characteristics of the polymeric dye can affect the fluorescent properties of the molecule.

[0074] Any convenient polymer dye may be utilized in the subject devices and methods. In some cases, the polymer dye is a multichromophore having a structure capable of harvesting light to amplify the fluorescent output of the fluorophore. In some cases, the polymer dye can harvest light and efficiently convert it to emitted light at a longer wavelength. In some cases, the polymer dye has a light-harvesting multichromophore system that can efficiently transfer energy to a nearby emissive species (e.g., a "signaling chromophore"). Energy transfer mechanisms include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer (FRET)), quantum charge exchange (Dexter energy transfer), and the like. In some cases, these energy transfer mechanisms are relatively short-range, i.e., the proximity of the light-harvesting multichromophore system to the signaling chromophore allows for efficient energy transfer. Under conditions for efficient energy transfer, when the number of individual chromophores in the light-harvesting multichromophore system is large, amplification of the emission from the signaling chromophore occurs, i.e., the emission from the signaling chromophore is stronger when the incident light (excitation light) is at a wavelength absorbed by the light-harvesting multichromophore system than when the signaling chromophore is directly excited by the pump light.

[0075] The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains many closely spaced conjugated segments. Thus, conjugated polymers are efficient at harvesting light and enable light amplification via Förster energy transfer.

[0076] Polymeric dyes of interest include those described by Gaylord et al. in U.S. Patent Application Publication Nos. 2004 / 0142344, 2008 / 0293164, 2008 / 0064042, 2010 / 0136702, 2011 / 0256549, 2011 / 0257374, 2012 / 0028828, 2012 / 0252986, and 2013 / 0190193, the entire disclosures of which are incorporated herein by reference, as well as those described by Gaylord et al., J. Am. Chem. Soc., 2001, 123, the entire disclosures of which are incorporated herein by reference. (26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607.

[0077] In some embodiments, the polymeric dye comprises a conjugated polymer having a plurality of first optically active units forming a conjugated system, the first optically active units having a first absorption wavelength at which they absorb light to form an excited state (e.g., as described herein). The conjugated polymer (CP) may be a polycationic, polyanionic, and / or charge-neutral conjugated polymer.

[0078] The polymeric dye may have any convenient length. In some cases, the specific number of monomeric repeat units or segments of the polymeric dye may be in the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments.

[0079] The polymeric dye may have any convenient molecular weight (MW). In some cases, the MW of the polymeric dye may be expressed as an average molecular weight. In some cases, the polymeric dye has an average molecular weight in the range of 500 to 500,000, e.g., 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or 50,000 to 100,000. In some embodiments, the polymeric dye has an average molecular weight of 70,000.

[0080] In some cases, the polymeric dye has the structure:

[0081] [ka]

[0082] In the structure, CP1, CP2, CP3, and CP4 are independently conjugated polymer segments or oligomeric structures, one or more of CP1, CP2, CP3, and CP4 are bandgap-lowering n-conjugated repeat units, each n and each m is independently 0 or an integer in the range of 1 to 10,000, and p is an integer in the range of 1 to 100,000.

[0083] In some cases, the polymeric dye has the structure:

[0084] [ka]

[0085] In the structure, each R 1 are independently solubilizing groups or linker dyes, and L 1 and L 2 is an optional linker, and each R 2 are independently H or an aryl substituent, and each A 1 and each A 2 are independently H, an aryl substituent, or a fluorophore; G 1 and G 2are each independently selected from the group consisting of an end group, a π-conjugated segment, a linker, and a linked specific binding member, each n and each m is independently 0 or an integer in the range of 1 to 10,000, and p is an integer in the range of 1 to 100,000. Solubilizing groups of interest include PEOZ solubilizing groups as described herein.

[0086] In some cases, the polymeric dye includes a conjugated segment having one of the following structures as part of the polymer backbone:

[0087] [ka]

[0088] In the structure, each R 3 are independently an optionally substituted alkyl or aryl group, Ar is an optionally substituted aryl or heteroaryl group, and each n is an integer in the range of 1 to 10,000. 3 is an optionally substituted alkyl group. In one embodiment, R 3 is an optionally substituted aryl group. 3 is substituted with polyethylene glycol, a dye, a chemoselective functional group, or a specific binding moiety. In some cases, Ar is substituted with polyethylene glycol, a dye, a chemoselective functional group, or a specific binding moiety.

[0089] In some cases, the polymeric dye has the structure:

[0090] [ka]

[0091] In the structure, each R 1 are independently solubilizing groups or linker dye groups, and each R 2 are independently H or an aryl substituent, and each L 1 and each L 3 are independently any linker, and each A1 and each A 3 are independently H, a fluorophore, a functional group, or a specific binding moiety (e.g., an antibody); n and m are each independently 0 or an integer in the range of 1 to 10,000, and n+m>1.

[0092] The polymer dye may have one or more desirable spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. (See, for example, Chattopadhyay et al., "Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments," Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymer dye has an absorption curve in the range of 280 nm to 475 nm. In certain embodiments, the polymer dye has an absorption maximum (excitation maximum) in the range of 280 nm to 475 nm. In some embodiments, the polymer dye absorbs incident light having a wavelength in the range of 280 nm to 475 nm. In some embodiments, the polymeric dye has an emission maximum wavelength within the range of 400 nm to 850 nm, e.g., 415 nm to 800 nm. Specific examples of emission maxima of interest include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some embodiments, the polymeric dye has an emission maximum wavelength within a range selected from the group consisting of 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 421 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 570 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 602 nm. In some embodiments, the polymer dye has a maximum emission wavelength of 650 nm. In some embodiments, the polymer dye has a maximum emission wavelength of 711 nm. In some embodiments, the polymer dye has a maximum emission wavelength of 786 nm. In some embodiments, the polymer dye has a maximum emission wavelength of 421 nm ± 5 nm. In some embodiments, the polymer dye has a maximum emission wavelength of 510 nm ± 5 nm.In some embodiments, the polymeric dye has an emission maximum wavelength of 570 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 602 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 650 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 711 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 786 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0093] In some cases, polymeric dyes are 1 x 10 6 cm -1 M -1 or more, for example, 2 x 10 6 cm -1 M -1 That's it, 2.5 x 10 6 cm -1 M -1 That's it, 3 x 10 6 cm -1 M -1 That's it, 4 x 10 6 cm -1 M -1 That's it, 5 x 10 6 cm -1 M -1 That's it, 6 x 10 6 cm -1 M -1 That's it, 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1In some embodiments, the polymeric dye has a quantum yield of 0.05 or greater, e.g., 0.1 or greater, 0.15 or greater, 0.2 or greater, 0.25 or greater, 0.3 or greater, 0.35 or greater, 0.4 or greater, 0.45 or greater, 0.5 or greater, or greater. In some cases, the polymeric dye has a quantum yield of 0.1 or greater. In some cases, the polymeric dye has a quantum yield of 0.3 or greater. In some embodiments, the polymeric dye has a quantum yield of 1×10 6 In some embodiments, the polymeric dye has an extinction coefficient of 2×10 or greater and a quantum yield of 0.3 or greater. 6 It has an extinction coefficient of 0.5 or more and a quantum yield of 0.5 or more.

[0094] Specific polymer dyes that may be used include, but are not limited to, BD Horizon Brilliant™ Dyes, such as BD Horizon Brilliant™ Violet Dye (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant™ Ultraviolet Dye (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant™ Blue Dye (e.g., BB515) (BD Biosciences, San Jose, CA).

[0095] Tandem Dyes Also provided is a tandem dye comprising a donor fluorophore, an acceptor fluorophore, and a PEOZ copolymer having a water-solubilizing group. A tandem dye is a compound having two covalently attached, distinct fluorophores, which may be covalently attached to each other directly or via a linking group. One of the fluorophores functions as a donor fluorophore, and the other functions as an acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescence resonance energy transfer (FRET) pair. Such a FRET pair functions as a unique dye with the excitation characteristics of the donor fluorophore and the emission characteristics of the acceptor fluorophore.

[0096] Excitation of the donor can result in energy transfer to and emission from the covalently bound acceptor fluorophore. Mechanisms for energy transfer between donor chromophores and from donor chromophores to the linked acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer (FRET)), quantum charge exchange (Dexter energy transfer), and the like. These energy transfer mechanisms can be relatively short-range; i.e., the proximity of the chromophores of the light-harvesting multichromophore system to each other and / or to the acceptor fluorophore allows for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the acceptor fluorophore can occur; emission from the emitting acceptor fluorophore is more intense when the incident light (pump light) is at a wavelength that is absorbed by and transmitted from the chromophore of the light-harvesting chromophore than when the emitting acceptor fluorophore is directly excited by the incident light (pump light). "Efficient" energy transfer means that 10% or more, for example 20% or more, 30% or more, 40% or more, or 50% or more of the energy acquired by the donor chromophore is transferred to the acceptor. "Amplification" means that when excited by energy transfer from the donor light-harvesting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more compared to direct excitation of the acceptor fluorophore with incident light of equivalent intensity. The signal may be measured using any convenient method. In some cases, a signal of 1.5 times or more refers to the intensity of emitted light. In some cases, a signal of 1.5 times or more refers to an increase in the signal-to-noise ratio. In some embodiments of the tandem dye, the emission of the acceptor fluorophore when excited by the chromophore is at least 1.5 times greater than when the acceptor fluorophore is directly excited with incident light, for example at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 8 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or more than that when the acceptor fluorophore is directly excited with incident light.

[0097] In some cases, the tandem dye exhibits an effective Stokes shift in the range of 25 nm to 300 nm, e.g., 50 nm to 250 nm, or 75 nm to 200 nm. In some cases, when the light-harvesting chromophore is directly excited by incident light, the effective Stokes shift is 25 nm or more, e.g., 50 nm or more, 75 nm or more, 100 nm or more, e.g., 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 250 nm or more.

[0098] The emission of the tandem dye can have a quantum yield of 0.03 or greater, for example, 0.04 or greater, 0.05 or greater, 0.06 or greater, 0.07 or greater, 0.08 or greater, 0.09 or greater, 0.1 or greater, 0.15 or greater, 0.2 or greater, 0.3 or greater, or greater. In some cases, the polymeric tandem dye has a quantum yield of 5×10 5 cm -1 M -1 For example, 6 x 10 5 cm -1 M -1 That's it, 7 x 10 5 cm -1 M -1 That's it, 8 x 10 5 cm -1 M -1 That's it, 9 x 10 5 cm -1 M -1 or more, for example, 1×10 6 cm -1 M -1 That's it, 1.5 x 10 6 cm -1 M -1 That's it, 2 x 10 6 cm -1 M -1 That's it, 2.5 x 10 6 cm -1 M -1 That's it, 3 x 10 6 cm -1 M -1 That's it, 4 x 10 6 cm -1 M -1 That's it, 5 x 10 6 cm -1 M-1 That's it, 6 x 10 6 cm -1 M -1 That's it, 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 In some embodiments, the tandem dye has an extinction coefficient of 5×10 or greater. 5 M -1 cm -1 In some embodiments, the tandem dye has a molar extinction coefficient of 1×10 or greater. 6 M -1 cm -1 It has a molar extinction coefficient of at least 1000 ppm.

[0099] At least one of the donor fluorophore and the acceptor fluorophore, and in some cases both, comprises an organic dye. For example, the donor fluorophore organic dye can be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrromethene boron difluoride, naphthalimide, thiazine dyes, and acridine dyes. In some cases, the acceptor fluorophore organic dye can be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrromethene boron difluoride, naphthalimide, thiazine dyes, and acridine dyes.

[0100] Organic dyes of interest that may be used as donors or acceptors include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 516, Alexa Fluor 518, Alexa Fluor 519, Alexa Fluor 520, Alexa Fluor 530, Alexa Fluor 540, Alexa Fluor 550, Alexa Fluor 560, Alexa Fluor 570, Alexa Fluor 580, Alexa Fluor 590, Alexa Fluor 600, Alexa Fluor 610, Alexa Fluor 620, Alexa Fluor 630, Alexa Fluor 640, Alexa Fluor 650, Alexa Fluor 660, Alexa Fluor 670, Alexa Fluor 680, Alexa Fluor 690, Alexa Fluor 691, Alexa Fluor 692, Alexa Fluor 693, Alexa Fluor 694, Alexa Fluor 695, Alexa Fluor 696, Alexa Fluor 697, Alexa Fluor 698, Alexa Fluor 699 Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPYFL, BODIPYFL-Br.sub.2, BODIPY 530 / 550, BODIPY558 / 568, BODIPY 564 / 570, BODIPY576 / 589, BODIPY 581 / 591, BODIPY630 / 650, BODIPY 650 / 665, BODIPYR6G, BODIPYTMR, BODIPYTR, Dyonomics dyes (e.g., DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831), dipyrromethene boron difluoride (BODIPY), biotin CF555, diethylaminocoumarin, and derivatives thereof.

[0101] As described above, the dye may comprise a non-conjugated polymer backbone. Similarly, in some embodiments, the tandem dye may comprise a non-conjugated polymer backbone having a non-conjugated repeat unit. In some embodiments, the non-conjugated repeat unit comprises a plurality of amino acid residues. The water-solubilizing group of the PEOZ copolymer may be attached to the donor fluorophore, the acceptor fluorophore, and / or the non-conjugated polymer backbone.

[0102] As described above, the dyes may include fluorophores that are conjugated polymers. Thus, in some cases, the donor fluorophore of a tandem dye may include a conjugated polymer. As described above with respect to the dyes, in some cases, the conjugated polymer may have a series of optionally substituted aryl and / or heteroaryl groups. Aryl groups of interest include fluorene and phenyl, while heteroaryl groups of interest include thiophene, pyridine, and BODIPY groups. In such cases, the PEOZ water-solubilizing group may be attached to the conjugated polymer and / or the acceptor fluorophore.

[0103] Labeled specific binding members Aspects of the present disclosure further include labeled specific binding members, including, for example, a dye or tandem dye as described above, along with the specific binding member. Alternatively, the specific binding member is labeled, for example, with a dye or tandem dye to allow for detection of a target analyte that binds to the specific binding member.

[0104] composition The present disclosure further provides compositions comprising a PEOZ copolymer. In some cases, the composition comprises 10 or more individual copolymer molecules, i.e., the composition comprises 10 or more PEOZ copolymers. In some embodiments, the composition comprises 100 or more, e.g., 1,000 or more, 10,000 or more, 100,000 or more, or 1,000,000 or more PEOZ copolymers.

[0105] In some embodiments, the molecular weights of the PEOZ copolymers in the composition may be relatively similar to one another. As used herein, the terms "dispersity" (D) and "polydispersity index" (PDI) are used interchangeably and refer to a measure of the distribution of molecular weights within the composition. Specifically, D = PDI = M w / M n and M w is the mass-average molar mass, and M n is the number average molar mass. Smaller D values ​​indicate a more uniform distribution of molecular weights, while larger D values ​​indicate a more heterogeneous distribution of molecular weights. A D value of exactly 1.0 means that the distribution is completely uniform. In some cases, M w is determined by a method selected from the group consisting of static light scattering, small angle neutron scattering, X-ray scattering, and sedimentation velocity. n is determined by a method selected from the group consisting of gel permeation chromatography, viscosity measurement (e.g., using the Mark-Houwink equation), and colligative methods (e.g., vapor pressure osmometry). w and M n The formula for is shown below, and N i is the molecular weight M i is the number of molecules.

[0106]

number

[0107] Thus, in some cases, the PEOZ copolymers in the composition have relatively similar molecular weights, e.g., the dispersity (D) of the copolymers is 1.5 or less, e.g., 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, 1.05 or less, or 1.02 or less.

[0108] Method for producing PEOZ copolymer First method for producing PEOZ copolymers A first method for producing a PEOZ copolymer is provided, the first method comprising: (a) producing an (ethylene glycol)-oxazoline (EOZ) dimer having a nucleophilic hydroxyl group, and upon producing (i) reacting a first EOZ monomer having a protected hydroxyl group with a second EOZ monomer having a protected hydroxyl group to produce an EOZ dimer having two protected hydroxyl groups; (ii) selectively deprotecting one hydroxyl group of the EOZ dimer to produce an EOZ dimer having a nucleophilic hydroxyl group; (b) forming an EOZ dimer having an electrophilic leaving group; (i) reacting a third EOZ monomer having a protected hydroxyl group with a fourth EOZ monomer having a protected hydroxyl group to produce an EOZ dimer having two protected hydroxyl groups; (ii) selectively deprotecting one hydroxyl group of the EOZ dimer; (iii) converting the deprotected hydroxyl group of the EOZ dimer into an electrophilic leaving group; (c) An EOZ dimer having a nucleophilic hydroxyl group is reacted with an EOZ dimer having an electrophilic leaving group to produce an EOZ tetramer, which is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0109] In step (a)(i) of this method, a first EOZ monomer is reacted with a second EOZ monomer to form a dimer having two protected hydroxyl groups, for example, by nucleophilic substitution between a nucleophilic group (e.g., hydroxyl) of the first monomer and an electrophilic leaving group (e.g., triflate) of the second monomer.

[0110] In step (a)(ii), one of the hydroxyl groups is selectively deprotected. Therefore, the first hydroxyl protecting group has different properties from the second hydroxyl protecting group. For example, one hydroxyl can be protected with TBDMS, while the other hydroxyl is protected with a benzyl group. The TBDMS group can be removed with TBATB (e.g., Firouzabadiet et al., Synthetic Communications, 1996, 26, doi:10.1080 / 00397919608003713), but acids (e.g., camphorsulfonic acid, acetic acid) and bases (e.g., HF-pyridine, TBAF) can also be used to perform such selective deprotection. The benzyl group can be removed with H2 / Pd(O), CrO3 / acetic acid, ozone, N-bromosuccinimide (NBS), or N-iodosuccinimide (NIS).

[0111] Thus, in step (a), an EOZ dimer having a nucleophilic hydroxyl group is produced.

[0112] In step (b), a third EOZ monomer and a fourth EOZ monomer are reacted to produce another EOZ dimer having two protected hydroxyl groups. As in step (a), the hydroxyls can be protected with different protecting groups (e.g., benzyl and TBDMS). In step (b)(ii), one hydroxyl group is selectively deprotected, e.g., as described above. In step (b)(iii), the deprotected hydroxyl group can be converted to an electrophilic group, e.g., by reaction with a base (e.g., NaH) and Tf-Cl to produce an OTf (i.e., triflate) leaving group.

[0113] In step (c), two EOZ dimers are reacted with each other by nucleophilic substitution of the nucleophilic hydroxyl group of the first dimer with the electrophilic leaving group (e.g., OTf) of the second dimer to produce an EOZ tetramer.

[0114] This procedure can be repeated to produce larger polymers. For example, a sample of the PEOZ tetramer produced by step (c) can be modified to have nucleophilic hydroxyl groups. In addition, a second sample of the PEOZ tetramer can be modified to have electrophilic groups (e.g., OTf). The two samples of PEOZ tetramer can then be reacted to produce a PEOZ octamer. Thus, the present disclosure provides a general method for synthesizing PEOZ x-mers, in which a PEOZ a-mer having nucleophilic hydroxyl groups is reacted with a PEOZ b-mer having electrophilic leaving groups (e.g., OTf). x is equal to the sum of a and b.

[0115] In some cases, the reaction is carried out in a solvent, such as an aqueous or organic solvent. In some cases, the organic solvent is an alkane solvent (e.g., hexane, octane), diethyl ether, dimethyl sulfoxide (DMSO), or tetrahydrofuran (THF). In some cases, the reaction is carried out without a solvent, i.e., neat.

[0116] Such methods can produce relatively monodisperse distributions, for example, due to the stepwise procedure. In some cases, the PEOZ copolymer compositions produced by this method have a dispersity (D) of 1.5 or less, e.g., 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, 1.05 or less, or 1.02 or less.

[0117] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0118] Second Method for Producing PEOZ Copolymers There is also provided a method for producing a PEOZ copolymer, comprising: reacting a compound of formula (Ia) with a compound of formula (Ib) to form a compound of formula (Ic);

[0119] [ka]

[0120] deprotecting the compound of formula (Ic) to form a compound of formula (Id);

[0121] [ka]

[0122] converting a compound of formula (Ic) to a compound of formula (Ie),

[0123] [ka]

[0124] reacting a compound of formula (Id) with a compound of formula (Ie) to form a compound of formula (If);

[0125] [ka]

[0126] wherein PG1 and PG2 are each independently a hydroxyl protecting group; OX is a leaving group, R is an alkyl group.

[0127] In some embodiments, the hydroxyl protecting group PG1 and the hydroxyl protecting group PG2 are independently selected from benzyl and TBDMS. In some embodiments, PG1 and PG2 are different hydroxyl protecting groups. In some embodiments, deprotection to remove the TBDMS group is performed by contact with TBATB, an acid (e.g., camphorsulfonic acid, acetic acid), or a base (e.g., HF-pyridine, TBAF). In some embodiments, benzyl protecting groups are removed by contact with H2 / Pd(O), CrO3 / acetic acid, ozone, N-bromosuccinimide (NBS), or N-iodosuccinimide (NIS).

[0128] In some embodiments, R is selected from the group consisting of H, alkyl, and substituted alkyl. In some embodiments, R is alkyl, such as methyl.

[0129] In some cases, R contains one or more water-solubilizing groups to enhance the solubility of the copolymer (e.g., as shown in Figures 6-8). In some embodiments, R is a substituted alkyl group containing a polyethylene glycol group or another PEOZ copolymer. In some embodiments, R is a substituted alkyl group containing a sulfonic acid group (i.e., -SO3 group), which may provide increased solubility due to its charge.

[0130] In some cases, R comprises a reactive moiety or a reactive moiety-protecting group (e.g., as shown in Figures 6-8). In some embodiments, the reactive moiety is configured to form a covalent bond with another group via a chemoselective reaction, e.g., a click chemistry reaction. Exemplary reactive moieties include a succinimidyl group, an azide group, or a tetrazine group.

[0131] The R group can further comprise a specific binding moiety, such as an antibody, a fragment of an antibody, or a lipid, for incorporation into the liposomal membrane or cell membrane.

[0132] Such methods can produce relatively monodisperse distributions, for example, due to the stepwise procedure. In some cases, the PEOZ copolymer compositions produced by this method have a dispersity (D) of 1.5 or less, e.g., 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, 1.05 or less, or 1.02 or less.

[0133] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0134] A third method for producing PEOZ copolymers A third method for producing a PEOZ copolymer is provided, comprising: providing a first (ethylene glycol)-oxazoline (EOZ) monomer having a nucleophilic hydroxyl group attached to a substrate; reacting a first EOZ monomer with a second EOZ monomer having an electrophilic leaving group and a protected hydroxyl group to produce an EOZ dimer having a protected hydroxyl group and attached to the substrate; deprotecting the protected hydroxyl group to produce an EOZ dimer having a nucleophilic hydroxyl group bound to the substrate; The substrate-bound EOZ dimer is reacted with a third EOZ monomer bearing an electrophilic leaving group and a protected hydroxyl group to produce a substrate-bound EOZ trimer bearing a protected hydroxyl group, which is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0135] In some cases, the substrate is a particle, such as a nanoparticle, microparticle, or bead. The substrate may comprise a material selected from the group consisting of a metal, a metalloid, a ceramic, and a polymer.

[0136] Optionally, the method further includes releasing the PEOZ copolymer from the substrate, for example, by contact with an acid, base, or nucleophile, such as, for example, optionally, by contact with methyl iodide (i.e., Mel) and a base (e.g., NaOH, KOH).

[0137] Such methods can produce relatively monodisperse distributions, for example, due to the stepwise procedure. In some cases, the PEOZ copolymer compositions produced by this method have a dispersity (D) of 1.5 or less, e.g., 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, 1.05 or less, or 1.02 or less.

[0138] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0139] Method for increasing compound solubility using PEOZ copolymers The present disclosure provides a method for increasing the solubility of a compound with a PEOZ copolymer compared to the solubility of the compound without the PEOZ component. As such, the PEOZ copolymer can be considered a water-solubilizing group. In some cases, the method includes attaching the water-solubilizing group of the PEOZ copolymer to the compound. The PEOZ copolymer can be attached to the compound by a covalent or non-covalent bond (e.g., ionic bond).

[0140] In some embodiments, the compound solubilized by the PEOZ group is a dye comprising a fluorophore, e.g., as described above. In other cases, the compound solubilized is a tandem dye comprising a donor fluorophore and an acceptor fluorophore, e.g., as described above. In some cases, the compound is a specific binding member labeled with, e.g., a dye or tandem dye.

[0141] In some cases, the compound to be solubilized is an active pharmaceutical ingredient (API), while in other cases, the compound is selected from the group consisting of a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier, and a pharmaceutically acceptable adjuvant.

[0142] For example, some known compounds are linked to polyethylene glycol (PEG) groups and are referred to as "PEGylated" compounds. PEOZ copolymers can be linked instead of or in addition to PEG groups using the methods described herein. For example, Harris et al. have described polypeptide pharmaceuticals with PEG groups linked thereto (NatureReviews Drug Discovery, 2003, 2, 214, doi:10.1038 / nrd1033), and therefore, polypeptide pharmaceuticals can be linked to PEOZ groups instead of such PEG groups according to the methods of the present disclosure. Other compounds that can be functionalized with PEOZ instead of or in addition to PEG include dyes (e.g., Wuet al. (doi: 10.1016 / j.ejmech.2018.10.046) and Collado et al. (doi: 10.1039 / C3RA46235H)), cytokines and therapeutic proteins (Francis et al., doi: 10.1016 / s0925-5710(98)00039-5), small molecule drugs (Li et al., 10.1016 / j.progpolymsci.2012.07.006), excipients (e.g., the PEGylated excipients included in the Pfizer COVID-19 vaccine, as described by Cabanillas et al. (doi: 10.1111 / all.14711)), liposomes (Heger et al., doi: 10.1016 / j.mvr.2009.02.006). In such cases, compounds (e.g., dyes, cytokines, therapeutic proteins, small molecule drugs, excipients, or liposomes) can be attached to the PEOZ copolymer to increase the solubility of the compound.

[0143] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0144] Methods for labeling target molecules Further provided are methods for labeling a target molecule, which involve contacting the target molecule with a dye or tandem dye as described herein to covalently bind the target molecule to a reactive moiety on the dye or tandem dye, thereby producing a labeled target molecule.

[0145] Methods of interest for labeling targets include, but are not limited to, the methods and reagents described in Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013. The contacting step may be carried out in aqueous solution. In some cases, the reactive moiety has an amino functionality and the target molecule has an active ester functionality, such as an NHS ester or sulfo-NHS ester, or vice versa. In some cases, the reactive moiety has a maleimide functionality and the target molecule has a thiol functionality, or vice versa. In some cases, the reactive moiety has an alkyne (e.g., cyclooctyne) functionality and the target molecule has an azide functionality, or vice versa, which may be conjugated via click chemistry.

[0146] Any convenient target molecule may be selected for labeling using the subject methods. Target molecules of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins such as fusion proteins; modified proteins such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, sumoylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term "target protein" refers to all members of a target family and fragments thereof. A target protein may be any protein of interest, such as a therapeutic or diagnostic target, including, but not limited to, hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony-stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method or any convenient synthetic method, or that can be purchased commercially. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In some cases, the specific binding member is an antibody. Optionally, the specific binding member is an antibody fragment or binding derivative thereof. Optionally, the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an scFv, a diabody, and a triabody.

[0147] In some cases, the method comprises a separation step in which the labeled target molecule is separated from the reaction mixture, e.g., excess reagents or unlabeled target. A variety of methods may be used to separate the target from the sample, e.g., by immobilization on a support, precipitation, chromatography, etc.

[0148] Optionally, the method further comprises detecting and / or analyzing the labeled target molecule. Optionally, the method further comprises fluorescently detecting the labeled target molecule. Any convenient method for detecting and / or analyzing the labeled target molecule may be used in conjunction with the subject methods and compositions. Methods for analyzing targets of interest used in the subject methods include, but are not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. Detection methods of interest include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in-situ hybridization (FISH), protein mass spectrometry, flow cytometry, and the like.

[0149] Detection may be direct via the polymer-tandem dye or indirectly via a secondary detection system. Secondary detection systems may be based on any one or combination of several different principles, including, but not limited to, antibody-labeled anti-species antibodies and other forms of immunological or non-immunological cross-linking and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology, or nucleic acid probe / anti-nucleic acid probe). Suitable reporter molecules may be those known in the fields of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cellular immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, counting, and / or signal output quantification. Two or more antibodies, of specific and / or non-specific nature, may be labeled and used simultaneously or sequentially to improve target detection, discrimination, and / or analysis.

[0150] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0151] Method for evaluating a sample for the presence of a target analyte Methods are provided for assessing a sample for the presence of a target analyte using a labeled specific binding member comprising a PEOZ copolymer with a WSG. In some embodiments, the methods include: (a) contacting the sample with a labeled specific binding member that specifically binds to the target analyte to produce a labeled sample; (b) Analyzing the labeled composition for the presence of labeled specific binding member-target analyte binding complexes to assess whether the target analyte is present in the sample.

[0152] Labeled specific binding members used in embodiments of the methods of the present invention include specific binding members conjugated to dyes or tandem dyes, as described above. In the following sections, depending on whether the labeled specific binding member is used as a primary or secondary label, the target analyte may be a target molecule of interest or a reagent bound to the target molecule, e.g., a primary antibody. Any convenient method may be used to contact a sample with a labeled specific binding member that specifically binds to the target analyte to generate an analytical composition. In some cases, the sample is contacted with the labeled specific binding member under conditions under which the labeled specific binding member, if present, specifically binds to the target analyte. To allow the labeled specific binding member to specifically bind to the target analyte, an appropriate medium that maintains the biological activity of the sample components and the single domain antibody may be used. The medium may be a balanced salt solution, such as saline, PBS, or Hank's balanced salt solution, conveniently supplemented with fetal bovine serum, human platelet lysate, or other factors, along with an acceptable buffer at low concentrations, e.g., 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. A variety of media, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, and the like, optionally supplemented with fetal bovine serum or human platelet lysate, are commercially available and may be used depending on the nature of the target analyte. The final components of the medium, which may be a solution, may be selected depending on the components of the sample involved. The temperature at which specific binding of the labeled specific binding member to the target analyte occurs may vary and, in some cases, may be within the range of 5°C to 50°C (e.g., as described above), e.g., 10°C to 40°C, 15°C to 40°C, or 20°C to 40°C, e.g., 20°C, 25°C, 30°C, 35°C, or 37°C. In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the specific binding member and / or the target analyte. In some cases, the temperature is 25°C, 30°C, 35°C, or 37°C. In some cases, the temperature at which specific binding occurs is room temperature (eg, 25°C), 30°C, 35°C, or 37°C.Any convenient incubation time for specific binding may be selected to allow for the production of the desired amount of binding complex, and may optionally be 1 minute (min) or more, e.g., 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or 6 hours or more.

[0153] Any convenient specific binding member may be used for the labeled specific binding member used in the methods of the present invention. Specific binding members of interest include, but are not limited to, specific binding members that specifically bind to cell surface proteins of various cell types, including, but not limited to, stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory cells, dendritic cells, B cells, e.g., memory B cells, antigen-specific B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells bearing a convenient cell surface marker or antigen that may be captured by a convenient specific binding member conjugate. In some embodiments, the target cells are selected from HIV-bearing cells from whole blood, bone marrow, or umbilical cord blood, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+). Any convenient cell surface protein or cell marker may be targeted for specific binding to the conjugates used in the subject methods, hi some embodiments, the target cell comprises a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, target cells may comprise cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0154] Any convenient target may be selected for evaluation using the subject methods. Targets of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins such as fusion proteins; modified proteins such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, sumoylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term "target protein" refers to all members of a target family and fragments thereof. A target protein may be any protein of interest, such as a therapeutic or diagnostic target, including, but not limited to, hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony-stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method or any convenient synthetic method, or that can be purchased commercially. In some embodiments, the polymer-dye conjugate comprises an antibody or antibody fragment. Any convenient target analyte that specifically binds to the antibody or antibody fragment of interest may be targeted in the subject method.

[0155] In some embodiments, the target analyte binds to a cell. Optionally, the target analyte is a cell surface marker of the cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. Optionally, the target analyte is an intracellular target, and the method further comprises treating the cell to allow the labeled specific binding member access to the intracellular target, for example, by permeabilizing or lysing the cell. Thus, the labeled specific binding member used in the method of the invention may target a cell surface or intracellular antigen. Alternatively, the labeled specific binding member used in the method of the invention may target a primary antibody that, in turn, specifically binds to the target cell surface or intracellular antigen.

[0156] In some embodiments, the sample may comprise a heterogeneous cell population from which target cells are isolated. Optionally, the sample comprises peripheral whole blood, peripheral whole blood from which red blood cells have been lysed prior to cell isolation, umbilical cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenized tissue. Optionally, the sample comprises hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample comprises tumor cells in peripheral blood. Optionally, the sample is a sample containing (or suspected of containing) viral cells (e.g., HIV).

[0157] Labeled specific binding members are used in the subject methods, e.g., to label target cells, particles, targets, or analytes with polymer-tandem fluorescent dyes. For example, labeled specific binding members are used in labeling cells to be processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. Labeled specific binding members may include, for example, specific binding members, e.g., antibodies or binding fragments thereof, that specifically bind to cell surface proteins of various cell types (e.g., as described herein). Labeled specific binding members may be used to investigate various biological (e.g., cellular) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. Labeled specific binding members may be used in any application that includes (or may include) antibody-mediated labeling of cells, particles, or analytes.

[0158] In some aspects of the method, an analytical composition, i.e., a sample contacted with a labeled specific binding member, is analyzed for the presence of a labeled specific binding member-target analyte binding complex to assess whether the target analyte is present in the sample. Once the sample is contacted with the labeled specific binding member, any convenient method may be used to analyze the resulting analytical composition for the presence of a labeled specific binding member-target analyte binding complex. A labeled specific binding member-target analyte binding complex is a binding complex formed when the labeled specific binding member specifically binds to the target analyte, if present (or a primary binding member for the target antigen, e.g., a primary antibody, depending on the embodiment). When analyzing the analytical composition, a fluorescent signal from the binding complex, if present, may be detected. In some cases, the analysis involves a separation step to separate the target analyte from the sample, if present. A variety of methods can be used to separate the target analyte from the sample, such as immobilization on a support. Analytical methods of interest include, but are not limited to, any convenient methods and assay formats that use specific binding member pairs, such as avidin-biotin or hapten-antihapten antibody. Methods and analytical formats of interest that may be adapted for use with the subject compositions include, but are not limited to, flow cytometry, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0159] In certain embodiments, the methods further comprise contacting the sample with a second specific binding member that specifically binds to the target analyte. Optionally, the second specific binding member is carrier-bound. Any convenient carrier may be utilized for immobilizing components of the subject methods (e.g., the second specific binding member). Optionally, the carrier is a particle, such as a magnetic particle. Optionally, the second specific binding member and the polymer dye conjugate form a sandwich complex that may be isolated and detected, if present, using any convenient method. In some embodiments, the methods further comprise analyzing the polymer dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte, by flow cytometry. Analyzing for the presence of the labeled specific binding member-target analyte binding complex may provide an analytical result (e.g., qualitative or quantitative analytical data) that can be used to assess whether the target analyte is present in the sample.

[0160] Any convenient support may be utilized in the subject methods to immobilize any convenient component of the method, e.g., labeled specific binding members, targets, secondary specific binding members, etc. Supports of interest include, but are not limited to, solid substrates that may have a variety of configurations, such as sheets, beads, or other structures, e.g., plates with wells; beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, chromatography supports, etc. In some cases, the support is selected from the group consisting of particles, planar solid substrates, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography supports. The support may be incorporated into a system that provides cell isolation assisted by any convenient method, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the support is used in an automated liquid handling system, e.g., a flow cytometer, for high-throughput isolation of cells.

[0161] In some embodiments of the method, the separation step involves applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet may be used as the source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, such as a permanent magnet or an electromagnet. In some cases, immobilizing the magnetic particles means that the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e., the magnet.

[0162] The separation step may further include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing method may be used, for example, washing the immobilized support with a biocompatible buffer that maintains the specific binding interaction between the polymeric dye and the specific binding member. Separating and optionally washing the unbound material of the sample from the support may enrich the population of target cells and remove undesired cells and materials.

[0163] In certain embodiments, the methods detect labeled target analytes. Detecting labeled target analytes may involve exciting the polymeric fluorescent tandem dye with one or more lasers, followed by detecting fluorescent emission from the polymeric fluorescent tandem dye using one or more photodetectors. Any convenient instrument or method can be used to detect labeled targets, including, but not limited to, flow cytometry, FACS systems, fluorescence microscopy; plate readers for detecting fluorescence, luminescence, ultraviolet, and / or visible light; high-performance liquid chromatography (HPLC); and mass spectrometry. When fluorescently labeled components are used in the methods and compositions of the present disclosure, it is recognized that the subject methods can be performed using various types of fluorescence detection systems. In some cases, high-throughput screening can be performed, such as systems using 96-well or larger microtiter plates. Various methods for analysis using fluorescent materials can be used, for example, those described in Lakowicz, J.R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D.L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Co., Inc. (1978), pp. 296-361.

[0164] Fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through excitation optics. The excitation optics cause the excitation radiation to excite the sample. In response to the excitation of the sample, fluorescently labeled targets in the sample emit radiation having a wavelength different from the excitation wavelength. Collection optics then collect the emitted light from the sample. The device may include a temperature controller to maintain the sample at a specific temperature during scanning. In some cases, a multi-axis translation stage moves a microtiter plate holding multiple samples to position various wells for exposure. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data collection may be managed by an appropriately programmed digital computer. The computer may also convert data collected during analysis into another format for presentation.

[0165] In some embodiments, the method of evaluating a sample for the presence of a target analyte further comprises detecting fluorescence with a flow cytometer. In some embodiments, the method of evaluating a sample for the presence of a target analyte further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer-dye conjugate-target analyte binding complexes in the contacted sample and assess whether the target analyte is present. Microscopy of interest for use in the subject methods includes laser scanning confocal microscopy.

[0166] The methods described herein may include multiple steps. Each step may be performed with a predetermined time between steps, as desired. Thus, the time between steps may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, e.g., 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following completion of the previous step, e.g., a waiting period ranging from several minutes to overnight.

[0167] kit Embodiments of the present invention further include kits for use in practicing the subject methods. The dyes, tandem dyes, labeled specific binding members, or combinations thereof of the present invention can be included as starting materials or as reagents in kits provided for use in, for example, the methods described above. Such dyes, tandem dyes, and labeled specific binding members can be provided with containers. Any convenient container can be utilized, such as a tube, bottle, or well of a multiwell strip or plate, a box, a bag, an insulated container, or the like. The subject kits can further include, as desired, one or more elements selected from a primer specific binding member for a given target analyte, a specific binding member bound to a carrier, cells, a carrier, a biocompatible aqueous elution buffer, controls (positive and / or negative), and the like, and instructions for use. A given kit can include reagents suitable for the detection of a single target analyte or multiple reagents suitable for the detection of two or more different target analytes, e.g., if a given kit is configured for multiplexed detection applications.

[0168] In certain embodiments, the kit is used in assessing a sample for the presence of a target analyte, such as an intracellular target. As such, in some cases, the kit includes one or more components suitable for permeabilizing or lysing cells. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multiwell strip or plate).

[0169] In some embodiments, the kit further comprises reagents for performing flow cytometry analysis. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting chromophores with cell samples, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may further comprise one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. Additionally, the kit may comprise a cell permeabilization reagent, such as methanol, acetone, or a detergent, such as Triton, NP-40, saponin, Tween 20, digitonin, leucoperm, or any combination or buffer thereof. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kits.

[0170] The compositions of the kit may be provided in liquid compositions, e.g., any suitable buffer. Alternatively, the compositions of the kit may be provided in dry compositions (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry compositions. In some embodiments, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0171] Additionally, one or more elements may be combined in a single container, such as a glass or plastic vial, tube, or bottle. In some cases, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all of the elements (and their separate containers) reside. The kit may further include packaging, separate from or attached to the kit container, printed with information about the kit, its elements, and / or instructions for use.

[0172] In addition to the above elements, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Yet another means is a computer-readable medium on which the information is recorded, such as a diskette, CD, DVD, portable flash drive, etc. Yet another means that may be present is a website address usable via the internet to access the information at a remote location. Any convenient means may be provided in the kit.

[0173] Example The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp (base pairs), kb (kilobases), pl (picoliters), s or sec (seconds), min (minutes), h or hr (hours), aa (amino acid), nt (nucleotide), etc.

[0174] Example 1: Solution phase synthesis of PEOZ copolymer As shown in Figure 2 , a series of solution-phase chemical reactions were carried out to produce poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers from two different monomers.

[0175] Both starting monomers were protected analogs of N,N-bis(2-hydroxyethyl)propionamide. The monomer on the left side of Figure 2 had one free hydroxyl group and one -O-TBDMS group, which was a hydroxyl group protected with tert-butyldimethylsilyl (TBDMS). The monomer on the right side of Figure 2 had an -O-CH-CH group, which may also be referred to as an -OBz ​​group and may be interpreted as a protected hydroxyl group. The monomer on the right side also had an -OTf group, where -OTf refers to a triflate group and has the formula -OSOCF, which may be referred to as a leaving group.

[0176] Dimers were generated by reacting two monomers in solution, for example, by nucleophilic substitution, such that a new covalent bond was formed between the free hydroxyl group and the triflate group. As shown in Figure 2, the PG2 group in the dimer refers to the benzyl-protected hydroxyl group.

[0177] A first sample of the dimer was selectively deprotected to remove the TBDMS group (labeled "Deprotection 1"), while the PG2 group (i.e., the benzyl group) was not deprotected. The TBDMS group was removed with TBATB (e.g., Firouzabadiet et al., Synthetic Communications, 1996, 26, doi:10.1080 / 00397919608003713), although acids (e.g., camphorsulfonic acid, acetic acid) and bases (e.g., HF-pyridine, TBAF) can also be used to perform such selective deprotection. This selectively deprotected first sample was then reacted with Tf-Cl to generate the -OTf group.

[0178] A second sample of the dimer was selectively deprotected (labeled "Deprotection 2") to remove the PG2 (benzyl) group without removing the PG1 (i.e., TBDMS) group. H2 / Pd(O) was used to remove the benzyl group, although CrO3 / acetic acid, ozone, N-bromosuccinimide (NBS), and N-iodosuccinimide (NIS) can also be used.

[0179] The selectively deprotected first and second samples of the dimer are then reacted to generate a new covalent bond between the free hydroxyl group and the triflate group, resulting in a tetramer.

[0180] As shown in Figure 2, the steps involving selective deprotection, addition of a triflate group to one sample, and then reaction of the two samples can be repeated to produce larger copolymers, for example, copolymers having 8, 16, 32 or more repeating units.

[0181] Example 2: Solid-supported synthesis of PEOZ copolymer As shown in Figure 3, the synthesis begins by reacting the 2-(4-(hydroxymethyl)phenyl)-N-methylacetamide group attached to the solid resin support with an N,N-bis(2-hydroxyethyl)propionamide analog bearing one TBDMS-protected hydroxyl group and one carboxylic acid, thereby forming a new covalent bond between the free hydroxyl and carboxylic acid moieties, e.g., via transesterification. Thus, the synthesis step yields a solid-supported monomer bearing a terminal TBDMS-protected hydroxyl group.

[0182] The TBDMS protecting group was then removed to generate free hydroxyl groups. The solid-supported monomer bearing the free hydroxyl groups was reacted with the solution-phase monomer bearing the OTf and OTBDMS groups to generate new bonds at the free hydroxyl and OTf sites. The resulting dimer was solid-supported and had terminal TBDMS-protected hydroxyl groups.

[0183] The procedure of deprotecting the OTBDMS group and reacting the free hydroxyl group with a monomer containing an OTf leaving group was repeated, and n units were added to the PEOZ copolymer by repeating this procedure n times.

[0184] After the desired number of monomer units had been added, the solid-supported PEOZ copolymer was reacted with methyl iodide (MeI) and then with a base to cleave the resin. The resulting compound was a PEOZ copolymer with terminal methoxy and carboxylic acid groups.

[0185] Example 3: Synthesis of Monomer Starting Materials As shown in Figure 4, commercially available 3-(benzyloxy)propanal was reacted with commercially available 2-aminoethan-1-ol to produce 2-((2-(benzyloxy)ethyl)amino)ethan-1-ol. This compound was then reacted with acetic acid in the presence of EDC in DMF at 80 °C according to the method of Gokhale et al. (Biomacromolecules, 2013, 14, 2489) to produce the N-(2-(benzyloxy)ethyl)-N-(2-hydroxyethyl)acetamide monomer. This compound can be interpreted as a PEOZ monomer in which the amine is replaced with a -C(O)Me group and one of the hydroxyl groups is protected with a benzyl group.

[0186] Additionally, as further shown in Figure 4, a TBDMS-protected analog was synthesized by reacting TBDMS-protected 3-(l1-oxidaneyl)propanal with commercially available 2-aminoethan-1-ol to produce the TBDMS-protected analog of 2-((2-(l1-oxidaneyl)ethyl)amino)ethan-1-ol. This compound can be considered the TBDMS-protected analog of the corresponding benzyl-protected compound shown at the top of Figure 4. The TBDMS-protected compound was reacted with acetic acid according to the method of Gokhale et al. to produce the corresponding TBDMS-protected compound.

[0187] Example 4: Second solution-phase synthesis of PEOZ copolymer As shown in Figure 5, PEOZ copolymers were synthesized from two different monomer starting compounds.

[0188] A monomer with a free hydroxyl and a benzyl-protected hydroxyl was reacted with NaH and TfCl to convert the free hydroxyl to an OTf group, and the resulting compound was reacted with the free hydroxyl of a second monomer to produce a dimer with one terminal OTBDMS group and one terminal OBz group.

[0189] One sample of the dimer was selectively deprotected to remove the benzyl group by reaction with H / Pd(O) in a manner similar to that described in Example 1 and Figure 2. A second sample of the dimer was reacted with TBATB in methanol to deprotect the OTBDMS group, followed by reaction with NaH and TfCl to introduce the OTf moiety.

[0190] The two samples were then reacted in the presence of NaH to generate tetramers by creating covalent bonds between the free hydroxyl sites and the OTf moieties through a nucleophilic substitution reaction.

[0191] The procedure of selective deprotection and reaction of the hydroxyl groups with OTf groups was repeated to generate the octameric compound.

[0192] The terminal hydroxyl groups were reacted with NaH and MeI to generate terminal methoxyl groups, while the terminal O-TBDMS groups were deprotected with TBATB to generate free hydroxyl groups. The final compound was a PEOZ octamer copolymer with terminal hydroxy and methoxy groups.

[0193] Example 5: End and Side Groups of PEOZ Copolymer Figure 6 shows PEOZ copolymers with various end groups, including methoxy, carboxylic acid, triflate (i.e., OTf), and succinimidyl groups. Figure 6 also shows embodiments in which the side chain of the amino group is an acetyl group or an acetyl group substituted with a polyethylene glycol group. The carboxylic acid and succinimidyl groups can be considered functional linkers that can form bonds with other groups.

[0194] Figure 7 shows examples of side and end groups of PEOZ copolymers. Figure 7 shows tetrazine, succinimidyl, and azide (N3) groups that can be used for conjugation. SO3 groups can be used to enhance solubility. - A side group bearing a group is further shown, along with a lipid tail that can be used for incorporation into liposomes or cell membranes.

[0195] Figure 8 shows how the amino groups attached to the side chain groups can be polymeric groups such as PEG or PEOZ groups to further enhance solubility. The embodiment in Figure 8 shows dimeric, tetrameric, and octameric PEG groups along with tetrameric PEOZ side chains.

[0196] Regardless of the scope of the appended claims, the present disclosure is further defined by the following notes.

[0197] Appendix 1. Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0198] Appendix 2. Having the structure of formula (I):

[0199] [ka]

[0200] m is an integer in the range of 1 to 20; n is an integer in the range of 2 to 10,000; R is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azido, carboxy, cyano, ether, halogen, hydroxy, nitro, thiol, thioether, thioketo, borate, -SO2, -SO3-, a reactive moiety, and a reactive moiety protecting group; 2. The PEOZ copolymer of claim 1, wherein each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxy, a reactive moiety, and a reactive moiety protecting group.

[0201] Item 3. The PEOZ copolymer of item 2, wherein m is an integer in the range of 1 to 6.

[0202] Item 4. The PEOZ copolymer of item 3, wherein m is 1 or 2.

[0203] Appendix 5. Having the structure of formula (II):

[0204] [ka]

[0205] PEOZ copolymers as described in Appendix 4.

[0206] Item 6. The PEOZ copolymer of any one of Items 1 to 5, wherein R is selected from the group consisting of H, alkyl, and substituted alkyl.

[0207] Item 7. The PEOZ copolymer of any one of Items 1-6, wherein each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, ether, hydroxy, and a reactive moiety protecting group.

[0208] Appendix 8. Fluorophores, and Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers with water-solubilizing groups Contains pigments.

[0209] Appendix 9. The dye according to Appendix 8, which is an organic dye.

[0210] Item 10. The dye of Item 9, wherein the organic dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0211] Item 11. The dye of item 9 or 10, wherein the water-solubilizing group of the PEOZ copolymer is attached to an organic dye.

[0212] Appendices 12. The dye of any one of Appendices 9 to 11, comprising a non-conjugated polymer backbone having non-conjugated repeat units.

[0213] Item 13. The dye of Item 12, wherein the non-conjugated repeat unit comprises a plurality of amino acid residues.

[0214] Appendix 14. The dye of any one of appendices 12 and 13, comprising an organic dye attached to a non-conjugated polymer backbone.

[0215] Applicant's note 15. The dye of any one of Applicants' notes 12 to 14, wherein the water-solubilizing groups of the PEOZ copolymer are attached to a non-conjugated polymer backbone.

[0216] Applicant's note 16: The dye of any one of Applicants' notes 12 to 14, wherein the water-solubilizing group of the PEOZ copolymer is attached to an organic dye.

[0217] Item 17. The dye of item 8, comprising a conjugated polymer.

[0218] Item 18. The dye of item 17, wherein the water-solubilizing group of the PEOZ copolymer is attached to a conjugated polymer.

[0219] Item 19. The dye of item 17, wherein the water-solubilizing group of the PEOZ copolymer is attached to an organic dye that is attached to a conjugated polymer.

[0220] Appendix 20. Donor fluorophores, an acceptor fluorophore, and Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers with water-solubilizing groups Tandem dyes, including:

[0221] Clause 21. The tandem dye of clause 20, wherein at least one of the donor fluorophore and the acceptor fluorophore comprises an organic dye.

[0222] 22. The tandem dye of claim 21, wherein the organic dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0223] Item 23. The tandem dye of item 22, wherein the organic dye is dipyrromethene boron difluoride.

[0224] Appendices 24. The tandem dye of any one of Appendices 20 to 23, comprising a non-conjugated polymer backbone having non-conjugated repeat units.

[0225] Item 25. The tandem dye of item 24, wherein the non-conjugated repeat unit comprises a plurality of amino acid residues.

[0226] Appendices 26. The tandem dye of any one of Appendices 21 to 25, wherein both the donor fluorophore and the acceptor fluorophore comprise organic dyes.

[0227] Applicant's note 27. The tandem dye of any one of Applicants' notes 23 to 26, wherein the water-solubilizing groups of the PEOZ copolymer are attached to a non-conjugated polymer backbone.

[0228] Item 28. The tandem dye of any one of items 23 to 26, wherein the water-solubilizing group of the PEOZ copolymer is attached to a donor fluorophore.

[0229] Applicant's note 29: The tandem dye of any one of Applicants' notes 23 to 26, wherein the water-solubilizing group of the PEOZ copolymer is attached to an acceptor fluorophore.

[0230] Item 30. The tandem dye of any one of Items 20-22, wherein the donor fluorophore comprises a conjugated polymer.

[0231] Item 31. The tandem dye of item 30, wherein the water-solubilizing group of the PEOZ copolymer is attached to a conjugated polymer.

[0232] Item 32. The tandem dye of item 30, wherein the water-solubilizing group of the PEOZ copolymer is attached to an acceptor fluorophore.

[0233] Appendix 33. A dye according to any one of appendices 8 to 19, or a tandem dye according to any one of appendices 20 to 32, and Specific Binding Members a labeled specific binding member comprising:

[0234] Clause 34. A method for evaluating a sample for the presence of a target analyte, comprising: (a) contacting the sample with a labeled specific binding member according to Appendix 33 that specifically binds to the target analyte to produce a labeled sample; (b) analyzing the labeled composition for the presence of labeled specific binding member-target analyte binding complexes to assess whether the target analyte is present in the sample.

[0235] Appendix 35. A method for labeling a target molecule, comprising: 32. A method of producing a labeled target molecule by contacting the target molecule with a dye according to any one of claims 8 to 19, or a tandem dye according to any one of claims 20 to 32, to covalently bond the target molecule to a reactive moiety on the dye or tandem dye.

[0236] 36. The method of claim 36, wherein the target molecule is selected from the group consisting of DNA, RNA, an antibody, or a fragment thereof.

[0237] Appendix 37. A dye according to any one of appendices 8 to 19, a tandem dye according to any one of appendices 20 to 32, a labeled specific binding member according to appendix 33, or a combination thereof; and container The kit includes:

[0238] Clause 38. A kit according to clause 37, comprising a labelled specific binding member according to clause 33 and a dye according to any one of clauses 8 to 19.

[0239] Appendix 39. The kit of Appendix 37, comprising a tandem dye of any one of Appendixes 20-32 and a dye of any one of Appendixes 8-19.

[0240] Appendix 40. A method for producing a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, comprising: (a) producing an (ethylene glycol)-oxazoline (EOZ) dimer having a nucleophilic hydroxyl group, and upon producing (i) reacting a first EOZ monomer having a protected hydroxyl group with a second EOZ monomer having a protected hydroxyl group to produce an EOZ dimer having two protected hydroxyl groups; (ii) selectively deprotecting one hydroxyl group of the EOZ dimer to produce an EOZ dimer having a nucleophilic hydroxyl group; (b) forming an EOZ dimer having an electrophilic leaving group; (i) reacting a third EOZ monomer having a protected hydroxyl group with a fourth EOZ monomer having a protected hydroxyl group to produce an EOZ dimer having two protected hydroxyl groups; (ii) selectively deprotecting one hydroxyl group of the EOZ dimer; (iii) converting the deprotected hydroxyl group of the EOZ dimer into an electrophilic leaving group; (c) A method of reacting an EOZ dimer having a nucleophilic hydroxyl group with an EOZ dimer having an electrophilic leaving group to produce an EOZ tetramer, which is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0241] Appendix 41. A method for producing a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, comprising: reacting a compound of formula (Ia) with a compound of formula (Ib) to form a compound of formula (Ic);

[0242] [ka]

[0243] deprotecting the compound of formula (Ic) to form a compound of formula (Id);

[0244] [ka]

[0245] converting a compound of formula (Ic) to a compound of formula (Ie),

[0246] [ka]

[0247] reacting a compound of formula (Id) with a compound of formula (Ie) to form a compound of formula (If);

[0248] [ka]

[0249] wherein PG1 and PG2 are each independently a hydroxyl protecting group; OX is a leaving group, The method wherein R is an alkyl group.

[0250] Item 42. A method for producing a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, comprising: providing a first (ethylene glycol)-oxazoline (EOZ) monomer having a nucleophilic hydroxyl group attached to a substrate; reacting a first EOZ monomer with a second EOZ monomer having an electrophilic leaving group and a protected hydroxyl group to produce an EOZ dimer having a protected hydroxyl group and attached to the substrate; deprotecting the protected hydroxyl group to produce an EOZ dimer having a nucleophilic hydroxyl group bound to the substrate; The method comprises reacting the substrate-bound EOZ dimer with a third EOZ monomer having an electrophilic leaving group and a protected hydroxyl group to produce a substrate-bound EOZ trimer having a protected hydroxyl group, wherein the EOZ trimer is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0251] Appendix 43. A method for increasing the solubility of a compound using a water-solubilizing group of a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, comprising: A method of attaching the water-solubilizing groups of a PEOZ copolymer to a compound.

[0252] Applicant's statement 44. The method of claim 43, wherein the compound is a dye comprising a fluorophore or a tandem dye comprising a donor fluorophore and an acceptor fluorophore.

[0253] Clause 45. The method of clause 43, wherein the compound is a specific binding member.

[0254] Item 46. The method of item 43, wherein the compound is an active pharmaceutical ingredient (API).

[0255] Item 47. The method of item 43, wherein the compound is selected from the group consisting of a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier, and a pharmaceutically acceptable adjuvant.

[0256] Announcement 48.10 or more containing poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, A composition wherein the dispersity of 10 or more PEOZ copolymers is 1.5 or less.

[0257] Item 49. The composition of item 48, wherein the dispersity is 1.1 or less.

[0258] In at least some of the embodiments described above, one or more elements used in an embodiment may be used interchangeably in another embodiment unless the substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter as defined by the appended claims.

[0259] In general, those skilled in the art will understand that the terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "includes" should be interpreted as "including, but not limited to"). Those skilled in the art will further understand that where a specific number of introduced claim recitations are intended, such intention will be explicitly set forth in the claim, and that in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments containing only one such recitation, even if that claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. Furthermore, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., the literal recitation "two recitations" means at least two recitations or more than two recitations, without other modifiers).Furthermore, when a conventional expression similar to "at least one of A, B, and C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). When a conventional expression similar to "at least one of A, B, or C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). It will be further understood by those skilled in the art that virtually any disjunctive word and / or disjunctive phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0260] Additionally, when features or aspects of the disclosure are described in Markush format, those skilled in the art will recognize that the disclosure is further described in terms of any individual member or subgroup of members of the Markush group.

[0261] As will be understood by those skilled in the art, for all purposes, including those described in the specification, all ranges disclosed herein further encompass any and all possible subranges and combinations of these subranges. Any recited range can be readily recognized as being sufficiently descriptive to allow that same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be readily divided into a lower third, middle third, upper third, etc. As will be further understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited number and that can subsequently be divided into subranges as previously described. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 elements refers to groups having 1, 2, or 3 elements. Similarly, a group having 1 to 5 elements refers to groups having 1, 2, 3, 4, or 5 elements, etc.

[0262] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0263] Accordingly, the foregoing merely illustrates the essence of the present invention. It is clear that those skilled in the art will be able to devise various configurations that embody the essence of the present invention and are within the spirit and scope of the present invention, although not explicitly described or shown herein. Furthermore, all examples and conditional language set forth herein are intended primarily to aid the reader in understanding the essence of the present invention and the concepts provided by the inventors to advance the art, and should not be construed as limiting the examples and conditions specifically set forth. Furthermore, all statements herein that describe the essence, aspects, and embodiments of the present invention, as well as specific examples of the present invention, are intended to encompass both structural and functional equivalents of the present invention. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be publicly disclosed, regardless of whether such disclosure is explicitly set forth in the claims.

[0264] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" appears at the beginning of such limitation in the claim; if such precise phrase is not used in a claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0265] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 428,508, filed November 29, 2022, the disclosure of which is incorporated herein by reference.

Claims

1. Poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

2. having the structure of formula (I): 【Chemistry 1】 m is an integer ranging from 1 to 20; n is an integer ranging from 2 to 10,000; R is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azido, carboxy, cyano, ether, halogen, hydroxy, nitro, thiol, thioether, thioketo, borate, -SO 2 , -SO 3 -, a reactive moiety, and a reactive moiety protecting group; 2. The PEOZ copolymer of claim 1, wherein each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxy, a reactive moiety, and a reactive moiety protecting group.

3. 3. The PEOZ copolymer of claim 2, wherein m is an integer ranging from 1 to 6.

4. 4. The PEOZ copolymer of claim 3, wherein m is 1 or 2.

5. having the structure of formula (II): 【Chemistry 2】 5. The PEOZ copolymer of claim 4.

6. A PEOZ copolymer according to any one of claims 1 to 5, wherein R is selected from the group consisting of H, alkyl and substituted alkyl.

7. 7. The PEOZ copolymer of any one of claims 1 to 6, wherein each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, ether, hydroxy, and a reactive moiety protecting group.

8. fluorophores, and The poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer according to any one of claims 1 to 7, having water-solubilizing groups. Contains pigments.

9. The dye of claim 8 comprising an organic dye or a conjugated polymer.

10. donor fluorophore, an acceptor fluorophore, and The poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer according to any one of claims 1 to 9, having water-solubilizing groups. Tandem dyes, including:

11. A dye according to any one of claims 1 to 9 or a tandem dye according to claim 10, and Specific Binding Members a labeled specific binding member comprising:

12. 1. A method for evaluating a sample for the presence of a target analyte, comprising: (a) contacting a sample with a labeled specific binding member of claim 11 that specifically binds to a target analyte to produce a labeled sample; (b) analyzing the labeled composition for the presence of a labeled specific binding member-target analyte binding complex to assess whether said target analyte is present in the sample.

13. 1. A method for labeling a target molecule, comprising:

11. A method of producing a labeled target molecule by contacting a target molecule with the dye of claim 8 or 9, or the tandem dye of claim 10, to covalently bind the target molecule to a reactive moiety of the dye or the tandem dye.

14. A dye according to claim 8 or 9, a tandem dye according to claim 10, a labelled specific binding member according to claim 11, or a combination thereof; and container The kit includes:

15. 1. A method for increasing the solubility of a compound using water-solubilizing groups of a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, comprising: A method comprising attaching a water-solubilizing group of a PEOZ copolymer to said compound.

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