Polyethylene glycol-polyoxazoline (PEOZ) copolymers for dissolving drugs, dyes and biopharmacy

Abstract

A polyethylene glycol-polyoxazoline (PEOZ) copolymer is provided. In some cases, the PEOZ polymer has repeating units of the structure of-CH2N (C (O) R) CH2CH2OCH2CH2-. The PEOZ copolymers may be used in a variety of applications, e.g., to increase the solubility of various compounds, such as drugs, dyes and biopharmaceuticals. Also provided are methods of making the PEOZ copolymers, which, in some cases, can produce monodispersed compositions of the PEOZ copolymers.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 428,508, filed on November 29, 2022; the disclosure of which is incorporated herein by reference. Background of the Invention

[0003] Polyethylene glycol (PEG) groups are attached to small molecules, nucleotides, peptides, proteins, liposomes, and nanoparticles to improve solubility, stability, and pharmacokinetic properties (Chen et al., ACS Nano, 2021, 15, 14022). In some cases, polymer-drug conjugates are formed using PEG, such as PEG groups with a molecular weight of 0.3 - 60 kDa per polymer (Kong et al., Frontiers in Bioengineering and Biotechnology, 2022, 10, 879988). PEG has also been used in bioconjugation and nanomedicine to extend blood circulation time and improve drug efficacy (Thi et al., Polymers, 2020, 12, 298).

[0004] However, some untreated individuals may have pre-existing antibodies that can bind to PEG and induce an immune response (Chen et al., supra). At least 25 different drugs or compositions contain PEG groups, including the mRNA-1273 vaccine for COVID-19 produced by Moderna, Inc. (ibid). Additionally, it has been observed that treating subjects with drugs containing PEG groups can lead to the production of anti-PEG antibodies, which may be undesirable (Thi et al., supra).

[0005] Furthermore, many common methods for synthesizing PEG and other polymers produce polydisperse compositions with a wide molecular weight distribution. This polydispersity may adversely inhibit control over polymer properties. Summary of the Invention

[0006] Polyethylene glycol-polyoxazoline (PEOZ) copolymers are provided. In some cases, the PEOZ polymer has a repeating unit with the structure -CH 2 N(C(O)R)CH 2 CH 2 OCH 2 CH 2 -. The PEOZ copolymers can be used in a variety of applications, for example, for increasing the solubility of various compounds such as drugs, dyes, and biopharmaceuticals. The present invention also provides methods for preparing PEOZ copolymers, which in some cases can generate monodisperse compositions of the PEOZ copolymers. Brief Description of the Drawings

[0007] Figure 1 Shows the general structures of polyethylene glycol (PEG), polyoxazoline (POZ), and polyethylene glycol - polyoxazoline (PEOZ) copolymers.

[0008] Figure 2 Shows an exemplary solution - phase synthesis of PEOZ copolymers.

[0009] Figure 3 Shows an exemplary solid - supported synthesis of PEOZ copolymers.

[0010] Figure 4 Shows the chemical synthesis of ethylene glycol - oxazoline monomers.

[0011] Figure 5 Shows a second solution - phase synthesis of PEOZ copolymers.

[0012] Figure 6 Shows PEOZ copolymers with different side groups and end groups.

[0013] Figure 7 Shows more examples of PEOZ copolymers with different side groups and end groups.

[0014] Figure 8 Shows PEOZ copolymers with PEG or PEOZ side chains. Detailed Description

[0015] "Alkyl" refers to a monovalent, branched or linear, acyclic saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n - propyl, isopropyl, n - butyl, isobutyl, tert - butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, the alkyl group has 1 to 24 carbon atoms, such as 1 to 12, 1 to 6, or 1 to 3 carbon atoms.

[0016] "Alkenyl" refers to a monovalent, branched or linear, acyclic hydrocarbon group containing a carbon - carbon double bond. Exemplary alkenyl groups include vinyl, n - propenyl, isopropenyl, n - butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl.

[0017] "Alkynyl" refers to a monovalent, branched or linear, acyclic hydrocarbon group containing a carbon - carbon triple bond. Exemplary alkynyl groups include ethynyl and n - propynyl.

[0018] "Cycloalkyl" refers to a monovalent, cyclic saturated hydrocarbon group. Similarly, "cycloalkenyl" refers to a monovalent, cyclic group having a carbon - carbon double bond, and "cycloalkynyl" refers to a monovalent, cyclic group having a carbon - carbon triple bond.

[0019] "Heterocyclic group" refers to a monocyclic, cyclic group containing a heteroatom (e.g., O, S, N) as a ring atom and being non-aromatic (i.e., distinguishing a heterocyclic group from a heteroaryl). Examples of heterocyclic groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0020] "Aryl" refers to an aromatic group containing at least one aromatic ring, where each atom in the ring is a carbon atom, i.e., the ring atoms are not heteroatoms (e.g., O, S, N). In some cases, the aryl has a second aromatic ring, e.g., fused to the first aromatic ring. Exemplary aryls are phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone.

[0021] "Heteroaryl" refers to an aromatic group containing at least one aromatic ring, where at least one atom on the aromatic ring is a heteroatom (e.g., O, S, N). Exemplary heteroaryls include those obtained by removing one hydrogen atom from pyridine, pyrimidine, furan, thiophene, or benzothiophene.

[0022] The term "substituted" means removing one or more hydrogens from an atom (e.g., from a C or N atom) and replacing them with different groups. For example, a hydrogen atom on a phenyl (-C 6 H 5 ) group can be replaced by a methyl group to form -C 6 H 4 CH 3 group. Thus, -C 6 H 4 CH 3 group can be regarded as a substituted aryl. As another example, two hydrogen atoms on the second carbon of a propyl (-CH 2 CH 2 CH 3 ) group can be replaced by an oxygen atom to form -CH 2 C(O)CH 3 group, which can be regarded as a substituted alkyl. However, replacing a hydrogen atom on a propyl (-CH 2 CH 2 CH 3 ) group with a methyl group (e.g., to obtain -CH 2 CH(CH 3 )CH 3 ) is not considered "substituted" as used herein because both the starting group and the ending group are alkyls. However, if the propyl is replaced by a methoxy group, thus obtaining -CH 2 CH(OCH 3 )CH 3If the group contains such a substituent group, the entire group is no longer considered an "alkyl group" but a "substituted alkyl group". Therefore, for a group to be considered a substituent, the type of the substituting group is different from the original group. In addition, unless described as substituted, a group is assumed to be unsubstituted. For example, the terms "alkyl group" and "unsubstituted alkyl group" are used interchangeably herein.

[0023] Exemplary substituents include alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxy, nitro, sulfonate, and their substituted forms.

[0024] In some cases, a substituent itself can be further substituted by one or more groups. For example, the group -C 6 H 4 CH 2 CH 3 can be considered a substituted aryl, namely an aryl substituted by an ethyl group, where the ethyl group is an alkyl group. In addition, the ethyl group itself can be substituted by a pyridyl group to form -C 6 H 4 CH 2 CH 2 C 5 H 5 N, where -C 6 H 4 CH 2 CH 2 C 5 H 5 N can also be considered a substituted aryl as used in the terms herein. In some cases, a substituent is not substituted by any other group.

[0025] Bifunctional groups are also described herein, which are in contrast to monofunctional groups such as the above-mentioned alkyl and aryl groups. The term "alkylene" refers to the bifunctional form of an alkyl group, i.e., an alkylene is a bifunctional, branched or linear, cyclic or acyclic saturated hydrocarbon group. Exemplary alkylene groups include dimethylene (-CH 2 -, also known as methylene), 1,2-dimethylethane (-CH 2 CH 2 -), and 1,1-dimethylethane (i.e., the CHCH 3 fragment, where the first atom has two single bonds connecting to two different other groups). The term "arylene" refers to the bifunctional form of an aryl group. For example, 1,4-diphenylene refers to the C 6 H 4 fragment, where two hydrogens para to each other are removed and replaced by single bonds connecting to other groups. The terms "alkenylene", "alkynylene", "heteroarylene", and "heterocycloalkylene" are also used herein.

[0026] "Acyl" refers to a group of the formula -C(O)R, where R is an alkyl, alkenyl, alkynyl or a substituted form thereof. For example, acetyl has the formula -C(O)CH 3 . "Carbonyl" refers to the divalent group of the formula -C(O)-.

[0027] "Alkoxy" refers to a group of the formula -O(alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl and other groups.

[0028] "Amino" refers to the group -NR X R Y , where R X and R Y are each independently H or a non-hydrogen substituent. Exemplary non-hydrogen substituents include alkyl (e.g., methyl, ethyl and isopropyl).

[0029] "Carbonyl" refers to the divalent group of the formula -C(O)-.

[0030] "Carboxy" is used interchangeably with carboxyl and carboxylate, and refers to the -CO 2 H group and its salts.

[0031] "Ether" refers to the divalent group of the formula -O-. For example, if the ether group is attached to an alkyl, the whole group is an alkoxy (e.g., -OCH 3 or methoxy). If the ether group is attached to a carbonyl, the whole group is an ester group of the formula -OC(O)-.

[0032] "Halogenated" and "halogen" refer to chlorine, bromine, fluorine and iodine groups.

[0033] "Nitro" refers to a group of the formula -NO 2 .

[0034] Unless otherwise specified, the mention of an atom shall include all isotopes of that atom. For example, the mention of H shall include 1 H, 2 H (i.e., D or deuterium) and 3 H (i.e., tritium), and the mention of C shall include 12 C and all other carbon isotopes (e.g., 13 C). Unless otherwise specified, a group shall include all possible stereoisomers.

[0035] The terms "reactive structural moiety", "chemoselective functional group", "chemoselective tag", and "conjugate tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases after optionally activating 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 active esters, and groups that can react with each other by click chemistry, such as azides and alkynes (e.g., cyclooctynyl), tetrazines, trans-cyclooctenes, dienes and dienophiles, and azides, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyls, hydrazides, hydrazino groups, aldehydes, ketones, azido groups, alkynyl groups, phosphines, epoxides, succinimides, etc.

[0036] As used herein, the term "sample" refers to a material or mixture of materials, in some cases in liquid form, containing one or more analytes of interest. In some embodiments, the term is used in its broadest sense and refers to any plant, animal, or bacterial material that contains cells or produces cellular metabolites, such as tissue or fluid isolated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or components of an in vitro cell culture, as well as samples from the environment. The term "sample" can also refer to a "biological sample". As used herein, the term "biological sample" refers to an entire organism or a subset of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate, or extract prepared from an entire organism or a subset of its tissues, cells, or components or a part or portion thereof, including but not limited to plasma, serum, spinal fluid, lymphatic fluid, external sections of the skin, respiratory tract, intestine, and urogenital tract, tears, saliva, milk, blood cells, tumors, and organs. In some embodiments, the sample has been taken from an animal or a plant. A biological sample can include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of eukaryotic and prokaryotic organisms that has at least one nucleus and a cell membrane. In some embodiments, the cells include prokaryotic cells, such as cells from bacteria. In other embodiments, the cells include eukaryotic cells, such as cells obtained from a biological sample of an animal, a plant, or a fungus.

[0037] The terms "vector-bound" and "linked to a vector" are used interchangeably and refer to a structural moiety (e.g., a specific binding member) that is covalently or non-covalently linked to a vector of interest. Covalent linkage can involve a 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 target structural moieties (e.g., a vector and a specific binding member). In some cases, non-covalent linkage can involve specific binding between two structural moieties of interest (e.g., two affinity moieties such as a hapten and an antibody or a biotin moiety and streptavidin, etc.). In some cases, non-covalent linkage can involve absorption to a matrix.

[0038] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides of 2-50 amino acids in length and polypeptides of 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 having a modified peptide backbone (where the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone). Polypeptides can be of any convenient length, such as 2 or more amino acids, such as 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, such as up to 500 or 1000 or more amino acids. A "peptide" can contain 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the peptide is 5 to 30 amino acids in length.

[0039] The terms "polyethylene oxide", "PEO", "polyethylene glycol", and "PEG" are used interchangeably and refer to a polymer containing repeating units of the formula -(CH 2----O--)n - polymer groups or derivatives thereof of the described chain. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15 or 10 to 15. It should be understood that the PEG polymer group can be of any convenient length and can contain various end groups and / or other substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide end groups and / or substituents. The PEG group is also described by S. Zalipsky in "Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates", Bioconjugate Chemistry 1995, 6(2), 150 - 165; by Zhu et al. in "Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy", Chem. Rev., 2012, 112(8), pp 4687 - 4735, "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", J.M. Harris, Ed., Plenum Press, New York, N.Y. (1992); and "Poly(ethylene glycol) Chemistry and Biological Applications", J.M. Harris and S. Zalipsky, Eds., ACS (1997); and International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964, and U.S. Patents 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0040] As used herein, the term "isolated" means that the structure of interest is at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, or even at least 99% free of other components associated with the structure prior to purification.

[0041] As used herein, the terms "assess", "determine", "measure", "evaluate", and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0042] As used herein, the term "separation" refers to the physical separation of two elements (e.g., by size or affinity, etc.) and the degradation of one element while the other remains intact.

[0043] The term "linker" or "linkage" refers to a connecting structural moiety that connects two groups and has a backbone of 100 or fewer atoms. The linker or linkage can be a covalent bond connecting two groups or a chain of 1 to 100 atoms, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, where the linker can be linear, branched, cyclic, or a single atom. In some cases, the linker is a branched linker, which refers to a connecting structural moiety that connects three or more groups. In some cases, one, two, three, four, five, or more carbon atoms of the linker backbone can optionally be replaced by sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker backbone includes a connecting functional group, such as an ether, thioether, amino group, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the backbone atoms can be saturated or unsaturated, and in some cases, there is no more than one, no more than two, or no more than three unsaturated bonds in the linker backbone. The linker can include one or more substituents, such as an alkyl group, an aryl group, or an alkenyl group. The linker can include, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, alkyl groups, which can be straight-chain or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone can include a cyclic group, such as an aryl group, a heterocycle, or a cycloalkyl group, where 2 or more atoms (e.g., 2, 3, or 4 atoms) of the cyclic group are included in the backbone. The linker can be cleavable or non-cleavable.

[0044] As used herein, the terms "water-soluble group", "water-solubilizing group", and "WSG" are used interchangeably and refer to a group or substituent that is well soluble in an aqueous environment (e.g., under physiological conditions) and imparts better water solubility to the molecule to which it is attached. Compared to a control tandem dye or its components lacking a WSG, a WSG can increase the solubility of a tandem dye or its components (e.g., a donor fluorophore or an acceptor fluorophore) in an aqueous medium. In some cases, compared to a control compound in which the WSG is replaced by a hydrogen atom, a WSG can increase the solubility of a compound (e.g., a dye, a tandem dye, or a labeled specific binding member). In some cases, a WSG can increase the solubility in an aqueous medium (e.g., distilled water) by 1% or more, such as by 10% or more, 25% or more, 50% or more, 100% or more, or 500% or more. A water-soluble group can be any convenient hydrophilic group as long as it dissolves well in an aqueous environment.

[0045] A water-soluble group (WSG) can impart a water solubility of more than 10 mg / mL to the subject dye or polymeric tandem dye, such as more than 20 mg / mL, more than 30 mg / mL, more than 40 mg / mL, more than 50 mg / mL, more than 60 mg / mL, more than 70 mg / mL, more than 80 mg / mL, more than 90 mg / mL, or more than 100 mg / mL. In some cases, a branched nonionic water-soluble group (WSG) can enable the subject dye or polymeric tandem dye to have a solubility in water (e.g., an aqueous buffer) of 20 mg / mL or higher, such as 30 mg / mL or higher, 40 mg / mL or higher, 50 mg / mL or higher, 60 mg / mL or higher, 70 mg / mL or higher, 80 mg / mL or higher, 90 mg / mL or higher, 100 mg / mL or higher, and even higher. It is understood that water-soluble dipyrromethene dyes can form discrete water-solvated nanoparticles in an aqueous system under some conditions. In some cases, the water-solvated nanoparticles have anti-aggregation properties and can be used in various bioassays.

[0046] The term "specific binding" refers to the direct binding between two molecules, for example due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions, including interactions such as salt bridges and water bridges. Specific binding members describe the members of a pair of molecules that have binding specificity for each other. The members of a specific binding pair can be naturally derived or produced wholly or in part synthetically. One member of the molecular pair has a region on its surface or in a cavity that specifically binds and is thus complementary to the specific spatial and polar organization of the other member of the molecular pair. Thus, the members of the pair have the property of specifically binding to each other. Examples of specific binding member pairs are antigen - antibody, biotin - avidin, hormone - hormone receptor, receptor - ligand, enzyme - substrate. The specific binding members of a binding pair exhibit high affinity and binding specificity for binding to each other. Generally, the affinity between a pair of specific binding members is characterized by a K d (dissociation constant) of 10 -6 M or lower, for example 10 -7 M or lower, including 10 -8 M or lower, for example 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -13 M or lower, 10 -14 M or lower, including 10 -15 M or lower. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In one embodiment, the affinity is determined by surface plasmon resonance (SPR), such as used in a Biacore system. The affinity of one molecule for another is determined by measuring the binding kinetics of the interaction, for example at 25 °C. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In one embodiment, the affinity is determined by surface plasmon resonance (SPR), for example, as used in a Biacore system. The affinity of one molecule for another is determined by measuring the binding kinetics of the interaction, for example at 25 °C.

[0047] The specific binding member can be a protein. As used herein, the term "protein" refers to a structural moiety composed of amino acid residues. The protein structural moiety can be a polypeptide. In some cases, the protein specific binding member is an antibody. In some embodiments, the protein specific binding member is an antibody fragment, such as an antibody binding fragment that specifically binds a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein composed of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes (e.g., in humans) include the kappa (κ), lambda (λ), and heavy chain loci, which together constitute numerous variable region genes, as well as the constant region genes mu (μ), delta (δ), gamma (γ), sigma (σ), and alpha (α), which encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain consists of "framework" regions (FRs) interrupted by three hypervariable regions (also called "complementary determining regions" or "CDRs"). The ranges of the framework regions and CDRs have been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The framework region sequences of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs. The CDRs are primarily responsible for binding to the antigen epitope. The term "antibody" is intended to include full-length antibodies and can refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism, for experimental, therapeutic, or other purposes, as further defined below.

[0048] Antibody fragments of interest include, but are not limited to, Fab, Fab′, F(ab′) 2 , Fv, scFv, or other antigen-binding sequences of an antibody, either produced by modifying the whole antibody or synthesized de novo using recombinant DNA technology. Antibodies can be monoclonal or polyclonal and may have other specific activities against cells (e.g., antagonist, agonist, neutralizing antibody, inhibitory antibody, or stimulatory antibody). It is understood that antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0049] In certain embodiments, the specific binding member is a Fab fragment, F(ab′) 2Fragment, scFv, diabody or triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0050] "Active pharmaceutical ingredient" (API), "active agent", "pharmacologically active agent" and "drug" are used interchangeably herein and refer to a chemical material or compound that induces a desired pharmacological and / or physiological effect by local and / or systemic action when administered to an organism (e.g., a human or non-human animal).

[0051] "Pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers and adjuvants that can be used to prepare pharmaceutical compositions, which are generally safe, non-toxic, and have neither biological adverse effects nor other adverse effects, and include excipients, diluents, carriers and adjuvants that can be used for veterinary use as well as human drug use. "Pharmaceutically acceptable excipients, diluents, carriers and adjuvants" as used in the specification and claims includes one or more such excipients, diluents, carriers and adjuvants.

[0052] "Plurality" includes at least 2 members. In some cases, the plurality can have 5 or more, such as 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.

[0053] A numerical range includes the numbers defining the range.

[0054] Detailed Description

[0055] Polyethylene glycol - polyoxazoline (PEOZ) copolymers are provided. In some cases, the PEOZ polymer has a repeating unit with the structure –CH 2 N(C(O)R)CH 2 CH 2 OCH 2 CH 2 –. The PEOZ copolymers can be used in a variety of applications, e.g., for increasing the solubility of various compounds such as drugs, dyes, and biopharmaceuticals. Methods for preparing PEOZ copolymers are also provided, which in some cases can produce a monodisperse composition of the PEOZ copolymer.

[0056] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as these embodiments can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is defined only by the appended claims.

[0057] When numerical ranges are provided, it should be understood that unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of the range and any other stated value or intermediate value within the stated range (to one-tenth of the unit of the lower limit) is encompassed within the scope of the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed within the scope of the present invention, except for any specifically excluded limit values within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of the included limits are also included within the scope of the present invention.

[0058] Some of the numerical values of the ranges given herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number preceding it as well as a number that is close to or approximates the number preceding it. In determining whether a number is close to or approximates a particular recited number, the unrecited number that is close to or approximates may be a number that provides substantially the same functionality as the particular recited number in the context in which it is presented.

[0059] 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 may also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described only.

[0060] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference and to disclose and describe the methods and / or materials associated with the cited publications. 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. In addition, the provided publication dates may differ from the actual publication dates and may need to be independently verified.

[0061] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as antecedent basis for the use of exclusive terms such as "solely", "only", etc. in the recitation of claim elements or the use of "negative" limitations.

[0062] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any one of several other embodiments without departing from the scope or spirit of the invention. Any of the described methods can be performed in the order of the described events or any other order that is logically feasible.

[0063] Although the devices and methods have been or will be described for purposes of grammatical fluency and functional explanation, it must be clearly understood that unless expressly set forth under 35 USC § 112, the claims should not be construed as necessarily limited to any construction that is "device" or "step" limited, but rather should be accorded, under the doctrine of equivalents, the full scope of the meaning and equivalents of the definition provided by the claims, and in the case where the claims are expressly set forth under 35 USC § 112, the full statutory equivalents under 35 USC § 112 should be obtained.

[0064] PEOZ copolymer

[0065] As described above, poly(ethylene glycol)-poly(oxazoline) (PEOZ) copolymers are provided. The chemical structures of ethylene glycol and oxazoline are shown below. The PEOZ copolymer comprises ethylene glycol and oxazoline monomer units. In some cases, the PEOZ copolymer is a copolymer having monomer units derived from ethylene glycol and oxazoline or analogs thereof. The structures of ethylene glycol and oxazoline are shown below:

[0066]

[0067] For example, Figure 1 The chemical structures of the PEOZ copolymer and the poly(ethylene glycol) (PEG) polymer and the poly(oxazoline) (POZ) polymer according to an embodiment of the invention are shown. In some embodiments, the PEOZ copolymer has the structure of formula (I):

[0068]

[0069] Wherein:

[0070] m is an integer from 1 to 20;

[0071] n is an integer from 2 to 10000;

[0072] R is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, carboxyl, cyano, ether, halogen, hydroxy, nitro, thiol, thioether, thione, borate, -SO 2 、-SO 3-, a group consisting of a reactive structural moiety and a protecting group for the reactive structural moiety; and

[0073] Each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxy, a reactive structural moiety, and a protecting group for the reactive structural moiety.

[0074] As described above, m can vary and, in some cases, is an integer 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):

[0075]

[0076] The variable m can also vary and, in some cases, is an integer in the range of 2 to 10,000, such as 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, such as 5 to 10,000, 10 to 10,000, or 25 to 10,000.

[0077] R can vary. 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.

[0078] In some cases, R includes one or more water-soluble groups, e.g., to increase the solubility of the copolymer (e.g., as Figures 6 - 8 shown). In some embodiments, R is a substituted alkyl containing a polyethylene glycol group or another PEOZ copolymer. In some embodiments, R is a substituted alkyl containing a sulfonic acid group (i.e., -SO 3 - group), and this sulfonic acid group can confer increased solubility due to its charge.

[0079] In some cases, R includes a reactive structural moiety or a protecting group for the reactive structural moiety (e.g., as Figures 6 - 8as shown). In some embodiments, the reactive structural moiety is configured to form a covalent bond with another group through a chemoselective reaction (e.g., click chemistry reaction). Exemplary reactive structural moieties include thiol and maleimide or iodoacetamide, amine and carboxylic acid or its active ester, and groups that can react with each other through click chemistry, such as azide and alkyne (e.g., cyclooctynyl), tetrazine, trans-cyclooctene, diene and dienophile, and azide, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, and hydroxyl, hydrazide, hydrazino, aldehyde, ketone, azido, alkynyl, phosphine, epoxide, and succinimide.

[0080] The R group may also include a specific binding moiety or lipid for binding into the cell membrane or the membrane of a liposome. In some cases, the R group is a specific binding member, such as an antibody, an antibody fragment (e.g., Fab fragment, F(ab′) 2 fragment, scFv, diabody or triabody), antigen, biotin group or avidin group.

[0081] In some embodiments, each Y group is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, ether, hydroxyl, and a protecting group for the reactive structural moiety. In some cases, the Y group may include a water-soluble group, a reactive structural moiety, a protecting group for the reactive structural moiety, a specific binding structural moiety, or a combination thereof.

[0082] Dye

[0083] Also provided are dyes comprising a fluorophore and a water-soluble group of a PEOZ copolymer. The terms "fluorophore" and "chromophore" are used interchangeably herein.

[0084] In some cases, the dyes include organic dyes. The organic dyes can vary, and the organic dyes that can be modified with the PEOZ group of the present invention include, but are not limited to: cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, boron dipyrromethene difluoride, naphthalimide, thiazine dyes, and acridine dyes. The organic dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxy-p-methylaminophenol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin-chlorophyll 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, 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, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, Dyonomics dyes (such as DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831), boron dipyrromethene difluoride (BODIPY), Biotium CF 555, diethylaminocoumarin, and its derivatives.In some cases, the water-soluble groups of the PEOZ copolymer are combined with an organic dye.

[0085] In some embodiments, the dye further comprises a non-conjugated polymer backbone comprising non-conjugated repeating units. "Non-conjugated" means that at least a portion of the repeating units comprises saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds) that exclude π-conjugation or delocalized electronic structures that extend along the polymer backbone from one repeating unit to the next. It should be understood that even if a repeating unit may not be conjugated to an adjacent repeating unit, such a repeating unit may comprise one or more independent unsaturated groups (e.g., alkenyl or alkynyl groups) and / or aryl or heteroaryl groups containing unsaturated bonds, which may be part of the backbone. In some cases, each repeating unit of the polymer backbone comprises a side chain comprising a linked side group or a chemoselective tag for attachment to a side group.

[0086] In some embodiments, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from peptides, peptoids, hydrocarbon polymers, and PEG polymers. In certain cases, the linear polymer is a peptide. In certain cases, the linear polymer is a peptoid. In certain cases, the polymer is a hydrocarbon polymer. In certain other cases, the non-conjugated polymer is a PEG polymer. For additional details regarding non-conjugated polymer backbones useful in embodiments of the present invention, see PCT application serial number PCT / US2019 / 024662 (published as WO2019 / 191482) and PCT application serial number PCT / US2020 / 019510 (published as WO2020 / 222894); the disclosures of these applications are incorporated herein by reference.

[0087] In some cases, the non-conjugated polymer backbone is a peptide having 2 to 100 amino acids, such as 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, such as 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 having 5 to 30 amino acids, such as 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

[0088] The non-conjugated repeating units can have any convenient configuration, such as linear, branched, or dendritic configurations. The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some cases, the dye includes multiple side-chain chromophores, each independently attached to a non-conjugated repeating unit of the polymer backbone. The configuration of the side groups can be installed during or after the synthesis of the non-conjugated polymer backbone. The attachment of the side groups can be achieved in a random configuration, a block configuration, or in a sequence-specific manner by stepwise synthesis, depending on the particular synthesis method used.

[0089] In some cases, the non-conjugated repeating units contain multiple amino acid residues. In some cases, the dye includes an organic dye bound to the non-conjugated polymer backbone. The water-soluble groups of the PEOZ copolymer can be bound 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 that can be used in the methods and systems of the present invention are diverse. In some cases of the present method, the polymeric dye includes a conjugated polymer. A conjugated polymer (CP) is characterized by a delocalized electronic structure that includes a backbone having alternating unsaturated bonds (e.g., double bonds 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 can impart an extended linear structure to the polymeric dye, with limited bond angles between the repeating units of the polymer. For example, proteins and nucleic acids, although also polymers, do not form extended rod structures in some cases but instead fold into higher-order three-dimensional shapes. Additionally, the CP can form a "rigid rod" polymer backbone and experience a limited torsional (e.g., twisting) angle between the monomer repeating units along the polymer backbone chain. In some cases, the polymeric dye includes a CP having a rigid rod structure. The structural characteristics of the polymeric dye can affect the fluorescence properties of the molecule.

[0090] Any convenient polymeric dye can be used in the present device and method. In some cases, the polymeric dye is a multi-chromophore that has a structure capable of collecting light to amplify the fluorescence output of a fluorophore. In some cases, the polymeric dye is capable of collecting light and efficiently converting it into emitted light of a longer wavelength. In some cases, the polymeric dye has a light-harvesting multi-chromophore system that can efficiently transfer energy to a nearby luminescent species (e.g., a "signal chromophore"). The energy transfer mechanisms include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge transfer (Dexter energy transfer), etc. In some cases, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the light-harvesting multi-chromophore system to the signal chromophore provides efficient energy transfer. Under the conditions of efficient energy transfer, when the number of individual chromophores in the light-harvesting multi-chromophore system is large, the emission of the signal chromophore is amplified; that is, when the wavelength of the incident light (“excitation light”) is absorbed by the light-harvesting multi-chromophore system, the emission of the signal chromophore is stronger than when the signal chromophore is directly excited by the pump light.

[0091] The multi-chromophore can 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. Since the effective conjugation length is much shorter than the length of the polymer chain, the backbone contains a large number of closely adjacent conjugated segments. Thus, conjugated polymers can efficiently collect light and achieve optical amplification through energy transfer.

[0092] Polymer dyes of interest include, but are not limited to, those described by Gaylord et al. in U.S. Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, the disclosures of which are hereby incorporated by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123(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, the disclosures of which are hereby incorporated by reference in their entirety.

[0093] In some embodiments, the polymer dye includes a conjugated polymer that includes a plurality of first optically active units that form a conjugated system and have a first absorption wavelength (e.g., as described herein), at which the first optically active units absorb light to form an excited state. The conjugated polymer (CP) can be a polycationic, polyanionic, and / or electrically neutral conjugated polymer.

[0094] The polymeric dye can have any convenient length. In some cases, the specific number of monomeric repeating units or segments of the polymeric dye can fall within 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 for example 100 to 100,000, 200 to 100,000 or 500 to 50,000 units or segments.

[0095] The polymeric dye can have any convenient molecular weight (MW). In some cases, the MW of the polymeric dye can be expressed as an average molecular weight. In some cases, the average molecular weight of the polymeric dye is from 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000 or even an average molecular weight of 50,000 to 100,000. In some embodiments, the average molecular weight of the polymeric dye is 70,000.

[0096] In some cases, the polymeric dye comprises the following structure:

[0097]

[0098] wherein CP 1 、CP 2 、CP 3 and CP 4 are independently a conjugated polymer segment or an oligomeric structure, wherein one or more of CP 1 、CP 2 、CP 3 and CP 4 are n-conjugated repeating units that reduce the band gap, and each n and each m are independently 0 or an integer from 1 to 10,000, and p is an integer from 1 to 100,000.

[0099] In some cases, the polymeric dye comprises the following structure:

[0100]

[0101] wherein each R 1 is independently a solubilizing group or a linking dye; L 1 and L 2 are optional linkers; each R 2 is independently H or an aryl substituent; each A 1 and A 2 are independently H, an aryl substituent or a fluorophore; G 1 and G 2each independently selected from the group consisting of a terminal group, a π-conjugated chain segment, a linker, and a conjugated specific binding member; each n and each m are independently an integer from 0 or 1 to 10,000; and p is an integer from 1 to 100,000. Solubilizing groups of interest include the PEOZ solubilizing groups described herein.

[0102] In some cases, the polymeric dye includes a conjugated chain segment having one of the following structures as part of the polymer backbone:

[0103]

[0104] wherein each R 3 is independently an optionally substituted alkyl or aryl; Ar is an optionally substituted aryl or heteroaryl; and each n is an integer from 1 to 10,000. In some embodiments, R 3 is an optionally substituted alkyl. In some embodiments, R 3 is an optionally substituted aryl. In some cases, R 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.

[0105] In some cases, the polymeric dye includes the following structure:

[0106]

[0107] wherein each R 1 is independently a solubilizing group or a linking dye group; each R 2 is independently H or an aryl substituent; each L 1 and L 3 are independently an optional linker; each A 1 and A 3 are independently H, a fluorophore, a functional group, or a specific binding moiety (e.g., an antibody); and n and m are each independently 0 or an integer from 1 to 10,000, where n + m > 1.

[0108] The polymeric dye may have one or more desired spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, extinction coefficient, quantum yield, etc. (see, e.g., 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 polymeric dye has an absorption curve between 280 nm and 475 nm. In some embodiments, the polymeric dye has a maximum absorption (excitation maximum) in the range of 280 nm to 475 nm. In some embodiments, the polymeric dye absorbs incident light having a wavelength in the range of 280 nm to 475 nm. In some embodiments, the emission maximum wavelength of the polymeric dye ranges from 400 nm to 850 nm, such as 415 nm to 800 nm, where specific examples of emission maximum wavelengths 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 cases, the emission maximum wavelength range of the polymeric dye is selected from 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 emission maximum wavelength of the polymeric dye is 421 nm. In some cases, the emission maximum wavelength of the polymeric dye is 510 nm. In some cases, the emission maximum wavelength of the polymeric dye is 570 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 602 nm. In some cases, the emission maximum wavelength of the polymeric dye is 650 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 711 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 786 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 421 nm ± 5 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 510 nm ± 5 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 570 nm ± 5 nm. In some cases, the emission maximum wavelength of the polymeric dye is 602 nm ± 5 nm. In some embodiments, the emission maximum wavelength of the polymeric dye is 650 nm ± 5 nm. In some cases, the emission maximum wavelength of the polymeric dye is 711 nm ± 5 nm. In some cases, the emission maximum wavelength of the polymeric dye is 786 nm ± 5 nm.In some embodiments, the maximum emission wavelength of the polymeric dye is selected from 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0109] In some cases, the polymeric dye has an extinction coefficient of 1x10 6 cm -1 M -1 or greater, such as 2x10 6 cm -1 M -1 or greater, 2.5x10 6 cm -1 M -1 or greater, 3x10 6 cm -1 M -1 or greater, 4x10 6 cm -1 M -1 or greater, 5x10 6 cm -1 M -1 or greater, 6x10 6 cm -1 M -1 or greater, 7x10 6 cm -1 M -1 or greater, or 8x10 6 cm -1 M -1 or greater. In some embodiments, the quantum yield of the polymeric dye is 0.05 or higher, such as 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.25 or higher, 0.3 or higher, 0.35 or higher, 0.4 or higher, 0.45 or higher, 0.5 or higher, or even higher. In some cases, the quantum yield of the polymeric dye is 0.1 or higher. In some cases, the quantum yield of the polymeric dye is 0.3 or higher. In some cases, the quantum yield of the polymeric dye is 0.5 or higher. In some embodiments, the extinction coefficient of the polymeric dye is 1x10 6 or greater, and the quantum yield is 0.3 or higher. In some embodiments, the extinction coefficient of the polymeric dye is 2x10 6 or greater, and the quantum yield is 0.5 or higher.

[0110] Specific polymeric dyes that can be used include, but are not limited to, BD Horizon Brilliant TM dyes, such as BDHorizon Brilliant TMPurple dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant TM UV dyes (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant TM Blue dyes (e.g., BB515) (BD Biosciences, San Jose, CA).

[0111] Tandem dyes

[0112] Tandem dyes are also provided, which comprise a donor fluorophore, an acceptor fluorophore, and a PEOZ copolymer water-soluble group. A tandem dye is a compound having two covalently linked different fluorophores, which can be covalently linked directly to each other or covalently linked to each other through a linking group. One of the fluorophores serves as the donor fluorophore and the other fluorophore serves as the acceptor fluorophore. The donor fluorophore and the acceptor fluorophore together form a fluorescence resonance energy transfer (FRET) pair. Such a FRET pair behaves as a unique dye having the excitation characteristics of the donor fluorophore and the emission characteristics of the acceptor fluorophore.

[0113] Excitation of the donor can result in energy transfer to and emission from the covalently linked acceptor fluorophore. The energy transfer mechanism between the donor chromophore and the linked acceptor signal fluorophore includes, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be over relatively short distances; that is, the chromophores of the light-harvesting multi-chromophore system are close to each other and / or close to the acceptor fluorophore, providing efficient energy transfer. Under conditions of efficient energy transfer, amplification of the emission of the acceptor fluorophore can occur when the wavelength of the incident light ("pump light") is absorbed by the chromophore of the light-harvesting chromophore and transferred from it, and the emission of the luminescent acceptor fluorophore is more intense than when the luminescent acceptor fluorophore is directly excited by the pump light. "Efficient" energy transfer means that 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more of the energy collected by the donor chromophore is transferred to the acceptor. "Amplification" means that when the acceptor fluorophore is excited by energy transfer from the donor light-harvesting chromophore system, the signal from the acceptor fluorophore is 1.5 times or greater than when directly excited by incident light of the same intensity. The signal can be measured using any convenient method. In some cases, a signal of 1.5 times or greater refers to the intensity of the emitted light. In some cases, a signal of 1.5 times or greater refers to the increased signal-to-noise ratio. In some embodiments of the tandem dye, the emission of the acceptor fluorophore when excited by the chromophore is 1.5 times or more greater than when directly excited by the incident light, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even greater than when directly exciting the acceptor fluorophore with the incident light.

[0114] In some cases, the tandem dye exhibits an effective Stokes shift in the range of 25 nm to 300 nm, such as 50 nm to 250 nm or 75 nm to 200 nm. In some cases, when the light-harvesting chromophore is directly excited by the incident light, the effective Stokes shift is 25 nm or greater, such as 50 nm or greater, 75 nm or greater, 100 nm or greater, such as 110 nm or greater, 120 nm or greater, 130 nm or greater, 140 nm or greater, 150 nm or greater, 160 nm or greater, 170 nm or greater, 180 nm or greater, 190 nm or greater, 200 nm or greater, 250 nm or greater.

[0115] The emission of the tandem dye can have a quantum yield of 0.03 or higher, such as 0.04 or higher, 0.05 or higher, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.09 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.3 or higher or even higher quantum yield. In some cases, the polymeric tandem dye has 5x10 5 cm -1 M -1or a greater extinction coefficient, such as 6x10 5 cm -1 M -1 or greater, 7x10 5 cm -1 M -1 or greater, 8x10 5 cm -1 M -1 or greater, 9x10 5 cm -1 M -1 or greater, such as 1x10 6 cm -1 M -1 or greater, 1.5x10 6 cm -1 M -1 or greater, 2x10 6 cm -1 M -1 or greater, 2.5x10 6 cm -1 M -1 or greater, 3x10 6 cm -1 M -1 or greater, 4x10 6 cm -1 M -1 or greater, 5x10 6 cm -1 M -1 or greater, 6x10 6 cm -1 M -1 or greater, 7x10 6 cm -1 M -1 or greater, or 8x10 6 cm -1 M -1 or greater. In some embodiments, the tandem dye has a molar extinction coefficient of 5x10 5 M -1 cm -1 or greater. In some embodiments, the tandem dye has a molar extinction coefficient of 1x10 6 M -1 cm -1 or greater.

[0116] At least one of the donor fluorophore and the acceptor fluorophore comprises an organic dye, and in some cases both comprise an organic dye. For example, the organic dye of the donor fluorophore may be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrene, boron dipyrromethene difluoride, naphthalimide, thiazine dyes, and acridine dyes. In some cases, the organic dye of the acceptor fluorophore may be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrene, boron dipyrromethene difluoride, naphthalimide, thiazine dyes, and acridine dyes.

[0117] Organic dyes of interest that can be used as donors and acceptors include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxy-p-methylaminophenol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin-chlorophyll 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, 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, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, Dyonomics dyes (e.g., DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831), boron dipyrromethene difluoride (BODIPY), Biotium CF 555, diethylaminocoumarin and its derivatives.

[0118] As described above, the dye can include a non-conjugated polymer backbone. Similarly, in some embodiments, the tandem dye can include a non-conjugated polymer backbone that includes non-conjugated repeating units. In some embodiments, the non-conjugated repeating units include a plurality of amino acid residues. The PEOZ copolymer water-soluble group can be bound to the donor fluorophore, the acceptor fluorophore, and / or the non-conjugated polymer backbone.

[0119] As described above, the dye can include a fluorophore that is a conjugated polymer. Thus, in some cases, the donor fluorophore of the tandem dye can include a conjugated polymer. As described above with respect to the dye, in some cases, the conjugated polymer can include 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-soluble group can be bound to the conjugated polymer and / or the acceptor fluorophore.

[0120] Labeled specific binding member

[0121] Aspects of the present disclosure also include labeled specific binding members that include a dye or tandem dye (such as those described above) and a specific binding member. In other words, the specific binding member is labeled with a dye or tandem dye, for example, to allow detection of a target analyte that binds to the specific binding member.

[0122] Composition

[0123] The present invention also provides a composition 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 PEOZ copolymers, such as 1,000 or more, 10,000 or more, 100,000 or more, or 1,000,000 or more.

[0124] In some embodiments, the molecular weights of the PEOZ copolymers in the composition can be relatively similar to each other. As used herein, the terms "dispersity" (D) and "polydispersity index" (PDI) are used interchangeably and refer to a measure of the molecular weight distribution within the composition. Specifically, D = PDI = Mw / Mn, where Mw is the weight-average molar mass and Mn is the number-average molar mass. A smaller D value corresponds to a more uniform molecular weight distribution, while a larger D value corresponds to a more non-uniform molecular weight distribution. A D value of exactly 1.0 refers to a completely uniform distribution. In some cases, Mw is determined by a method selected from static light scattering, small-angle neutron scattering, X-ray scattering, and sedimentation velocity. In some embodiments, Mn is determined by a method selected from gel permeation chromatography, viscometry (e.g., via the Mark-Houwink equation), and colligative methods (e.g., vapor pressure osmometry). The equations for Mw and Mn are shown below, where Ni is the number of molecules with molecular weight Mi.

[0125]

[0126] Thus, in some cases, the PEOZ copolymers in the composition have relatively similar molecular weights, e.g., where the dispersity (D) of the copolymer is 1.5 or lower, e.g., 1.4 or lower, 1.3 or lower, 1.2 or lower, 1.1 or lower, 1.05 or lower, or 1.02 or lower.

[0127] Method for preparing PEOZ copolymer

[0128] The first method for preparing PEOZ copolymer

[0129] A first method for preparing a PEOZ copolymer is provided, the method comprising:

[0130] (a) generating a (ethylene glycol)-oxazoline (EOZ) dimer comprising a nucleophilic hydroxyl group, said generating comprising:

[0131] (i) reacting a first EOZ monomer comprising a protected hydroxyl group with a second EOZ monomer comprising a protected hydroxyl group to form an EOZ dimer comprising two protected hydroxyl groups;

[0132] (ii) selectively deprotecting one hydroxyl group of the EOZ dimer to thereby form an EOZ dimer comprising a nucleophilic hydroxyl group; (b) generating an EOZ dimer comprising an electrophilic leaving group, said generating comprising:

[0133] (i) reacting a third EOZ monomer comprising a protected hydroxyl group with a fourth EOZ monomer comprising a protected hydroxyl group to form an EOZ dimer comprising two protected hydroxyl groups;

[0134] (ii) selectively deprotecting one hydroxyl group of the EOZ dimer;

[0135] (iii) Convert the deprotected hydroxyl group of the EOZ dimer into an electrophilic leaving group;

[0136] (c) React an EOZ dimer containing a nucleophilic hydroxyl group with an EOZ dimer containing an electrophilic leaving group to form an EOZ tetramer, namely a polyethylene glycol - polyoxazoline (PEOZ) copolymer.

[0137] Step (a)(i) of the method involves reacting a first EOZ monomer with a second EOZ monomer to connect these monomers into a dimer having two protected hydroxyl groups. For example, the connection can be achieved by a nucleophilic substitution reaction between a nucleophilic group (such as a hydroxyl group) on the first monomer and an electrophilic leaving group (such as trifluoromethanesulfonate) on the second monomer.

[0138] In step (a)(ii), one of the hydroxyl groups is selectively deprotected. Thus, the first hydroxyl protecting group and the second hydroxyl protecting group have different properties. For example, one hydroxyl group can be protected with TBDMS, while the other hydroxyl group can be protected with benzyl. The TBDMS group can be removed with TBATB (e.g., Firouzabadi et al., Synthetic Communications, 1996, 26, doi: 10.1080 / 00397919608003713), but this selective deprotection can also be carried out using acids (such as camphorsulfonic acid, acetic acid) and bases (such as HF - pyridine, TBAF). Benzyl can be removed by H 2 / Pd(O), CrO 3 / acetic acid, ozone, N - bromosuccinimide (NBS) or N - iodosuccinimide (NIS).

[0139] Thus, step (a) generates an EOZ dimer having a nucleophilic hydroxyl group.

[0140] Step (b) includes reacting a third and a fourth EOZ monomer to form another EOZ dimer having two protected hydroxyl groups. Similar to step (a), the hydroxyl groups can be protected by different protecting groups (such as benzyl and TBDMS). Step (b)(ii) involves selectively deprotecting one of the hydroxyl groups, for example as described above. Step (b)(iii) involves converting the deprotected hydroxyl group into an electrophilic group, for example, reacting with a base (such as NaH) and Tf - Cl can generate an OTf (i.e., trifluoromethanesulfonate) leaving group.

[0141] In step (c), the two EOZ dimers react with each other to form an EOZ tetramer by nucleophilic substitution between the nucleophilic hydroxyl group of the first dimer and the electrophilic leaving group (such as OTf) of the second dimer.

[0142] The process can be repeated to generate larger polymers. For example, a sample of the PEOZ tetramer produced in step (c) can be modified to have nucleophilic hydroxyl groups. Additionally, a second sample of the PEOZ tetramer can be modified to have an electrophilic group (e.g., OTf). Then the two PEOZ tetramer samples can be reacted to generate a PEOZ octamer. Thus, the present disclosure provides a general method for synthesizing PEOZ x-polymers, the method comprising reacting a PEOZ a-polymer having nucleophilic hydroxyl groups with a PEOZ b-polymer having an electrophilic leaving group (e.g., OTf), where x is equal to the sum of a and b.

[0143] In some cases, the reaction is carried out in a solvent, such as an aqueous solvent or an 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 can be carried out without a solvent, i.e., neat.

[0144] Such methods can produce a relatively monodisperse distribution, e.g., due to their stepwise procedure. In some cases, the composition of the PEOZ copolymer produced by this method has 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.

[0145] The methods described herein can include multiple steps. Each step can be carried out after a predetermined time between steps as needed. Thus, the time between carrying out each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and including 5 hours or longer. In some embodiments, each subsequent step is carried out immediately after completion of the previous step. In other embodiments, the steps can be carried out after an incubation or waiting time after completion of the previous step, e.g., a waiting time of several minutes to overnight.

[0146] A second method for preparing a PEOZ copolymer

[0147] There is also provided a method for preparing a PEOZ copolymer, the method comprising:

[0148] Reacting a compound of formula (Ia) with a compound of formula (Ib) to produce a compound of formula (Ic)

[0149]

[0150] Deprotecting the compound of formula (Ic) to produce a compound of formula (Id)

[0151]

[0152] Convert the compound of formula (Ic) to the compound of formula (Ie)

[0153]

[0154] React the compound of formula (Id) with the compound of formula (Ie) to form the compound of formula (If)

[0155]

[0156] wherein:

[0157] PG 1 and PG 2 are each independently a hydroxyl protecting group;

[0158] OX is a leaving group; and

[0159] R is an alkyl group.

[0160] In some cases, the hydroxyl protecting groups PG 1 and PG 2 are independently selected from benzyl and TBDMS. In some embodiments, PG 1 and PG 2 are different hydroxyl protecting groups. In some cases, deprotection to remove the TBDMS group involves contacting with TBATB, an acid (e.g., camphorsulfonic acid, acetic acid) or a base (e.g., HF-pyridine, TBAF). In some embodiments, removal of the benzyl protecting group involves contacting with H 2 / Pd(O), CrO 3 / acetic acid, ozone, N-bromosuccinimide (NBS) or N-iodosuccinimide (NIS).

[0161] In some embodiments, R is selected from H, alkyl and substituted alkyl. In some cases, R is an alkyl group, such as methyl.

[0162] In some cases, R includes one or more water-soluble groups to increase the solubility of the copolymer (e.g., as Figures 6 - 8 shown). 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., -SO 3 - group), which can confer increased solubility due to its charge.

[0163] In some cases, R includes a reactive moiety or a reactive moiety protecting group (e.g., as Figures 6 - 8as shown). In some embodiments, the reactive structural moiety is configured to form a covalent bond with another group through a chemoselective reaction (e.g., click chemistry reaction). Exemplary reactive structural moieties include succinimidyl, azido, or tetrazinyl groups.

[0164] The R group may also include a specific binding moiety, such as an antibody, antibody fragment, or lipid, for binding into the cell membrane or the membrane of a liposome.

[0165] Such methods can generate a relatively monodisperse distribution, e.g., due to their stepwise procedures. In some cases, the composition of the PEOZ copolymer generated by this method has a dispersity (D) of 1.5 or lower, e.g., 1.4 or lower, 1.3 or lower, 1.2 or lower, 1.1 or lower, 1.05 or lower, or 1.02 or lower.

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

[0167] A third method for preparing a PEOZ copolymer

[0168] A third method for preparing a PEOZ copolymer is provided, the method comprising:

[0169] providing a first (ethylene glycol)-oxazoline (EOZ) monomer linked to a matrix and comprising a nucleophilic hydroxyl group;

[0170] reacting the first EOZ monomer with a second EOZ monomer comprising an electrophilic leaving group and a protected hydroxyl group to form a matrix-linked EOZ dimer comprising a protected hydroxyl group;

[0171] deprotecting the protected hydroxyl group to form a matrix-linked EOZ dimer comprising a nucleophilic hydroxyl group;

[0172] reacting the matrix-linked EOZ dimer with a third EOZ monomer comprising an electrophilic leaving group and a protected hydroxyl group to form a matrix-linked EOZ trimer comprising a protected hydroxyl group, wherein the EOZ trimer is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0173] In some cases, the matrix is a particle, such as a nanoparticle, microparticle, or bead. The matrix may comprise a material selected from metals, metalloids, ceramics, and polymers.

[0174] In some cases, the method further includes releasing the PEOZ copolymer from the matrix, for example, by contacting with an acid, a base, or a nucleophile. For example, in some cases, the release includes contacting with methyl iodide (i.e., MeI) and a base (e.g., NaOH, KOH).

[0175] Such methods can generate a relatively monodisperse distribution, for example, due to their stepwise procedures. In some cases, the composition of the PEOZ copolymer generated by the method has a dispersity (D) of 1.5 or lower, such as 1.4 or lower, 1.3 or lower, 1.2 or lower, 1.1 or lower, 1.05 or lower, or 1.02 or lower.

[0176] The methods described herein can include multiple steps. Each step can be performed after a predetermined time between steps as needed. Thus, the time between performing each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and including 5 hours or longer. In some embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, the steps can be performed after an incubation or waiting time after completion of the previous step, such as a waiting time of several minutes to overnight.

[0177] Method for increasing the solubility of a compound having a PEOZ copolymer

[0178] The present disclosure provides a method for increasing the solubility of a compound having a PEOZ copolymer, compared to the solubility of a compound that does not have a PEOZ component. Thus, the PEOZ copolymer can be considered a water-soluble group. In some cases, the method includes the step of binding the PEOZ copolymer water-soluble group to the compound. The PEOZ copolymer can be covalently or non-covalently (e.g., ionically) bound to the compound.

[0179] In some embodiments, the compound solubilized by the PEOZ group is a dye, for example, as described above, which includes a fluorophore. In other cases, the compound solubilized is a tandem dye, which includes a donor fluorophore and an acceptor fluorophore, for example, as described above. In some cases, the compound is a specific binding member, for example, labeled with a dye or a tandem dye.

[0180] In some cases, the compound solubilized is an active pharmaceutical ingredient (API). In other cases, the compound is selected from pharmaceutically acceptable excipients, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers, and pharmaceutically acceptable adjuvants.

[0181] For example, some known compounds have attached polyethylene glycol (PEG) groups and are thus referred to as "PEGylated" compounds. Using the methods described herein, PEOZ copolymers can be bonded in place of the PEG groups or, in addition to the PEG groups, PEOZ copolymers can be bonded. For example, Harris et al. described polypeptide drugs attached with PEG groups (Nature Reviews Drug Discovery, 2003, 2, 214, doi:10.1038 / nrd1033). Thus, according to the methods of the present disclosure, polypeptide drugs can be bonded with PEOZ groups instead of the PEG groups. Other compounds that can be functionalized with PEOZ instead of or in addition to PEG include dyes (e.g., Wu et 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., PEGylated excipients in Pfizer's COVID-19 vaccine as discussed by Cabanillas et al. (doi:10.1111 / all.14711)), liposomes (Heger et al., doi:10.1016 / j.mvr.2009.02.006). In such cases, the compounds (e.g., dyes, cytokines, therapeutic proteins, small molecule agents, excipients or liposomes) can be combined with PEOZ copolymers to increase the solubility of the compounds.

[0182] The methods described herein can include multiple steps. Each step can be performed after a predetermined time has elapsed between steps as needed. Thus, the time between performing each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and include 5 hours or longer. In some embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, the steps can be performed after an incubation or waiting time after the previous step is completed, such as a waiting time of several minutes to overnight.

[0183] Methods for labeling target molecules

[0184] The present invention also provides methods for labeling target molecules. Such methods include contacting the target molecule with a dye or tandem dye described herein such that the target molecule covalently binds to a reactive structural moiety of the dye or tandem dye, thereby producing a labeled target molecule.

[0185] Methods of interest for labeling a target include, but are not limited to, the methods and reagents described in Hermanson's Biocoupler Techniques, Third Edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some cases, the reactive structural moiety includes an amino functional group, while the target molecule includes a reactive ester functional group, such as an NHS ester or a sulfo-NHS ester, and vice versa. In some cases, the reactive structural moiety includes a maleimide functional group, while the target molecule includes a thiol functional group, and vice versa. In some cases, the reactive structural moiety includes an alkyne (e.g., cyclooctynyl) functional group, while the target molecule includes an azide functional group, and vice versa, which can be conjugated by click chemistry.

[0186] Using the present method, any convenient target molecule can be selected for labeling. 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, glycosylated, ubiquitinated, sumoylated, 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 protein family and fragments thereof. The target protein can be any protein of interest, such as a therapeutic or diagnostic target, including, but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic factors, colony-stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression method or using any convenient synthetic method, or 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. In some cases, the specific binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2 fragments, scFv, diabodies, and triabodies.

[0187] In some cases, the present method includes a separation step, in which the labeled target molecule is separated from the reaction mixture (e.g., excess reagent or unlabeled target). A variety of methods can be used to separate the target from the sample, such as by immobilization on a support, precipitation, chromatography, etc.

[0188] In some cases, the method further includes detecting and / or analyzing a labeled target molecule. In some cases, the method further includes fluorescently detecting a labeled target molecule. Any convenient method can be utilized to combine the method and the composition for detecting and / or analyzing a labeled target molecule. Methods for analyzing a target of interest that can be used in the present method include, but are not limited to, flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. 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, etc.

[0189] Detection can be achieved directly through polymeric tandem dyes or indirectly through a secondary detection system. The latter can be based on any one or a combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immune or non-immune bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technologies, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can 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 quantification of signal output. More than one specific and / or non-specific antibody can be labeled and used simultaneously or sequentially to enhance target detection, recognition, and / or analysis.

[0190] The methods described herein can include multiple steps. Each step can be performed after a predetermined time between steps as needed. Thus, the time between performing each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and includes 5 hours or longer. In some embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, the steps can be performed after an incubation or waiting time after completion of the previous step, such as a waiting time of several minutes to overnight.

[0191] Method for assessing the presence of a target analyte in a sample

[0192] There is provided a method for assessing the presence of a target analyte in a sample using a labeled specific binding member comprising a PEOZ copolymer WSG. In some embodiments, the method comprises:

[0193] (a) contacting the sample with a labeled specific binding member that specifically binds to the target analyte to generate a labeled sample; and

[0194] (b) Detecting the presence of a labeled specific binding member - target analyte binding complex in the composition of the detection label to evaluate the presence of the target analyte in the sample.

[0195] The labeled specific binding members for use in the embodiments of the methods of the present invention include the specific binding members conjugated to dyes or tandem dyes as described above. In the following sections, the target analyte can be a target molecule of interest or a reagent that binds to the target molecule, e.g., a primary antibody, depending on whether the labeled specific binding member is used as a primary label or a secondary label. Any convenient method can be used to contact the sample with the labeled specific binding member that specifically binds to the target analyte to produce an assay composition. In some cases, the sample is contacted with the labeled specific binding member under conditions where the labeled specific binding member specifically binds to the target analyte, if present. To enable the labeled specific binding member to specifically bind to the target analyte, an appropriate culture medium can be used to maintain the biological activity of the sample components and the single-domain antibody. The culture medium can be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum, human platelet lysate, or other factors, in combination with a low concentration of an acceptable buffer, e.g., 5 - 25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. A variety of culture media are commercially available and can be used according to the nature of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., supplemented with fetal bovine serum or human platelet lysate in some cases. The final composition of the culture medium (which can be a solution) can be selected according to the composition of the sample included. The temperature at which the labeled specific binding member specifically binds to the target analyte can vary and may be between 5°C and 60°C in some cases, e.g., 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, e.g., 20°C, 25°C, 30°C, 35°C, or 37°C (e.g., as described above). 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 (e.g., 25°C), 30°C, 35°C, or 37°C. Any convenient specific binding incubation time can be selected to allow the formation of the desired amount of binding complex, and in some cases, it can be 1 minute (min) or longer, e.g., 2 minutes or longer, 10 minutes or longer, 30 minutes or longer, 1 hour or longer, 2 hours or longer, or even 6 hours or longer.

[0196] Any convenient specific binding member can be used as the specific binding member employed in the method of the present invention. Specific binding members of interest include, but are not limited to, those that specifically bind to cell surface proteins of various cell types, including but not limited to stem cells, such as pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory cells, dendritic cells, B cells, such as memory B cells, antigen-specific B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (such as HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and red blood cells. Target cells of interest include cells having convenient cell surface markers or antigens that can be captured by convenient specific binding member conjugates. In some embodiments, the target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood. Any convenient cell surface protein or cell marker can be targeted for specific binding to the conjugates employed in the subject method. In some embodiments, the target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cells can include 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 α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0197] Any convenient target can be selected and evaluated 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, glycosylated, ubiquitinated, ubiquitin-like, or acetylated proteins, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. The term "target protein" as used herein refers to all members of a target protein family and fragments thereof. The target protein can 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, osteogenic 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 purchased from the market. In some embodiments, the polymeric dye conjugate includes an antibody or antibody fragment. In the subject methods, any convenient target analyte that specifically binds to the antibody or antibody fragment of interest can be targeted.

[0198] In some embodiments, the target analyte is cell-associated. In some cases, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens. In some cases, the target analyte is an intracellular target, and the method further includes treating the cell to enable the labeled specific binding member to access the intracellular target, such as by permeabilizing or lysing the cell. Thus, the labeled specific binding member used in the methods of the invention can target cell surface or intracellular antigens. Alternatively, the labeled specific binding member used in the methods of the invention can target a primary antibody that in turn specifically binds to a target cell surface or intracellular antigen.

[0199] In some embodiments, the sample can include a heterogeneous cell population from which target cells are isolated. In some cases, the sample includes whole peripheral blood, whole peripheral blood in which red blood cells have been lysed prior to cell isolation, cord blood, bone marrow, density gradient-purified peripheral blood mononuclear cells, or homogeneous tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or cord blood. In some embodiments, the sample includes tumor cells in peripheral blood. In some cases, the sample is a sample that includes (or is suspected of including) viral cells (e.g., HIV).

[0200] Labeled specific binding members can be used in the present method, e.g., for labeling target cells, particles, targets, or analytes with polymeric tandem fluorophores. For example, the labeled specific binding members can be used to label cells to be processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. The labeled specific binding members can include specific binding members, such as antibodies or binding fragments thereof, that specifically bind to, e.g., cell surface proteins of a variety of cell types (e.g., as described herein). The labeled specific binding members can be used to study a variety of biological (e.g., cellular) properties or processes, such as the cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. The labeled specific binding members can be used in any application that involves (or can involve) antibody-mediated labeling of cells, particles, or analytes.

[0201] Aspects of the present method include assaying an assay composition, i.e., a sample contacted with a labeled specific binding member, to determine 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 has been contacted with the labeled specific binding member, any convenient method can be utilized to assay for the presence of the labeled specific binding member - target analyte binding complex in the resulting assay composition. The labeled specific binding member - target analyte binding complex is a binding complex generated when the labeled specific binding member specifically binds to the target analyte (or, according to an embodiment, a primary binding member such as a primary antibody to a target antigen), if present. Assaying the assay composition can include detecting a fluorescent signal from the binding complex, if present. In some cases, the assay includes a separation step in which the target analyte, if present, is separated from the sample. A variety of methods can be used to separate the target analyte from the sample, e.g., by immobilization on a support. Assay methods of interest include, but are not limited to, any convenient method and assay format in which pairs of specific binding members are of interest, such as avidin - biotin or hapten - anti - hapten antibody. Methods and assay formats of interest that can be applied to the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme - linked immunosorbent assays (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0202] In some embodiments, the method further comprises contacting the sample with a second specific binding member that specifically binds to the target analyte. In some cases, the second specific binding member is carrier-bound. Any convenient carrier can be used to immobilize the components of the method (e.g., the second specific binding member). In some cases, the carrier is a particle, such as a magnetic particle. In some cases, the second specific binding member and the polymer dye conjugate form a sandwich complex, and any convenient method can be used to separate and detect the complex (if present). In some embodiments, the method further comprises flow cytometry analysis of the polymer dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte. Determining the presence of the labeled specific binding member-target analyte binding complex can provide an assay result (e.g., qualitative or quantitative assay data), which can be used to evaluate the presence of the target analyte in the sample.

[0203] Any convenient carrier can be used in the method to immobilize any convenient component of the method, such as the labeled specific binding member, the target, the second specific binding member, etc. Carriers of interest include, but are not limited to: solid matrices, where the matrix can have various configurations, such as sheets, beads, or other structures, such as plates with pores; beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, needles, microarray surfaces, chromatographic supports, etc. In some cases, the carrier is selected from the group consisting of particles, planar solid matrices, fibrous meshes, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic supports. The carrier can be incorporated into a system that aids in cell separation by any convenient method, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the carrier can be used in an automated liquid handling system for high-throughput cell separation, such as a flow cytometer.

[0204] In some embodiments of the method, the separation step comprises applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet can be used as the source of the external magnetic field (e.g., a 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 aggregate near the surface closest to the source of the magnetic field gradient (i.e., the magnet).

[0205] Separation can also include one or more optional washing steps to remove unbound material of the sample from the carrier. Any convenient washing method can be used, e.g., washing the immobilized carrier with a biocompatible buffer to retain the specific binding interaction between the polymer dye and the specific binding member. Separating and optionally washing the unbound material of the sample from the carrier can yield an enriched population of target cells, where unwanted cells and materials can be removed.

[0206] In some embodiments, the method includes detecting a labeled target analyte. Detecting the labeled target analyte can include exciting a polymeric fluorescent tandem dye with one or more lasers and then detecting the fluorescence emission of the polymeric fluorescent tandem dye using one or more optical detectors. Any convenient instrument and method can be used to detect the labeled target, including but not limited to flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When fluorescently labeled components are used in the methods and compositions of the present disclosure, it will be appreciated that different types of fluorescence detection systems can be used to practice the subject methods. In some cases, high throughput screening can be performed, such as using a system with a 96-well or larger microtiter plate. A variety of methods can be utilized to assay fluorescent materials, such as those described in the following: 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, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0207] The 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, the fluorescently labeled target in the sample emits radiation having a wavelength different from the excitation wavelength. Collection optics then collect the emission from the sample. The apparatus can include a temperature controller to maintain the sample at a specific temperature while scanning the sample. In some cases, a multi-axis translation stage moves a microtiter plate containing multiple samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data collection can be managed by a suitably programmed digital computer. The computer can also convert the data collected during the analysis into another format for presentation.

[0208] In some embodiments, methods for assessing the presence of a target analyte in a sample further include detecting fluorescence in a flow cytometer. In some embodiments, methods for assessing the presence of a target analyte in a sample further include imaging a sample contacted with a labeling composition using a fluorescence microscope. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contacted sample to assess the presence of the target analyte. Microscopic methods of interest for use in the present methods include laser scanning confocal microscopy.

[0209] The methods described herein can include multiple steps. Each step can be performed after a predetermined time between steps as needed. Thus, the time between performing each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and including 5 hours or longer. In some embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, the steps can be performed after an incubation or waiting time after completion of the previous step, such as a waiting time of several minutes to overnight.

[0210] Kit

[0211] Aspects of the present invention also include kits for practicing the methods of the present invention. Dyes, tandem dyes, labeled specific binding members, or combinations thereof can be included in the kit as reagents, as starting materials or for use in, for example, the methods described above. Such dyes, tandem dyes, and labeled specific binding members can be provided with a container. Any convenient container can be used, such as a tube, bottle, or a well in a multi-well strip or plate, a cassette, a bag, an insulated container, etc. The kits of the present invention can also include one or more components selected from primer specific binding members for a given target analyte, vector binding specific binding members, cells, vectors, biocompatible aqueous elution buffers, controls (positive and / or negative), etc., and instructions for use (as needed). A given kit can include reagents suitable for detecting a single target analyte, or multiple reagents suitable for detecting two or more different target analytes, for example, when a given kit is configured for multiplex detection applications.

[0212] In some embodiments, the kit can be used to assess the presence of a target analyte in a sample, such as an intracellular target. Thus, in some cases, the kit includes one or more components suitable for permeabilizing or lysing cells. One or more additional components of the kit can be provided in separate containers (e.g., separate test tubes, bottles, or wells in a multi-well strip or plate).

[0213] In some aspects, the kit further includes reagents for performing flow cytometry analysis. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting cell samples with chromophores, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. Additionally, the kit may include cell permeabilization reagents, such as methanol, acetone, or detergents (e.g., triton, NP-40, saponin, tween 20, digitonin, leukocyte permeabilizing agent, 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 this kit.

[0214] The compositions in the kit may be provided in the form of a liquid composition, such as any suitable buffer. Alternatively, the compositions in the kit may be provided in the form of a dry composition (e.g., may be lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry composition. In some aspects, the kit may include aliquots of the compositions provided in separate containers (e.g., separate test tubes, bottles, or wells in a multi-well strip or plate).

[0215] In addition, one or more components may be combined into 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 components (and their separate containers) are present. The kit may also include a package separate from or attached to the kit container, on which information about the kit, kit components, and / or instructions for using the kit are printed.

[0216] In addition to the above components, the kit of the present invention may further include instructions for implementing the methods of the present invention. These instructions may exist in multiple forms in the kit of the present invention, and one or more of these forms may be present in the kit. One form in which these instructions may exist is as printed information on a suitable medium or substrate, such as one or more sheets of paper with printed information, in the kit packaging, in the package insert, etc. Another way is a computer-readable medium, such as a floppy disk, CD, DVD, portable flash drive, etc., on which the information is recorded. Another way is a website address through which information on a remote site can be accessed via the Internet. Any convenient way may be present in the kit.

[0217] Examples

[0218] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art on how to make and use the present invention. It is not intended to limit the scope of the invention as conceived by the inventors, nor is it intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (such as amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise specified, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations can be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide; and so on.

[0219] Example 1: Solution-phase synthesis of PEOZ copolymer

[0220] A series of solution-phase chemical reactions were carried out to generate a poly(ethylene glycol)-poly(oxazoline) (PEOZ) copolymer from two different monomers, as Figure 2 shown.

[0221] Both starting monomers are protected analogs of N,N-bis(2-hydroxyethyl)propionamide. Figure 2 The left monomer has a free hydroxyl group and a -O-TBDMS group, which is a hydroxyl group protected by tert-butyldimethylsilyl (TBDMS). Figure 2 The right monomer has a -O-CH 2 -C 6 H 5 group, which can also be referred to as the -OBz group and can be understood as a protected hydroxyl group. The right monomer also has a -OTf group, where -OTf refers to the trifluoromethanesulfonate group, and its chemical formula is -OSO 2 CF 3 and can be called a leaving group.

[0222] The two monomers react in solution such that a new covalent bond is formed between the site of the free hydroxyl group and the site of the trifluoromethanesulfonate group, thereby generating a dimer through a nucleophilic substitution reaction, as Figure 2 shown, and the PG 2 group of the dimer refers to a benzyl-protected hydroxyl group.

[0223] Selective deprotection is carried out on the first sample of the dimer to remove the TBDMS group (labeled as "deprotection 1"), but not the PG 2(i.e., benzyl). The TBDMS group was removed using TBATB (e.g., Firouzabadi 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 effect this selective deprotection. Next, the first sample of this selective deprotection was reacted with Tf-Cl to generate the -OTf group.

[0224] A second sample of the dimer was selectively deprotected (labeled "deprotection 2") to remove the PG 2 (benzyl) group without removing the PG 1 (i.e., TBDMS) group. Using H 2 / Pd(O) to remove the benzyl, but CrO 3 / acetic acid, ozone, N-bromosuccinimide (NBS), and N-iodosuccinimide (NIS) can also be used.

[0225] Then the first and second samples of the selectively deprotected dimer were reacted to create a new covalent bond between the sites of the free hydroxyl and the trifluoromethanesulfonate group, where the resulting compound is a tetramer.

[0226] The steps of selective deprotection, adding the trifluoromethanesulfonate group to one sample, and then reacting the two samples can be repeated to generate larger copolymers, e.g., copolymers having 8, 16, 32, or more repeat units, as Figure 2 shown.

[0227] Example 2: Solid-phase synthesis of PEOZ copolymers

[0228] As Figure 3 shown, the synthesis began by reacting the 2-(4-(hydroxymethyl)phenyl)-N-methylacetamide group attached to a solid resin support with an N,N-bis(2-hydroxyethyl)propanamide analogue having one TBDMS-protected hydroxyl and one carboxylic acid, thereby forming a new covalent bond between the sites of the free hydroxyl and the carboxylic acid by, for example, transesterification. Thus, the synthesis step resulted in the generation of a solid support monomer having a terminal TBDMS-protected hydroxyl.

[0229] Next, the TBDMS protecting group was removed to generate a free hydroxyl, and the solid-supported monomer containing the free hydroxyl was reacted with a solution-phase monomer containing an OTf group and an OTBMDS group to form a new bond at the sites of the free hydroxyl and the OTf group, and the resulting dimer was solid-supported and had a terminal TBDMS-protected hydroxyl.

[0230] The order of deprotection of the repeating OTBDMS groups and reaction of the free hydroxyl groups with monomers containing an OTf leaving group is repeated, where repeating this order n times results in the addition of n units to the PEOZ copolymer.

[0231] After adding the desired number of monomer units, the immobilized PEOZ copolymer is reacted with methyl iodide (MeI) and then with a base to cause resin cleavage. The resulting compound is a PEOZ copolymer with terminal methoxy and terminal carboxylic acid groups.

[0232] Example 3: Synthesis of monomer starting materials

[0233] As Figure 4 shown, commercially available 3-(benzyloxy)propanal is reacted with commercially available 2-aminoethan-1-ol to form 2-((2-(benzyloxy)ethyl)amino)ethan-1-ol. Then, according to the method of Gokhale et al. (Biomacromolecules, 2013, 14, 2489), this compound is reacted with acetic acid in DMF in the presence of EDC at 80 °C to form the N-(2-(benzyloxy)ethyl)-N-(2-hydroxyethyl)acetamide monomer. This compound can be interpreted as a PEOZ monomer, where the amine is replaced by a -C(O)Me group and one of the hydroxyl groups is protected by a benzyl group.

[0234] In addition, as Figure 4 shown, the TBDMS-protected analogue is synthesized by reacting TBDMS-protected 3-(l1-oxidaneyl)propanal with commercially available 2-aminoethan-1-ol to form the TBDMS-protected analogue of 2-((2-(l1-oxidaneyl)ethyl)amino)ethan-1-ol. This compound can be regarded as Figure 4 the TBDMS-protected analogue of the corresponding benzyl-protected compound in the above figure. According to the method of Gokhale et al., the TBDMS-protected compound is reacted with acetic acid to form the corresponding TBDMS-protected compound.

[0235] Example 4: Second solution-phase synthesis of PEOZ copolymer

[0236] As Figure 5 shown, the PEOZ copolymer is synthesized starting from two different monomer starting compounds.

[0237] The monomer with free hydroxyl and benzyl-protected hydroxyl is reacted with NaH and TfCl to convert the free hydroxyl into an OTf group. The resulting compound is reacted with the free hydroxyl of the second monomer to form a dimer with one terminal OTBDMS group and one terminal OBz group.

[0238] In accordance with Example 1 and Figure 2In a similar manner, a sample of the dimer is selectively deprotected by reaction with H 2 / Pd(O) to remove the benzyl group. A second sample of the dimer is reacted with TBATB in methanol to deprotect the OTBDMS group, and then it is reacted with NaH and TfCl to install the OTf moiety.

[0239] Next, the two samples are reacted in the presence of NaH to form a covalent bond between the free hydroxyl site and the OTf site through a nucleophilic substitution reaction, thereby forming a tetramer.

[0240] The sequence of selective deprotection and reaction of the hydroxyl group with the OTf group is repeated to form an octamer compound.

[0241] The terminal hydroxyl group is reacted with NaH and MeI to form a terminal methoxy group, while the terminal O-TBDMS group is deprotected with TBATB to form a free hydroxyl group. The final compound is a PEOZ octamer copolymer with terminal hydroxyl and methoxy groups.

[0242] Example 5: End groups and side groups of PEOZ copolymers

[0243] Figure 6 PEOZ copolymers with various end groups are shown, including methoxy, carboxylic acid group, trifluoromethanesulfonate (i.e., OTf) group, and succinimide group. Figure 6 Embodiments with side chains at the amino group being acetyl or acetyl substituted with a polyethylene glycol group are also shown. The carboxylic acid and succinimide groups can be regarded as functional linkers that can form bonds with other groups.

[0244] Figure 7 Examples of side groups and end groups on the PEOZ copolymer are shown. Figure 7 Tetrazine, succinimide group, and azide (N 3 ) group that can be used for conjugation are shown. Side groups with SO 3 - groups are also shown for increasing solubility and lipid tails that can be used to bind to liposomes or cell membranes.

[0245] Figure 8 How the amino group to which the side group is attached becomes a polymer group, such as a PEG or PEOZ group, to further improve solubility is shown. Figure 8 Embodiments show PEG groups that are dimers, tetramers, and octamers, and PEOZ side chains that are tetramers.

[0246] Notwithstanding the additional claims, the present disclosure is also defined by the following clauses:

[0247] 1. Polyethylene glycol - polyoxazoline (PEOZ) copolymer.

[0248] 2. The copolymer according to Clause 1, wherein the copolymer has a structure of formula (I):

[0249]

[0250] Wherein:

[0251] m is an integer from 1 to 20;

[0252] n is an integer from 2 to 10000;

[0253] R is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, carboxyl, cyano, ether, halogen, hydroxyl, nitro, thiol, thioether, thione, borate, -SO 2 、-SO 3 -, reactive structural moiety, and the group consisting of reactive structural moiety protecting groups; and

[0254] Each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxyl, reactive structural moiety, and reactive structural moiety protecting groups.

[0255] 3. The copolymer according to Clause 2, wherein m is an integer from 1 to 6.

[0256] 4. The copolymer according to Clause 3, wherein m is 1 or 2.

[0257] 5. The copolymer according to Clause 4, wherein the PEOZ copolymer has a structure of formula (II):

[0258]

[0259] 6. The copolymer according to any one of Clauses 1-5, wherein R is selected from the group consisting of H, alkyl, and substituted alkyl.

[0260] 7. The copolymer according to any one of Clauses 1-6, wherein each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, ether, hydroxyl, and reactive structural moiety protecting groups.

[0261] 8. A dye, comprising:

[0262] A fluorophore; and

[0263] Polyethylene glycol - polyoxazoline (PEOZ) copolymer water - soluble group.

[0264] 9. The dye according to clause 8, wherein the dye comprises an organic dye.

[0265] 10. The dye according to clause 9, wherein the organic dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, boron dipyrromethene difluoride, naphthalimide, thiazine dyes, and acridine dyes.

[0266] 11. The dye according to any one of clauses 9 - 10, wherein the PEOZ copolymer water - soluble group is bound to the organic dye.

[0267] 12. The dye according to any one of clauses 9 - 11, which further comprises a non - conjugated polymer backbone containing non - conjugated repeating units.

[0268] 13. The dye according to clause 12, wherein the non - conjugated repeating units comprise a plurality of amino acid residues.

[0269] 14. The dye according to any one of clauses 12 - 13, wherein the dye comprises an organic dye bound to the non - conjugated polymer backbone.

[0270] 15. The dye according to any one of clauses 12 - 14, wherein the PEOZ copolymer water - soluble group is bound to the non - conjugated polymer backbone.

[0271] 16. The dye according to any one of clauses 12 - 14, wherein the PEOZ copolymer water - soluble group is bound to the organic dye.

[0272] 17. The dye according to clause 8, wherein the dye comprises a conjugated polymer.

[0273] 18. The dye according to clause 17, wherein the PEOZ copolymer water - soluble group is bound to the conjugated polymer.

[0274] 19. The dye according to clause 17, wherein the PEOZ copolymer water - soluble group is bound to the organic dye bound to the conjugated polymer.

[0275] 20. A tandem dye, comprising:

[0276] A donor fluorophore;

[0277] An acceptor fluorophore; and

[0278] Polyethylene glycol - polyoxazoline (PEOZ) copolymer water - soluble group.

[0279] 21. The tandem dye according to clause 20, wherein at least one of the donor fluorophore and the acceptor fluorophore comprises an organic dye.

[0280] 22. The tandem dye according to clause 21, wherein the organic dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrene, boron dipyrromethene difluoride, naphthalimide, thiazine dyes, and acridine dyes.

[0281] 23. The tandem dye according to clause 22, wherein the organic dye is boron dipyrromethene difluoride.

[0282] 24. The tandem dye according to any one of clauses 20 - 23, which further comprises a non-conjugated polymer backbone containing non-conjugated repeating units.

[0283] 25. The tandem dye according to clause 24, wherein the non-conjugated repeating units comprise a plurality of amino acid residues.

[0284] 26. The tandem dye according to any one of clauses 21 - 25, wherein both the donor fluorophore and the acceptor fluorophore comprise organic dyes.

[0285] 27. The tandem dye according to any one of clauses 23 - 26, wherein the PEOZ copolymer water-soluble group is bound to the non-conjugated polymer backbone.

[0286] 28. The tandem dye according to any one of clauses 23 - 26, wherein the PEOZ copolymer water-soluble group is bound to the donor fluorophore.

[0287] 29. The tandem dye according to any one of clauses 23 - 26, wherein the PEOZ copolymer water-soluble group is bound to the acceptor fluorophore.

[0288] 30. The tandem dye according to any one of clauses 20 - 22, wherein the donor fluorophore comprises a conjugated polymer.

[0289] 31. The tandem dye according to clause 30, wherein the PEOZ copolymer water-soluble group is bound to the conjugated polymer.

[0290] 32. The tandem dye according to clause 30, wherein the PEOZ copolymer water-soluble group is bound to the acceptor fluorophore.

[0291] 33. A labeled specific binding member, comprising:

[0292] a dye according to any one of clauses 8 - 19 or a tandem dye according to any one of clauses 20 - 32; and

[0293] a specific binding member.

[0294] 34. A method for evaluating the presence of a target analyte in a sample, the method comprising:

[0295] (a) contacting the sample with a labeled specific binding member as described in clause 33 to produce a labeled sample, the labeled specific binding member specifically binding to the target analyte; and

[0296] (b) detecting the presence of a labeled specific binding member - target analyte binding complex in the labeled composition to evaluate the presence of the target analyte in the sample.

[0297] 35. A method for labeling a target molecule, the method comprising:

[0298] contacting the target molecule with a dye as described in any one of clauses 8 - 19 or a tandem dye as described in any one of clauses 20 - 32 to covalently bind the target molecule to a reactive structural moiety of the dye or tandem dye, thereby producing a labeled target molecule.

[0299] 36. The method according to clause 36, wherein the target molecule is selected from the group consisting of DNA, RNA, an antibody, or a fragment thereof.

[0300] 37. A kit, comprising:

[0301] a dye as described in any one of clauses 8 - 19, a tandem dye as described in any one of clauses 20 - 32, a labeled specific binding member as described in clause 33, or a combination thereof; and

[0302] a container.

[0303] 38. The kit according to clause 37, wherein the kit comprises a labeled specific binding member as described in clause 33 and a dye as described in any one of clauses 8 - 19.

[0304] 39. The kit according to clause 37, wherein the kit comprises a tandem dye as described in any one of clauses 20 - 32 and a dye as described in any one of clauses 8 - 19.

[0305] 40. A method for preparing a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, the method comprising:

[0306] (a) generating an (ethylene glycol)-oxazoline (EOZ) dimer comprising a nucleophilic hydroxyl group, the generating comprising:

[0307] (i) reacting a first EOZ monomer comprising a protected hydroxyl group with a second EOZ monomer comprising a protected hydroxyl group to produce an EOZ dimer comprising two protected hydroxyl groups;

[0308] (ii) Selectively deprotect one hydroxyl group of the EOZ dimer to generate an EOZ dimer containing a nucleophilic hydroxyl group;

[0309] (b) Generate an EOZ dimer containing an electrophilic leaving group, and the generation includes:

[0310] (i) React a third EOZ monomer containing a protected hydroxyl group with a fourth EOZ monomer containing a protected hydroxyl group to generate an EOZ dimer containing two protected hydroxyl groups;

[0311] (ii) Selectively deprotect one hydroxyl group of the EOZ dimer;

[0312] (iii) Convert the deprotected hydroxyl group of the EOZ dimer into an electrophilic leaving group;

[0313] (c) React an EOZ dimer containing a nucleophilic hydroxyl group with an EOZ dimer containing an electrophilic leaving group to generate an EOZ tetramer, i.e., a polyethylene glycol - polyoxazoline (PEOZ) copolymer.

[0314] 41. A method for preparing a poly(ethylene glycol) - polyoxazoline (PEOZ) copolymer, the method comprising: reacting a compound of formula (Ia) with a compound of formula (Ib) to generate a compound of formula (Ic)

[0315]

[0316] Deprotect the compound of formula (Ic) to obtain a compound of formula (Id)

[0317]

[0318] Convert the compound of formula (Id) into a compound of formula (Ie)

[0319]

[0320] React the compound of formula (Id) with the compound of formula (Ie) to generate a compound of formula (If)

[0321]

[0322] Wherein:

[0323] PG 1 and PG 2 Are each independently a hydroxyl protecting group;

[0324] OX is a leaving group; and

[0325] R is an alkyl group.

[0326] 42. A method for preparing a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, the method comprising:

[0327] providing a first (ethylene glycol)-oxazoline (EOZ) monomer attached to a matrix and comprising a nucleophilic hydroxyl group;

[0328] reacting the first EOZ monomer with a second EOZ monomer comprising an electrophilic leaving group and a protected hydroxyl group to form a matrix-attached EOZ dimer comprising a protected hydroxyl group;

[0329] deprotecting the protected hydroxyl group to form a matrix-attached EOZ dimer comprising a nucleophilic hydroxyl group;

[0330] reacting the matrix-attached EOZ dimer with a third EOZ monomer comprising an electrophilic leaving group and a protected hydroxyl group to form a matrix-attached EOZ trimer comprising a protected hydroxyl group, wherein the EOZ trimer is a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer.

[0331] 43. A method for increasing the solubility of a compound having a water-soluble group of a poly(ethylene glycol)-polyoxazoline (PEOZ) copolymer, the method comprising:

[0332] combining the water-soluble group of the PEOZ copolymer with the compound.

[0333] 44. The method according to clause 43, wherein the compound is a dye comprising a fluorophore or a tandem dye comprising a donor fluorophore and an acceptor fluorophore.

[0334] 45. The method according to clause 43, wherein the compound is a specific binding member.

[0335] 46. The method according to clause 43, wherein the compound is an active pharmaceutical ingredient (API).

[0336] 47. The method according to clause 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.

[0337] 48. A composition comprising:

[0338] ten or more poly(ethylene glycol)-polyoxazoline (PEOZ) copolymers,

[0339] wherein the dispersity of the ten or more PEOZ copolymers is 1.5 or less.

[0340] 49. The composition according to clause 48, wherein the dispersity is 1.1 or less.

[0341] In at least some of the previously described embodiments, one or more of the elements used in an embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the above-described methods and structures without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0342] Those skilled in the art will understand that, generally speaking, the terms used herein, especially the terms in the appended claims (e.g., the subject matter of the appended claims), are generally intended to be "open" terms (e.g., the term "comprising" should be interpreted as "comprising but not limited to", the term "having" should be interpreted as "having at least", the term "including" should be interpreted as "including but not limited to", etc.). Those skilled in the art should also understand that if the intention is to introduce a specific number of claim recitations, such intention will be explicitly stated in the claims, and if there is no such statement, there is no such intention. For example, for the sake of understanding, the following appended claims may contain the use of introductory phrases such as "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that introducing a claim recitation by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim recitation to an embodiment containing only one such recitation, even if the same claim includes an introductory phrase such as "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as "at least one" or "one or more"); the same is true for the use of the definite article to introduce a claim recitation. In addition, even if a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted as meaning at least the stated number (e.g., a simple recitation of "two recitations" without any other modifiers means at least two recitations, or two or more recitations). In addition, in cases where a convention similar to "at least one of A, B, and C, etc." is used, generally the meaning of such construction is understood by those skilled in the art (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In cases where a convention similar to "at least one of A, B, or C, etc." is used, generally the meaning of such construction is understood by those skilled in the art (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that in fact, whether in the specification, the claims, or the drawings, any disjunctive word and / or phrase representing two or more alternative terms should be understood as contemplating the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" should be understood as including the possibility of "A" or "B" or "A and B".

[0343] In addition, when a feature or aspect of the present disclosure is described in the form of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in the form of any single member or subgroup of members of the Markush group.

[0344] Those skilled in the art will understand that, for any purpose, such as in providing a written description, all ranges disclosed herein also cover any possible sub-ranges and combinations of their sub-ranges. Any listed range can be readily understood as fully describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all language such as "up to", "at least", "greater than", "less than", etc., includes the recited numbers and refers to ranges that can then be broken down into sub-ranges as described above. Finally, those skilled in the art will understand that a range includes each individual member. Thus, for example, a group having 1 - 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1 - 5 articles refers to a group having 1, 2, 3, 4, or 5 articles, and so on.

[0345] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art that some changes and modifications can be made thereto according to the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0346] Accordingly, the foregoing merely illustrates the principles of the invention. It is to be understood that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. In addition, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Moreover, all statements herein, including the principles, aspects, and embodiments of the invention and its specific examples, are intended to cover structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0347] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, a reference to 35 USC § 112(f) or 35 USC § 112(6) as a claim limitation is expressly defined only when the claim limitation begins with the exact phrase "means for" or the exact phrase "step for"; if such exact phrase is not used in the claim limitation, then 35 USC § 112(f) or 35 USC § 112(6) is not referenced.

Claims

1. Poly(ethylene glycol)-poly(oxazoline) (PEOZ) copolymer.

2. The copolymer according to claim 1, wherein the copolymer has a structure of formula (I): Wherein: m is an integer from 1 to 20; n is an integer from 2 to 10000; R is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, carboxyl, cyano, ether, halogen, hydroxy, nitro, thiol, thioether, thione, borate, -SO 2 -, -SO 3 -, a reactive structural moiety, and a reactive structural moiety protecting group; and Each Y is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, ether, halogen, hydroxyl, reactive structural moiety, and reactive structural moiety protecting group.

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

4. The copolymer according to claim 3, wherein m is 1 or 2.

5. The copolymer according to claim 4, wherein the PEOZ copolymer has a structure of formula (II):

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

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

8. A dye, comprising: A fluorophore; and The poly(ethylene glycol)-poly(oxazoline) (PEOZ) copolymer water-solubilizing group according to any one of claims 1-7.

9. The dye according to claim 8, wherein the dye comprises an organic dye or a conjugated polymer.

10. A tandem dye, comprising: A donor fluorophore; An acceptor fluorophore; and The poly(ethylene glycol)-poly(oxazoline) (PEOZ) copolymer water-solubilizing group according to any one of claims 1-9.

11. A labeled specific binding member, comprising: The dye according to any one of claims 1-9 or the tandem dye according to claim 10; and A specific binding member.

12. A method for evaluating the presence of a target analyte in a sample, the method comprises: (a) contacting the sample with the labeled specific binding member according to claim 11 to produce a labeled sample, the labeled specific binding member specifically binding to the target analyte; and (b) detecting the presence of the labeled specific binding member-target analyte binding complex in the labeled composition to evaluate the presence of the target analyte in the sample.

13. A method for labeling a target molecule, the method comprises: contacting the target molecule with the dye according to any one of claims 8-9 or the tandem dye according to claim 10 so that the target molecule covalently binds to the reactive structural moiety of the dye or the tandem dye, thereby producing a labeled target molecule.

14. A kit, comprising: The dye according to any one of claims 8-9, the tandem dye according to claim 10, the labeled specific binding member according to claim 11, or a combination thereof; and A container.

15. A method for increasing the solubility of a compound having a water-soluble group of a polyethylene glycol-polyoxazoline (PEOZ) copolymer, the method comprising: combining the water-soluble group of the PEOZ copolymer with the compound.

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