Metal-Containing Polymers for Mass Cytometry

Abstract

The present disclosure relates to compounds of formula I, elemental tags including soft metals, and methods and kits for performing mass cytometry. [Formula 1] TIFF2024525625000116.tif69159

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims the benefit of priority from U.S. Patent Application No. 63 / 219,787, filed July 8, 2021, and U.S. Patent Application No. 63 / 359,182, filed July 7, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to metal-containing polymers, particularly soft metal-containing polymers, as elemental tags for mass cytometry. [Background technology]

[0003] Metal-containing polymers are one of the important classes of polymers developed in the twentieth century. 1,2 By incorporating different metal units into conventional organic polymers, functional polymers with novel magnetic, optical, electronic, catalytic and bioactive properties can be obtained, finding wide applications in diverse areas such as sensing, catalysis, bioimaging, drug delivery, anticancer agents and biocides. 3~7 Recently, a novel single-cell proteomics technique known as mass cytometry was developed to address the limitations of the multiplexing capabilities of conventional flow cytometry due to spectral overlap. 8 A feature of this technique is the use of metal-tagged antibodies combined with inductively coupled plasma time-of-flight mass spectrometry (ICP-MS) detection. By tagging antibodies with isotopes of different heavy metal ions, the expression of multiple biomarkers can be investigated in individual cells by simultaneously monitoring the signal in different mass channels. The development of mass cytometry offers new opportunities where metal-containing polymers can be used as elemental mass tags for high-parameter single-cell analysis.

[0004] Over the past 15 years, several metal chelating polymers have been developed with pendant aminocarboxylate chelators such as diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for mass cytometry applications. 9~13 These chelators are particularly effective at binding hard metal ions such as lanthanides, yttrium, and bismuth. Using these polymer tags, researchers can measure over 40 different biomarkers per cell. These chelators are less effective at binding ions of softer metals such as Pd or Pt. One study investigated a series of metal chelating polymers with a wide variety of aminocarboxylate pendant groups as carriers for platinum or palladium ions in mass cytometry applications. 14 Metal-containing polymers can be prepared, but they fail to stain the target biomarkers and appear to lose the heavy metal through interactions with soft ligands (presumably thiols) associated with cells. However, there remains a great demand to introduce more new metals into mass cytometry, to increase the number of mass channels that can be used, and thus to expand the capabilities of mass cytometry. Summary of the Invention

[0005] Described herein are metal chelating polymers with heterocyclic pendant groups such as dipicolylamine (DPA) and imidazole suitable for binding soft metal ions including Re, Hg, or Ag. These polymers have been shown to be effective for mass cytometry applications. As shown in the embodiments herein, metal-tagged antibodies provide accurate quantification for single-cell immunophenotyping and can be used in combination with commercially available reagents for mass cytometry immunoassays. Polymers containing DPA chelating groups have been used in 4-plex assays of PBMCs and shown to be capable of quantifying cell populations. DPA is an effective metal chelator for many different polarizable heavy metal ions. Thus, these results introduce a new mass unit into mass cytometry.

[0006] The resulting chelates exhibit excellent stability towards ligand substitution and d 6 Decomposition due to low-spin electron configuration is shown. 17,19,20 It can be appreciated that stability may be observed with other soft metals of similar electronic configuration. Since each soft metal element has multiple naturally occurring isotopes, the soft metal chelating polymers enable novel mass channels for mass cytometry applications.

[0007] Metal-containing polymers used in mass cytometry applications may have one or more of the following characteristics: First, the polymer may have a relatively narrow distribution of chain lengths, so that each labeled antibody carries a similar number of metal ions. Second, the metal may be bound in such a way that there is little or no exchange during storage in the application (e.g., in lyophilized form) and / or during use in aqueous solution over the time period over which mass cytometry experiments may be performed. Third, the polymer may contain functional groups for antibody conjugation. Finally, the polymer may be water-soluble, since bioassays are performed in aqueous media. Meeting the above characteristics simultaneously represents an integrated challenge.

[0008] In one aspect, the present disclosure provides a compound of formula I

[0009] [ka]

[0010] (In the formula, A is a polymer backbone, and optionally the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof; each B is independently a nitrogen-containing 5-7 membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof; L is absent or a linker; Each L 2 is independently absent or a linker; Each R 2 is a first modifying group independently selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof; X is a functional group selected from esters, ethers and amides; Each L 1 is independently absent or a linker; Each R 1 is independently H, C1-C8 alkyl, C3-C8 cycloalkyl, OH, C1-C10 alkoxy, C1-C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof; n is an integer from 0 to 50; m is an integer from 0 to 40; p is an integer from 0 to 30; and q is an integer greater than 0. This includes compounds of the formula:

[0011] In another embodiment, the disclosure includes a compound of formula I, wherein the compound of formula I is chelated to one or more metals M, and the compound is represented by formula II

[0012] [ka]

[0013] or a derivative or salt thereof.

[0014] In another embodiment, the disclosure includes compositions comprising one or more compounds of formula I and one or more metals M.

[0015] In another embodiment, the disclosure includes a compound of Formula I or Formula II for use in mass cytometry.

[0016] In another aspect, the present disclosure includes an element tag comprising a linear or branched polymer comprising a plurality of chelating groups, at least one of the chelating groups being chelated to a soft metal atom of a soft metal, the soft metal being monoisotopic.

[0017] In another aspect, the present disclosure provides an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; and An element tag comprising a linear or branched polymer containing multiple chelating groups containing two nitrogen-containing five- or six-membered heterocycles, each chelating group containing or capable of binding at least one soft metal atom of an isotopic composition. The kit includes: The kit does not contain any radioactive soft metals.

[0018] In another aspect, the present disclosure provides: providing an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; providing an element tag comprising a linear or branched polymer comprising a plurality of chelating groups each independently comprising two nitrogen-containing five- or six-membered heterocycles, each chelating group being capable of binding at least one soft metal atom of the isotopic composition; and Binding the soft metal atom of the isotopic composition to one or more chelating groups of the elemental tag. The method includes the steps of: The soft metal atoms are non-radioactive.

[0019] In another aspect, the present disclosure includes a method for analysis of an analyte in a biological sample, the method comprising: (i) incubating an elementally tagged affinity reagent with the analyte, the elementally tagged affinity reagent comprising an affinity reagent tagged with an elemental tag, the elemental tag comprising a linear or branched polymer having a plurality of chelating groups each independently comprising two nitrogen-containing five- or six-membered heterocycles, the elemental tag further comprising a plurality of soft metal atoms of a single isotope of the soft metal; (where: Each chelating group of the element tag contains or is capable of binding at least one soft metal atom; The soft metal atoms are non-radioactive, and Affinity reagents specifically bind analytes. (ii) separating unbound element-tagged affinity reagents from bound element-tagged affinity reagents; and (iii) analyzing the elemental tags attached to the affinity reagents that are bound to the analyte by mass spectrometry atomic spectrometry. Includes.

[0020] Exemplary embodiments of the present disclosure are further described in conjunction with the drawings. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a 1H-NMR (600 MHz) spectrum of compound I-1. [Diagram 2] Figure 2(a) shows the 1H-NMR (600 MHz) spectrum of the aromatic region of the Re-loaded polymer, and Figure 2(b) shows the FTIR spectra of the Re salt, compound I-1, and Re-loaded compound II-1. [Diagram 3] Figures 3(a)-3(e) are biaxial scatter plots of 170Er-CD3 versus 187Re-CD20 in human PBMCs at different titers, and Figure 3(f) is a biaxial scatter plot of 170Er-CD3 versus 147Sm-CD20 in human PMBCs at optimal titer. [Figure 4] FIG. 4(a) is the 1H-NMR (600 MHz) spectrum of the RAFT reaction mixture, and FIG. 4(b) is the GPC trace of poly(PFPA) synthesized by RAFT polymerization of PFPA monomer. [Diagram 5] FIG. 5 shows the 1H-NMR (600 MHz) spectrum of poly(PFPA) and the 19F-NMR (564 MHz) spectrum of poly(PFPA). [Figure 6] FIG. 6 shows 1H-NMR (600 MHz) spectra of compound 1-2 (top), compound 1-3 (center), and compound 1-4 (bottom). [Figure 7] FIG. 7 is a series of 19F-NMR (564 MHz) spectra showing the aminolysis of polyPFPA using a lysine-based rhenium chelator. [Figure 8] FIG. 8 shows the 1H-NMR (600 MHz) spectrum of polymer 2-2 and the 19F-NMR (564 MHz) spectrum of polymer 2-2. [Figure 9] FIG. 9 is the UV-visible spectrum of polymer 2-2 and DDMAT CTA. [Figure 10] FIG. 10 is a 1H-NMR (600 MHz) spectrum of polymer 2-3 obtained by PEGylation of polymer 2-2. [Figure 11] Figure 11(a) shows the 1H-NMR (600 MHz) spectrum of Bis-Mal-PEG6, and Figure 11(b) shows the 1H-NMR (600 MHz) spectrum of a mixture of Bis-Mal-PEG6 and a rhenium salt. [Figure 12] FIG. 12 is an image of lyophilized rhenium-loaded polymeric compound II-1. [Figure 13] Figure 13(a) is the UV-visible spectrum of Re-loaded polymer compound II-1 in PBS. Figure 13(b) is the FPLC chromatogram of pure CD20 antibody. Figure 13(c) is the FPLC chromatogram of antibody-polymer conjugate. [Figure 14A] FIG. 14(a) is the 1H-NMR (600 MHz) spectrum of polymer 3-2 / I-5, and FIG. 14(b) is the 1H-NMR (600 MHz) spectrum of compound I-1. [Figure 14B] FIG. 14(a) is the 1H-NMR (600 MHz) spectrum of polymer 3-2 / I-5, and FIG. 14(b) is the 1H-NMR (600 MHz) spectrum of compound I-1. [Figure 15] FIG. 15 is the FTIR spectrum of polymer 3-2 / I-5. [Figure 16] FIG. 16 is a 1H-NMR (600 MHz) spectrum of the Pt-supported polymer 4-1 / II-2. [Figure 17] FIG. 17 is a series of dual axis scatter plots of 170Er-CD3 versus 195Pt-CD20 in human PBMCs and 170Er-CD3 versus 147Sm-CD20 in T and B lymphocytes at different titers. [Figure 18] FIG. 18 is the 1H-NMR (600 MHz) spectrum of polymer 2-3. [Figure 19] FIG. 19 is a 1H-NMR (600 MHz) spectrum of the Hg-loaded polymer 5-1 / II-3. [Figure 20] FIG. 20 is a 1H-NMR (600 MHz) spectrum of Ag-loaded polymer 6-1 / II-4. [Figure 21]FIG. 21 shows the H-NMR (600 MHz) spectra of (a) Pt-supported polymer 14-3 / II-6 (arrows indicate the chemical shift change of pyridyl protons after metallization with Pt) and (b) Hg-supported polymer 14-2 / II-5 (arrows indicate the chemical shift change of pyridyl protons after metallization with Hg). [Figure 22] FIG. 22 is the 1H-NMR (600 MHz) spectrum of compound 11-4. [Figure 23] FIG. 23 is the 1H-NMR (600 MHz) spectrum of compound I-11. [Figure 24] FIG. 24 is a 1H-NMR (600 MHz) spectrum of compound I-12. [Diagram 25] 25 shows the results of a mass cytometry immunoassay of identification of CD20+ B cells from PBMCs by rhenium-tagged zwitterionic solubility modifiers comprising polymers of the present disclosure conjugated to CD20 antibodies at various concentrations of polymer conjugate. Maxpar™ 147Sm-CD20 conjugate was used as a positive control. [Figure 26] 26 shows the results of a mass cytometry immunoassay of CD8+ T cells identification from PBMCs by rhenium-tagged zwitterionic solubility modifiers comprising polymers of the present disclosure conjugated to CD8a antibodies at various concentrations of polymer conjugates. Maxpar™ 146Nd-CD8a conjugate was used as a positive control. [Figure 27] 27 is a graph showing signal distribution histograms of 187Re and 147Sm signals obtained from non-T / B cells (CD3-CD20-) in PBMCs using rhenium-tagged zwitterionic solubility modifiers comprising polymers of the present disclosure conjugated to CD20 antibodies at various concentrations of polymer conjugate. Maxpar™ 147Sm-CD20 conjugate was used as a control. [Figure 28]28 is a graph showing signal distribution histograms of 187Re and 146Nd signals obtained from B cells (CD3-CD20+) in PBMCs using rhenium-tagged zwitterionic solubility modifiers comprising a polymer of the present disclosure conjugated to a CD8a antibody at various concentrations of polymer conjugate. Maxpar™ 146Nd-CD8a conjugate was used as a control. [Figure 29] FIG. 29 is a graph showing the results from non-specific binding tests of both PEG-modified rhenium polymers (Group A, polymer concentrations of 1 ug / mL, 2 ug / mL and 5 ug / mL) and zwitterion-modified rhenium polymers (Group B, polymer concentrations of 1 ug / mL, 2 ug / mL and 5 ug / mL). [Diagram 30] FIG. 30 is the 1H-NMR (600 MHz) spectrum of compound 16-3. [Diagram 31] 31 is a series of graphs showing results from non-specific binding testing of glutathione-modified polymers of the present disclosure versus non-glutathione-modified polymers of the present disclosure. Maxpar™ was used as a positive control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] I. Definition Unless otherwise indicated, the definitions and embodiments set forth in this and other paragraphs are intended to apply to all embodiments and aspects of the disclosure described herein for which they are appropriate, as understood by one of skill in the art.

[0023] As used herein, the terms "compound of the disclosure" or "compound of the present disclosure" and the like refer to compounds of Formula I or Formula II, their salts, solvates and / or derivatives.

[0024] The term "and / or" as used herein means that the listed items are present or used individually or in combination. In effect, the term means that "at least one" or "one or more" listed items are used or present. The term "and / or" with respect to its pharma- ceutically acceptable salts and / or solvates means that the compounds of the present disclosure are present as individual salts and hydrates, as well as in combination with, for example, solvates of salts of the compounds of the present disclosure.

[0025] As used in this disclosure, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present a particular aspect having one compound, or two or more additional compounds.

[0026] In embodiments that include an "additional" or "second" component, such as an additional or second compound, the second component, as used herein, is chemically distinct from the other component or the first component. For example, the second component and the first component may have the same chelating agent, but the metal chelated to the second component may be different from the metal chelated to the first component. The "third" component is distinct from the other, first, and second components, and furthermore, the recited or "additional" components are similarly distinct.

[0027] As used in this disclosure and the claims, the words "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "include" and "includes") or "containing" (and all forms of containing, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0028] As used herein, the term "consisting of" and its derivatives are intended to be closed terminology specifying the presence of stated features, elements, components, groups, integers, and / or steps, and excluding the presence of other, unstated features, elements, components, groups, integers, and / or steps.

[0029] As used herein, the term "consisting essentially of" is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps and that do not materially affect the basic and novel characteristics of these features, elements, components, groups, integers, and / or steps.

[0030] As used herein, the term "suitable" means that the selection of a particular compound or conditions will depend on the particular synthetic operation being performed, the identity of the molecule being converted, and / or the particular use of the compound, but that the selection is within the skill of one trained in the art.

[0031] In the embodiment of the present disclosure, the compounds described herein may have at least one asymmetric center. When compounds retain one or more asymmetric centers, they may exist as diastereomers. It should be understood that all such isomers and their mixtures in any ratio are included within the scope of the present disclosure. While the stereochemistry of a compound may be as shown in any given compound listed herein, it should be further understood that such compounds may also include compounds of the present disclosure with a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of alternative stereochemistry. It is intended that any optical isomer, whether as separated, purified or partially purified optical isomer or as a racemic mixture thereof, is included within the scope of the present disclosure.

[0032] The compounds of the present disclosure may also exist in different tautomeric forms, and all tautomeric forms of the compounds, as well as mixtures thereof, are intended to be included within the scope of the present disclosure.

[0033] This description refers to numerous chemical terms and abbreviations used by those of skill in the art. Nonetheless, definitions of selected terms are provided for clarity and consistency.

[0034] As used herein, the terms "about," "substantially," and "approximately" refer to a reasonable amount of deviation of the modified term such that the end result is not significantly altered. These terms of degree should be interpreted as including at least or up to ±5% deviation of the modified term, unless this deviation negates the meaning of the word it modifies or the context would suggest otherwise to one of ordinary skill in the art.

[0035] The term "alkyl" as used herein, whether used alone or as part of another group, refers to a straight or branched chain saturated alkyl group. The number of carbon atoms possible in the alkyl group is indicated by the prefix "Cn1-n2". For example, the term C1-10 alkyl refers to an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0036] The term "alkylene," whether used alone or as part of another group, refers to a straight or branched saturated alkylene group, i.e., a saturated carbon chain containing substituents at both ends. The number of carbon atoms possible in the alkylene group is indicated by the prefix "Cn1-n2." For example, the term C2-6 alkylene refers to an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.

[0037] The term "available," as in "available hydrogen atom" or "available atom," refers to an atom that is known to one of ordinary skill in the art to be replaceable by a substituent.

[0038] As used herein, the terms "amine" or "amino," whether used alone or as part of another group, refer to a group of general formula NR'R'', where R' and R'' are each independently selected from hydrogen or C1-6 alkyl.

[0039] The term "cycloalkyl" as used herein, whether used alone or as part of another group, refers to a saturated carbocyclic group containing one or more rings. The number of carbon atoms possible in the cycloalkyl group is indicated by the numerical prefix "Cn1-n2". For example, the term C3-10 cycloalkyl refers to a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0040] The term "aryl" as used herein, whether used alone or as part of another group, refers to a carbocyclic group containing at least one aromatic ring. In one embodiment of the present disclosure, the aryl group, such as phenyl, indanyl or naphthyl, contains 6, 9 or 10 carbon atoms.

[0041] As used herein, the terms "heterocycle," "heterocyclic," and the like, whether used alone or as part of another group, refer to a cyclic group that contains at least one aromatic or non-aromatic ring in which one or more atoms are heteroatoms selected from O, S, and N. Heterocyclic groups are either saturated or unsaturated (i.e., contain one or more double bonds). When a heterocyclic group contains the prefix Cn1-n2, this prefix designates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably one to five, ring atoms are replaced with a heteroatom as defined above.

[0042] As used herein, the terms "heteroaryl," "heteroaromatic," and the like, whether used alone or as part of another group, refer to a cyclic group that contains at least one heteroaromatic ring in which one or more elements are heteroatoms selected from O, S, and N. When a heteroaryl group contains the prefix Cn1-n2, this prefix designates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably one to five, ring atoms are replaced with a heteroatom as defined above.

[0043] All cyclic groups, including aryl and cyclo groups, contain one or more rings (i.e., are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spiro-fused, or joined through a bond.

[0044] A first ring "fused" to a second ring means that the first ring and the second ring share two adjacent atoms between them.

[0045] A first ring "bridged" to a second ring means that the first ring and the second ring share two non-adjacent atoms between them.

[0046] A first ring that is "spirofused" to a second ring means that the first ring and the second ring share one atom between them.

[0047] The term "halo" as used herein refers to a halogen atom and includes fluoro, chloro, bromo and iodo.

[0048] The term "optionally substituted" refers to a group, structure, or molecule that is unsubstituted or substituted with one or more substituents.

[0049] As used herein, the term "atm" refers to the ambient environment.

[0050] As used herein, the term "MS" refers to mass spectrometry.

[0051] As used herein, the term "aq." refers to aqueous.

[0052] The term "protecting group" or "PG" as used herein refers to a chemical moiety that protects or masks reactive parts of a molecule to prevent side reactions at those reactive parts of the molecule while manipulating or reacting different parts of the molecule. After the manipulation or reaction is completed, the protecting group is removed under conditions that do not degrade or decompose the remaining parts of the molecule. The selection of an appropriate protecting group can be made by those skilled in the art. Many conventional protecting groups are known in the art and are described, for example, in "Protective Groups in Organic Chemistry" McOmie, JFW Ed., Plenum Press, 1973, Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd Edition, 1999, and Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (Americas).

[0053] The term "inert organic solvent" as used herein refers to a solvent that is generally considered to be non-reactive with the functional groups present in the compounds being combined together in a given reaction, as it will not interfere with or inhibit the desired synthetic transformation. Organic solvents are typically non-polar and will dissolve compounds that are insoluble in aqueous solutions.

[0054] As used herein, the term "cell" refers to a cell or multiple cells, including cells either in cell culture or, as the case may be, in a subject.

[0055] The term "solvate" as used herein means a compound, or a salt or derivative of a compound, in which molecules of a suitable solvent are incorporated into the crystal lattice. Examples of suitable solvents may include ethanol, water, etc. When water is the solvent, the molecule is referred to as a "hydrate."

[0056] The term "antibody" as used herein is intended to include any and all antibodies and fragments thereof, including monoclonal antibodies, polyclonal antibodies, and chimeric antibodies and binding fragments thereof. Antibodies may be from recombinant sources and / or produced in transgenic animals. Antibodies may be fragmented using conventional techniques. For example, F(ab')2 fragments may be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments may be treated to reduce disulfide bonds to generate Fab' fragments. Papain digestion may result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments may also be synthesized by recombinant techniques. Antibody fragment as used herein refers to binding fragments.

[0057] The term "oligonucleotide" as used herein refers to a nucleic acid comprising a sequence of nucleotide or nucleoside monomers consisting of naturally occurring and non-naturally occurring bases, sugars, and intersugar (backbone) linkages, including single-stranded and double-stranded molecules, RNA and DNA. Oligonucleotides can be long (e.g., more than 1000 monomers and up to 10K monomers), medium size (e.g., 200-1000 nucleotides, inclusive), or short, e.g., less than 200 monomers, 100 monomers, 50 monomers, including non-naturally occurring monomers. The term "oligonucleotide" includes, for example, single-stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA, reverse transcribed from RNA), messenger RNA (mRNA), "antisense oligonucleotides" and "miRNA" as well as oligonucleotide analogs such as "morpholino oligonucleotides", "phosphorothioate oligonucleotides", or any oligonucleotide or analog thereof known to those of skill in the art.

[0058] The terms "element tag", "tag" and the like as used herein refer to a chemical moiety that includes one element or multiple elements that has one or many isotopes (such as soft metals) attached to a supporting molecular structure or that can bind said element or isotopes. An element tag can also include a means for attaching the element tag to a molecule of interest or a target molecule (e.g., a biomolecule such as an analyte). Different element tags may be distinguished based on the elemental composition of the tag. An element tag can include many copies of a given isotope and can have a reproducible number of copies of each isotope in each tag. An element tag is functionally distinguishable from multiple other element tags in the same sample because its elemental or isotopic composition is different from that of the other tags.

[0059] The term "ICP-MS" as used herein refers to an inductively coupled plasma mass spectrometer-a highly sensitive mass spectrometry based elemental analyzer. Different ICP-MS configurations are primarily distinguished by the mass selection technique used, which can be, for example, quadrupole or time-of-flight (ICP-TOF) or magnetic sector (high resolution ICP-MS). There are many commercially available ICP-MS models with a wide range of configurations, capabilities and modifications.

[0060] The term "polymer" as used herein refers to a substance composed of molecules characterized by multiple repeats of one or more species or groups of atoms (building blocks) bonded together in sufficient amounts to provide a set of properties that do not change significantly with the addition or deletion of one or a few building blocks. (IUPAC definition, see E.S. White, J. Chem. Inf. Comput. Sci. 1997, 37, 171-192). A polymer molecule can be thought of in terms of its backbone, the connected bonds of atoms that run the length of the molecule, and the pendant groups attached to the backbone portion of each building block. The pendant groups are often chemically and functionally distinct from the main chain. Pendant groups that have a high affinity for metal ions can function as chelating groups or ligands for those ions. In some cases, the polymers can have from about 10 to about 300 units.

[0061] The term "copolymer" as used herein refers to a polymer composed of two or more chemically distinct building blocks. A "linear polymer" is a polymer characterized by a linear sequence of building blocks. A "block copolymer" is a linear polymer having a sequence of a common type of building block unit attached to an sequence of a different type of building block unit. A "branched polymer" is a polymer having additional polymer chains (branches) emanating from the polymer backbone. Generally, the longest linear sequence is referred to as the "backbone". A branched polymer in which the chemical composition of the building blocks of the branches differs from that of the backbone is called a "graft copolymer".

[0062] The term "star polymer" as used herein refers to a polymer having multiple linear polymer chains emanating from a common building block or core. The term "hyperbranched polymer" as used herein refers to a multiply branched polymer in which the backbone atoms are arranged in a tree shape. These polymers are related to "dendrimers" and have three distinguishing architectural features: an initiator core, an inner layer (generation) made up of repeating units radially attached to the initiator core, and an outer surface of terminal functionality attached to the outermost generation. "Dendrimers" differ from hyperbranched polymers by their unusual symmetry, high branching, and maximized (telechelic) terminal functionality.

[0063] As used herein, the term "metal-tagged polymer" (also "polymeric metal tag carrier", or "metal-polymer conjugate", or "chelating derivatized polymer"), etc., refers to a wide variety of element tags consisting of a polymer backbone carrying at least one pendant chelating group having a metal atom bound thereto. These metal-tagged polymers can be, but are not limited to, linear, star, branched, or hyperbranched homopolymers or copolymers, and block or graft copolymers.

[0064] As used herein, the term "metal-binding pendant group" is a pendant group on a polymer that is capable of binding a metal or an isotope of a metal. It may also be referred to as a chelator.

[0065] As used herein, the term "chelation" refers to the process of binding a ligand, chelator, chelator or chelating agent to a metal ion to form a metal complex, a chelate. In contrast to a single monodentate ligand, such as HO or NH, a multidentate chelator forms multiple bonds with the metal ion.

[0066] As used herein, the term "metal" refers to elements having one of the following atomic numbers: 3, 4, 11-13, 19-33, 37-52, 55-84, 87-102.

[0067] The term "soft metals" as used herein refers to metals that are considered soft according to Pearson's theory of hard and soft Lewis acids and bases.

[0068] When referring to a single isotope, it is assumed to refer to a substantially single isotope of a metal. For example, a single isotope can include trace amounts of other isotopes of the metal and / or trace amounts of another metal. For example, a substantially single isotope can refer to an isotope having an isotopic purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%, or having 100% purity of the isotope. For example, a single isotope can include about 95% or more than 95% of an isotope and about 5% or less than 5% of other isotopes. In some embodiments, a single isotope can include about 97% or more than 97% of an isotope and about 3% or less than 3% of other isotopes. In some embodiments, a single isotope can include about 98% or more than 98% of an isotope and about 2% or less than 2% of other isotopes. In some embodiments, a single isotope may comprise about 99% or greater than 99% of an isotope and about 1% or less than 1% of other isotopes. In some embodiments, a single isotope may comprise about 99.5% or greater than 99.5% of an isotope and about 0.5% or less than 0.5% of other isotopes. In some embodiments, a single isotope may comprise about 99.9% or greater than 99.9% of an isotope and about 0.1% or less than 0.1% of other isotopes. In some embodiments, a single isotope comprises 100% of an isotope.

[0069] The terms Mn, Mw and PDI (polydispersity index): Mw / Mn are used to indicate the number and average molecular weight, respectively, and the polydispersity index describes the molecular weight distribution.

[0070] The recitations herein of numerical ranges by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0071] Moreover, it is intended that the definitions and embodiments described in a particular section are applicable to other embodiments herein, as described as appropriate, as understood by those skilled in the art. For example, in the following sections, different aspects of the present disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects, unless expressly indicated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

[0072] For ranges set forth herein, subranges are also contemplated, e.g., in increments of 0.1 therebetween. For example, if a range is 0 ppm to about 5 ppm, then 0.1 ppm to about 5 ppm, 0 ppm to about 4.9 ppm, 0.1 ppm to about 4.9 ppm, etc. are also contemplated.

[0073] II. Compounds, Compositions and Kits In one aspect, the present disclosure provides a compound of formula I

[0074] [ka]

[0075] (In the formula, A is a polymer backbone, and optionally the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof; each B is independently a nitrogen-containing 5-7 membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof; L is absent or a linker; Each L 2 is independently absent or a linker; Each R 2is a first modifying group independently selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof; X is a functional group selected from esters, ethers and amides; Each L 1 is independently absent or a linker; Each R 1 are independently H, C1-C8 alkyl, C1-C8 alkyl, C2-C8 alkenyl, C3-C8 cycloalkyl, OH, C1-C10 alkoxy, C1-C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof; n is an integer from 0 to 50; m is an integer from 0 to 40; p is an integer from 0 to 30; and q is an integer greater than 0. This includes compounds of the formula:

[0076] In another embodiment, the disclosure includes a compound of formula I, wherein the compound of formula I is chelated to one or more metals M, and the compound is represented by formula II

[0077] [ka]

[0078] or a derivative or salt thereof.

[0079] In another aspect, the disclosure includes a composition comprising one or more compounds of formula I or one or more compounds of formula II and a solvent.

[0080] In another embodiment, the disclosure includes a compound of Formula I or Formula II for use in mass cytometry.

[0081] In some embodiments, the polymer backbone A can include from 10 to 300 monomer units.

[0082] In some embodiments, n is an integer of 0 to 20, 1 to 10, or 0 to 7. In some embodiments, m is an integer of 0 to 30, 0 to 20, 0 to 10, or 0 to 4. In some embodiments, p is an integer of 0 to 20, 0 to 10, 0 to 5, or 0 to 3. In some embodiments, q is an integer greater than 0. For example, q is an integer of 2 to 300, 2 to 200, 2 to 150, 2 to 100, 4 to 80, 4 to 60, 4 to 20, 4 to 12, or 10 to 60. For example, q is an integer that is at least 2, at least 4, or at least 10. For example, q is an integer that is up to 300, up to 250, up to 200, up to 150, up to 100, up to 80, up to 60, up to 20, up to 12, or up to 10. In some embodiments, q may be 1 or more but less than 20 to avoid steric hindrance and / or reduce background, such as when used to stain tissue for imaging mass cytometry or to label intracellular targets in suspension mass cytometry. In some embodiments, n is an integer between 0 and 7; m is an integer between 0 and 4; p is an integer between 0 and 3; and q is an integer greater than 0. For example, n is 2, 3, 4, or 5. In some embodiments, m is 0, 1, or 2. In some embodiments, p is 1 or 2.

[0083] As used herein, the term "modifying group", such as a first modifying group or a second modifying group, refers to a group, moiety, structure, and / or substituent that, when attached to a chemical structure, such as a chemical substance or a polymer, modifies, changes, regulates, or alters the functionality and / or properties of the chemical structure, such as a chemical substance or a polymer. For example, the modifying group can modify, change, regulate, or alter the solubility, reactivity, and / or hydrophobicity of a chemical substance, or the affinity of a chemical substance to another chemical substance. In some embodiments, the modifying group can be a solubility regulator and / or a reactive functional group.

[0084] As used herein, "solubility modifier" refers to a group, moiety, structure and / or substituent that, when attached to a chemical substance or a chemical structure, such as an oligomer or polymer, modifies, alters, regulates or changes the solubility of the chemical substance or chemical structure in water. For example, the solubility modifier can include a water-soluble polymer, such as polyethylene glycol (PEG), a zwitterionic polymer, or a charged polymer. Zwitterionic polymers can include poly(sulfobetaine methacrylate) (PSBMA) and poly(carboxybetaine methacrylate) (PCBMA). In some embodiments, each R 2The solubility modifier of the first modifying group and the solubility modifier of the second modifying group each independently comprise a polyethylglycol (PEG), a sugar, an oligosaccharide, or a zwitterionic polymer such as poly(carboxylbetaine) methacrylate or poly(sulfobetaine) methacrylate (PBSMA). The solubility modifier may increase the solubility of the polymer (e.g., a metal-loaded polymer described herein), such as a two-fold increase in the amount of polymer that can be in solution (e.g., an aqueous solution, a buffered solution at a pH between 6 and 8 or therebetween) compared to the absence of the solubility modifier. In certain aspects, the solubility modifier may increase the solubility of the polymer (e.g., a metal-loaded polymer described herein) compared to the absence of the solubility modifier. In some embodiments, the oligomer can have up to 10 monomer units. In some embodiments, the solubility modifier can include a polymer having about 10 to about 5000 units. For example, the solubility modifier can be a PEG group. For example, the PEG group can have about 10 to about 350 units, about 10 to about 300 units, about 10 to about 250 units, about 10 to about 200 units, about 10 to about 150 units, or about 110 units of ethylene glycol. For example, the PEG group can have at least 10, at least 20, or at least 30 units of ethylene glycol. For example, the PEG group can have up to 300, up to 250, up to 200, up to 150, up to 100, or up to 50 units of ethylene glycol. For example, the PEG group can have an Mn of about 5000 g / mol to about 10000 g / mol.

[0085] In certain aspects, solubility regulators can also reduce the non-specific binding of the compounds of the present disclosure to targets in samples.In some embodiments, certain solubility regulators have been shown to be more effective in reducing non-specific binding.For example, zwitterionic solubility regulators have been shown to show less non-specific binding than PEG solubility regulators.

[0086] In some embodiments, at least a portion or all of the metal M may be coordinated to one or more solubility modifiers. For example, the solubility modifier may be a ligand of the metal M. In some embodiments, the solubility modifier may include a thiol small molecule. For example, the thiol small molecule may be selected from glutathione, cysteine, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonate, and combinations thereof. In some embodiments, the solubility modifier is glutathione. It may be understood that the solubility modifier may be coordinated to the metal M by a ligand exchange reaction.

[0087] As used herein, a "reactive functional group" refers to a group of atoms or a single atom that interacts or reacts with another group of atoms or a single atom to form a chemical interaction between the two groups of atoms or between the two atoms. For example, one or more reactive functional groups may be combined with a chemical or When attached onto a chemical structure such as a polymer, it modifies or changes the reactivity of the chemical or chemical structure such as a polymer, allowing the chemical or chemical structure to interact or react with a group of atoms on another chemical or chemical structure such as a biomolecule. In some cases, it may be understood that a given reactive functional group can interact or react with a specific functional group to form a chemical interaction. For example, it is known that azides are suitable for click chemistry and maleimides can react with thiols. In some embodiments, the chemical interaction is covalent or ionic. For example, the chemical interaction is covalent. In some embodiments, the reactive functional group is for binding to one or more biomolecules. In some embodiments, each R 2The reactive functional group of the first modifying group and the reactive functional group of the second modifying group are each independently selected from a carboxylic acid, an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, a maleimide, a thiol, an azide, a dibenzocyclooctyne (DBCO), a trans-cyclooctene (TCO), a tetrazine, a furan, a hydrazide, or an aldehyde.

[0088] It can be understood that reactive functional groups can be reversibly protected or capped with suitable protecting groups until they are required for further reaction. For example, thiols can be capped with thiol capping groups such as maleimides or other groups known in the art. For example, thiol-containing polymers can be temporarily protected or temporarily present as disulfide dimers and can be reduced using known methods (e.g., DTT reduction) to expose thiol groups as needed. Thus, the reactive functional groups described herein are also considered to include the protected versions of reactive functional groups.

[0089] As used herein, when a metal is non-radioactive, it means that the metal is essentially non-radioactive. For example, a radioactive metal can have a decay rate that is suitable for use in a radiodetection assay, whereas a non-radioactive metal can have a decay rate that is not suitable for a radiodetection assay or is below the detection limit of a typical radiodetection assay used in the field of radiolabeling. In some embodiments, a non-radioactive metal can have a half-life of more than about 150,000 years, more than 200,000 years, or more than about 210,000 years. For example, 99 It is known that the Tc isotope has a half-life of 210,000 years and is therefore considered to be non-radioactive for the purposes of this disclosure.

[0090] In some embodiments, each B is independently a 5- or 6-membered heterocycle. For example, each B can be independently substituted or unsubstituted tetrahydropyrrole. In some embodiments, each B is independently a nitrogen-containing 5- or 6-membered heteroaryl, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof, and optionally one or more B is coordinated to a soft metal and / or conjugated to one or more biomolecules.

[0091] For example, each B is independently pyridine or imidazole, optionally substituted with one or more polar functional groups selected from C1-C5 alkyl, C2-C5 alkenyl, COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof, and optionally one or more B is coordinated to a soft metal and / or conjugated to one or more biomolecules.

[0092] It is believed that two B's attached to the same nitrogen are not essentially the same chelating group. Nevertheless, apart from ease of synthesis, two B's attached to the same nitrogen may be the same.

[0093] In some embodiments, one or more B's are coordinated to the soft metal. It is believed that not all of the B's of the compound of formula I need be coordinated to the metal. For example, in some embodiments, about 30% to about 95%, about 40% to about 90%, about 50% to about 85% of the B's are coordinated to the metal. In some embodiments, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, or at least or about 85% of the B's are coordinated to the metal. In some embodiments, up to 95%, up to 90%, up to 85%, up to 80%, up to 75%, up to 70%, or up to 65% of the B's are coordinated to the metal. In some embodiments, about 75% to about 80% of the B's are coordinated to the metal. In some embodiments, all of the B's are coordinated to the metal. Without wishing to be bound by theory, it can be understood that two B's attached to the same nitrogen atom can chelate to the same metal atom in a bidentate manner.

[0094] In certain embodiments, B is an optionally substituted pyridine.

[0095] In some embodiments, B is a substituted or unsubstituted imidazole. In some cases, B is a di- or tetra-substituted imidazole. Suitable imidazole-based chelators include those described in Maresca et al., Bioconjugate Chem., 2010, 21, 1032, the contents of which are incorporated by reference in their entirety.

[0096] In some embodiments, R 1 is a biomolecule. For example, R 1 may be an antibody. In some embodiments, R 1 is an affinity reagent.

[0097] In some embodiments, R 2is a biomolecule. For example, R 2 may be an antibody. In some embodiments, R 2 is an affinity reagent.

[0098] In some embodiments, X is an amide. For example, X is -C(O)NR 4 -OR-NR 4 C(O)-(wherein, R 4 is H or C1-C4 alkyl).

[0099] In certain embodiments, X is -C(O)NR 4 and the compound has the formula Ia

[0100] [ka]

[0101] It has the structure:

[0102] In some embodiments, X is -NR 4 C(O)-, and the compound has the formula Ib

[0103] [ka]

[0104] It has the structure:

[0105] In certain embodiments, X is -C(O)NR 4 and the compound has the formula Ic

[0106] [ka]

[0107] (In the formula, each R 3are independently selected from H, C1-C5 alkyl, C2-C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkyl phosphonate, alkyl ether, or polyether. It has the structure:

[0108] In certain embodiments, X is -C(O)NR 4 and the compound has the formula Id or Ie

[0109] [ka]

[0110] It has the structure:

[0111] In some embodiments, X is -NR 4 C(O)-, and the compound has the formula If

[0112] [ka]

[0113] (In the formula, each R 3 are independently selected from H, C1-C5 alkyl, C2-C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkyl phosphonate, alkyl ether, or polyether. It has the structure:

[0114] In some embodiments, X is -NR 4 C(O)—, and the compound has formula Ig or formula Ih

[0115] [ka]

[0116] It has the structure:

[0117] In some embodiments, R4 is H. In some embodiments, R 4 is C1-C3 alkyl.

[0118] In some embodiments, the compound of formula I has the formula Ii

[0119] [ka]

[0120] (In the formula, A 1 is a monomer of A, and r is from about 3 to about 300, from about 3 to about 250, from about 3 to about 200, from about 3 to about 150, from about 3 to about 100, from about 3 to about 50, from about 6 to about 30, or from about 10 to about 25. In some embodiments, r is up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 30, or up to 25. In some embodiments, r is at least 3, at least 6, or at least 10. In some embodiments, the polymer backbone A is a linear polymer or copolymer.

[0121] In some embodiments, the compound of formula I is represented by formula Ij

[0122] [ka]

[0123] (In the formula, A 1 and A 2 are each a monomer of A, r is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25, and the polymer backbone A is a linear copolymer. In some embodiments, r is up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 30, or up to 25. In some embodiments, r is at least 3, at least 6, or at least 10.

[0124] In some embodiments, each R 3 is H, -(CH2) 1~3 COOH, -(CH2) 1~3 O(CH2) 1~2 CH3, -(CH2) 2~4 OH, -(CH2) 2~5 and independently selected from -P(O)(OCH2CH3)2, or -CH2CH(OMe)2.

[0125] In some embodiments, A is selected from polyacrylates, polyacrylamides, polyethers, polyamino acids, polyvinylamines, poly(2-oxazolines), polyethylene glycols, polysaccharides, dendrimers, copolymers thereof, or combinations thereof.

[0126] For example, A can be a polyamino acid, such as optionally substituted polyglutamic acid, polyaspartic acid, polylysine, poly(2,4-dimethylaminobutyric acid) (polyDab), poly(2,4-diaminopimelic acid) (polyDap), derivatives thereof, or combinations thereof.

[0127] It is contemplated that the polymer backbone A of the compounds of the present disclosure may be a copolymer. For example, it may be a copolymer that includes PEG.

[0128] In some embodiments, the polymer backbone is a linear polymer. For example, the compound of formula I can have the structure shown below:

[0129] [ka]

[0130] In some embodiments, the polymer backbone is a branched polymer, such as a hyperbranched polymer, or a grafted polymer. Representative representations of compounds of formula I include the structures shown below.

[0131] [ka]

[0132] For each a (e.g. a1, a2, …, a r ) are monomer units of the polymer backbone. The polymer backbone of the compound of formula I can be a homopolymer or a copolymer. The copolymer can include a graft copolymer or a block copolymer. Each monomer unit (e.g., a, a1, a2, ..., a r It can be understood that a) can be the same, e.g., in a homopolymer, or different, e.g., in a copolymer. For example, a2 can be the same monomer as a1, or a different monomer. Similarly, a3 can be the same monomer as a2 and / or a1, or a different monomer.

[0133] At least one monomer unit a has the structure

[0134] [ka]

[0135] which can be chelated to a metal, such as a soft metal. In some embodiments, each monomer unit of the polymer backbone is

[0136] [ka]

[0137] In some other embodiments, some, but not all, of the monomer units of the polymer backbone are linked to

[0138] [ka]

[0139] is bound to.

[0140] In some embodiments, the first modifying group R 2 can be present at the termini of the polymer backbone. For example, both ends of the polymer backbone can be terminated with an optional linker L 2 via the first modifying group R 2 Each first modifying group R 2 and each linker L 2 are independently defined herein. In some embodiments, some, but not all, of the termini of the polymer backbone may be linked with an optional linker, L 2 via the first modifying group R 2 Each first modifying group R 2 and each linker L 2 are independently defined herein.

[0141] In some embodiments, the polymer backbone may be a copolymer of monomers containing different pendant groups. For example, in a polyacrylamide backbone, it is envisioned that an acrylamide monomer may be attached to a pendant or modifying group of a chelator, such as a solubility-modifying group or reactive functional group. Representative polymer compounds of the present disclosure having a copolymer backbone are shown below.

[0142] [ka]

[0143] In some embodiments, the degree of polymerization (DP) can be approximately 1-1000 (1-2000 backbone atoms). Larger polymers are also possible with the same functionality within the scope of the present invention as would be understood by one skilled in the art. Typically, the degree of polymerization is 10-250. The polymers may be amenable to synthesis by routes that result in relatively narrow polydispersity. The polymers may be synthesized by atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization or ring-opening polymerization, which should result in Mw / Mn values ​​in the range of 1.1-1.2. Alternative strategies include anionic polymerization, which results in polymers with Mw / Mn of approximately 1.02-1.05. Thus, the polymers may have a polydispersity index of 1.02-1.5, for example 1.02-1.2, 1.02-1.05, or 1.2-1.5. These methods allow control of the end groups through the selection of initiators or terminators. This allows the synthesis of polymers to which linkers can be attached. A strategy can be adopted to prepare polymers containing functional pendant groups on repeating units to which ligand-bound transition metal units (e.g., soft metal units) can be attached in a later step. This embodiment has several advantages. It avoids the complications that can arise when carrying out the polymerization of ligand-containing monomers. Furthermore, the polymer backbone is a known one that can be applied to most, if not all, soft metal-containing polymers. Thus, the polymers can have a common average chain length and chain length distribution.

[0144] In some embodiments, each linker independently comprises or is independently selected from a C-C alkylamine, a C-C alkylene, a C-C cycloalkyl, a C-C heterocycloalkyl, a 5- or 6-membered aryl or heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, C(O), C(O)O, an amide, an amine, a thioether, a maleimide-thiol conjugate, a polyethylene glycol (PEG), or a mixture thereof, and optionally an amine. Each of aryl, alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.

[0145] In some embodiments, each L 2 is independently comprised or selected from a C-C alkylene, a C-C alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, and optionally each alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from a C-C alkyl, a C-C alkenyl, a C-C cycloalkyl, a C-C heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, CN, or a mixture thereof.

[0146] In some embodiments, each L 1is independently comprised or selected from a C-C alkylene, a C-C alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, and optionally each alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from a C-C alkyl, a C-C alkenyl, a C-C cycloalkyl, a C-C heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, CN, or a mixture thereof.

[0147] In some embodiments, L is absent or a C3-C8 alkylamine.

[0148] It is understood that in some embodiments, the linker can include a functional group that attaches the linker to the remainder of the compound.

[0149] Biomolecules may be classified as proteins, oligonucleotides, lipids, carbohydrates, or small molecules, or combinations thereof. Alternatively or additionally, biomolecules may be classified by their functionality. Biomolecules are not particularly limited, and different functionalizations may be used to conjugate biomolecules to the compounds of the present disclosure. For example, oligonucleotides may be single-stranded DNA molecules, and in some cases, cDNAs that hybridize to target nucleic acid analytes (e.g., sample nucleic acid biomolecules) or oligonucleotides under stringent conditions may be aptamers. For example, biomolecules may be oligonucleotides that specifically hybridize to target oligonucleotides, such as target mRNAs present in the sample (e.g., hybridize to sample oligonucleotides). Hybridization may be of a sequence that is more than 8, more than 10, more than 15, or more than 20 nucleotides.

[0150] In certain embodiments, biomolecules may be classified according to their functionality. For example, a biomolecule may be an affinity reagent, an antigen (e.g., an analyte specifically bound by an affinity reagent), or an enzyme substrate. An affinity reagent may be an antibody (e.g., or a fragment thereof), an aptamer, a receptor (e.g., or a portion thereof), or any other biomolecule that specifically binds a target (e.g., an avidin, such as streptavidin, which specifically binds biotin). For example, an element tag may be associated with an antibody and used to detect and / or analyze the presence of its target antigen in a sample, such as the presence of a cytokine, a viral protein, a cancer biomarker, etc. In certain methods and kits, an element tag may be functionalized with avidin for binding another biomolecule functionalized with biotin (e.g., to allow the compounds of the present disclosure to be compatible with any of a number of different assays). An antigen may be a protein (or a peptide sequence thereof) that contains an epitope that is specifically bound by an affinity reagent, such as an antibody. For example, the compounds of the present disclosure may bind to viral antigens (e.g., viral protein sequences) and may be used to detect the presence of antibodies in a sample that specifically bind viral antigens, as further described herein. Enzyme substrates may be any substrate that is acted upon by a specific enzyme, for example, by an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. For example, the substrate may be a protein (e.g., or a peptide sequence thereof) that is a substrate for enzymes such as proteases, phosphatases, kinases, methyltransferases, demethylases, etc. Non-protein substrates include, for example, double-stranded oligonucleotides that contain restriction sequences or sites for DNA repair (such as nicks) that can be cleaved by restriction enzymes, oligonucleotide sequences that contain sequences targeted by DNA methyltransferases, or any non-protein substrates known to those skilled in the art.For example, a compound of the disclosure may be bound to a substrate and exposed to a sample containing an enzyme that modifies the substrate, and modification (or lack thereof) of the substrate may be detected (e.g., as further described herein).

[0151] In some embodiments, the one or more biomolecules are each independently selected from a small molecule, a polypeptide, an oligonucleotide, a lipid, a carbohydrate, or a mixture thereof.

[0152] In some embodiments, the one or more biomolecules are each independently an affinity reagent, and in some cases the affinity reagent is an antibody.

[0153] In some embodiments, the affinity reagent is or includes an antibody or a binding fragment thereof. The antibody can be, for example, a biotinylated antibody or binding fragment, and can be directly or indirectly attached to a compound of the present disclosure.

[0154] In some embodiments, the compound of formula I is

[0155] [ka] TIFF2024525625000021.tif233159TIFF2024525625000022.tif72159

[0156] (In the formula, r is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3wherein each is as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.

[0157] In some embodiments, the compound of formula I is

[0158] [ka] TIFF2024525625000024.tif185159TIFF2024525625000025.tif186159

[0159] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3 wherein each is as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.

[0160] In some embodiments, the compound of formula I is

[0161] [ka] TIFF2024525625000027.tif201159TIFF2024525625000028.tif195159TIFF2024525625000029.tif92159

[0162] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3 wherein each is as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.

[0163] In some embodiments, the compound of formula I is

[0164] [ka] TIFF2024525625000031.tif183159TIFF2024525625000032.tif228159TIFF2024525625 000033.tif223159TIFF2024525625000034.tif218159TIFF2024525625000035.tif92159

[0165] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25; R 3 is as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.

[0166] In some embodiments, the compound of formula I is

[0167] [ka] TIFF2024525625000037.tif233159TIFF2024525625000038.tif206159TIFF2024525625000039.tif112159

[0168] is selected from.

[0169] In some embodiments, the compound of formula II is

[0170] [ka] TIFF2024525625000041.tif242139TIFF2024525625000042.tif242155TIFF20245256250 00043.tif219159TIFF2024525625000044.tif137159TIFF2024525625000045.tif190159

[0171] (Wherein R is H,

[0172] [ka]

[0173] or other suitable thiol capping groups known in the art). In some embodiments, R represents a second compound of formula II that forms a dimer via a disulfide bond.

[0174] It can be seen that bis-heterocyclic chelators such as DPA, bis((1-methyl-imidazol-4-yl)methyl)amine, bis((1-methyl-imidazol-2-yl)methyl)amine, bis((1H-imidazol-4-yl)methyl)amine and bis((1H-imidazol-2-yl)methyl)amine can stably chelate metals, particularly soft metals. Polymeric compounds of the present disclosure that include one or more pendant groups, including bis-heterocyclic chelators such as DPA, bis((1-methyl-imidazol-4-yl)methyl)amine, bis((1-methyl-imidazol-2-yl)methyl)amine, bis((1H-imidazol-4-yl)methyl)amine and bis((1H-imidazol-2-yl)methyl)amine, can chelate metals, including soft metals. When chelated to a metal, the compounds of the present disclosure introduce new mass channels into applications such as mass cytometry that are represented by stable isotopes of the metal.

[0175] The examples herein show representative chelates formed using the polymeric compounds of the present disclosure with metals including Re, Pt, Hg, and Ag. It is known that chelating agents such as DPA can form stable chelates with other soft metals in non-polymeric environments. Thus, the polymeric compounds of the present disclosure carrying chelating agents such as DPA, bis((1-methyl-imidazol-4-yl)methyl)amine, bis((1-methyl-imidazol-2-yl)methyl)amine, bis((1H-imidazol-4-yl)methyl)amine, and bis((1H-imidazol-2-yl)methyl)amine can chelate to other soft metals as well.

[0176] For example, Seubert et al. ["Chimeric GNA / DNA metal-mediated base pairs", Chem. Commun., 2011, 47, 11041-11043] reported a computational analysis of Au(III) chelated to DPA. Messori et al. [J. Med. Chem. 2000, 43, 3541-3548] reported a series of Au(III) complexes with diamines and triamines.

[0177] A molybdenum complex with DPA [Mo(dipic)(CO)3] has been reported by van Staveren et al., Labelling of [Leu5]-enkephalin with organometallic Mo complexes by solid-phase synthesis, Chem. Commun., 2002, 1406-1407.

[0178] [Ru III We reported that [Ru(2,2'-dipicolylamine)(OH2)3](OTf)2 can react with dipicolylamine in aqueous solution, thereby reducing Ru(III) to Ru(II). [Ru(2,2'-dipicolylamine)(OH2)3](OTf)2 is stable up to 150 °C.

[0179] Lonnon et al. [Rhodium, palladium and platinum complexes of tris(pyridylalkyl)amine and tris(benzimidazolylmethyl)amine N4-tripodal ligands, Dalton Trans., 2006, 3785-3797, DOI:10.1039 / b602556k] prepared the crystal structures of Rh, Pd, and Pt using tris(2-pyridylmethyl)amine.

[0180] Song et al. [Cadmium(II) complexes containing N0-substituted N,N-di(2-picolyl)amine: The formation of monomeric versus dimeric complexes is affected by the N'-substitution group on the amine moiety, J. Organometallic Chem. 783(2015)55e63, doi.org / 10.1016 / j.jorganchem.2015.02.011] showed that N-alkyldipicolylamine derivatives were effective in the synthesis of Cd 2+ It was shown that Zn tends to form a dimeric complex with

[0181] It is therefore believed that the polymeric compounds of the present disclosure, including heterocyclic chelators such as DPA and bis((1H-imidazol-2-yl-)methyl)amine, can be used to chelate many different soft metals and open up many new mass channels for mass cytometry applications based on stable metal isotopes. A non-limiting summary is shown in Table 1 below.

[0182] [Table 1]

[0183] In some embodiments, M is a soft metal. For example, M can be selected from Re, Pt, Pd, Nb, Tc, Hg, Ag, Au, Mo, Ru, Rh, Cd, W, Os, or mixtures thereof.

[0184] In some embodiments, M is non-radioactive. It is believed that the compounds of the present disclosure can be used in radiation detection assays. Thus, when the compounds are used in radiation detection assays, M can be radioactive.

[0185] In some embodiments, M is isotopically enriched, e.g., M does not contain naturally occurring mixtures of isotopes.

[0186] In another aspect, the disclosure includes compounds of formula II as defined herein for use in mass cytometry.

[0187] In another aspect, the present disclosure includes an element tag comprising a linear or branched polymer comprising a plurality of chelating groups, at least one of the chelating groups being chelated to a soft metal atom of a soft metal, the soft metal being monoisotopic.

[0188] In some embodiments, the element tag is a compound of the present disclosure.

[0189] In another aspect, the present disclosure provides a method for the preparation of a an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; and An element tag comprising a linear or branched polymer containing multiple chelating groups containing two nitrogen-containing five- or six-membered heterocycles, each chelating group containing or capable of binding at least one soft metal atom of an isotopic composition. The kit includes:

[0190] In some embodiments, the kit does not include any radioactive soft metals.

[0191] In some embodiments, the isotopic composition does not include naturally occurring mixtures of isotopes.

[0192] It is contemplated that elemental tags can be functionalized to bind biomolecules, for example, elemental tags can be covalently attached to biomolecules.

[0193] In some embodiments, the kit further comprises a biomolecule.

[0194] For example, the biomolecule can be an oligonucleotide. For example, the biomolecule can be an antibody or other affinity reagent.

[0195] In some cases, each chelating group contains at least one soft metal atom of isotopic composition.

[0196] In some embodiments, the isotopic composition is a soft metal solution provided separately from the elemental tags, and each chelating group is capable of binding at least one soft metal atom of the isotopic composition.

[0197] In some embodiments, the kit further comprises an additional isotopic composition, for example, the additional isotopic composition comprises a plurality of additional soft metal atoms of an additional single isotope of the soft metal that is different from the single isotope of the soft metal of the isotopic composition.

[0198] In some embodiments, the kit further comprises an additional element tag comprising an additional linear or branched polymer that comprises a plurality of additional chelating groups.

[0199] In some embodiments, each chelating group of a linear or branched polymer of an elemental tag comprises at least one soft metal atom of an isotopic composition, and each additional chelating group of an additional linear or branched polymer of an additional elemental tag comprises at least one additional soft metal atom of an additional isotopic composition.

[0200] In some embodiments, each element tag is covalently attached to a different antibody.

[0201] In one embodiment, each chelating group is capable of binding at least one soft metal atom of the isotopic composition, each chelating group is selected from dipicolylamine or bis((1H-imidazol-2-yl)methyl)amine, and each imidazole is optionally substituted with one or more polar functional groups selected from C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof.

[0202] In some embodiments, the kit further comprises a reagent for covalent attachment of the element tag to the antibody.

[0203] In some embodiments, each element tag is independently a compound of formula I described herein or a compound of formula II described herein.

[0204] The kits described in the above embodiments may have any of the additional aspects described herein, such as element tags that include one or more solubility modifiers (e.g., on the same pendant group as the chelating group).

[0205] III. Methods and Uses In another aspect, the present disclosure provides: providing an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; providing an element tag comprising a linear or branched polymer comprising a plurality of chelating groups, each of which independently comprises two nitrogen-containing five- or six-membered heterocycles, each of which is capable of binding at least one soft metal atom of the isotopic composition; and Binding the soft metal atom of the isotopic composition to one or more chelating groups of the elemental tag. The present invention includes a method comprising the steps of:

[0206] In some embodiments, the soft metal atoms are non-radioactive.

[0207] In some embodiments, the isotopic composition does not include naturally occurring mixtures of isotopes.

[0208] In some embodiments, the method may further include providing an additional isotopic composition comprising a plurality of additional soft metal atoms of an additional single isotope of the non-radioactive soft metal that is different from the single isotope of the non-radioactive soft metal of the isotopic composition.

[0209] For example, the method further comprises providing an additional element tag comprising an additional linear or branched polymer that includes a plurality of chelating groups.

[0210] In some embodiments, each chelating group of the linear or branched polymer of the elemental tag comprises at least one soft metal atom of an isotopic composition, and each additional chelating group of the additional linear or branched polymer of the additional elemental tag comprises at least one additional soft metal atom of an additional isotopic composition.

[0211] In some embodiments, the method further comprises: Providing a biomolecule; and Covalently linking a biomolecule to an elemental tag Includes.

[0212] In another aspect, the present disclosure provides a method for analysis of an analyte in a biological sample, comprising: (i) incubating an elementally tagged affinity reagent with the analyte, the elementally tagged affinity reagent comprising an affinity reagent tagged with an elemental tag, the elemental tag comprising a linear or branched polymer having a plurality of chelating groups each independently comprising two nitrogen-containing five- or six-membered heterocycles, the elemental tag further comprising a plurality of soft metal atoms of a single isotope of the soft metal; (where: Each chelating group of the element tag contains or is capable of binding at least one soft metal atom; Affinity reagents specifically bind analytes. (ii) separating unbound element-tagged affinity reagents from bound element-tagged affinity reagents; and (iii) analyzing the elemental tags attached to the affinity reagents that are bound to the analyte by mass spectrometry atomic spectrometry. The method includes the steps of:

[0213] In some embodiments, the soft metal atoms are non-radioactive.

[0214] In some embodiments, the soft metal does not contain a naturally occurring mixture of isotopes.

[0215] In some embodiments, the step of incubating the element-tagged affinity reagent with the analyte comprises: The method includes incubating two or more differential elementally tagged affinity reagents with two or more analytes, where the elementally tagged affinity reagents specifically bind to the two or more analytes to generate two or more differentially tagged analytes, and analyzing the elemental tags bound to the affinity reagents includes analyzing the differential elemental tags bound to the two or more analytes by mass spectrometry atomic spectrometry.

[0216] In some cases, the affinity reagent is further labeled with a fluorescent label.

[0217] In some embodiments, the mass spectrometric atomic spectroscopy is ICP-MS. In one embodiment, the mass spectrometric atomic spectroscopy is by a mass spectrometer-based flow cytometer.

[0218] In some embodiments, the affinity reagent is an antibody.

[0219] In some embodiments, the affinity reagent specifically binds biotin.

[0220] In some embodiments, the affinity reagent is an oligonucleotide.

[0221] In some embodiments, the element-tagged affinity reagent is configured to bind to an analyte in a biological sample, the biological sample comprising a cell. In some embodiments, the element-tagged affinity reagent is configured to bind to an analyte in a biological sample, the soft metal is an element that does not naturally occur in the biological sample.

[0222] In some embodiments, the soft metal is selected from Re, Pt, Pd, Nb, Tc, Hg, Ag, Au, Mo, Ru, Rh, Cd, W, Os, or mixtures thereof.

[0223] In some embodiments, the element tag is a compound of formula I, as described herein, or a compound of formula II, as described herein.

[0224] The methods of the above embodiments may have any of the additional aspects described herein, such as element tags that include one or more solubility modifiers (e.g., on the same pendant group as the chelating group).

[0225] The present disclosure also provides the following embodiments:

[0226] Embodiment 1. A compound of formula I

[0227] [ka]

[0228] (In the formula, A is a polymer backbone, and optionally the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof; each B is independently a nitrogen-containing 5- to 7-membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof; and L is absent or a linker; Each L 2 is independently absent or a linker; Each R 2 is a first modifying group independently selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof; X is a functional group selected from esters, ethers and amides; Each L 1 is independently absent or a linker; Each R 1 is independently H, C1-C8 alkyl, C2-C8 alkenyl, C3-C8 cycloalkyl, OH, C1-C10 alkoxy, C1-C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof; n is an integer from 0 to 7; m is an integer from 0 to 4; p is an integer from 0 to 3; and and q is an integer greater than 0.

[0229] Embodiment 2. The compound of embodiment 1, wherein each B is independently a nitrogen-containing 5- or 6-membered heteroaryl, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof, and optionally one or more B is coordinated to a soft metal and / or conjugated to one or more biomolecules.

[0230] Embodiment 3. The compound of embodiment 1 or 2, wherein each B is independently pyridine or imidazole, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof, and optionally one or more B is coordinated to a soft metal and / or conjugated to one or more biomolecules.

[0231] Embodiment 4. The compound of any one of embodiments 1 to 3, wherein one or more B is coordinated to a soft metal.

[0232] Embodiment 5. R 1 or R 2 The compound of any one of embodiments 1 to 4, wherein is a biomolecule, and optionally the biomolecule is an affinity reagent such as an antibody.

[0233] Embodiment 6 The compound of any one of embodiments 1 to 5, wherein X is an amide.

[0234] Embodiment 7. X is -C(O)NR 4 -OR-NR 4 C(O)- and R 4 is H or C1-C4 alkyl.

[0235] Embodiment 8. X is -C(O)NR 4 and the compound has formula Ia

[0236] [ka]

[0237] The compound of any one of embodiments 1 to 7, having the structure:

[0238] Embodiment 9. X is -NR 4 C(O)-, and the compound has the formula Ib

[0239] [ka]

[0240] The compound of any one of embodiments 1 to 7, having the structure:

[0241] Embodiment 10. X is -C(O)NR 4 and the compound has the formula Ic

[0242] [ka]

[0243] (In the formula, each R 3 are independently selected from H, C1-C5 alkyl, C2-C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkyl phosphonate, alkyl ether, or polyether. The compound of any one of embodiments 1 to 7, having the structure:

[0244] Embodiment 11. The compound has formula Id or formula Ie

[0245] [ka]

[0246] The compound of embodiment 10, having the structure:

[0247] Embodiment 12. X is -NR 4 C(O)-, and the compound has the formula If

[0248] [ka]

[0249] (In the formula, each R 3are independently selected from H, C1-C5 alkyl, C2-C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkyl phosphonate, alkyl ether, or polyether. The compound of any one of embodiments 1 to 7, having the structure:

[0250] Embodiment 13. The compound has Formula Ig or Formula Ih.

[0251] [ka]

[0252] 13. The compound of embodiment 12, having the structure:

[0253] Embodiment 14. Each R 3 H, -(CH2) 1~3 COOH, -(CH2) 1~3 O(CH2) 1~2 CH3, -(CH2) 2~4 OH, -(CH2) 2~5 The compound of any one of embodiments 10 to 13, independently selected from P(O)(OCH2CH3)2, or -CH2CH(OMe)2.

[0254] Embodiment 15. The compound of any one of embodiments 1 to 14, wherein n is 2, 3, 4, or 5.

[0255] Embodiment 16. The compound of any one of embodiments 1 to 15, wherein m is 0, 1, or 2.

[0256] Embodiment 17. The compound of any one of embodiments 1 to 16, wherein p is 1 or 2.

[0257] Embodiment 18. The compound of any one of embodiments 1 to 17, wherein A is selected from polyacrylates, polyacrylamides, polyethers, polyamino acids, polyvinylamines, poly(2-oxazolines), polyethylene glycols, polysaccharides, dendrimers, copolymers thereof, or combinations thereof.

[0258] Embodiment 19 The compound of embodiment 18, wherein A is a polyamino acid.

[0259] Embodiment 20. The compound of embodiment 18 or 19, wherein the polyamino acid is polyglutamic acid, polyaspartic acid, polylysine, poly(2,4-dimethylaminobutyric acid) (polyDab), poly(2,4-diaminopimelic acid) (polyDap), derivatives thereof, or combinations thereof.

[0260] Embodiment 21. Each linker independently comprises or is independently selected from a C-C alkylamine, a C-C alkylene, a C-C cycloalkyl, a C-C heterocycloalkyl, a 5- or 6-membered aryl or heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, C(O), C(O)O, an amide, an amine, a thioether, a maleimide-thiol conjugate, a polyethylene glycol (PEG), or a mixture thereof, and optionally includes an amine, an alkylene, an aryl, an alkyl, a cycloalkyl ... The compound of any one of embodiments 1 to 20, wherein each of the aryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.

[0261] Embodiment 22. Each L 2 22. The compound of any one of embodiments 1 to 21, wherein: independently comprises or is independently selected from a C-C alkylene, a C-C alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, and optionally wherein each of the alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from a C-C alkyl, a C-C alkenyl, a C-C cycloalkyl, a C-C heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, CN, or a mixture thereof.

[0262] Embodiment 23. Each L 1 23. The compound of any one of embodiments 1 to 22, wherein: independently comprises or is independently selected from a C-C alkylene, a C-C alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, and optionally wherein each of the alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from a C-C alkyl, a C-C alkenyl, a C-C cycloalkyl, a C-C heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C-C cycloalkylaryl, a C-C cycloalkylheteroaryl, CN, or a mixture thereof.

[0263] Embodiment 24. The compound of any one of embodiments 1 to 23, wherein L is absent or a C3-C8 alkylamine.

[0264] Embodiment 25. Each R 225. The compound of any one of embodiments 1 to 24, wherein the solubility modifier of the first modifying group and the solubility modifier of the second modifying group each independently comprise a polyethylene glycol (PEG), a sugar, an oligosaccharide, or a zwitterionic polymer such as poly(carboxylbetaine) methacrylate or poly(sulfobetaine) methacrylate (PBSMA).

[0265] Embodiment 26 The compound of any one of embodiments 1 to 25, wherein the reactive functional group is for binding to one or more biomolecules.

[0266] Embodiment 27. Each R 2 27. The compound of any one of embodiments 1 to 26, wherein the reactive functional group of the first modifying group and the reactive functional group of the second modifying group are each independently selected from a carboxylic acid, a maleimide, a thiol, an azide, a dibenzocyclooctyne (DBCO), a trans-cyclooctene (TCO), a tetrazine, a furan, or an aldehyde.

[0267] Embodiment 28. The compound of any one of embodiments 1 to 27, wherein the one or more biomolecules are each independently selected from a small molecule, a polypeptide, an oligonucleotide, a lipid, a carbohydrate, or a mixture thereof.

[0268] Embodiment 29. The compound of embodiment 28, wherein the one or more biomolecules are each independently an affinity reagent, and optionally the affinity reagent is an antibody.

[0269] Embodiment 30. The compound comprises:

[0270] [ka] TIFF2024525625000056.tif242159TIFF2024525625000057.tif66159

[0271] (wherein r is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3 are each as defined in any one of embodiments 10 to 14. The compound of embodiment 1, selected from:

[0272] Embodiment 31. The compound comprises:

[0273] [ka] TIFF2024525625000059.tif187159TIFF2024525625000060.tif178159

[0274] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 200, about 6 to about 30, or about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3 are each as defined in any one of embodiments 10 to 14. The compound of embodiment 1, selected from:

[0275] Embodiment 32. The compound comprises:

[0276] [ka] TIFF2024525625000062.tif200159TIFF2024525625000063.tif183159TIFF2024525625000064.tif78159

[0277] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 200, about 6 to about 30, or about 10 to about 25; R 1 , R 2 , L 1 , L 2 and R 3 are each as defined in any one of embodiments 10 to 14. The compound of embodiment 1, selected from:

[0278] Embodiment 33. The compound comprises:

[0279] [ka] TIFF2024525625000066.tif184159TIFF2024525625000067.tif227159TIFF2024525625000068.tif224159 TIFF2024525625000069.tif212159TIFF2024525625000070.tif176159TIFF2024525625000071.tif118159

[0280] (In the formula, s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30; r is about 3 to about 200, about 6 to about 30, or about 10 to about 25; R 3 is as defined in any one of embodiments 10 to 14. The compound of embodiment 1, selected from:

[0281] Embodiment 34. A compound of formula I as defined in any one of embodiments 1 to 33, chelated to one or more metals M and having formula II

[0282] [ka]

[0283] A compound of formula I having the structure: or a derivative or salt thereof.

[0284] Embodiment 35 The compound of embodiment 34, wherein M is a soft metal.

[0285] Embodiment 36. The compound of embodiment 34 or 35, wherein M is selected from Re, Pt, Pd, Nb, Tc, Hg, Ag, Au, Mo, Ru, Rh, Cd, W, Os, or mixtures thereof.

[0286] Embodiment 37 The compound of any one of embodiments 34 to 36, or the composition of any one of embodiments 35 to 37, wherein M is non-radioactive.

[0287] Embodiment 38 The compound of any one of embodiments 34 to 37, or the composition of any one of embodiments 35 to 38, wherein M is isotopically enriched.

[0288] Embodiment 39. A composition comprising one or more compounds of formula I, each independently as defined in any one of embodiments 1 to 33, or one or more compounds of formula II, each independently as defined in any one of embodiments 34 to 38, and a solvent.

[0289] Embodiment 40. A compound of formula I as defined in any one of embodiments 1 to 33 or a compound of formula II as defined in any one of embodiments 34 to 38 for use in mass cytometry.

[0290] Embodiment 41. An element tag comprising a linear or branched polymer comprising a plurality of chelating groups, each of which is capable of binding a soft metal, the soft metal being monoisotopic, and at least one of the chelating groups being chelated to a soft metal atom of the soft metal.

[0291] Embodiment 42. A kit comprising: an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; and an element tag comprising a linear or branched polymer comprising a plurality of chelating groups comprising two nitrogen-containing five- or six-membered heterocycles, each chelating group of the element tag comprising or capable of binding at least one soft metal atom of the isotopic composition; Includes; In some cases, it does not contain any radioactive soft metals, kit.

[0292] Embodiment 43 The kit of embodiment 42, wherein the isotopic composition does not include naturally occurring mixtures of isotopes.

[0293] Embodiment 44 The kit of embodiment 42 or 43, wherein the element tag is functionalized to bind a biomolecule.

[0294] Embodiment 45 The kit of embodiment 42 or 43, wherein the element tag is covalently attached to the biomolecule.

[0295] Embodiment 46 The kit of any one of embodiments 42 to 44, further comprising a biomolecule.

[0296] Embodiment 47 The kit of any one of embodiments 43 to 46, wherein the biomolecule is an oligonucleotide.

[0297] Embodiment 48 The kit of any one of embodiments 43 to 46, wherein the biological molecule is an antibody.

[0298] Embodiment 49. The kit of any one of embodiments 42 to 48, wherein each chelating group comprises at least one soft metal atom of isotopic composition.

[0299] Embodiment 50. The kit of any one of embodiments 42 to 48, wherein the isotopic composition is a soft metal solution provided separately from the elemental tags, and each chelating group is capable of binding at least one soft metal atom of the isotopic composition.

[0300] Embodiment 51. The kit of any one of embodiments 42 to 50, further comprising an additional isotopic composition, the additional isotopic composition comprising a plurality of additional soft metal atoms of an additional single isotope of the soft metal that is different from the single isotope of the soft metal of the isotopic composition.

[0301] Embodiment 52. The kit of embodiment 51, further comprising an additional element tag comprising an additional linear or branched polymer comprising a plurality of additional chelating groups.

[0302] Embodiment 53. The kit of embodiment 52, wherein each chelating group of the linear or branched polymer of the elemental tag comprises at least one soft metal atom of an isotopic composition, and each additional chelating group of the additional linear or branched polymer of the additional elemental tag comprises at least one additional soft metal atom of an additional isotopic composition.

[0303] Embodiment 54 The kit of any one of embodiments 42 to 53, wherein each element tag is covalently attached to a different antibody.

[0304] Embodiment 55. The kit of any one of embodiments 42 to 54, wherein each chelating group is capable of binding at least one soft metal atom of the isotopic composition, each chelating group is selected from dipicolylamine or bis((1H-imidazol-2-yl)methyl)amine, and each imidazole is optionally substituted with one or more polar functional groups selected from C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or combinations thereof.

[0305] Embodiment 56 The kit of any one of embodiments 42 to 55, further comprising a reagent for covalently binding the element tag to the antibody.

[0306] Embodiment 57. The kit of any one of embodiments 42 to 56, wherein each element tag is independently a compound of formula I as defined in any one of embodiments 1 to 33 or a compound of formula II as defined in any one of embodiments 34 to 38.

[0307] Embodiment 58. A method comprising: providing an isotopic composition comprising a plurality of soft metal atoms of a single isotope of the soft metal; providing an element tag comprising a linear or branched polymer comprising a plurality of chelating groups each independently comprising two nitrogen-containing five- or six-membered heterocycles, each chelating group being capable of binding at least one soft metal atom of the isotopic composition; and Binding the soft metal atom of the isotopic composition to one or more chelating groups of the elemental tag. Includes; The method wherein the soft metal atoms are non-radioactive.

[0308] Embodiment 59. The method of embodiment 58, wherein the isotopic composition does not include naturally occurring mixtures of isotopes.

[0309] Embodiment 60. The method of embodiment 59, further comprising providing an additional isotopic composition, the additional isotopic composition comprising a number of additional soft metal atoms of an additional single isotope of the non-radioactive soft metal that is different from the single isotope of the non-radioactive soft metal of the isotopic composition.

[0310] Embodiment 61. The method of any one of embodiments 58 to 60, further comprising the step of providing an additional element tag comprising an additional linear or branched polymer comprising a plurality of chelating groups.

[0311] Embodiment 62. The method of any one of embodiments 58 to 61, wherein each chelating group of the linear or branched polymer of the elemental tag comprises at least one soft metal atom of an isotopic composition, and each additional chelating group of the additional linear or branched polymer of the additional elemental tag comprises at least one additional soft metal atom of an additional isotopic composition.

[0312] 63. In addition: Providing a biomolecule; and Covalently linking a biomolecule to an elemental tag 63. The method of any one of embodiments 58 to 62, comprising:

[0313] Embodiment 64. A method for the analysis of an analyte in a biological sample, comprising: (i) incubating an elementally tagged affinity reagent with the analyte, the elementally tagged affinity reagent comprising an affinity reagent tagged with an elemental tag, the elemental tag comprising a linear or branched polymer having a plurality of chelating groups each independently comprising two nitrogen-containing five- or six-membered heterocycles, the elemental tag further comprising a plurality of soft metal atoms of a single isotope of the soft metal; (where: Each chelating group of the element tag contains or is capable of binding at least one soft metal atom; The soft metal atoms are non-radioactive, and Affinity reagents specifically bind analytes. (ii) separating unbound element-tagged affinity reagents from bound element-tagged affinity reagents; and (iii) analyzing the elemental tags attached to the affinity reagents that are bound to the analyte by mass spectrometry atomic spectrometry. A method comprising:

[0314] Embodiment 65. The method of embodiment 64, wherein the soft metal does not contain naturally occurring mixtures of isotopes.

[0315] Embodiment 66. The step of incubating an element-tagged affinity reagent with a sample comprises: the step of incubating two or more differential elementally tagged affinity reagents with two or more analytes, where the elementally tagged affinity reagents specifically bind to the two or more analytes to produce two or more differentially tagged analytes, and the step of analyzing the elemental tags bound to the affinity reagents includes analyzing the differential elemental tags bound to the two or more analytes by mass spectrometry atomic spectrometry; The method of embodiment 64 or 65.

[0316] Embodiment 67 The method of any one of embodiments 64 to 66, wherein the affinity reagent is further labeled with a fluorescent label.

[0317] Embodiment 68 The method of any one of embodiments 64 to 67, wherein the mass spectrometric atomic spectroscopy is ICP-MS.

[0318] Embodiment 69. The method of any one of embodiments 64 to 67, wherein the mass spectrometric atomic spectroscopy is by a mass spectrometer-based flow cytometer.

[0319] Embodiment 70 The method of any one of embodiments 64 to 69, wherein the affinity reagent is an antibody.

[0320] Embodiment 71 The method of any one of embodiments 64 to 70, wherein the affinity reagent specifically binds biotin.

[0321] Embodiment 72 The method of any one of embodiments 64 to 69, wherein the affinity reagent is an oligonucleotide.

[0322] Embodiment 73. The method of any one of embodiments 64 to 72, wherein the element-tagged affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells.

[0323] Embodiment 74. The method of any one of embodiments 64 to 73, wherein the soft metal is selected from Re, Pt, Pd, Nb, Tc, Hg, Ag, Au, Mo, Ru, Rh, Cd, W, Os, or mixtures thereof.

[0324] Embodiment 75 The method of any one of embodiments 64 to 74, wherein the soft metal is an element that is not naturally present in the biological sample.

[0325] Embodiment 76 The method of any one of embodiments 54 to 75, wherein the element tag is or comprises a compound of formula I, as defined in any one of embodiments 1 to 33, or a compound of formula II, as defined in embodiment 34.

[0326] The above disclosure generally describes the present disclosure. A more complete understanding may be obtained by reference to the following specific examples. These examples are set forth merely for illustrative purposes and are not intended to limit the scope of the present application. Changes in form and substitution of equivalents are contemplated where circumstances may suggest or provide expedience. Although certain terms have been used herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0327] Working Example While the present disclosure has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the disclosure is not limited to the disclosed embodiments. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0328] Example 1 Preparation of chelating polymer compound I-1 Polymers have been synthesized that have dipicolylamine (DPA) chelators attached to pendant groups capable of chelating rhenium, which pendant groups increase the number of mass channels useful for mass cytometry. The synthesis of the metal-chelator polymers and the materials used are described.

[0329] A representative chelating polymer compound I-1 of the present disclosure was prepared according to Scheme 2. A representative activated ester polymer 2-1 was reacted with a representative Lys-DPA chelator 1-4. The resulting polymer 2-2 was then conjugated to a modifying group including PEG and maleimide to obtain compound I-1. The chelator 1-4 was synthesized according to Scheme 1. It can be understood that other compounds of the present disclosure can be produced using similar methods, techniques and principles, with appropriate modifications, as described below.

[0330] material Triethylamine (TEA, catalog no. 471283), acryloyl chloride (catalog no. 549797), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT, catalog no. 723010), 2,2'-azobis(2-methylpropionitrile) (AIBN, catalog no. 44109), Nε-Boc-L-lysine (catalog no. 359661), sodium triacetoxyborohydride (STAB, catalog no. 316393), 2-pyridinecarboxaldehyde (catalog no. P62003), HCl (4 M in dioxane, catalog no. 345547), tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP, catalog no. 646547) were obtained from Sigma-Aldrich. Pentafluorophenol was purchased from Matrix Scientific (catalog number 006058). mPEG6-NH2 (catalog number 281204) was obtained from ChemPep. Bis-Mal-PEG6 (catalog number BP-22152) was obtained from Broadpharm. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, catalog number D461245) was purchased from Toronto Research Chemicals. All organic solvents (anhydrous) were obtained from commercial sources and used without further purification.

[0331] Synthesis of lysine-based rhenium chelators. As shown in Scheme 1, (i) direct reductive alkylation of a Boc-protected lysine precursor 20 The chelators 1-4 were prepared in three steps by (ii) Boc deprotection with hydrochloric acid (HCl in dioxane), followed by (iii) conversion of the amine hydrochloride to the free base using NaOH. The reaction intermediates and resulting products were 1 They were characterized by 1 H-NMR, which confirmed their structures (Figure 6).

[0332] [ka]

[0333] To a mixture of Nε-Boc-L-lysine 1-1 (2 g) and STAB (4.5 g) in dichloroethane (45 mL) was added 2-pyridinecarboxaldehyde (1.85 g, dissolved in 5 mL of dichloroethane) at 0 °C under nitrogen. The suspension was stirred at room temperature for about 5 h and became homogeneous bright yellow. The reaction mixture was decomposed with deionized water (30 mL) and diluted with dichloroethane (100 mL). The separated organic phase was washed with deionized water (30 mL × 4), dried over Na2SO4, and concentrated by rotary evaporation to give tert-butyloxycarbonyl (Boc)-protected lysine-based chelator 1-2 (2.5 g). 1 H NMR(600MHz,CDCl3)δ8.53(ddd,J=5.1,1.8,0.9Hz,2H),7.65(td,J=7.7,1.8Hz,2H),7.32(dt,J=7.9,1.1Hz,2H),7.20(ddd,J=7.6,5.0,1.2Hz,2H),4. 70(s,1H),4.10(d,J=3.8Hz,4H),3.47(dd,J=7.8,6.5Hz,1H),3.16~3.07(m ,2H),2.04~1.92(m,1H),1.88~1.72(m,1H),1.52~1.45(m,4H),1.43(s,9H).

[0334] To remove the Boc group, hydrogen chloride solution (4 M in dioxane, 7 mL) was slowly added to a solution of the above compound 1-2 (1.5 g) in DCM (5 mL) at 0° C. The resulting solution was stirred for 24 h at room temperature. A yellow precipitate formed during the reaction. The solvent was discarded and the precipitate was dissolved in methanol (5 mL) and precipitated in diethyl ether (15 mL). The precipitate was collected by centrifugation (2700×g, 10 min). This dissolution-precipitation cycle was repeated once more. Finally, the precipitate was dried in a vacuum oven at room temperate for 24 h to obtain the Boc-deprotected lysine-based DPA chelator as the amine salt 1-3 (1.3 g). 1 H NMR(600MHz,D2O)δ8.68(ddd,J=6.0,1.6,0.7Hz,2H),8.49(td,J=7.9,1.6H z,2H),8.05(dt,J=8.0,1.0Hz,2H),7.92(ddd,J=7.5,5.9,1.3Hz,2H),4.42( q,J=16.5Hz,4H),3.57~3.51(m,1H),2.96(t,J=7.7Hz,2H),1.92(ddd,J=9. 9,8.4,5.8Hz,1H),1.89~1.82(m,1H),1.69~1.61(m,2H),1.52~1.44(m,2H).

[0335] To convert the amine salt to the free base, concentrated NaOH solution (5 M) was slowly added to the solution of the amine salt (0.5 g, dissolved in 2 mL of water) at 0° C. until a pH of about 13 was reached. The basic solution was then lyophilized to give a light brown solid. Dichloromethane (5 mL) was added to dissolve the free base. Undissolved solids were removed by centrifugation (16000×g, 10 min). The supernatant was collected, concentrated, and dried under vacuum at room temperature for 24 h to give the final lysine-based DPA chelator as the free base 1-4 (0.3 g). 1H NMR(500MHz,D2O)δ8.24(ddd,J=5.0,1.8,0.9Hz,2H),7.60(td,J=7.7,1.8Hz,2H),7.37(dt,J=7.9,1.2Hz,2H),7.14(ddd,J=7.6,5.0,1.2Hz, 2H), 3.95(d,J=14.6Hz,2H),3.79(d,J=14.6Hz,2H),3.21(dd,J=8.5,6.3Hz,1H),2.59(t,J=6.8Hz,2H),1.75~1.64(m,2H),1.45~1.26(m,4H).

[0336] Synthesis of pentafluorophenyl acrylate (PFPA) monomer To prepare the activated ester polymer 2-1, pentafluorophenyl acrylate (PFPA) monomer was first synthesized by slowly adding triethylamine (18.3 mL) to a solution of pentafluorophenol (20 g in 130 mL of dichloromethane) under nitrogen at 0° C., followed by the addition of 10.6 mL of acryloyl chloride. The reaction mixture was stirred at 0° C. for 2 h and then at room temperature overnight. The salts were removed by filtration. The solution was concentrated by rotary evaporation and then purified using silica gel column chromatography with hexane as the eluent. 1 H NMR (500MHz, CDCl3) δ6.71(dd,J=17.3,1.0Hz,1H),6.37(dd,J=17.3,10.6Hz,1H),6.17(dd,J=10.5,0.9Hz,1H). 19 F NMR (564MHz, CDCl3) δ-153.17~-153.27(m),-158.74(t,J=21.5Hz),-163.11(td,J=23.0,22.5,5.4Hz).

[0337] RAFT polymerization of PFPA monomer The synthesis of the activated ester polymer 2-1, poly(pentafluorophenyl acrylate) (PPFPA), was achieved via reversible addition-fragmentation chain transfer (RAFT) polymerization. 21,22Polymerization was carried out at 70 °C in 1,4-dioxane (6.0 mL) equipped with a stir bar using 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (DDMAT, 0.28 mmol) as chain transfer agent (CTA) and 2,2'-azobis(2-methylpropionitrile) (AIBN, 0.028 mmol) as thermal initiator. The solution was degassed by three freeze-pump-thaw cycles, after which the flask was sealed and placed in a preheated oil bath (70 °C) for 9 h. After polymerization, the solution was cooled to room temperature with cold water and exposed to air. The polymer was precipitated in excess cold hexane (30 mL). The resulting polymer was dissolved in chloroform (5 mL) and precipitated again in hexane (30 mL). This dissolution-precipitation process was repeated three times. After drying overnight in vacuum at room temperature, the final polymer, poly(PFPA)2-1, was obtained as a yellow powder.

[0338] A monomer-to-CTA-to-initiator molar ratio ([M]:[CTA]:[I]) of 30:1:0.1 was selected, and the molecular weight of the resulting polymer 2-1 was adjusted to an apparent number average molecular weight (M n GPC ) and a relatively narrow molecular weight distribution (D = 1.25) obtained at a conversion of about 73% (Figure 4).

[0339] 1 The degree of polymerization (DP) was determined by comparing the integral of the PPFPA backbone peak (δ = 3.11 ppm) with that of the methyl units for the dodecyl chain ends (δ = 0.88 ppm) using H NMR spectroscopy (Figure 5S2). The corresponding DP of polymer 2-1 was approximately 20. 19 The F NMR spectrum showed three broad peaks with an integration ratio of 2:1:2 at −153.2, −156.8, and −162.3 ppm corresponding to the pentafluorophenyl groups along the polymer backbone (Figure 5).

[0340] Aminolysis of poly(PFPA) with lysine-based rhenium chelators Polymer 2-1 was then treated with a slight excess of lysine-based chelator 1-4 (1.6 equivalents relative to the PFPA repeat unit) at room temperature to give polymer 2-2 (Scheme 2, step a). As shown in Scheme 1 above, chelator 1-4 was prepared in three steps. The aminolysis reaction was carried out as follows: 19 The reaction was monitored by F NMR spectroscopy. Over time, the broad signals corresponding to the PFPA units along the backbone disappeared, while sharp signals corresponding to the released pentafluorophenol appeared (Figure 7). After stirring overnight at room temperature (13 h), 100 μL of ethanolamine was added to the reaction mixture, and the reaction mixture was stirred for 3 h. The polymer was precipitated in diethyl ether (50 mL) and then dissolved in HO. The excess chelating agent was removed using a spin filter (Amicon, Ultra-15, 3 kDa) and washed twice with HO, three times with PBS buffer, and three times with HO. After lyophilization, polymer 2-2 (polyDPA) was obtained as a light brown powder.

[0341] [ka]

[0342] After purification, polymer 2-2 1 H NMR shows a series of resonances corresponding to the incorporation of chelators 1-4, while 19 The F spectrum showed no signal (Figure 8). These NMR studies suggest complete modification of the PFPA units with nearly quantitative incorporation of the chelator 1-4 along the polymer backbone. Furthermore, the UV-visible spectrum of polymer 2-2 in methanol showed negligible absorbance at 309 nm, suggesting cleavage of the trithiocarbonate group during aminolysis (Figure 9). However, a sharp absorbance peak located at 262 nm was observed, corresponding to absorption from the 2-pyridyl group of the chelator.

[0343] The carboxylic acid (COOH) moiety of 1-4 can serve two purposes. First, the carboxylate ion is expected to improve the water solubility of the metal-loaded polymer. Second, the metal-polymer complex can carry a net positive charge. A polymer with a positive charge at each pendant group can interact nonspecifically with cells, which generally have a negatively charged outer membrane. The carboxylate provides a potential counterion, so each pendant group is zwitterionic. The rhenium-loaded polymer 2-2 was found to have low solubility in water or PBS buffer. To further improve the water solubility, polymer 2-2 was modified with a short methoxypolyethylene glycol (mPEG6-NH2) as shown in Scheme 2, step b. The goal here is not only to increase the water solubility of the polymer, but also to provide a PEG corona to protect the positively charged complex from interaction with cells.

[0344] PEGylation of polymer 2-2 with mPEG6-NH2 First, polymer 2-2 (50.3 mg) was treated with an excess of (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) (DMTMM, 288 mg in 1 mL of HO, approximately 8 molar equivalents for each carboxylic acid group) in PB buffer (4 mL, 0.2 M, pH 8.0) (Scheme 2, step b). The reaction mixture was stirred at room temperature for 5 min to activate the carboxylic acid functional groups. Following this, an excess of mPEG6-NH2 (approximately 7 molar equivalents for each carboxylic acid group) was quickly added and the reaction solution was stirred overnight (15 h) at room temperature. Subsequently, a PEGylated version of polymer 2-2, referred to herein as polymer 2-3, was obtained. Polymer 2-3 was purified using a spin filter (Amicon, Ultra-15, 10 kDa) and washed three times with HO, twice with PBS buffer, and three times with HO. After lyophilization, the final polymer, PolyDPA-mPEG62-3, was obtained as a light brown solid.

[0345] Polymer 2-3 1H NMR spectrum confirms PEGylation with the appearance of new peaks at 3.32 ppm and 3.59 ppm, corresponding to the methoxy groups and ethylene glycol repeat units of mPEG6 (Figure 10). Rhenium loading experiments confirmed that the metal-loaded polymer 2-3 was soluble in both water and PBS buffer.

[0346] Conjugation of maleimide functional groups to polymer 2-3 Polymer 2-3 was further modified to incorporate maleimide functional groups (Scheme 2, step c). First, any disulfide bonds that may have formed in the previous step were reduced with tris(2-carboxyethyl)phosphine (TCEP). To a solution of polymer 2-3 (10 mg) in HO (1 mL), TCEP (80 μL, 0.5 M) was added. The concentration of TCEP in the reaction solution was about 50 mM. The reaction mixture was stirred at room temperature for 1 h. Afterwards, the polymer was washed twice with acetic acid solution (about 5 mM, pH 3.5) using a spin filter (Amicon, Ultra-15, 10 kDa) to remove excess TCEP.

[0347] The newly reduced thiol groups were then reacted with excess bismaleimide (Bis-Mal-PEG6) to give the maleimide-functionalized compound I-1. Immediately after this, the concentrated polymer solution (~300 μL) was transferred to a 2-dram glass vial and 0.5 mL of PB buffer (0.2 M, pH 7.0) was added, followed by the addition of Bis-Mal-PEG6 (22 mg in 120 μL of DMF). The reaction solution was stirred at room temperature for 90 min. Compound I-1 was then purified using a spin filter (Amicon, Ultra-15, 10 kDa) and washed twice with HO, once with PB buffer (0.2 M, pH 7.0), and three times with HO. After washing, the concentrated polymer solution was centrifuged at 12000 × g for 10 min to remove undissolved solids. The supernatant was removed and lyophilized to give the final polymer, polyDPA-mPEG6-Mal compound I-1, as a light brown solid.

[0348] Compound I-1 1The H NMR spectrum showed the appearance of a new peak at 6.89 ppm corresponding to maleimide (Figure 1). The ratio of the integral of this peak at 8.27 ppm to the integral of the chelator protons was 1:20, suggesting near quantitative incorporation of the maleimide functionality into the polymer.

[0349] Rhenium loading on compound I-1 Metal loading was achieved with a slight excess of the rhenium salt [NEt4]2[Re(CO)3Br3] 24 This was achieved by incubating compound I-1 (2.1 mg in 2 mL of anhydrous MeOH) in a 2-dram glass vial with rhenium-containing chelators (3 mg, 1.2x for each chelator, dissolved in 0.5 mL of anhydrous MeOH). The reaction mixture was incubated at 37°C for 2 hours without stirring. After incubation, the solution was poured into an Amicon spin filter (Ultra-15, 10 kDa) pre-filled with HO (12 mL). The polymer was washed three times with HO. After washing, the polymer solution was lyophilized to obtain the rhenium-loaded polymer.

[0350] Figure 2(a) shows the reaction of Re-loaded compound I-1 (compound II-1) in the aromatic region. 1 The figure represents a portion of the H NMR spectrum. Compared to the unsupported polymer, all pyridyl proton signals were shifted downfield due to the electron-withdrawing inductive effect of Re(I). Importantly, the maleimide groups remained under these reaction conditions (Figure 11). The successful loading was further confirmed by Fourier transform near infrared (FTIR) spectroscopy, as shown in Figure 2(b). The rhenium salt was detected at 1848 and 1998 cm -1 Compound I-1 showed two strong carbonyl absorptions at 1650 and 1095 cm, which correspond to the C=O stretching of the amide group and the vibration of the ether C-O-C bond in PEG, respectively. -1 It showed an absorption band. 25 After loading, two new absorption bands corresponding to carbonyl groups were observed at 1908 and 2030 cm -1 and fac-[Re(CO)3] +The rhenium-loaded polymers can be lyophilized for long-term storage and redissolved in buffer prior to the bioconjugation reaction (lyophilized sample shown in FIG. 12).

[0351] Example 2 The Re-bearing polymer compound II-1 was labeled with a primary antibody and used in a mass cytometry immunoassay.

[0352] Antibody labeling of Re-bearing polymers To evaluate the performance of representative element-tagged compound II-1 in mass cytometry immunoassays, the primary antibody, CD20, was labeled with a polymer tag following standard Maxpar™ antibody labeling protocols. Briefly, the antibody was partially reduced with TCEP, washed in a spin filter, and then mixed with excess polymer, and the mixture was incubated at 37° C. for 1 hour. The antibody-polymer conjugate was purified by fast protein liquid chromatography to remove excess unconjugated polymer ( FIG. 13 ).

[0353] Antibody titer measurement experiment Titering experiments were then performed in human peripheral blood mononuclear cells (PBMCs) to evaluate the performance of the purified conjugates. 154 Sm-CD45, 160 Gd-CD14, 170 Er-CD3 and 187 / 185 Human PBMCs were stained with a 4-plex antibody panel (Fludig Maxpar™ Reagents) including Re-CD20. 187 / 185 Re-CD20 147 An alternative staining panel substituting Sm-CD20 was used as a positive control. 187 / 185 The Re-CD20 conjugate was titrated at concentrations of 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL and 2.5 μg / mL. As shown in FIG. 187 / 185 Re-CD20 is the CD20 marker from the remaining cell subsets in PBMCs. + This allows for clear separation of B cell subsets. 187 / 185Re-CD20 (Figure 3(b)) or at optimal titers 147 By using either rhenium-tagged antibodies (Figure 3(f)), we achieved highly comparable proportions of the major cell subsets within PBMCs. These results suggest that rhenium-tagged antibodies provide accurate quantification for single-cell immunophenotyping experiments and can be used in combination with commercially available reagents for mass cytometry immunoassays.

[0354] Example 3 A chelating agent having one or more platinum atoms chelated with DPA Polymers were synthesized with dipicolylamine (DPA) chelators attached to pendant groups (the pendant groups can chelate platinum, useful for mass cytometry). The synthesis of representative metal-chelator polymers with platinum and materials used are described.

[0355] material Polymer 3-2 was prepared (Scheme 3) using polymer 2-2 in the same manner as in Example 1. Azido-PEG6-NH2 (catalog number 76172) and potassium tetrachloroplatinate (K2PtCl4, catalog number 520853) were obtained from Sigma-Aldrich.

[0356] PEGylation of polymer 2 using azide-PEG6-NH2 The resulting polymer 3-2 was characterized using 1H-NMR (Figure 14). FTIR further confirms the successful incorporation of the azide group (Figure 15). To a solution of end-capped polymer 3-1 (20 mg in 2 mL of PB buffer, 0.2 M, pH 8.0), a solution of DMTMM (124 mg in 0.5 mL of H2O, approximately 8 times for each COOH) was added. The solution was stirred at room temperature for 5 min to activate the COOH groups. After 5 min, a mixture of PEG containing mPEG6-NH2 (54 mg, 3.5 times equivalent for each COOH) and azide-PEG6-NH2 (64 mg, 3.5 times equivalent for each COOH) was added. The reaction mixture was stirred at room temperature overnight. The resulting polymer 3-2 was purified using a spin filter (Amicon, Ultra-5, 10 kDa) and washed three times with HO, two times with PB buffer (0.2 M, pH 7.6), and three times with HO. The polymer solution was then lyophilized overnight to obtain the final product 3-2.

[0357] [ka]

[0358] Platinum Loading onto Polymer 3-2 Platinum loading onto polymer 3-2 was carried out according to scheme 4). 1 H-NMR was used to confirm the successful chelation of Pt (Figure 16). To a solution of polymer 3-2 (5.8 mg in 5.5 mL anhydrous MeOH) in a 2-dram (~7 mL) glass vial, a solution of K2PtCl4 (200 μL, 50 mM in DMSO, 1.2 equivalents for each chelator) was added. The vial was wrapped in aluminum foil to avoid exposure to daylight. The reaction mixture was incubated at 45 °C in an oil bath for 2 h without stirring. After incubation, the solution was poured into an Amicon spin filter (Ultra-15, 10 kDa) pre-filled with H2O (10 mL). The resulting solution of polymer 4-1 (compound II-2) was washed three times with NaCl solution (20 mM) and once with H2O. After washing, the polymer solution was lyophilized to obtain Pt-loaded polymer 4-1.

[0359] [ka]

[0360] Example 4 The Pt-loaded polymer was labeled with a primary antibody and used in a mass cytometry immunoassay.

[0361] Antibody labeling of Pt-loaded polymers To evaluate the performance of polymer element tag 4-1 / II-2 in mass cytometry immunoassay, the primary antibody, CD20, was labeled with polymer element tag 4-1 / II-2. To a solution of dibenzocyclooctyne (DBCO) modified with CD20 Ab (160 μg, 1.6 mg / mL), a solution of Pt-loaded polymer 4-1 was added (0.27 mg in 50 μL of H2O, approximately 13-fold molar excess over Ab). The reaction mixture was vortexed for 2 h at room temperature and then overnight at 4 °C. The conjugate was purified by washing five times with PBS using a spin filter (Amicon, Ultra-0.5, 100 kDa).

[0362] Antibody titer measurement experiment Antibody titration experiments were performed as described below. Figure 17 shows that CD20-PolyPt was effective in separating B cells from T cells at titers of either 0.5 μg / mL or 1.0 μg / mL.

[0363] Different Maxpar® MCP-Ab conjugates nat Antibody staining cocktail (70 μL) was prepared by mixing with Pt-Ab conjugate. nat For immunoassays using Pt-CD20, four antibody staining cocktails were prepared. One cocktail contained Maxpar® MCP-Ab conjugate (i.e. 154 Sm-CD45, 160 Gd-CD14, 170 Er-CD3 and 147Sm-CD20) was used as a positive control. The other three cocktails contained only Maxpar® MCP-Ab conjugates (i.e., 154 Sm-CD45, 160 Gd-CD14 and 170 Er-CD3) and nat Each cocktail consisted of both Pt-CD20 conjugates and nat The concentrations of Pt-CD20 conjugates were varied with titers of 0.5 μg / mL, 1.0 μg / mL and 2.5 μg / mL, respectively.

[0364] For the staining process, the PBMC suspension (approximately 3 million cells in 30 μL of Maxpar® cell staining buffer, Fc-blocked) was added to the antibody cocktail (70 μL). The mixture was gently vortexed and incubated at room temperature for 30 min. After incubation, the cells were washed twice with cell staining buffer and then fixed with 1.6% formaldehyde / PBS solution at room temperature for 10 min. The fixed cells were pelleted and cell intercalation solution (Ir-intercalator, 1 mL, final concentration: 125 nM) was added. The cells were then incubated overnight at 4° C. After incubation, the cells were washed twice with cell staining buffer and twice with Maxpar® cell acquisition solution. The pelleted cells were suspended in cell acquisition solution (1 million cells per mL) containing EQ™ quaternary calibration beads and subjected to mass cytometry analysis.

[0365] Uptake of CD20-PolyPt conjugates by mononuclear cells was also observed.

[0366] Example 5 A chelating agent having one or more mercury atoms chelated by DPA Polymers have been synthesized that have dipicolylamine (DPA) chelators attached to pendant groups that can chelate mercury useful for mass cytometry. The synthesis of representative mercury-chelator polymers and materials used are described.

[0367] material PolyDPA polymer (2-3) was prepared as described in Example 1. Mercuric acetate (catalog number 176109) and methanol (catalog number 322415) were purchased from Sigma-Aldrich.

[0368] Hg loading onto polyDPA (polymer 2-3) To a solution of polymer 2-3 (2.5 mg of polymer in 2 mL of methanol) in a 2-dram glass vial, a solution of Hg salt (1.5 mg of Hg(OAc)2 in 0.5 mL of methanol, 1.4-fold excess over DPA chelator) was added, followed by gentle swirling. The resulting solution was incubated at 40° C. for 2 h without stirring (Scheme 5). After incubation, the solution was poured into a spin filter (Amicon Ultra 4, 10 kDa) pre-filled with water (1.5 mL). The resulting solution of polymer 5-1 / II-3 was washed three times with water (2700 g, 20 min) and then lyophilized overnight to obtain the final product. For NMR measurements, polymer 5-1 / II-3 was redissolved in D2O. 1 H-NMR was used to confirm successful mercury chelation (Figures 18 and 19).

[0369] [ka]

[0370] Example 6 A chelating agent having one or more silver atoms chelated with DPA Polymers have been synthesized that have dipicolylamine (DPA) chelators attached to pendant groups that can chelate silver useful for mass cytometry. The synthesis of representative silver-chelator polymers and materials used are described.

[0371] material Polymer 2-3 was prepared as described in Example 1. Silver perchlorate (catalog number 226548) and methanol (catalog number 322415) were purchased from Sigma-Aldrich.

[0372] Ag loading onto polyDPA (polymer 2-3) To a solution of polymer 2-3 (2.5 mg of polymer in 2 mL of methanol) in a 2-dram glass vial, a solution of Ag salt (1.0 mg of AgClO4 in 0.5 mL of methanol, 1.2-fold excess relative to the DPA chelator) was added, followed by gentle swirling. The resulting solution was incubated at 40° C. for 2 h without stirring (Scheme 6). After incubation, the solution was poured into a spin filter (Amicon Ultra 4, 10 kDa) pre-filled with water (1.5 mL). The resulting solution of polymer 6-1 / II-4 was washed three times with water (2700 g, 20 min) and then lyophilized overnight to obtain the final product. For NMR measurements, polymer 6-1 / II-4 was redissolved in D2O. 1 H-NMR was used to confirm successful silver chelation (Figures 18 and 20).

[0373] [ka]

[0374] equipment 1 H NMR and 19 F NMR experiments were performed on an Agilent DD2 500 MHz spectrometer or an Agilent DD2 600 MHz spectrometer.

[0375] UV-visible measurements were performed on an Agilent Cary 300 UV-visible spectrophotometer.

[0376] FT-IR measurements were performed on a PerkinElmer Spectrum Two™ infrared spectrometer with an ATR accessory. All spectra were measured using a 1 cm -1 Resolution: 500-4000 cm -1was recovered within the range.

[0377] GPC measurements were performed on a Waters 515 HPLC equipped with a Viscotek VE3580 refractive index (RI) detector. Tetrahydrofuran (THF) containing 2.5 g / L tetra-n-butylammonium bromide (TBAB) was used as the eluent (35° C., flow rate=0.6 mL / min). The system was calibrated with PMMA standards.

[0378] FPLC experiments were carried out on an AKTA pure 25L system. For purification of polymer-antibody conjugates, a Superdex™ 200 10 / 300GL column was used.

[0379] Mass cytometry experiments were performed on a CyTOF® Helios™ system from Fluidigm Canada (Markham, ON). Data were acquired in FCS3.0 file format and processed by FlowJo software.

[0380] Example 7 Preparation of zwitterionic poly(sulfobetaine methacrylate) PSBMA as a solubility modifier. material 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTA reagent), 2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate (SBMA), 4,4′-azobis(4-cyanovaleric acid) (ACVA), and 2,2,2-trifluoroethanol (TFE) were obtained from Sigma-Aldrich.

[0381] Experimental details In a 10 mL Schlenk flask, add CTA (14.51 mg, 5.19 × 10 -2 mmol, 1.0 equiv.), SBMA (501 mg, 1.78 mmol, 35 equiv.), and ACVA (1.692 mg, 5.13 × 10 -3(mmol, 0.1 equiv.) was dissolved in TFE (2.63 mL). The polymerization solution was subjected to three freeze-pump-thaw cycles. The polymerization solution was heated at 70° C. for 8 h in a preheated oil bath. The polymerization was quenched by freezing the solution via plunging the Schlenk flask into liquid nitrogen. After thawing, the polymerization solution was stored at 4° C. overnight. An aliquot of the crude polymerization solution was 1 H-NMR analysis showed that the monomer conversion was about 70%. Excess solvent was evaporated using a rotary evaporator under vacuum. The crude polymer mixture was redissolved in TFE (about 1 mL), precipitated in methanol (about 13 mL), and centrifuged at 2700 rcf for 5 min; this process was repeated three times. The resulting polymer was then dried in vacuum overnight to obtain the desired polymer. The solid product was clearly separated into pink and white solids.

[0382] [ka]

[0383] PSBMA, such as polymer 7-1, can optionally be coupled to a polymeric chelator, such as compound 2-2, via a diamine linker, for example as shown in Scheme 8. For example, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) or other similar chemicals can be used as the coupling agent.

[0384] [ka]

[0385] Example 8 Preparation of imidazole-based chelating polymer It can be appreciated that imidazole-based chelators can be prepared using methods similar to those used in Example 1 for the preparation of DPA-based chelators. A representative synthesis is shown in Scheme 9. Other examples of lysine-imidazole chelators include those described in Maresca et al., Bioconjugate Chem., 2010, 21, 1032-1042, the contents of which are incorporated herein by reference in their entirety.

[0386] [ka]

[0387] The Nε-protected lysine 9-1 can be functionalized with two imidazole groups at the α-amino group by reductive amination and deprotected to arrive at the lysine-imidazole chelator 9-4. Once obtained, the lysine-imidazole chelator can be incorporated into a polymer scaffold by a procedure similar to that of Scheme 2. A representative method is shown in Scheme 10.

[0388] [ka]

[0389] Activated ester polymer 10-1 can be coupled to a lysine-imidazole chelator such as compound 9-4 to give chelating polymer 10-2 / compound I-7. In some cases, a modifying group such as a solubility regulator (e.g., PEG) can be coupled to give polymer 10-3 / compound I-8. Additionally, a reactive functional group (e.g., maleimide) can be coupled to polymer 10-3 to give polymer 10-4 / compound I-9.

[0390] Example 9 Preparation of DPA Chelator-Containing Polymers Using Zwitterionic Solubility Modifiers Two representative zwitterionic sulfobetaine solubility modifiers were prepared and conjugated to DPA chelator-containing polymers such as compound 12-1.

[0391] 3-((3-aminopropyl)-dimethylammonio)propane-1-sulfonate 11-4 was prepared based on a previously reported synthetic procedure, as shown in Scheme 11. (26)

[0392] [ka]

[0393] Compound 11-2: Di-tert-butyl dicarbonate (15.6 g, 71.5 mmol) was added to a solution of 3-(dimethylamino)-1-propylamine (4.9 g, 6 mL, 47.7 mmol) in 50 mL of 1,4-dioxane. The solution was stirred at 0° C. for 2 h and further stirred at room temperature overnight for 18 h. After 18 h, the solvent was removed in vacuo and 50 mL of MilliQ water was added to the crude product. The product was extracted three times with ethyl acetate (30 mL). The ethyl acetate was dried to give a pale yellow clear oil as compound 11-2. The product was used in the following synthetic step without further purification.

[0394] Compound 11-3: Compound 11-2 (1 eq, 2.5 g, 0.012 mol) was dissolved in 15 mL of anhydrous DMF. 1,3-propane sultone (1.4 eq, 2.113 g, 0.017 mol) was added to the solution of compound 11-2 and stirred at room temperature for 3 days. The crude product was dried in vacuum to remove DMF until a pale yellow viscous oil remained. The viscous oil was washed with diethyl ether (30 mL) followed by another wash with ethyl acetate (30 mL) to remove the remaining 1,3-propane sultone. The oil was lyophilized to give a white solid as compound 11-3.

[0395] Compound 11-4: Compound 11-3 (4 g, 0.012 mol) was dissolved in 50 mL of DCM and cooled to 0° C. to give a slightly cloudy, slightly white solution. After cooling to 0° C., 4M HCl in dioxane (5 mL) was added to the solution and left to stir for 1 h. After 1 h, the solution became clear and a solid white clumpy precipitate formed. The solvent was removed in vacuum, dried, and precipitated with DCM / isopropanol / MeOH (10:5:1 v / v ratio). The precipitated product was a sticky white clump, which was lyophilized. The final product, compound 11-4, was obtained as a white solid and stored in a vacuum sealed bag in the freezer. The elution of compound 11-4 was 1 The H NMR spectrum is shown in Figure 22.

[0396] Compound 12-2 / I-11 As shown in Scheme 12, sulfobetaine 11-4 was then coupled to the DPA-containing polymeric chelator 12-1 to give compound 12-2 / polymer I-11.

[0397] [ka]

[0398] DPA chelator-containing polymer 12-1 was dissolved in 0.4 mL of 0.2 M pH 8 sodium phosphate buffer. DMTMM (28.8 mg, 0.104 mmol, 7.3 equivalents per pendant group) was dissolved in 0.1 mL of MilliQ water and added to the polymer solution and allowed to pre-react for 5 min. After 5 min, sulfobetaine 11-4 (14.58 mg, 0.065 mmol, 5 equivalents per pendant group) was added to the DPA chelator-containing polymer 12-1 solution and the reaction mixture was stirred overnight at room temperature for 20 h. After 20 h, the reaction mixture was purified using a 3k MWCO Amicon Ultra 4 mL spin filter and washed five times with MilliQ water. The retentate was removed and lyophilized to give the product, 12-2. The 1H NMR spectrum of compound 12-2 / I-11 is shown in Figure 23.

[0399] Compound I-12 PBSMA7-1 was prepared as shown in Scheme 7 of Example 7. An ethylenediamine linker was then attached to 7-1 as shown in Scheme 13, and the resulting compound 13-1 was attached to the DPA chelator-containing polymer 12-1 to produce polymer I-12. The 1H NMR spectrum of compound I-12 is shown in Figure 24.

[0400] [ka]

[0401] DPA-chelator-containing polymer, compound 12-1 (Dp=20, M n = 6000 g / mol, 0.0005 mmol, 3 mg) was dissolved in 0.1 mL of 0.2 M, pH 8 sodium phosphate buffer. A solution of DMTMM (7.3 equivalents per pendant group, 20.20 mg, 0.073 mmol) in 0.1 mL of MilliQ water was added to the DPA-chelator-containing polymer solution and allowed to pre-react for 10 min. Compound 13-1 (M n = 5600 g / mol, 112 mg, 0.02 mmol, 2 equivalents per pendant amine) was added to the pre-reacted DPA-chelator-containing polymer, compound 12-1 solution and left stirring overnight at room temperature for approximately 20 h. The crude product was purified using an Amicon Ultra-4 mL 10 kDa MWCO, spin filter twice with MilliQ water, twice with 20 mM NaCl solution, and again three times with MilliQ water, and lyophilized to yield the polymer, compound 13-2 / I-12.

[0402] Alternatively, compound 13-1 can be prepared according to Scheme 13a.

[0403] [ka]

[0404] 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid 13a-1 (500 mg, 1.79 mmol, 1 equiv.), EDC (555.8 mg, 3.58 mmol, 2 equiv.), and NHS (412 mg, 3.58 mmol, 2 equiv.) were dissolved in a minimum amount of acetonitrile and vortexed for 15 min. N-Boc-ethylenediamine 13a-2 (430 mg, 2.685 mmol, 1.5 equiv.) was then added and stirred overnight. After 24 h, the reaction was dried with compressed air and then purified by silica gel flash column chromatography. The solvent system was 100% DCM to 1:1 DCM / EtOAc gradient elution. The purified product was dried in vacuum to give the desired product, N-boc CTA 13a-3, as a red-pink solid.

[0405] N-boc CTA 13a-3 (45 mg, 0.1067 mmol), SBMA monomer 13a-5 (1.4 g, 2.252 mmol), and 4,4'-azobis(4-cyanovaleric acid) initiator 13a-4 (3.038 mg, 0.0108 mmol) were dissolved in 4 mL of 2,2,2-trifluoroethanol and the solution was bubbled with nitrogen gas for 20 min. After the solution was bubbled with nitrogen gas for 20 min, the reaction was stirred at 70 °C for 6 h. After 6 h, the reaction was exposed to air and a small aliquot of the crude product was taken for H NMR, and trifluoroacetic acid (1.7 mL) was added to the remaining crude product and stirred overnight at room temperature to hydrolyze the Boc group. After 21 h, the sample was precipitated with 7:3 diethyl ether / methanol and pelleted by centrifugation at 2.7 k rcf for 15 min. The supernatant was discarded. The pellet was redissolved once with 2,2,2-trifluoroethanol, reprecipitated with 7:3 diethyl ether / methanol, and centrifuged again under the same conditions. This process was repeated twice. The sample was dried in vacuum to give compound 13-1.

[0406] Example 10 Preparation of metal-chelating DPA chelator-containing polymers using various solubility modifiers Three DPA chelator-containing polymers (Scheme 14) were prepared based on the methods described in Examples 3 and 9. Each polymer was metallized with platinum or mercury using K2PtCl4 or HgCl2, respectively. Platinum metallization was carried out in methanol at 45°C for 2 hours. Mercury metallization was carried out in methanol at room temperature for 1 hour. The success of the metallization was assessed by proton NMR by the change in the chemical shift of the pyridyl protons. The NMR spectra of compounds 14-2 / II-7 and 14-3 / II-8 are shown in Figure 21.

[0407] [ka]

[0408] DPA-chelating agent-containing polymer 12-1 (Dp=20, M n Poly(SBMA) 13-1 (M = 6000 g / mol, 0.0005 mmol, 3 mg) was dissolved in 0.1 mL of 0.2 M, pH 8 sodium phosphate buffer. A solution of DMTMM (7.3 equivalents per pendant group, 20.20 mg, 0.073 mmol) in 0.1 mL of MilliQ water was added to the DPA-chelator-containing polymer solution and allowed to pre-react for 10 min. Poly(SBMA) 13-1 (M n = 5600 g / mol, 112 mg, 0.02 mmol, 2 equivalents per pendant amine) was added to the pre-reacted DPA-chelator-containing polymer, compound 12-1 solution and left stirring overnight at room temperature for approximately 20 h. The crude product was purified using an Amicon Ultra-4 mL 10 kDa MWCO, spin filter twice with MilliQ water, twice with 20 mM NaCl solution, and again three times with MilliQ water, and lyophilized to yield the polymer, compound 13-2 / I-12.

[0409] [ka]

[0410] Compound 13-2 / I-12 (11.6 mg) was dissolved in methanol / 2,2,2-trifluoroethanol (1:1 volume ratio). A solution of HgCl2 (2 mg / mL, 7 mM, 360 mL) was added to the compound 13-2 / I-12 solution and stirred at room temperature. A red precipitate formed within 10 min and the reaction was left stirring at room temperature for 1 h. The red precipitate was pelleted by centrifugation at 10k rcf for 10 min. The supernatant was discarded and the pellet was further washed twice with MeOH. The red pellet was lyophilized to give compound 14-6 / II-11. (Methodology adopted from 27)

[0411] Compound 13-2 / I-12 (20 mg) was dissolved in aqueous MeOH (1:1 volume ratio). A solution of K2PtCl4 (50 mM, 360 mL) was added to compound 13-2 / I-12, which was then heated at 45 °C for 2 h, resulting in a yellow solution. The yellow solution was spin-filtered through an Amicon Ultra-15 mL 10k MWCO spin filter, washing twice with MilliQ water, three times with 20 mM NaCl solution, and once with MilliQ water. The sample was then lyophilized to give compound 14-7 / II-12.

[0412] The metallation reaction can also be carried out in 2,2,2-trifluoroethanol and MeOH / 2,2,2-trifluoroethanol as solvent systems.

[0413] Example 11 Mass Cytometry Testing of Rhenium-Containing Antibody Conjugate Polymers Using Zwitterionic Solubility Modifiers Rhenium-chelating polymers of the present disclosure containing zwitterionic solubility modifiers were conjugated to antibodies and assessed for non-specific binding using mass cytometry.

[0414] Antibody Conjugation To evaluate the performance of rhenium-tagged polymers with zwitterionic solubility modifiers in mass cytometry immunoassays, two primary antibodies, CD20 and CD8a, were labeled with polymer tags according to a modified Maxpar™ antibody labeling protocol. Briefly, the antibodies were partially reduced with TCEP, washed with a spin filter (30 kDa), and then mixed with an excess (20-fold) of DBCO-PEG4-maleimide. The mixture was incubated at 37° C. for 30 min. The DBCO-modified Ab was purified using a spin filter (30 kDa) and then mixed with the desired polymer mass tag. The mixture was incubated at 37° C. for 90 min. The excess polymer was removed with a spin filter (100 kDa).

[0415] Mass cytometry titration experiments Antibody titration experiments were then performed in human peripheral blood mononuclear cells (PBMCs) to evaluate the performance of the conjugates. 145 Nd-CD4, 146 Nd-CD8a, 165 Ho-CD16, 154 Sm-CD45, 160 Gd-CD14, 170 Er-CD3 and 147 Human PBMCs were stained with a 7-plex antibody panel (Fludig Maxpar™ Reagents) including Sm-CD20. For titration of rhenium conjugates, 147 Sm-CD20 and 146 Nd-CD8a 187 / 185 Re-CD20 and 187 / 185 A separate staining panel was used in which Re-CD8a was substituted. 187 / 185 The Re-CD20 / CD8a conjugate was titrated at concentrations of 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL and 2.0 μg / mL. As shown in FIG. 187 / 185 Re-CD20 is the CD20 marker from the remaining cell subsets in PBMCs. + Allows for clear separation of B cell subsets. 187 / 185Re-CD8a also allows clear separation of CD8+ T cell subsets from the remaining cell subsets in PBMC at all titers (Figure 26). Importantly, as shown in Figures 27 and 28, both conjugates showed minimal non-specific binding to other cell populations. The rhenium-tagged CD20 conjugate showed minimal non-specific binding to non-T / B cells (Figure 27). The rhenium-tagged CD8a conjugate showed minimal non-specific binding to B cells (Figure 28).

[0416] Comparison with PEG-modified Re-containing polymer To evaluate the non-specific binding properties of the zwitterionic solubility modifiers compared to other solubility modifiers, PEG-modified polymers were synthesized and chelated to Re metal based on the method described in Example 1. For mass cytometry experiments, both polymers were mixed with antibody staining cocktails at various concentrations (1ug / mL, 2ug / mL and 5ug / mL), and the resulting antibody cocktails were used to stain PBMCs according to the regular staining protocol described above. It was observed that the PEG-modified polymers showed non-specific binding to major cell subsets in PBMCs at 1ug / mL. (See Figure 29)

[0417] Polymers of the present disclosure modified with PEG solubility modifiers showed higher non-specific binding to PBMCs compared to polymers modified with zwitterionic solubility modifiers. (See FIG. 29.) Polymers modified with zwitterionic solubility modifiers showed minimal non-specific binding to major subsets of PBMCs at 5 ug / mL. Zwitterionic modified rhenium polymers showed significantly lower non-specific binding to PBMCs than PEG modified rhenium polymers.

[0418] Example 12 Preparation of H-Dap Dipicolylamine Chelator The H-Dap dipicolylamine-based chelator was prepared as shown in Scheme 16.

[0419] [ka]

[0420] H-Dap(Boc)-OMeHCl16-1 (0.5 g, 1.86 mmol, 1 equiv.) was dissolved in about 30 mL of anhydrous acetonitrile and purged with N2(g) while stirring for 30 min. Then, 2-picolyl chloride hydrochloride (2.2 equiv., 671.21 mg, 4.092 mmol), K2CO3 (3.2 equiv., 5.95 mmol, 822.6 mg) were added successively and the reaction was stirred at room temperature for 2 h. After stirring for 2 h, potassium iodide (1 equiv., 679.27 mg, 4.092 mmol) was added and the reaction was heated and stirred at reflux (about 85 °C). The reaction was then left to react overnight (about 18 h). The sample was dried in vacuum to remove acetonitrile. The dried crude product was redissolved in DCM. The DCM organic phase was washed with 3 × 100 mL of water. The organic phase was collected and dried in vacuum to give the product, H-Dap Boc-OMe dipicolylamine 16-2, as a brown solid.

[0421] H-Dap Boc-OMe dipicolylamine 16-2 (185 mg) was dissolved in 40 mL of a 1:1 mixture of MilliQ water and concentrated HCl (to approximately 6 M HCl). The sample was then refluxed at 110° C. overnight (reaction start time: 11:40 am). After 24 hours, the reaction mixture was dried in vacuum. The sample was redissolved in approximately 20 mL of MilliQ water and basified with 1 M NaOH to neutralize the acid (checked with pH paper). The sample was lyophilized overnight and then redissolved in DCM. The DCM phase was then centrifuged at 2.7 k rcf for 15 minutes to remove any salts from the neutralization. The product 16-3 was dried and recovered as a dark brown solid. The 1H NMR spectrum of compound 16-3 is shown in FIG. 30.

[0422] Chelator 16-3 was used in the preparation of the polymers of the present disclosure as described herein.

[0423] Example 13 Addition of Modulators to Metal-Containing Polymers via Ligand Exchange Modifiers such as solubility modifiers have been introduced into metal-containing polymers via the metal center by ligand exchange with small molecules such as glutathione (Schemes 17 and 18).

[0424] [ka]

[0425] [ka]

[0426] To solutions containing mercury- or platinum-containing polymers (3 mg in 250 uL), glutathione solution (325 mM, 50 uL) was added and the reactions were gently vortexed for 15 min at room temperature. Samples were then spin-filtered on an Amicon, Ultra-0.5, 3 kDa, 8 times with MilliQ water at 2.7 k rcf. Samples were lyophilized to give the polymers.

[0427] Other examples of small molecule thiols containing one or more thiol functional groups for the ligand exchange reactions described herein include, but are not limited to, cysteine, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonate.

[0428] It was observed that the polymers containing glutathione ligands were more soluble compared to the original polymers with halide ligands. For example, for the Hg polymer, undissolved species were observed in MilliQ water. These precipitates were filtered off using a 0.2um nylon syringe filter. The addition of glutathione allowed the precipitates to re-dissolve, after which they were purified by spin filtration using an Amicon, Ultra-0.5, 3kDa spin filter. 1 H NMR showed that the redissolved species was the DPA-sulfobetaine modified polymer. Thus, ligand exchange to introduce solubility modifiers at the metal center could enhance solubility.

[0429] Several glutathione-modified Pt-containing polymers of the present disclosure were prepared and compared to their counterparts without the glutathione ligand as a solubility modifier:

[0430] [Table 2]

[0431] The polymers were conjugated to antibodies and evaluated for non-specific binding using mass cytometry methods based on those described in Example 11. The results are shown in Figure 31. As shown by the mass cytometry results, the glutathione modified polymers showed less non-specific binding for all polymers tested.

[0432] While the present application has been described with reference to examples, it should be understood that the claims should not be limited to the embodiments described in the examples, but should be accorded the broadest interpretation consistent with the specification as a whole.

[0433] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event that any term in this application is found to be defined differently in a document incorporated herein by reference, the definition provided herein serves as the definition for that term.

[0434] Citations for literature mentioned herein [Table 3]

Claims

1. A compound of formula I 【Chemical 1】 wherein A is a polymer backbone, and optionally, the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof; each B is independently, optionally, a nitrogen-containing 5- to 7-membered heterocyclic ring substituted with one or more polar functional groups selected from C1-C6 alkyleneCOOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof; and L is absent or a linker; Each L ２ either does not exist independently or is a linker; Each R ２ is independently a first modifying group selected from a solubility regulator, a reactive functional group, a biomolecule, or a combination thereof; X is a functional group selected from ester, ether, and amide; Each L １ either does not exist independently or is a linker; Each R １ is independently H, C1-C8 alkyl, C2-C8 alkenyl, C3-C8 cycloalkyl, OH, C1-C10 alkoxy, C1-C10 alkylamine, solubility regulator, reactive functional group, biomolecule, and combinations thereof; n is an integer from 0 to 7; m is an integer from 0 to 4; p is an integer from 0 to 3; q is an integer greater than 0, and each repeat of q is attached to a different monomer unit of the polymer backbone A; and the solubility modifiers of each R2 and the solubility modifier of R1 each independently comprise a zwitterionic polymer such as polyethylene glycol (PEG), sugar, oligosaccharide, or poly(carboxybetaine) methacrylate or poly(sulfobetaine) methacrylate (PBSMA)).

2. each B is independently a nitrogen-containing 5- or 6-membered heteroaryl, and optionally, each B is independently, optionally, pyridine or imidazole substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and optionally, one or more Bs are coordinated to a soft metal and / or conjugated to one or more biomolecules, the compound according to claim 1.

3. R １ or R ２ is a biomolecule, and in some cases, the biomolecule is an affinity reagent such as an antibody, the compound according to claim 1.

4. X is -C(O)NR ４ - or -NR ４ C(O)-, and R ４ is H or C1-C4 alkyl, the compound according to claim 1.

5. X is -C(O)NR ４ - and the formula Ia 【Chemical 2】 having the structure of, or X is --NR ４ C(O)--, and formula Ib 【Chemical Formula 3】 having the structure of, or X is -C(O)NR4-, of formula Ic [Chemical Formula 4] having the structure of, or X is -NR4C(O)-, of formula If 【Chemical Formula 5】 having the structure of, or of formula Id or formula Ie 【Chemical Formula 6】 having the structure of, or of formula Ig or formula Ih 【Chemical Formula 7】 having the structure, wherein each R3 is independently selected from H, C1-C5 alkyl, C2-C5 alkenyl, C1-C6 alkyleneCOOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, or polyether, the compound according to claim 1.

6. Each R ３ is independently selected from H, -(CH ２ ) １～３ COOH, -(CH ２ ) １～３ O(CH ２ ) １～２ CH ３ , -(CH ２ ) ２～４ OH, -(CH ２ ) ２～５ P(O)(OCH ２ CH ３ ) ２ , or -CH ２ CH(OMe) ２ , and / or n is 2, 3, 4, or 5, m is 0, 1, or 2, or p is 1 or 2, the compound according to claim 5. **Claim 7** The compound according to claim 1, wherein A is selected from polyacrylate, polyacrylamide, polyether, polyamino acid, polyvinylamine, poly(2-oxazoline), polyethylene glycol, polysaccharide, dendrimer, copolymers thereof, or combinations thereof. **Claim 8** Each linker independently comprises or is independently selected from C3-C8 alkylamine, C3-C8 alkylene, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, 5- or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C(O), C(O)O, amide, amine, thioether, maleimide-thiol conjugate, polyethylene glycol (PEG), or mixtures thereof, and optionally, each of said amine, alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof, or Each L1 and / or L2 independently comprises or is independently selected from C3-C8 alkylene, C3-C8 alkylamine, ester, amine, amide, thioether, maleimide-thiol conjugate, PEG, or mixtures thereof, and optionally, each of said alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof, The compound according to claim 1. **Claim 9** The reactive functional group is for binding to one or more biomolecules, and optionally, the one or more biomolecules are each independently selected from small molecules, polypeptides, oligonucleotides, lipids, carbohydrates, affinity reagents, optionally antibodies, or mixtures thereof, the compound according to claim 1.

10. The compound is 【Chemical 8】 【Chem.】 【Chem.】 selected from 【Chemical Formula 9】 【Chem.】 [Chemical] 【Chem.】 selected from wherein s is from about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30, r is from about 3 to about 200, about 6 to about 30, or about 10 to about 25, and R is H, 【Chemical 10】 or a thiol cap group, or the compound is 【Chemical 11】 【Chem.】 [Chemical] 【Chem.】 [Chemical] [Chemical] 【Chem.】 selected from wherein s is from about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30, and r is from about 3 to about 200, about 6 to about 30, or about 10 to about 25, the compound according to claim 5.

11. The compound of formula I is chelated to one or more metals M and has the structure of formula II 【Chemical Formula 11】 or a derivative or salt thereof, one or more Bs are chelated to one or more metals M, M is a soft metal, and optionally, M is selected from Re, Pt, Pd, Nb, Tc, Hg, Ag, Au, Mo, Ru, Rh, Cd, W, Os, or mixtures thereof, the compound of formula I according to any one of claims 1 to 10.

12. A compound of formula II as defined in claim 11 for use in mass spectrometry.

13. A kit comprising an isotope composition comprising a plurality of soft metal atoms of a single isotope of a soft metal; and an elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups each comprising two nitrogen-containing 5- or 6-membered heterocycles (each chelating group of the elemental tag comprises at least one of the soft metal atoms of the isotope composition or can bind to at least one of the soft metal atoms of the isotope composition) comprising each elemental tag is independently a compound of formula II as defined in claim 11, the elemental tag is functionalized to bind to a biomolecule and / or is bound to a biomolecule, optionally a kit that does not contain any radioactive soft metals. **Claim 14**: The kit according to claim 13, wherein the biomolecule is an oligonucleotide or an antibody and / or further comprises an additional isotope composition, the additional isotope composition comprising a plurality of additional soft metal atoms of an additional single isotope of a soft metal different from the single isotope of the soft metal of the isotope composition. **Claim 15** A method comprising: providing an isotope composition comprising a plurality of soft metal atoms of a single isotope of a soft metal; providing an elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups each independently comprising two nitrogen-containing 5- or 6-membered heterocycles, each chelating group being capable of binding at least one of the soft metal atoms of the isotope composition; and binding the soft metal atoms of the isotope composition to one or more of the chelating groups of the elemental tag wherein the soft metal atoms are non-radioactive. **Claim 16** The method according to claim 15, wherein the isotope composition does not comprise a natural mixture of isotopes and optionally the method comprises providing an additional isotope composition, the additional isotope composition comprising a plurality of additional soft metal atoms of an additional single isotope of a non-radioactive soft metal different from the single isotope of the non-radioactive soft metal of the isotope composition. **Claim 17** A method for analyzing an analyte in a biological sample, comprising: ​ (i) incubating an element-tagged affinity reagent with the sample, wherein the element-tagged affinity reagent comprises an affinity reagent tagged with an element tag, and the element tag is a compound of formula II as defined in claim 11, comprising a linear or branched polymer having a plurality of chelating groups each independently comprising two nitrogen-containing 5- or 6-membered heterocycles, the element tag further comprising a plurality of soft metal atoms of a single isotope of a soft metal, and optionally incubating two or more differential element-tagged affinity reagents with two or more samples, wherein the element-tagged affinity reagent specifically binds to the two or more samples to produce two or more differentially tagged samples, and the step of analyzing the element tag attached to the affinity reagent comprises analyzing the differential element tags attached to the two or more samples by mass spectrometry atomic spectrometry; (wherein: each chelating group of the element tag comprises at least one of the soft metal atoms or is capable of binding at least one of the soft metal atoms, the soft metal atoms are non-radioactive, and the affinity reagent specifically binds the sample) (ii) separating unbound element-tagged affinity reagent from the bound element-tagged affinity reagent; and (iii) analyzing the element tag attached to the affinity reagent bound to the sample by mass spectrometry atomic spectrometry A method comprising.

18. The method according to claim 17, wherein the element-tagged affinity reagent is configured to bind to the sample in a biological sample, and the biological sample comprises cells.

19. The compound according to claim 11, wherein q is an integer from 2 to 50.

20. The compound according to claim 11, wherein M is further bound to a solubility modifier comprising a thiol ligand, and the thiol ligand is selected from glutathione, cysteine, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonate, and combinations thereof.

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