Dithiol chelators for metal conjugation to antibodies

Abstract

Metal-protein conjugates for use in mass cytometry and methods of making the same are provided. The metal-protein conjugates may have a metal directly bound to the protein, or have a metal attached to the protein through a linking moiety. The metal-protein conjugates include mercury-chelated triazole-containing antibody complexes and methods of their formation from derivatized antibodies and sulfonyl azides.

Description

DITHIOL CHELATORS FOR METAL CONJUGATION TO ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to USSN 63 / 384,829, filed on November 23, 2022, which is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] In mass cytometry, cells are labeled with mass-tagged biologically active materials (such as antibodies or oligonucleotides), and mass tags can be detected by mass spectrometry with single cell resolution.

[0003] These mass tags are commonly lanthanide chelating polymers loaded with enriched lanthanide isotopes for conjugation with antibodies. One disadvantage of lanthanides for conjugation with antibodies is that proteins such as bovine serum albumin (BSA, often utilized in the buffers for purified antibodies) can bind lanthanides, resulting in failed antibody conjugation due to adsorption of the lanthanide.

[0004] Alternative elements for mass tags include yttrium, zirconium, tantalum, lead, silver, cadmium, indium, bismuth, selenium, tellurium, cerium, praseodymium, neodymium, samarium and europium. The number of mass-tagged biologically active materials that can be distinguished is determined by the number of isotopes of different mass. Development of additional mass tags, and particularly mass tags which are conjugates with other metals such as mercury, would allow for expansion of the number and type of targets detectable in mass cytometry applications, as well as avoiding unwanted interactions with other components of antibody preparations such as buffers or stabilizers including glycerol.

[0005] The background description provided herein is for the purposes of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description thatmay not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.TECHNICAL FIELD

[0006] The present disclosure relates to reagents and their use for elemental mass spectrometry, including imaging mass spectrometry, of biological samples.SUMMARY

[0007] The present disclosure provides metal-protein conjugates for use in mass cytometry and methods of making the same. The metal-protein conjugates may have a metal directly bound to the protein, or have a metal attached to the protein through a linking moiety. The metal-protein conjugates include mercury-chelated triazole-containing antibody complexes and methods of their formation from derivatized antibodies and sulfonyl azides.

[0008] Accordingly, in a first aspect, the present disclosure encompasses a conjugate. In some embodiments, the conjugate includes a metal atom; a sulfhydryl-containing moiety; and a protein; where the protein is conjugated to the sulfhydryl-containing moiety through a bond or through a linking moiety, the linking moiety having a structure of Formula (I):-L'-A-L2- (I), where L1is a first linking group including an alkyl carbonyl, amide, amine, ether, or ester, and L1is covalently bound to the protein; L2is a second linking group including a nitrogencontaining moiety, and L2is covalently bound to the sulfhydryl-containing moiety; and A is a cyclic group; and where the metal atom chelates the sulfhydryl-containing moiety.

[0009] In some embodiments, the sulfhydryl-containing moiety is a thiol, a dithiol, a cysteine or an N-acetylcysteine group.

[0010] In some embodiments, the dithiol is a 1,2-dithiol, a 1 ,3-dithiol or a 1,4-dithiol.

[0011] In some embodiments, the dithiol is lipoic acid.

[0012] In some embodiments, the metal atom is Hg, Pb, Pt, Sb or As.

[0013] In some embodiments, the dithiol chelates the metal atom.

[0014] In some embodiments, two dithiols chelate one metal atom.

[0015] In some embodiments, four dithiols chelate one metal atom.

[0016] In some embodiments, the protein is an antibody.

[0017] In some embodiments, the protein is a chemical antibody.

[0018] In some embodiments, the chemical antibody is an aptamer, which can include one or more modifications.

[0019] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a human antibody or a humanized antibody.

[0020] In some embodiments, the antibody is a dibenzocyclooctyne-derivatized antibody.

[0021] In some embodiments, the nitrogen-containing moiety is an amino, azido or amido group.

[0022] In a second aspect, the present disclosure encompasses a method of forming a metal- chelated chemical antibody conjugate for mass cytometry. In some embodiments the method includes reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol- containing sulfonyl azide chelator; chelating a metal with the dithiol-containing sulfonyl azide chelator to form an azide-containing metal-chelator complex, wherein the metal is Hg, Pb, Pt, Sb or As; reacting a chemical antibody with a reagent to form an alkyne-containing chemical antibody derivative; and conjugating the azide-containing metal-chelator complex to the alkyne-containing chemical antibody derivative to form a metal -chelated chemical antibody conjugate.

[0023] In some embodiments, the chemical antibody is an aptamer.

[0024] In a third aspect, the present disclosure encompasses a method of forming a metal- chelated protein conjugate for mass cytometry. In some embodiments, the method includes reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelator; chelating a metal with the dithiol-containing sulfonyl azide chelator to form an azide-containing metal-chelator complex; reacting a protein with a reagent to form an alkyne-containing protein derivative; and conjugating the azide-containing metal-chelator complex to the alkyne-containing protein derivative to form a metal -chelated protein conjugate.

[0025] In some embodiments, the metal is Hg, Pb Pt, Sb or As.

[0026] In some embodiments, the protein is an antibody.

[0027] In some embodiments, conjugating the azide-containing metal-chelator complex to the alkyne-containing protein derivative includes reacting an alkyne of the alkyne-containing protein derivative with an azide of the azide-containing metal-chelator complex to form a tri azole.

[0028] In some embodiments, the dithiol-containing sulfonic acid is 2, 3 -dimercaptopropane 1 -sulfonic acid.

[0029] In some embodiments, the reagent is DBCO-NHS.

[0030] In a fourth aspect, the present disclosure encompasses a method of forming a metal- chelated protein conjugate for mass cytometry. In some embodiments, the method includes chelating a metal with lipoic acid to form a metal-chelator complex; and covalently bonding the metal-chelator complex to a protein with a coupling agent.

[0031] In some embodiments, the metal is Hg, Pb, Pt, Sb or As.

[0032] In some embodiments, the protein is an antibody.

[0033] In some embodiments, the protein is a chemical antibody.

[0034] In some embodiments, the chemical antibody is an aptamer, which can include one or more modifications.

[0035] In some embodiments, the coupling agent is an amide-containing coupling agent.

[0036] In some embodiments, the coupling agent is 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholin-4-ium chloride.

[0037] In a fifth aspect, the present disclosure encompasses a method of forming a metal- chelated protein conjugate for mass cytometry. In some embodiments, the method includes reacting a poly(amino acid) with ethylene diamine to form an amine-containing poly(amino acid); functionalizing the amine-containing poly(amino acid) with a polyethylene glycol succinimidyl ester to form a functionalized poly(amino acid); covalently binding lipoic acid to the functionalized poly(amino acid) to form a lipoic acid-modified and functionalized poly(amino acid); chelating a metal to the lipoic acid-modified and functionalized poly(amino acid) to form a metal-chelated lipoic acid-modified and functionalized poly(amino acid);reacting a protein with a reagent to form an alkyne-containing protein derivative; and conjugating the metal-chelated lipoic acid-modified and functionalized poly(amino acid) with the alkyne-containing protein derivative to form a metal-chelated protein conjugate.

[0038] In some embodiments, the metal is Hg, Pb, Pt, Sb or As.

[0039] In some embodiments, the protein is an antibody.

[0040] In some embodiments, the protein is a chemical antibody.

[0041] In some embodiments, the chemical antibody, which can include one or more modifications.

[0042] In some embodiments, the poly(amino acid) is poly(y-benzyl a,L-glutamate).

[0043] In some embodiments, the reagent is (2,5-dioxopyrrolidin-l-yl) 4-(2- azatricyclo[10.4.0.04’9]hexadeca-l(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

[0044] In a sixth aspect, the present disclosure encompasses a method of forming a metal- chelated protein conjugate for mass cytometry. In some embodiments, the method includes reacting a protein including thiol groups with a reactant to form a modified protein; reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelator; reacting the modified protein with a reagent to form an alkyne-containing protein derivative; conjugating the alkyne-containing protein derivative with the dithiol-containing sulfonyl azide chelator to form a protein-chelator conjugate; and chelating a metal with the protein-chelator conjugate.

[0045] In some embodiments, the metal is Hg, Pb, Pt, Sb or As.

[0046] In some embodiments, the protein is an antibody.

[0047] In some embodiments, the protein is a chemical antibody.

[0048] In some embodiments, the chemical antibody is an aptamer, which can include one or more modifications.

[0049] In some embodiments, the reactant is glutathione.

[0050] In some embodiments, the reagent is DBCO-NHS.

[0051] In a seventh aspect, the present disclosure encompasses a method for forming a metal-chelated protein. In some embodiments, the method includes providing a protein having at least one disulfide moiety; reducing the at least one disulfide moiety with a reducingagent to form a dithiol-containing protein; and complexing a metal to the dithiol-containing protein.

[0052] In some embodiments, the metal is Hg, Pb, Pt, Sb or As.

[0053] In some embodiments, the protein is an antibody.

[0054] In some embodiments, the protein is a chemical antibody.

[0055] In some embodiments, the chemical antibody is an aptamer, which can include one or more modifications.

[0056] In some embodiments, the reducing agent is dithiothreitol, 2-mercaptoethanol, 2- mercaptothylamine or tris-(2-carboxy ethyl) phosphine.

[0057] In an eighth aspect, the present disclosure encompasses a method for forming a metal-chelated protein. In some embodiments, the method includes introducing sulfhydryl groups to a protein by reacting the protein with a reagent to form a thiol-containing protein; and chelating a metal to the thiol-containing protein.

[0058] In some embodiments, the metal is Hg, Pb, Pt, Sb or As.

[0059] In some embodiments, the protein is an antibody.

[0060] In some embodiments, the protein is a chemical antibody.

[0061] In some embodiments, the chemical antibody is an aptamer, which can include one or more modifications.

[0062] In some embodiments, introducing sulfhydryl groups includes reacting active sites on the protein with the reagent.

[0063] In some embodiments, the active sites are amine groups.

[0064] In some embodiments, the reagent is 2-iminothiolane.DETAILED DESCRIPTION

[0065] In the following description, numerous specific details are set forth to provide a thorough understanding of the presented embodiments. The disclosed embodiments may be practiced without some or all of the specific details. In other instances, well-known process operations have not been described in detail to not unnecessarily obscure the disclosedembodiments. While the disclosed embodiments will be described in conjunction with the specific embodiments, it will be understood that it is not intended to limit the disclosed embodiments.Definitions

[0066] As used herein, the term “about” is understood to account for minor increases and / or decreases beyond a recited value, which changes do not significantly impact the desired function of the parameter beyond the recited value(s). In some cases, “about” encompasses + / - 10% of any recited value. As used herein, this term modifies any recited value, range of values, or endpoints of one or more ranges.

[0067] The term “acyl,” or “alkanoyl,” as used interchangeably herein, represents an alkyl group, as defined herein, or hydrogen attached to the parent molecular group through a carbonyl group, as defined herein. This group is exemplified by formyl, acetyl, propionyl, butanoyl, and the like. The alkanoyl group can be substituted or unsubstituted. For example, the alkanoyl group can be substituted with one or more substitution groups, as described herein for alkyl. In some embodiments, the unsubstituted acyl group is a C2-7 acyl or alkanoyl group. In particular embodiments, the alkanoyl group is -C(O)-Ak, in which Ak is an alkyl group, as defined herein.

[0068] By “aliphatic” is meant a hydrocarbon moiety having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (Ci- 10), and which includes saturated groups such as alkanes (or alkyl) and unsaturated groups such as alkenes (or alkenyl), alkynes (or alkynyl), and also includes cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Such a hydrocarbon can be unsubstituted or substituted with one or more groups, such as halogens or groups described herein for an alkyl group.

[0069] By “alkenyl” is meant an optionally substituted C2-24 alkyl group having one or more double bonds. The alkenyl group can be cyclic (e.g., C3-24 cycloalkenyl) or acyclic. The alkenyl group can also be substituted or unsubstituted. For example, the alkenyl group can be substituted with one or more substitution groups, as described herein for alkyl. Non-limitingunsubstituted alkenyl groups include C2-8 alkenyl, C2-6 alkenyl, C2-5 alkenyl, C2-4 alkenyl, or C2- 3 alkenyl. Exemplary, non-limiting alkenyl groups include vinyl or ethenyl (-CH=CH2), 1- propenyl (-CH=CHCH3), allyl or 2-propenyl (-CH2-CH=CH2), 1-butenyl (-CE^CElCEbCEk), 2-butenyl (-CEECE^CElCEk), 3-butenyl (e.g. -CEECEECE^CEk), 2-butenylidene (e.g., =CH- CH=CHCH3), and the like.

[0070] By “alkenylene” is meant a multivalent (e.g., bivalent) form of an alkenyl group, which is an optionally substituted C2-24 alkyl group having one or more double bonds. The alkenylene group can be cyclic (e.g., C3-24 cycloalkenyl) or acyclic. The alkenylene group can be substituted or unsubstituted. For example, the alkenylene group can be substituted with one or more substitution groups, as described herein for alkyl. Exemplary, non-limiting alkenylene groups include -CH=CH- or -CH=CHCH2-.

[0071] By “alkoxy” is meant -OR, where R is an optionally substituted alkyl group, as described herein. Exemplary alkoxy groups include methoxy, ethoxy, butoxy, trihaloalkoxy, such as trifluoromethoxy, etc. The alkoxy group can be substituted or unsubstituted. For example, the alkoxy group can be substituted with one or more substitution groups, as described herein for alkyl. Exemplary unsubstituted alkoxy groups include C1-3, C1-6, C1-12, Ci-16, Ci-is, C1-20, or Ci-24 alkoxy groups.

[0072] By “alkoxyalkyl” is meant an alkyl group, as defined herein, which is substituted with an alkoxy group, as defined herein. Exemplary unsubstituted alkoxyalkyl groups include between 2 to 12 carbons (C2-12 alkoxyalkyl), as well as those having an alkyl group with 1 to 6 carbons and an alkoxy group with 1 to 6 carbons (i.e., C1-6 alkoxy-Ci-6 alkyl).

[0073] By “alkyl” and the prefix “alk” is meant a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl (Me), ethyl (Et), n-propyl (n-Pr or nPr), isopropyl (i-Pr or iPr), cyclopropyl, n-butyl (n-Bu or nBu), isobutyl (i-Bu or iBu), s-butyl (s-Bu or sBu), t-butyl (t-Bu or tBu), cyclobutyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic (e.g., C3-24 cycloalkyl) or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkylgroup can include haloalkyl, in which the alkyl group is substituted by one or more halo groups, as described herein. In another example, the alkyl group can be substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) Ci-6 alkoxy (e.g., -O-Ak, wherein Ak is optionally substituted Ci-6 alkyl); (2) amino (e.g., -NRN1RN2, where each of RN1and RN2is, independently, H or optionally substituted alkyl, or RN1and RN2, taken together with the nitrogen atom to which each are attached, form a heterocyclyl group); (3) aryl; (4) arylalkoxy (e.g., -O-Lk-Ar, wherein Lk is a bivalent form of optionally substituted alkyl and Ar is optionally substituted aryl); (5) aryloyl (e.g., -C(O)-Ar, wherein Ar is optionally substituted aryl); (6) cyano (e.g., -CN); (7) carboxyaldehyde (e.g., -C(O)H); (8) carboxyl (e.g., -CO2H); (9) C3-8 cycloalkyl (e.g., a monovalent saturated or unsaturated non-aromatic cyclic C3-8 hydrocarbon group); (10) halo (e.g., F, Cl, Br, or I); (11) heterocyclyl (e.g., a 3-, 4-, 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four non-carbon heteroatoms, such as nitrogen, oxygen, phosphorous, sulfur, or halo); (12) heterocyclyloxy (e.g., -O-Het, wherein Het is heterocyclyl, as described herein); (13) heterocyclyloyl (e.g., -C(O)-Het, wherein Het is heterocyclyl, as described herein); (14) hydroxyl (e.g., -OH); (15) N-protected amino; (16) nitro (e.g., -NO2); (17) oxo (e.g., =0); (18) -CO2RA, where RAis selected from the group consisting of (a) C1-6 alkyl, (b) C4-18 aryl, and (c) (C4-18 aryl) C1-6 alkyl (e.g., -Lk-Ar, wherein Lk is a bivalent form of optionally substituted alkyl group and Ar is optionally substituted aryl); (19) -C(0)NRBRc, where each of RBand Rcis, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C4-18 aryl, and (d) (C4-18 aryl) C1-6 alkyl (e.g., -Lk-Ar, wherein Lk is a bivalent form of optionally substituted alkyl group and Ar is optionally substituted aryl); and (20) -NRGRH, where each of RGand RHis, independently, selected from the group consisting of (a) hydrogen, (b) an N-protecting group, (c) C1-6 alkyl, (d) C2-6 alkenyl (e.g., optionally substituted alkyl having one or more double bonds), (e) C2-6 alkynyl (e.g., optionally substituted alkyl having one or more triple bonds), (f) C4-18 aryl, (g) (C4-18 aryl) C1-6 alkyl (e.g., Lk-Ar, wherein Lk is a bivalent form of optionally substituted alkyl group and Ar is optionally substituted aryl), (h) C3-8 cycloalkyl, and (i) (C3-8 cycloalkyl) C1-6 alkyl (e.g., -Lk-Cy, wherein Lk is a bivalent form of optionally substituted alkyl group and Cy is optionally substitutedcycloalkyl, as described herein), wherein in one embodiment no two groups are bound to the nitrogen atom through a carbonyl group. The alkyl group can be a primary, secondary, or tertiary alkyl group substituted with one or more substituents (e.g., one or more halo or alkoxy). In some embodiments, the unsubstituted alkyl group is a C1-2, C1-3, C1-6, C1-12, Ci-16, Ci-is, Ci- 20, Ci-24, C2-3, C2-6, C2-12, C2-16, C2-18, C2-20, or C2-24 alkyl group.

[0074] By “alkylene” is meant a multivalent (e.g., bivalent) form of an alkyl group, as described herein. Exemplary alkylene groups include methylene, ethylene, propylene, butylene, etc. In some embodiments, the alkylene group is a C1-3, C1-6, C1-12, Ci-16, Ci-18, C1-20, Ci-24, C2-3, C2-6, C2-12, C2-16, C2-18, C2-20, or C2-24 alkylene group. The alkylene group can be branched or unbranched. The alkylene group can also be substituted or un substituted. For example, the alkylene group can be substituted with one or more substitution groups, as described herein for alkyl.

[0075] By “alkyleneoxy” is meant an alkylene group, as defined herein, attached to the parent molecular group through an oxygen atom.

[0076] By “alkylcarbonyl” is meant an alkyl group as previously defined appended to the parent molecular moiety through a carbonyl group. Exemplary, non-limiting alkylcarbonyl groups include methylcarbonyl, ethylcarbonyl, and isopropyl carbonyl among others.

[0077] By “alkynyl” is meant an optionally substituted C2-24 alkyl group having one or more triple bonds. The alkynyl group can be cyclic or acyclic and is exemplified by ethynyl, 1-propynyl, and the like. The alkynyl group can also be substituted or unsubstituted. For example, the alkynyl group can be substituted with one or more substitution groups, as described herein for alkyl. Non-limiting unsubstituted alkynyl groups include C2-8 alkynyl, C2- 6 alkynyl, C2-5 alkynyl, C2-4 alkynyl, or C2-3 alkynyl. Exemplary, non-limiting alkynyl groups include ethynyl (-C=CH), 1-propynyl (-OCCH3), 2-propynyl or propargyl (-CH2OCH), 1- butynyl (-OCCH2CH3), 2-butynyl (-CH2OCCH3), 3-butynyl (-CH2CH2OCH), and the like.

[0078] By “alkynylene” is meant a multivalent (e.g., bivalent) form of an alkynyl group, which is an optionally substituted C2-24 alkyl group having one or more triple bonds. The alkynylene group can be cyclic or acyclic. The alkynylene group can be substituted orunsubstituted. For example, the alkynylene group can be substituted with one or more substitution groups, as described herein for alkyl. Exemplary, non-limiting alkynylene groups include -C=C- or -OCCH2-.

[0079] By “amido” is meant -N(RN1)C(O)-, where RN1is H, optionally substituted alkyl, or optionally substituted aryl.

[0080] By “amino” is meant -NRN1RN2, where each of RN1and RN2is, independently, H, optionally substituted alkyl, or optionally substituted acyl, or optionally substituted aryl, or RN1and RN2, taken together with the nitrogen atom to which each are attached, form a heterocyclyl group, as defined herein.

[0081] By “aminoalkyl” is meant an alkyl group, as defined herein, substituted by an amino group, as defined herein.

[0082] By “aminoaryl” is meant an aryl group, as defined herein, substituted by an amino group, as defined herein.

[0083] By “ ammonium” is meant a group including a protonated nitrogen atom N+. Exemplary ammonium groups include -N+RN1RN2RN3where each of RN1, RN2, and RN3is, independently, H, optionally substituted alkyl, optionally substituted cycloalkyl, or optionally substituted aryl; or RN1and RN2, taken together with the nitrogen atom to which each are attached, form an optionally substituted heterocyclyl group or heterocycle; or RN1and RN2, taken together, form an optionally substituted alkylene or heteroalkylene (e.g., as described herein); or RN1and RN2and RN3, taken together with the nitrogen atom to which each are attached, form an optionally substituted heterocyclyl group or heterocycle, such as a heterocyclic cation.

[0084] By “aromatic” is meant a cyclic, conjugated group or moiety of, unless specified otherwise, from 5 to 15 ring atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized n- electron system. Typically, the number of out of plane 7t-electrons corresponds to the Huckel rule (4n+2). The point of attachment to the parent structure typically is through an aromaticportion of the condensed ring system. Such an aromatic can be unsubstituted or substituted with one or more groups, such as groups described herein for an alkyl or aryl group. Yet other substitution groups can include aliphatic, haloaliphatic, halo, nitrate, cyano, sulfonate, sulfonyl, or others.

[0085] By “aryl” is meant a group that contains any carbon-based aromatic group including, but not limited to, phenyl, benzyl, anthracenyl, anthryl, benzocyclobutenyl, benzocyclooctenyl, biphenylyl, chrysenyl, dihydroindenyl, fluoranthenyl, indacenyl, indenyl, naphthyl, phenanthryl, phenoxybenzyl, picenyl, pyrenyl, terphenyl, and the like, including fused benzo- C4-8 cycloalkyl radicals (e.g., as defined herein) such as, for instance, indanyl, tetrahydronaphthyl, fluorenyl, and the like. The term aryl also includes heteroaryl, which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term non-heteroaryl, which is also included in the term aryl, defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one, two, three, four, or five substituents, such as any described herein for alkyl.

[0086] By “arylalkoxy” is meant an arylalkylene group, as defined herein, attached to the parent molecular group through an oxygen atom. In some embodiments, the arylalkoxy group is -O-Ak-Ar, in which Ak is an optionally substituted alkylene, as defined herein, and Ar is an optionally substituted aryl, as defined herein.

[0087] By “(aryl)(alkyl)ene” is meant a bivalent form including an arylene group, as described herein, attached to an alkylene or a heteroalkylene group, as described herein. In some embodiments, the (aryl)(alkyl)ene group is -L-Ar- or -L-Ar-L- or -Ar-L-, in which Ar is an arylene group and each L is, independently, an optionally substituted alkylene group or an optionally substituted heteroalkylene group.

[0088] By “arylalkylene” is meant an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. In some embodiments, the arylalkylene group is -Ak-Ar, in which Ak is an optionally substituted alkylene, as definedherein, and Ar is an optionally substituted aryl, as defined herein. The arylalkylene group can be substituted or unsubstituted. For example, the arylalkylene group can be substituted with one or more substitution groups, as described herein for aryl and / or alkyl. Exemplary unsubstituted arylalkylene groups are of from 7 to 16 carbons (C7-16 arylalkylene), as well as those having an aryl group with 4 to 18 carbons and an alkylene group with 1 to 6 carbons (i.e., (C4-18 aryl)Ci-6 alkylene).

[0089] By “arylene” is meant a multivalent (e.g., bivalent, trivalent, tetravalent, etc.) form of an aryl group, as described herein. Exemplary arylene groups include phenylene, naphthylene, biphenylene, triphenylene, diphenyl ether, acenaphthenylene, anthrylene, or phenanthrylene. In some embodiments, the arylene group is a C4-18, C4-14, C4-12, C4-10, Ce-18, Ce-14, C6-12, or Ce-io arylene group. The arylene group can be branched or unbranched. The arylene group can also be substituted or unsubstituted. For example, the arylene group can be substituted with one or more substitution groups, as described herein for aryl.

[0090] By “aryleneoxy” is meant an arylene group, as defined herein, attached to the parent molecular group through an oxygen atom.

[0091] By “aryloxy” is meant an aryl group, as defined herein, attached to the parent molecular group through an oxygen atom.

[0092] By “aryloyl” is meant an aryl group that is attached to the parent molecular group through a carbonyl group. In some embodiments, an unsubstituted aryloyl group is a C7-11 aryloyl or C5-19 aryloyl group. In particular embodiments, the aryloyl group is -C(O)-Ar, in which Ar is an aryl group, as defined herein.

[0093] By “attaching,” “attachment,” or related word forms is meant any covalent or non- covalent bonding interaction between two components. Non-covalent bonding interactions include, without limitation, hydrogen bonding, ionic interactions, halogen bonding, electrostatic interactions, TI bond interactions, hydrophobic interactions, inclusion complexes, clathration, van der Waals interactions, and combinations thereof.

[0094] By “ azido” is meant -N3.

[0095] By “boranyl” is meant a -BR2 group, in which each R, independently, can be H, halo, or optionally substituted alkyl.

[0096] By “borono” is meant a -BOH2 group.

[0097] By “branched alkenyl” is meant an isomer of a straight chain alkenyl compound; one having alkyl groups bonded to the main carbon chain.

[0098] By “carbonyl” is meant a -C(O)- group, which can also be represented as >C=O.

[0099] By “carboxyl” is meant a -CO2H group.

[0100] By “carboxylate anion” is meant a -CO2 group.

[0101] By“ covalent bond” is meant a covalent bonding interaction between two components. Non-limiting covalent bonds include a single bond, a double bond, a triple bond, or a spirocyclic bond, in which at least two molecular groups are bonded to the same carbon atom.

[0102] By “ cyano” is meant -CN.

[0103] By “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (e.g., cycloalkyl or heterocycloalkyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0104] By “cycloalkyl” is meant a monovalent saturated or unsaturated non-aromatic or aromatic cyclic hydrocarbon group of from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, and the like. The cycloalkyl group can also be substituted or unsubstituted. For example, the cycloalkyl group can be substituted with one or more groups including those described herein for alkyl.

[0105] By “dicarbonyl” is meant any moiety or compound including two carbonyl groups, as defined herein. Non-limiting dicarbonyl moi eties include 1,2-dicarbonyl (e.g., Rcl-C(O)- C(O)RC2, in which each of RC1and RC2is, independently, optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group); 1,3 -dicarbonyl (e.g., Rcl-C(O)- C(RlaR2a)-C(O)RC2, in which each of RC1and RC2is, independently, optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group and in which each of Rlaand R2ais, independently, H or an optional substituent provided for alkyl, as defined herein);and 1,4-dicarbonyl (e.g., Rcl-C(O)-C(RlaR2a)-C(R3aR4a)-C(O)RC2, in which each of RC1and RC2is, independently, optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group and in which each of Rla, R2a, R3a, and R4ais, independently, H or an optional substituent provided for alkyl, as defined herein).

[0106] By “ electron withdrawing moiety” is meant a moiety capable of donating at least a portion of its electron density into the ring or functional group to which it is directly attached, such as by resonance.

[0107] By “halo” is meant F, Cl, Br, or I.

[0108] By “ halo containing substituent” is meant a group that contains a halo, such as a haloaliphatic or haloalkyl group.

[0109] By “haloaliphatic” is meant an aliphatic group, as defined herein, substituted with one or more halo.

[0110] By “haloalkenyl” is meant an alkenyl group, as defined herein, substituted with one or more halo.

[0111] By “haloalkynyl” is meant an alkynyl group, as defined herein, substituted with one or more halo.

[0112] By “haloalkyl” is meant an alkyl group, as defined herein, substituted with one or more halogen. Non-limiting unsubstituted haloalkyl groups include C1-2 haloalkyl, C1-3 haloalkyl, Ci-4 haloalkyl, C1-5 haloalkyl, C1-6 haloalkyl, C2-3 haloalkyl, C2-4 haloalkyl, C2-5 haloalkyl, C2-6 haloalkyl, or C3-6 haloalkyl. Other non-limiting haloalkyl groups include - CXyH3-y, wherein y is 1, 2, or 3, and wherein each X is, independently, halo (F, Cl, Br, or I); - CXzH2-zCXyH3-y, wherein z is 0, 1, or 2, wherein y is 0, 1, 2, or 3, and wherein each X is, independently, halo (F, Cl, Br, or I), in which at least one of z or y is not 0; -CFFCXyFb-y, wherein y is 1, 2, or 3, and wherein each X is, independently, halo (F, Cl, Br, or I); - CXziH2-ziCXz2H2-z2CXyH3-y, wherein each of zl and z2 is, independently, 0, 1, or 2, wherein y is 0, 1, 2, or 3, and wherein each X is, independently, halo (F, Cl, Br, or I), in which at least one of zl, z2, or y is not 0; and -CXzHi-z[CXyiH3-yi][CXy2H3-y2], wherein z is 0 or 1, wherein eachof yl and y2 is, independently, 0, 1, 2, or 3, and wherein each X is, independently, halo (F, Cl, Br, or I), in which at least one of z, yl, or y2 is not 0.

[0113] By “haloalkylene” is meant an alkylene group, as defined herein, substituted with one or more halo.

[0114] By “heteroaliphatic” is meant an aliphatic group, as defined herein, including at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized forms thereof within the group.

[0115] By “heteroalkyl” is meant an alkyl group, as defined herein, containing one, two, three, or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorous, sulfur, selenium, or halo).

[0116] By “heteroalkylene” is meant an alkylene group, as defined herein, containing one, two, three, or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorous, sulfur, selenium, or halo). The heteroalkylene group can be saturated or unsaturated (e.g., having one or more double bonds or triple bonds). The heteroalkylene group can be substituted or unsubstituted. For example, the heteroalkylene group can be substituted with one or more substitution groups, as described herein for alkyl.

[0117] By “heteroaryl” is meant a subset of heterocyclyl groups, as defined herein, which are aromatic, i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system.

[0118] The term “heterocycloalkyl” is a type of cycloalkyl group as defined above where at least one of the carbon atoms and its attached hydrogen atoms, if any, are replaced by O, S, N, or NH. The heterocycloalkyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, sulfonic acid, sulfinic acid, fluoroacid, phosphonic acid, ester, ether, halide, hydroxy, ketone, nitro, cyano, azido, silyl, sulfonyl, sulfinyl, or thiol, as described herein.

[0119] By “heterocycle” is meant a compound having one or more heterocyclyl moi eties. Non-limiting heterocycles include optionally substituted imidazole, optionally substitutedtriazole, optionally substituted tetrazole, optionally substituted pyrazole, optionally substituted imidazoline, optionally substituted pyrazoline, optionally substituted imidazolidine, optionally substituted pyrazolidine, optionally substituted pyrrole, optionally substituted pyrroline, optionally substituted pyrrolidine, optionally substituted tetrahydrofuran, optionally substituted furan, optionally substituted thiophene, optionally substituted oxazole, optionally substituted isoxazole, optionally substituted isothiazole, optionally substituted thiazole, optionally substituted oxathiolane, optionally substituted oxadiazole, optionally substituted thiadiazole, optionally substituted sulfolane, optionally substituted succinimide, optionally substituted thiazolidinedione, optionally substituted oxazolidone, optionally substituted hydantoin, optionally substituted pyridine, optionally substituted piperidine, optionally substituted pyridazine, optionally substituted piperazine, optionally substituted pyrimidine, optionally substituted pyrazine, optionally substituted triazine, optionally substituted pyran, optionally substituted pyrylium, optionally substituted tetrahydropyran, optionally substituted dioxine, optionally substituted dioxane, optionally substituted dithiane, optionally substituted trithiane, optionally substituted thiopyran, optionally substituted thiane, optionally substituted oxazine, optionally substituted morpholine, optionally substituted thiazine, optionally substituted thiomorpholine, optionally substituted cytosine, optionally substituted thymine, optionally substituted uracil, optionally substituted thiomorpholine dioxide, optionally substituted indene, optionally substituted indoline, optionally substituted indole, optionally substituted isoindole, optionally substituted indolizine, optionally substituted indazole, optionally substituted benzimidazole, optionally substituted azaindole, optionally substituted azaindazole, optionally substituted pyrazolopyrimidine, optionally substituted purine, optionally substituted benzofuran, optionally substituted isobenzofuran, optionally substituted benzothiophene, optionally substituted benzisoxazole, optionally substituted anthranil, optionally substituted benzisothiazole, optionally substituted benzoxazole, optionally substituted benzthiazole, optionally substituted benzthiadiazole, optionally substituted adenine, optionally substituted guanine, optionally substituted tetrahydroquinoline, optionally substituted dihydroquinoline, optionally substituted dihydroisoquinoline, optionally substituted quinoline, optionally substituted isoquinoline, optionally substituted quinolizine, optionally substituted quinoxaline,optionally substituted phthalazine, optionally substituted quinazoline, optionally substituted cinnoline, optionally substituted naphthyridine, optionally substituted pyridopyrimidine, optionally substituted pyridopyrazine, optionally substituted pteridine, optionally substituted chromene, optionally substituted isochromene, optionally substituted chromenone, optionally substituted benzoxazine, optionally substituted quinolinone, optionally substituted isoquinolinone, optionally substituted carbazole, optionally substituted dibenzofuran, optionally substituted acridine, optionally substituted phenazine, optionally substituted phenoxazine, optionally substituted phenothiazine, optionally substituted phenoxathiine, optionally substituted quinuclidine, optionally substituted azaadamantane, optionally substituted dihydroazepine, optionally substituted azepine, optionally substituted diazepine, optionally substituted oxepane, optionally substituted thiepine, optionally substituted thiazepine, optionally substituted azocane, optionally substituted azocine, optionally substituted thiocane, optionally substituted azonane, optionally substituted azecine, etc. Optional substitutions include any described herein for aryl. Heterocycles can also include cations and / or salts of any of these (e.g., any described herein, such as optionally substituted piperidinium, optionally substituted pyrrolidinium, optionally substituted pyrazolium, optionally substituted imidazolium, optionally substituted pyridinium, optionally substituted quinolinium, optionally substituted isoquinolinium, optionally substituted acridinium, optionally substituted phenanthridinium, optionally substituted pyridazinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted phenazinium, or optionally substituted morpholinium).

[0120] By “heterocyclyl” is meant a 3-, 4-, 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorous, sulfur, selenium, or halo). The 3-membered ring has zero to one double bonds, the 4- and 5-membered ring has zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds. The term “heterocyclyl” also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, and another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Heterocyclics include acridinyl, adenyl, alloxazinyl, azaadamantanyl, azabenzimidazolyl, azabicyclononyl, azacycloheptyl, azacyclooctyl, azacyclononyl, azahypoxanthinyl, azaindazolyl, azaindolyl, azecinyl, azepanyl, azepinyl, azetidinyl, azetyl, aziridinyl, azirinyl, azocanyl, azocinyl, azonanyl, benzimidazolyl, benzisothiazolyl, benzisoxazolyl, benzodiazepinyl, benzodiazocinyl, benzodihydrofuryl, benzodioxepinyl, benzodioxinyl, benzodioxanyl, benzodi oxocinyl, benzodioxolyl, benzodithiepinyl, benzodithiinyl, benzodioxocinyl, benzofuranyl, benzophenazinyl, benzopyranonyl, benzopyranyl, benzopyrenyl, benzopyronyl, benzoquinolinyl, benzoquinolizinyl, benzothiadiazepinyl, benzothiadiazolyl, benzothiazepinyl, benzothiazocinyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzothiazinonyl, benzothiazinyl, benzothiopyranyl, benzothiopyronyl, benzotriazepinyl, benzotriazinonyl, benzotriazinyl, benzotriazolyl, benzoxathiinyl, benzotrioxepinyl, benzoxadiazepinyl, benzoxathiazepinyl, benzoxathiepinyl, benzoxathiocinyl, benzoxazepinyl, benzoxazinyl, benzoxazocinyl, benzoxazolinonyl, benzoxazolinyl, benzoxazolyl, benzylsultamyl, benzylsultimyl, bipyrazinyl, bipyridinyl, carbazolyl (e.g., 4H-carbazolyl), carbolinyl (e.g., P- carbolinyl), chromanonyl, chromanyl, chromenyl, cinnolinyl, coumarinyl, cytdinyl, cytosinyl, decahydroisoquinolinyl, decahydroquinolinyl, diazabicyclooctyl, diazetyl, diaziridinethionyl, diaziridinonyl, diaziridinyl, diazirinyl, dibenzisoquinolinyl, dibenzoacridinyl, dibenzocarbazolyl, dibenzofuranyl, dibenzophenazinyl, dibenzopyranonyl, dibenzopyronyl (xanthonyl), dibenzoquinoxalinyl, dibenzothiazepinyl, dibenzothiepinyl, dibenzothiophenyl, dibenzoxepinyl, dihydroazepinyl, dihydroazetyl, dihydrofuranyl, dihydrofuryl, dihydroisoquinolinyl, dihydropyranyl, dihydropyridinyl, dihydroypyridyl, dihydroquinolinyl, dihydrothienyl, dihydroindolyl, dioxanyl, dioxazinyl, dioxindolyl, dioxiranyl, dioxenyl, dioxinyl, di oxobenzofuranyl, dioxolyl, dioxotetrahydrofuranyl, dioxothiomorpholinyl, dithianyl, dithiazolyl, dithienyl, dithiinyl, furanyl, furazanyl, furoyl, furyl, guaninyl, homopiperazinyl, homopiperidinyl, hypoxanthinyl, hydantoinyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl (e.g., IH-indazolyl), indolenyl, indolinyl, indolizinyl, indolyl (e.g., 1H- indolyl or 3H-indolyl), isatinyl, isatyl, isobenzofuranyl, isochromanyl, isochromenyl,isoindazoyl, isoindolinyl, isoindolyl, isopyrazolonyl, isopyrazolyl, isoxazolidiniyl, isoxazolyl, isoquinolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, morpholinyl, naphthindazolyl, naphthindolyl, naphthiridinyl, naphthopyranyl, naphthothiazolyl, naphthothioxolyl, naphthotri azolyl, naphthoxindolyl, naphthyridinyl, octahydroisoquinolinyl, oxabicycloheptyl, oxauracil, oxadiazolyl, oxazinyl, oxaziridinyl, oxazolidinyl, oxazolidonyl, oxazolinyl, oxazolonyl, oxazolyl, oxepanyl, oxetanonyl, oxetanyl, oxetyl, oxtenayl, oxindolyl, oxiranyl, oxobenzoisothiazolyl, oxochromenyl, oxoisoquinolinyl, oxoquinolinyl, oxothiolanyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenothienyl (benzothiofuranyl), phenoxathiinyl, phenoxazinyl, phthalazinyl, phthalazonyl, phthalidyl, phthalimidinyl, piperazinyl, piperidinyl, piperidonyl (e.g., 4-piperidonyl), pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolopyrimidinyl, pyrazolyl, pyridazinyl, pyridinyl, pyridopyrazinyl, pyridopyrimidinyl, pyridyl, pyrimidinyl, pyrimidyl, pyronyl, pyrrolidinyl, pyrrolidonyl (e.g., 2-pyrrolidonyl), pyrrolinyl, pyrrolizidinyl, pyrrolyl (e.g., 2H- pyrrolyl), pyrylium, quinazolinyl, quinolinyl, quinolizinyl (e.g., 4H-quinolizinyl), quinoxalinyl, quinuclidinyl, selenazinyl, selenazolyl, selenophenyl, succinimidyl, sulfolanyl, tetrahydrofuranyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroisoquinolyl, tetrahydropyridinyl, tetrahydropyridyl (piperidyl), tetrahydropyranyl, tetrahydropyronyl, tetrahydroquinolinyl, tetrahydroquinolyl, tetrahydrothienyl, tetrahydrothiophenyl, tetrazinyl, tetrazolyl, thiadiazinyl (e.g., 6H-l,2,5-thiadiazinyl or 2H,6H-l,5,2-dithiazinyl), thiadiazolyl, thianthrenyl, thianyl, thianaphthenyl, thiazepinyl, thiazinyl, thiazolidinedionyl, thiazolidinyl, thiazolyl, thienyl, thiepanyl, thiepinyl, thietanyl, thietyl, thiiranyl, thiocanyl, thiochromanonyl, thiochromanyl, thiochromenyl, thiodiazinyl, thiodiazolyl, thioindoxyl, thiomorpholinyl, thiophenyl, thiopyranyl, thiopyronyl, thiotriazolyl, thiourazolyl, thioxanyl, thioxolyl, thymidinyl, thyminyl, triazinyl, triazolyl, trithianyl, urazinyl, urazolyl, uretidinyl, uretinyl, uricyl, uridinyl, xanthenyl, xanthinyl, xanthionyl, and the like, as well as modified forms thereof (e.g., including one or more oxo and / or amino) and salts thereof. The heterocyclyl group can be substituted or unsubstituted. For example, the heterocyclyl group can be substituted with one or more substitution groups, as described herein for aryl.

[0121] By “heterocyclyldiyl” is meant a bivalent form of a heterocyclyl group, as described herein. In one instance, the heterocyclyldiyl is formed by removing a hydrogen from a heterocyclyl group. Exemplary heterocyclyldiyl groups include piperdylidene, quinolinediyl, etc. The heterocyclyldiyl group can also be substituted or unsubstituted. For example, the heterocyclyldiyl group can be substituted with one or more substitution groups, as described herein for heterocyclyl.

[0122] By “hydroxyalkyl” is meant an alkyl group, as defined herein, substituted with one or more hydroxyl.

[0123] By “hydroxyalkylene” is meant an alkylene group, as defined herein, substituted with one or more hydroxy.

[0124] By “hydroxyl” is meant -OH.

[0125] By “imino” is meant -NR-, in which R can be H or optionally substituted alkyl.

[0126] By “isocyanato” is meant -NCO.

[0127] By “isothiocyanate” is meant -N=C=S.

[0128] By “leaving group” is meant an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons, or an atom (or a group of atoms) that can be replaced by a substitution reaction. Examples of suitable leaving groups include H, halides, and sulfonates including, but not limited to, tritiate (-OTf), mesylate (-OMs), tosylate (-OTs), brosylate (-OBs), acetate, Cl, Br, and I.

[0129] By “nitro” is meant an -NO2 group.

[0130] By “oxo” is meant an =0 group.

[0131] By “ oxy” is meant -O-.

[0132] By “phosphate” is meant a group derived from phosphoric acid. One example of phosphate includes a -O-P(=O)(ORP1)(ORP2) or -O-[P(=O)(ORP1)-O]P3-RP2group, where each of RP1and RP2, is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, or optionally substituted arylalkylene, and where P3 is an integer from 1 to 5. Yet other examples of phosphate include orthophosphoric acid, pyrophosphoricacid, tripolyphosphoric acid, tetrapolyphosphoric acid, trimetaphosphoric acid, and / or phosphoric anhydride, or combinations thereof.

[0133] By “phosphono” or “phosphonic acid” is meant a -P(O)(OH)2 group.

[0134] By “ salt” is meant an ionic form of a compound or structure (e.g., any formulas, compounds, or compositions described herein), which includes a cation or anion compound to form an electrically neutral compound or structure. Salts are well known in the art. For example, non-toxic salts are described in Berge S M et al., “Pharmaceutical salts,” J. Pharm. Set. 1977 January; 66(1): 1-19; and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use,” Wiley -VCH, April 2011 (2nd rev. ed., eds. P. H. Stahl and C. G. Wermuth. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base group with a suitable organic acid (thereby producing an anionic salt) or by reacting the acid group with a suitable metal or organic salt (thereby producing a cationic salt). Representative anionic salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, chloride, citrate, cyclopentanepropionate, digluconate, dihydrochloride, diphosphate, dodecyl sulfate, edetate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, hydroxyethanesulfonate, hydroxynaphthoate, iodide, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylbromide, methylnitrate, methyl sulfate, mucate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theophyllinate, thiocyanate, triethiodide, toluenesulfonate, undecanoate, valerate salts, and the like. Representative cationic salts include metal salts, such as alkali or alkaline earth salts, e.g., barium, calcium (e.g., calcium edetate), lithium, magnesium, potassium, sodium, and the like; other metal salts, such as aluminum, bismuth, iron, and zinc; as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine,dimethylamine, trimethylamine, triethylamine, ethylamine, pyridinium, and the like. Other cationic salts include organic salts, such as chloroprocaine, choline, dibenzylethylenediamine, diethanolamine, ethylenediamine, methylglucamine, and procaine. Yet other salts include ammonium, sulfonium, sulfoxonium, phosphonium, iminium, imidazolium, benzimidazolium, amidinium, guanidinium, phosphazinium, phosphazenium, pyridinium, etc., as well as other cationic groups described herein (e.g., optionally substituted isoxazolium, optionally substituted oxazolium, optionally substituted thiazolium, optionally substituted pyrrolium, optionally substituted furanium, optionally substituted thiophenium, optionally substituted imidazolium, optionally substituted pyrazolium, optionally substituted isothiazolium, optionally substituted triazolium, optionally substituted tetrazolium, optionally substituted furazanium, optionally substituted pyridinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted triazinium, optionally substituted tetrazinium, optionally substituted pyridazinium, optionally substituted oxazinium, optionally substituted pyrrolidinium, optionally substituted pyrazolidinium, optionally substituted imidazolinium, optionally substituted isoxazolidinium, optionally substituted oxazolidinium, optionally substituted piperazinium, optionally substituted piperidinium, optionally substituted morpholinium, optionally substituted azepanium, optionally substituted azepinium, optionally substituted indolium, optionally substituted isoindolium, optionally substituted indolizinium, optionally substituted indazolium, optionally substituted benzimidazolium, optionally substituted isoquinolinum, optionally substituted quinolizinium, optionally substituted dehydroquinolizinium, optionally substituted quinolinium, optionally substituted isoindolinium, optionally substituted benzimidazolinium, and optionally substituted purinium). Yet other salts can include an anion, such as a halide (e.g., F , Cl", Br , or I"), a hydroxide (e.g., OH"), a borate (e.g., tetrafluorob orate (BF4 ), a carbonate (e.g., CO32" or HCO3 ), or a sulfate (e.g., SO ).

[0135] By “silyl” is meant a -SiRJR2R3or -SiR1R2- group. In some embodiments, each of R1, R2, and R3is, independently, H, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aromatic, optionally substituted heteroaromatic, or optionally substituted amino. In particular embodiments, each of R1, R2, and R3is,independently, H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted alkyl-aryl, optionally substituted aryl-alkyl, or optionally substituted amino. In other embodiments, the silyl group is -Si(R)a(0R)b(NR2)c, in which each R is, independently, H, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aromatic, or optionally substituted heteroaromatic; each of a, b, and c > 0; and a + b + c = 3. In particular embodiments, each R is, independently, H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkyl-aryl, or optionally substituted aryl-alkyl.

[0136] By “spirocyclyl” is meant an alkylene diradical, both ends of which are bonded to the same carbon atom of the parent group to form a spirocyclyl group and also a heteroalkylene diradical, both ends of which are bonded to the same atom. Non-limiting alkylene and heteroalkylene groups for use within a spirocyclyl group includes C2-12, C2-11, C2-10, C2-9, C2-8, C2-7, C2-6, C2-5, C2-4, or C2-3 alkylene groups, as well as C1-12, C1-11, C1-10, C1-9, C1-8, C1-7, C1-6, C1-5, Ci-4, C1-3, or C1-2 heteroalkylene groups having one or more heteroatoms.

[0137] By “sulfate” is meant a group derived from sulfuric acid. One example of sulfate includes a -O-S(=O)2(ORsl) group, where RS1is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, or optionally substituted arylalkylene.

[0138] By “sulfo” or “sulfonic acid” is meant an -S(O)2OH group.

[0139] By “sulfonyl” is meant an -S(O)2- or -S(O)2R group, in which R can be H, optionally substituted alkyl, or optionally substituted aryl. Non-limiting sulfonyl groups can include a trifluorom ethyl sulfonyl group (-SO2-CF3 or Tf).

[0140] Use of the above terms is meant to encompass substituted and unsubstituted moieties. Substitution may be by one or more groups such as alcohols, ethers, esters, amides, sulfones, sulfides, hydroxyl, nitro, cyano, carboxy, amines, heteroatoms, lower alkyl, lower alkoxy, lower alkoxycarbonyl, alkoxyalkoxy, acyloxy, halogens, trifluoromethoxy, trifluoromethyl, alkyl, aralkyl, alkenyl, alkynyl, aryl, cyano, carboxy, carboalkoxy, carboxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, alkylheterocyclyl, heterocyclylalkyl, oxo, arylsulfonyl and aralkyaminocarbonyl, or any of the substituents of the preceding paragraphs or any of those substituents either directly attached or by suitable linkers. The linkers are typically short chainsof 1-3 atoms containing any combination of -C-, -C(O)-, -NH-, -S-, -S(O)-, -O-, -C(O)- or - S(O)O. Rings may be substituted multiple times.

[0141] The term “lower” modifying “alkyl”, “alkenyl”, “alkynyl”, “alkoxy” or “alkoxycarbonyl” refers to a Ci-Ce unit for a particular functionality. For example, “lower alkyl” means Ci-Ce alkyl.

[0142] By “ substituted” is meant having one or more substituent moieties whose presence does not interfere with the desired function or reactivity. Examples of substituents alkyl, alkenyl, alkynyl, cycloalkyl (non-aromatic ring), Si(alkyl)s, Si(alkoxy)3, alkoxy, amino, alkylamino, alkenylamino, amide, amidine, guanidine, hydroxyl, thioether, alkylcarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, carbonate, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphate ester, phosphonato, cyano, halo, acylamino, imino, sulfhydryl, alkylthio, thiocarboxylate, dithiocarb oxy late, sulfate, sulfato, sulfonate, sulfamoyl, sulfonamide, nitro, nitrile, azido, heterocyclyl, ether, ester, silicon-containing moieties, thioester or a combination thereof. The substituents may themselves be substituted. For instance, an amino substituent may itself be mono or independently disubstituted by further substituents defined above, such as alkyl, alkenyl, alkynyl, and cycloalkyl (non-aromatic ring).

[0143] By “sulfide” is meant a thioether -S-R’, where R’ may be, but is not limited to, aliphatic groups.

[0144] By “sulfhydryl” is meant a thiol i.e. -SH.

[0145] By “thiocyanato” is meant -SCN.

[0146] By “thioester” is meant -SC(O)R’, where R’ may be, but is not limited to, aliphatic groups.

[0147] By “unsubstituted” is meant any open valence of an atom being occupied by hydrogen. Also, if an occupant of an open valence position on an atom is not specified, then it is hydrogen.

[0148] By “unsaturated” is meant a moiety that contains double or triple carbon-carbon bonds.

[0149] By “unsaturated substituent” is meant a double or triple bond containing aliphatic chain, cyclic, aryl or heteroaryl group.

[0150] A person of ordinary skill in the art would recognize that the definitions provided above are not intended to include impermissible substitution patterns (e.g. methyl substituted with five substituents and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0151] As used herein, mass tag includes any tag that includes an enriched heavy atom, such as an enriched metal isotope. Mass tags may include a polymer loaded with the enriched metal isotope and may optionally include a conjugated biologically active material. Mass tags may be distinguishable based on the atomic mass of their enriched metal isotope.

[0152] As used herein, mass cytometry is any method of detecting mass tags in a biological sample, such as simultaneously detecting a plurality of distinguishable mass tags with single cell resolution. Mass cytometry includes suspension mass cytometry and Imaging Mass Cytometry™ (IMC™) and Secondary Ion Mass Spectrometry (SIMS). Mass cytometry may atomize and ionize mass tags of a cellular sample by one or more of laser radiation, ion beam radiation, electron beam radiation, and / or inductively coupled plasma (ICP). Mass cytometry may simultaneously detect distinct mass tags from single cells, such as by time of flight (TOF) or magnetic sector mass spectrometry (MS). As used herein, DBCO-NHS refers to (2,5-dioxopyrrolidin-l-yl) 4-(2- azatricyclo[10.4.0.04’9]hexadeca-l(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

[0153] As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies , antibody fragments ( such as Fab , Fab ', F ( ab ' ) 2 , and Fv fragments), single chain Fv ( scFv ) mutants, multi-specific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion ofan antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins : IgA, IgD, IgE, IgG, and IgM, or subclasses ( isotypes ) thereof ( e.g. IgGl, IgG2 IgG3, IgG4, IgAl and IgA2 ), based on the identity of their heavy-chain constant domains referred to as alpha , delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well- known subunit structures and three- dimensional configurations.

[0154] A “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full - length monoclonal antibodies as well as antibody fragments ( such as Fab , Fab ' , F ( ab ' ) 2 , Fv ) , single chain ( scFv ) mutants , fusion proteins comprising an antibody portion , and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0155] The term “humanized antibody” refers to an antibody derived from a non - human (e.g., murine ) immunoglobulin , which has been engineered to contain minimal non - human (e.g. , murine ) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability.

[0156] The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least onehuman heavy and / or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0157] The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0158] As used herein, the term “chemical antibody” encompasses aptamers (e.g., protein aptamers or nucleic acid aptamers). One of skill in the art readily appreciates that a chemical antibody can be substituted for an antibody in any embodiments described herein.

[0159] The term “protein” refers to small proteins less than 100 amino acids long and large biomolecules or macromolecules that have one or more long chains of amino acid residues including globular, fibrous or membrane proteins such as antibodies.

[0160] The term “chelator” refers to a moiety that can coordinate (e.g., stably coordinate) a metal atom.Introduction and Context

[0161] Conventional detection reagents for biological assays frequently consist of a binding moiety having specificity for the molecule of interest, conjugated to a moiety with enzymatic or optical properties. To date, these determinations are generally facilitated through the use of radiological, fluorescent or enzymatic tags. Among methods of interest for analysis, flow cytometry provides the means for simultaneous multiparametric analysis of the physical and / or chemical characteristics of up to thousands of particles per second and is routinely used for research and clinical diagnostic applications, including both particle analysis and particle sorting. The analysis of cells is of particular interest. Modem instruments usually havemultiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for simultaneous analysis of multiple labeled antibodies and can more precisely identify a target population by their phenotypic markers. In traditional flow cytometry, fluorescently labeled particles such as live cells, fixed cells, beads, etc. are individually distinguished and separated based on their fluorescence and light scatter characteristics. The phenotype of the particles can be further investigated after they are isolated.

[0162] Such traditional flow cytometry methods are limited by the number of simultaneous parameters that can be measured on a single particle, and there are problems with overlap of fluorescence emissions during simultaneous measurement; and background fluorescence or enzymatic activity. As the number of simultaneous parameters increases, this spectral overlap severely convolutes analysis impinging on both the accuracy as well as sensitivity of the assay. In alternative methods of detection, atomic mass spectrometry measurements have been used in conjunction with stable isotope tags of rare elements.

[0163] Existing elemental tagging capture reagents for use in ICP-MS are based on chelators, such as ethylenediamine tetra-acetic acid (EDTA), tetraazacyclododecanetetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), for example a maleimide functionalized polymer of DOTA, with an average length of between 10 and 30 monomers. Such protocols allow conjugation to a typical antibody of 6 or 7 polymers, thereby conjugating an average of 200 tagging isotope atoms per antibody. The sensitivity of this method is directly related to the number of elemental isotope tags per detection reagent molecule. The number of polymers that can be attached is limited to the number of disulfide bonds that can be broken on the immunoglobulin without disrupting its function. The number of metal chelating units that can be conjugated to a detection reagent is also limited because increased numbers can interfere with the detection reagent or induce nonspecific interactions and thus interfering or inducing high background in an assay.Metal-Protein Conjugates

[0164] The metal-protein conjugates may be composed of a number of different metals including mercury (Hg), lead (Pb), platinum (Pt), antimony (Sb) or arsenic (As) in some embodiments. As used herein, the term “conjugate” refers to a metal-containing compound such as a coordination complex. In some embodiments, the metal is a thiophilic metal. The proteins may be antibodies in some embodiments, and the metal and the protein may be joined together by a variety of suitable conjugation means. For example, a metal may be conjugated to a protein through covalent binding (e.g., amine chemistry, thiol chemistry, phosphate chemistry), an enzymatic reaction, a redox reaction (such as with a metal halide), and affinity intermediate (e.g., streptavidin or biotin), or a form of click chemistry (such as strain promoted click chemistry or metal-catalyzed click chemistry).

[0165] As used herein, “click chemistry”, “click chemistry reaction” or “click reaction” refers to a reaction designed to have a high thermodynamic driving force that drives a reaction quickly and irreversibly to a high yield of a single reaction product, with high reaction specificity (in some cases, with both regio- and stereospecificity). The reactions typically produce few, if any, toxic byproducts, and many are amenable to performance at physiological conditions, that is to say in an aqueous buffer, at approximately neutral pH and tolerant of typical buffer salts, with the resultant product also being stable under physiological conditions. Those reactions which do not strictly meet these criteria are nonetheless characterized by their high yield, simplicity and ability to be performed in benign or easily removed solvents. Of additional importance is that the molecular reactions are bio- orthogonal, which is to say they proceed without interacting with any of the functionalities typically seen in biological systems.

[0166] In some embodiments, the metal and protein are directly joined together, as when the protein is appropriately functionalized or derivatized to includemoi eties which may chelate the metal. In other embodiments, the metal may be chelated by a chelating agent attached to the protein or a derivatized protein through a linking moiety. These metal-protein conjugates are useful for mass cytometry applications in certain embodiments.

[0167] Suitable conjugates include a metal atom, a sulfhydryl-containing moiety and a protein wherein the protein is conjugated to the sulfhydryl-containing moiety through a bond or through a linking moiety. The linking moiety comprising a structure of Formula (I):-LkA-L2- (I), where L1and L2are linking groups. As used herein, “linking groups” refers to a compound which can bind to two moieties. In some embodiments, L1is a first linking group which may be an alkyl carbonyl, amide, amine, ether, or ester, and L1may be covalently bound to the protein; L2is a second linking group including a nitrogen-containing moiety, and L2may be covalently bound to the sulfhydryl-containing moiety; and A is a cyclic group; and wherein the metal atom chelates the sulfhydryl-containing moiety.Metal- Protein Conjugates Attached Through Linking Moieties

[0168] A metal can be indirectly connected to a protein such as an antibody through an intermediary linking moiety. Selection of an appropriate linking moiety is governed by its ability to maximize metal-intermediate bonding while minimizing cross-reaction, which may include but is not limited to polymer repeats that could form bonds with metals or metalbonding agents. The synthetic route to the metal-antibody conjugate will depend upon the type of linking moiety to be utilized.

[0169] In one embodiment, a method for making a conjugate includes a three-step process in some embodiments. The first step of the method is to prepare 2,3 -dimercaptopropane- 1- sulfonyl azide (DMPS-A) from 2, 3 -dimercaptopropane- 1 -sulfonic acid (DMPS, compound 1).

[0170] One example of this preparation is illustrated in Scheme 1 below.Scheme 1

[0171] Following the synthesis and purification of DMPS-A, mercury chelation may be performed as the second step in the method to prepare a metal-protein conjugate. The metalchelator complex may be formed at neutral pH for stability purposes and to avoid formation of insoluble hydroxides that may occur under alkaline conditions. In one embodiment, the complex may be formed by dissolving Hg(NOs)2 in a small quantity of dilute nitric acid and 5mM Hg2+may be added to 50mM 4-(2 -hydroxy ethyl)- 1 -piperazineethane sulfonic acid (HEPES) buffer (pH 7.0). The DMPS-A from the first step may be added to the Hg- containing buffer in a 1 : 1 molar ratio (5 mM) and incubated at room temperature for 1 minute prior to use.

[0172] A DMPS-Hg complex prepared in accordance with the second step may have a ratio of DMPS to Hg of 1 : 1, 2: 1 or 4: 1 in some embodiments.

[0173] In the third step, the metal loaded DMPS chelator can be covalently conjugated to a protein such as an antibody by a DBCO-azide click chemistry reaction, as shown in Scheme 2 below.DBCO-deriyatised Azi d e:res i d u a lis.iiig DBC O-deriyatised antibody DMPS peptide antibodyScheme 2

[0174] In Scheme 2, the middle structure labelled as an azide-residualizing DMPS peptide is an azide-residualsing metal loaded DMPS chelator; while the structure on the right labelled as a DBCO-derivitised antibody is a metal tagged antibody.

[0175] Other alkynes which may be utilized to derivatize the antibody include monofluorinated cyclooctyne, difluorocyclooctyne, dimethoxyazacyclooctyne, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, bicyclononyne, 2, 3,6,7- tetramethoxy dibenzocyclooctyne, sulfonylated dibenzocyclooctyne, carboxymethylmonobenzocyclooctyne or pyrrolocyclooctyne.

[0176] In another embodiment, the metal-chelated protein conjugate may be prepared with lipoic acid. Lipoic acid is a mercury chelator. In some embodiments, a lipoic acid-Hg complex may have a 1 : 1 configuration, as seen below:complex 1

[0177] Complex 1 may be attached to an antibody using a coupling agent which is capable of linking the metal loaded chelator (complex 1) to the antibody. Suitable coupling agents may be carbodiimides such as di cyclohexylcarbodiimide or l-ethyl-3-(-3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); or phosphonium salts such as benzotri azole- l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP). In some embodiments, the coupling agent is an amide coupling agent such as 4-(4,6-dimethoxy- l,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (DMTMM).

[0178] An alternative method to form a metal-chelated protein conjugate with lipoic acid includes use of a functionalized polymer. For example, lipoic acid could be covalently bound to an azide-terminated, poly(y-benzyl L-glutamate (PBLG) -based polymer backbone. The polymer’s backbone may be prepared by aminolysis with ethylenediamine or 1,6- hexanediamine to introduce reactive amine groups. Subsequent functionalization of 50% of the polymer’s repeat units with a polyethylene glycol succinimidyl ester such as PEG24-NHS ester would yield a functionalized polymer of good water solubility. Lipoic acid would then be attached to the remaining polymer repeat units. Then the polymer would be loaded with metal, and the polymeric azide end-group would allow attachment to an antibody which has been treated with NHS-DBCO; thus forming the metal-chelated protein conjugate.

[0179] In another embodiment, the method to prepare a metal-chelated protein conjugate such as a mercury-antibody conjugate involves conjugation of an azide-containing chelator toan antibody prior to chelation of mercury. This technique may avoid cross-reactivity of azides that could occur in the chelator complex. This method includes the following steps in certain embodiments: (1) incubation of antibodies with glutathione to cap free thiol groups, (2) conjugation of DBCO-modified antibodies with DMPS-A using a DBCO-azide click reaction, (3) chelation of Hg using the chelator-antibody complex.Directly-Bound Metal-Protein Conjugates

[0180] In some embodiments, a metal chelation site or sites may be introduced directly onto an antibody, allowing direct labeling of antibodies with metal ions. Disulfide bonds (also referred to as disulfide bridges) may be found for example in the hinge region of an antibody. The native disulfide bridge may be reduced to two sulfhydryls using a reducing agent, such as dithiothreitol (DTT), or tris-2- carboxyethyl phosphine (TCEP). The proximity of the two thiol groups allows facile metal chelation.

[0181] In some embodiments, when the antibody does not include native disulfide bonds, they may be appended to the antibody.Kits and Kit Components

[0182] Aspects of the subject application include making a kit as discussed herein, or a portion thereof. Aspects of the subject application include use of a kit described herein, such as for mass cytometry or delivery of a radioactive isotope.

[0183] Kits, components of kits, and steps of making kits may include suitable storage mediums. For example, solvents and co-solubilizing agents may include, but are not limited to, water; sterile water for injection (SWFI); physiological saline; alcohols, e.g. ethanol, benzyl alcohol and the like; glycols and polyalcohols, e.g. propylene glycol, glycerin and the like; esters of polyalcohols, e.g. diacetin, triacetin and the like; polyglycols and polyethers, e.g. polyethylene glycol 400, propylene glycol methyl ethers and the like; dioxolanes, e.g. isopropylidene glycerol and the like; dimethyl isosorbide; pyrrolidone derivatives, e.g. 2- pyrrolidone, N-methyl-2-pyrrolidone, polyvinylpyrrolidone (cosolubilizing agent only) and the like; polyoxyethylenated fatty alcohols; esters ofpoly oxy ethyl enated fatty acids; polysorbates, e.g., TWEEN, polyoxyethylene derivatives of polypropylene glycols, e.g., PLURONICS.

[0184] Suitable stabilizing agents include, but are not limited to, one or more monosaccharides (e.g., galactose, fructose, and fucose), disaccharides (e.g., lactose), polysaccharides (e.g., dextran), cyclic oligosaccharides (e.g., alpha-, beta-, gammacyclodextrin), aliphatic polyols (e.g., mannitol, sorbitol, and thioglycerol), cyclic polyols (e.g. inositol), organic solvents (e.g., ethyl alcohol and glycerol) and / or aprotic solvents (pyridine, ethyl acetate, DMF, HMPA, and DMSO). The above solvent and / or stabilizing agents may be used in any step of the synthetic methods described above, or storage of any of the above reagents (e.g., to provide in a kit).

[0185] In certain aspects, a solution may be acidic. An acidic solution of the subject application may include a strong acid such as one or more of nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, hydrochloric acid, and chloric acid. The acid may be present at more than 0.01% (such as more than 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, or 5%) and / or less than 10% (such as less than 5%, 2%, 1%, 0.5%, 0.2%, or 0.1%). For example, the acid may be present at 0.05% to 2%. An acidic solution may have a pH at or below 6, at or below 5, at or below 4.5, or at or below 4. Lyophilized compositions of the subject application may have less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% moisture content (by weight).

[0186] At any step (e.g., when providing a conjugate discussed herein in a kit), the conjugate may be lyophilized. For example, the conjugate may be lyophilized with less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% moisture content (e.g., by mass). Such lyophilization may allow for storage in a kit prior to use in mass cytometry, and / or may allow for flexibility of assay design when lyophilization stabilizes the conjugate.

[0187] Kits may further include any additional components (e.g., buffers, filters, etc.)

[0188] Alternatively, or in addition, kits may include additional reagents for mass cytometry such as buffers, standards, cell barcodes, and / or reagents including heavy atoms of different masses (e.g., mass tags attached to biologically active materials, or provided for attachment to biologically active materials).

[0189] In certain aspects, the kit may include a plurality of antibodies (e.g., to different targets). Such a collection of antibodies may be provided together in a single panel. A panel may be provided in solution, or in a lyophilized mixture including less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% moisture by mass. CONCLUSION

[0190] Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing the processes, systems and apparatus of the present embodiments. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the embodiments are not to be limited to the details given herein.

Claims

CLAIMSWhat is claimed is:

1. A conjugate comprising: a metal atom; a sulfhydryl-containing moiety; and a protein; wherein the protein is conjugated to the sulfhydryl-containing moiety through a bond or through a linking moiety, the linking moiety comprising a structure of Formula (I):-LkA-L2- (I), wherein L1is a first linking group comprising an alkyl carbonyl, amide, amine, ether, or ester, and L1is covalently bound to the protein;L2is a second linking group comprising a nitrogen-containing moiety, and L2is covalently bound to the sulfhydryl-containing moiety; andA is a cyclic group; and wherein the metal atom chelates the sulfhydryl-containing moiety.

2. The conjugate of claim 1, wherein the sulfhydryl-containing moiety comprises a thiol, a dithiol, a cysteine or an N-acetylcysteine group.

3. The conjugate of claim 2, wherein the dithiol comprises a 1,2-dithiol, a 1 ,3-dithiol or a1,4-dithiol.

4. The conjugate of claim 2, wherein the dithiol comprises lipoic acid.

5. The conjugate of claim 1, wherein the metal atom comprises Hg, Pb Pt, Sb or As.

6. The conjugate of claim 2, wherein the dithiol chelates the metal atom.

7. The conjugate of claim 2, wherein two dithiols chelate one metal atom.

8. The conjugate of claim 2, wherein four dithiols chelate one metal atom.

9. The conjugate of claim 1, wherein the protein comprises an antibody.

10. The conjugate of claim 9, wherein the antibody comprises a monoclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a human antibody or a humanized antibody.

11. The conjugate of claim 9, wherein the antibody comprises a dibenzocyclooctyne- derivatized antibody.

12. The conjugate of claim 1, wherein the nitrogen-containing moiety comprises amino, azido or amido.

13. A method of forming a metal-chelated protein conjugate for mass cytometry comprising: reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelator; chelating a metal with the dithiol-containing sulfonyl azide chelator to form an azide- containing metal-chelator complex; reacting a protein with a reagent to form an alkyne-containing protein derivative; and conjugating the azide-containing metal-chelator complex to the alkyne-containing protein derivative to form a metal-chelated protein conjugate.

14. The method of claim 13, wherein the metal comprises Hg, Pb, Pt, Sb or As.

15. The method of claim 13, wherein the protein comprises an antibody.

16. A method of forming a metal-chelated chemical antibody conjugate for mass cytometry comprising: reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelator; chelating a metal with the dithiol-containing sulfonyl azide chelator to form an azide- containing metal-chelator complex, wherein the metal comprises Hg, Pb, Pt, Sb orAs; reacting a chemical antibody with a reagent to form an alkyne-containing chemical antibody derivative; and conjugating the azide-containing metal -chelator complex to the alkyne-containing chemical antibody derivative to form a metal-chelated chemical antibody conjugate.

17. The method of claim 16, wherein the chemical antibody comprises an aptamer.

18. The method of claim 13, wherein conjugating the azide-containing metal-chelator complex to the alkyne-containing protein derivative comprises reacting an alkyne of the alkyne- containing protein derivative with an azide of the azide-containing metal-chelator complex to form a triazole.

19. The method of claim 13, wherein the dithiol-containing sulfonic acid comprises 2,3- dimercaptopropane 1 -sulfonic acid.

20. The method of claim 13, wherein the reagent comprises DBCO-NHS.

21. A method of forming a metal-chelated protein conjugate for mass cytometry comprising:chelating a metal with lipoic acid to form a metal-chelator complex; and covalently bonding the metal -chelator complex to a protein with a coupling agent.

22. The method of claim 21, wherein the metal comprises Hg, Pb, Pt, Sb or As.

23. The method of claim 21, wherein the protein comprises an antibody.

24. The method of claim 21, wherein the coupling agent comprises an amide-containing coupling agent.

25. The method of claim 24, wherein the coupling agent comprises 4-(4,6-dimethoxy-l,3,5- triazin-2-yl)-4-methylmorpholin-4-ium chloride.

26. A method of forming a metal-chelated protein conjugate for mass cytometry comprising: reacting a poly(amino acid) with ethylene diamine to form an amine-containing poly(amino acid); functionalizing the amine-containing poly(amino acid) with a polyethylene glycol succinimidyl ester to form a functionalized poly(amino acid); covalently binding lipoic acid to the functionalized poly(amino acid) to form a lipoic acid-modified and functionalized poly(amino acid); chelating a metal to the lipoic acid-modified and functionalized poly(amino acid) to form a metal-chelated lipoic acid-modified and functionalized poly(amino acid); reacting a protein with a reagent to form an alkyne-containing protein derivative; and conjugating the metal-chelated lipoic acid-modified and functionalized poly(amino acid) with the alkyne-containing protein derivative to form a metal-chelated protein conjugate.

27. The method of claim 26, wherein the metal comprises Hg, Pb, Pt, Sb or As.

28. The method of claim 26, wherein the protein comprises an antibody.

29. The method of claim 26, wherein the poly(amino acid) is poly(y-benzyl a,L-glutamate).

30. The method of claim 26, wherein the reagent is (2,5-dioxopyrrolidin-l-yl) 4-(2- azatricyclo[10.4.0.04’9]hexadeca-l(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

31. A method of forming a metal-chelated protein conjugate for mass cytometry comprising: reacting a protein including thiol groups with a reactant to form a modified protein; reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelator; reacting the modified protein with a reagent to form an alkyne-containing protein derivative; conjugating the alkyne-containing protein derivative with the dithiol-containing sulfonyl azide chelator to form a protein-chelator conjugate; and chelating a metal with the protein-chelator conjugate.

32. The method of claim 31, wherein the metal comprises Hg, Pb, Pt, Sb or As.

33. The method of claim 31, wherein the protein comprises an antibody.

34. The method of claim 31, wherein the reactant comprises glutathione.

35. The method of claim 31, wherein the reagent comprises DBCO-NHS.

36. A method for forming a metal-chelated protein comprising: providing a protein comprising at least one disulfide moiety;reducing the at least one disulfide moiety with a reducing agent to form a dithiol- containing protein; and complexing a metal to the dithiol-containing protein.

37. The method of claim 36, wherein the metal comprises Hg, Pb, Pt, Sb or As.

38. The method of claim 36, wherein the protein comprises an antibody.

39. The method of claim 36, wherein the reducing agent comprises dithiothreitol, 2- mercaptoethanol, 2-mercaptothylamine or tris-(2-carboxy ethyl) phosphine.

40. A method for forming a metal-chelated protein comprising: introducing sulfhydryl groups to a protein by reacting the protein with a reagent to form a thiol-containing protein; and chelating a metal to the thiol-containing protein.

41. The method of claim 40, wherein the metal comprises Hg, Pb, Pt, Sb or As.

42. The method of claim 40, wherein the protein comprises an antibody.

43. The method of claim 42, wherein the antibody comprises a chemical antibody.

44. The method of claim 43, wherein the chemical antibody comprises an aptamer.

45. The method of claim 40, wherein introducing sulfhydryl groups comprises reacting active sites on the protein with the reagent.

46. The method of claim 45, wherein the active sites comprise amines.

47. The method of claim 40, wherein the reagent comprises 2-iminothiolane.

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