Dithiol Chelators for Metal Conjugation to Antibodies

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 384,829, filed November 23, 2022, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] In mass cytometry, cells are labeled with mass-tagged bioactive agents (eg, antibodies or oligonucleotides), which can be detected by mass spectrometry with single-cell resolution.

[0003] These mass tags are generally lanthanide chelating polymers carrying enriched lanthanide isotopes for conjugation to antibodies. One drawback of lanthanides for antibody conjugation is that proteins, such as bovine serum albumin (BSA, a common buffer for purified antibodies), can bind to the lanthanides, resulting in failure of antibody conjugation due to lanthanide adsorption.

[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 substances that can be identified is determined by the number of isotopes with different masses. The development of additional mass tags, especially those conjugated with other metals such as mercury, will enable the number and variety of targets that can be detected in mass cytometry applications to be expanded, and will avoid undesirable interactions with other components of antibody formulations, such as buffers or stabilizers containing glycerol.

[0005] The description of the background art provided herein is for purposes of generally presenting the context of the present disclosure. The work of the presently named inventors, to the extent described in this background art section, as well as aspects of the present disclosure that may not qualify as prior art at the time of filing, are not admitted expressly or impliedly as prior art to the present disclosure.

[0006] Technical Field The present disclosure relates to reagents and their uses for elemental mass spectrometry, including imaging mass spectrometry, of biological samples. Summary of the Invention

[0007] The present disclosure provides metalloprotein conjugates for use in mass cytometry and methods for producing the same. The metalloprotein conjugates may have a metal bound directly to the protein or may have a metal bound to the protein via a linking moiety. Metalloprotein conjugates include mercury-chelating triazole-containing antibody complexes and methods for their formation from derivatized antibodies and sulfonyl azides.

[0008] Thus, in a first aspect, the present disclosure encompasses a conjugate. In some embodiments, the conjugate comprises a metal atom; a sulfhydryl-containing moiety; and a protein; the protein is conjugated to the sulfhydryl-containing moiety via a bond or via a linking moiety, the linking moiety having the formula (I): -L 1 -AL 2 - (I) (In the formula, L 1 is a first linking group comprising an alkylcarbonyl, an amide, an amine, an ether, or an ester; L 1 is covalently bound to the protein; L 2 is a second linking group containing a nitrogen-containing moiety, and L 2 is covalently linked to the sulfhydryl-containing moiety; and A is a cyclic group. having the structure The metal atom is chelated to the sulfhydryl-containing moiety.

[0009] In some embodiments, the sulfhydryl-containing moiety is a thiol, dithiol, cysteine, or 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 to a metal atom.

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

[0015] In some embodiments, four dithiols chelate to 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 and 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 for forming a metal chelate chemistry antibody conjugate for mass cytometry.In some embodiments, the method comprises the steps of: 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 the chemistry antibody with a reagent to form an alkyne-containing chemistry antibody derivative; and conjugating the azide-containing metal chelator complex to the alkyne-containing chemistry antibody derivative to form a metal chelate chemistry antibody conjugate.

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

[0024] In a third aspect, the present disclosure encompasses a method for forming a metal chelating protein conjugate for mass cytometry. In some embodiments, the method includes the steps of 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 chelating 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 comprises reacting the alkyne of the alkyne-containing protein derivative with the azide of the azide-containing metal chelator complex to form a triazole.

[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 includes a method for forming a metal chelating 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 binding the metal chelator complex to a protein using 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 and 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-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride.

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

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

[0043] In some embodiments, the reagent is (2,5-dioxopyrrolidin-1-yl)4-(2-azatricyclo[10.4.0.0 4,9 ]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

[0044] In a sixth aspect, the present disclosure encompasses a method for forming a metal-chelating protein conjugate for mass cytometry. In some embodiments, the method includes the steps of reacting a protein containing a thiol group 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 and 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 includes a method for forming a metal-chelating protein. In some embodiments, the method includes the steps of providing a protein having at least one disulfide moiety; reacting the at least one disulfide moiety with a reducing agent to form a dithiol-containing protein; and complexing a metal with 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 and can include one or more modifications.

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

[0057] In an eighth aspect, the present disclosure includes a method for forming a metal-chelating protein. In some embodiments, the method includes the steps of introducing a sulfhydryl group into the 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 and can include one or more modifications.

[0062] In some embodiments, introducing a sulfhydryl group comprises reacting an active site of the protein with a reagent.

[0063] In some embodiments, the active site is an amine group.

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

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

[0066] definition As used herein, the term "about" is understood to describe small increases and / or decreases beyond the recited value, where the change does not significantly affect the desired function of the parameter beyond the recited value. In some cases, "about" encompasses + / - 10% of any recited value. As used herein, the term modifies any recited value, range of values, or one or more end points of a range.

[0067] The terms "acyl" or "alkanoyl," as used interchangeably herein, refer to an alkyl group, as defined herein, or a hydrogen attached to a parent molecular group, as defined herein, through a carbonyl group. This group is exemplified by formyl, acetyl, propionyl, butanoyl, and the like. Alkanoyl groups can be substituted or unsubstituted. For example, alkanoyl groups can be substituted with one or more of the substituents described herein for alkyl. In some embodiments, an unsubstituted acyl group is C 2-7 It is an acyl or alkanoyl group. In certain embodiments, the alkanoyl group is -C(O)-Ak, where Ak is an alkyl group as defined herein.

[0068] "Aliphatic" means a group consisting of at least 1 carbon atom and at least 50 carbon atoms (C 1-50 ), e.g., 1 to 25 carbon atoms (C 1-25 ), or 1 to 10 carbon atoms (C 1-10 ) and includes saturated groups such as alkanes (or alkyls) and unsaturated groups such as alkenes (or alkenyls), alkynes (or alkynyls), and cyclic versions thereof, as well as straight and branched chain configurations and all stereoisomers and positional isomers. Such hydrocarbons can be unsubstituted or substituted with one or more groups, such as halogens or those described herein for alkyl groups.

[0069] "Alkenyl" means an optionally substituted C alkyl group having one or more double bonds. 2-24 The alkenyl group is a cyclic group (e.g., C 3-24 Alkenyl groups can be substituted or unsubstituted. For example, alkenyl groups can be substituted with one or more of the substituents described herein for alkyl. Non-limiting unsubstituted alkenyl groups include C 2-8 Alkenyl, C 2-6 Alkenyl, C 2-5 Alkenyl, C 2-4Alkenyl, or C 2-3 Representative non-limiting alkenyl groups include vinyl or ethenyl (-CH=CH), 1-propenyl (-CH=CHCH), allyl or 2-propenyl (-CH-CH=CH), 1-butenyl (-CH=CHCHCH), 2-butenyl (-CHCH=CHCH), 3-butenyl (e.g., -CHCHCH=CH), 2-butenylidene (e.g., =CH-CH=CHCH), and the like.

[0070] "Alkenylene" means a polyvalent (e.g., divalent) form of the alkenyl group, optionally substituted C 2-24 Alkenylene groups are cyclic (e.g., C 3-24 The alkenylene group may be substituted or unsubstituted. For example, the alkenylene group may be substituted with one or more substituents described herein for alkyl. Representative, non-limiting alkenylene groups include -CH=CH- or -CH=CHCH2-.

[0071] "Alkoxy" means -OR, where R is an optionally substituted alkyl group as described herein. Representative alkoxy groups include methoxy, ethoxy, butoxy, trihaloalkoxy, such as trifluoromethoxy, and the like. Alkoxy groups can be substituted or unsubstituted. For example, an alkoxy group can be substituted with one or more of the substituents described herein for alkyl. Representative unsubstituted alkoxy groups include C 1-3 , C 1-6 , C 1-12 , C 1-16 , C 1-18 , C 1-20 , or C 1-24 Alkoxy groups are included.

[0072] "Alkoxyalkyl" means an alkyl group, as defined herein, substituted with an alkoxy group, as defined herein. Representative unsubstituted alkoxyalkyl groups include those containing 2 to 12 carbon atoms (C 2-12 alkoxyalkyl), and those having an alkyl group having 1 to 6 carbons and an alkoxy group having 1 to 6 carbons (i.e., C 1-6 Alkoxy-C 1-6 alkyl).

[0073] The terms "alkyl" and "alk" refer to 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. An alkyl group can be cyclic (e.g., C 3-24 The alkyl group may be a cyclic or non-cyclic alkyl group (cycloalkyl). The alkyl group may be branched or unbranched. The alkyl group may also be substituted or unsubstituted. For example, the alkyl group may include a haloalkyl, where the alkyl group is substituted with one or more halo groups as described herein. In another example, the alkyl group is: (1) C 1-6 Alkoxy (e.g., —O-Ak, where Ak is an optionally substituted C 1-6 (2) amino (e.g., -NR N1 R N2 (In the formula, R N1 and R N2 are each independently H or optionally substituted alkyl, or R N1 and R N2(3) aryl; (4) arylalkoxy (e.g., -O-Lk-Ar, where Lk is a divalent form of an optionally substituted alkyl and Ar is an optionally substituted aryl); (5) aryloyl (e.g., -C(O)-Ar, where Ar is an optionally substituted aryl); (6) cyano (e.g., -CN); (7) carboxaldehyde (e.g., -C(O)H); (8) carboxyl (e.g., -COH); (9) C 3-8 Cycloalkyl (e.g., monovalent saturated or unsaturated non-aromatic cyclic C 3-8 (10) halo (e.g., F, Cl, Br, or I); (11) heterocyclyl (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring containing 1, 2, 3, or 4 non-carbon heteroatoms, e.g., nitrogen, oxygen, phosphorus, sulfur, or halo, unless otherwise specified); (12) heterocyclyloxy (e.g., -O-Het, where Het is heterocyclyl as described herein); (13) heterocyclyloyl (e.g., -C(O)-Het, where Het is heterocyclyl as described herein); (14) hydroxyl (e.g., -OH); (15) N-protected amino; (16) nitro (e.g., -NO); (17) oxo (e.g., =O); (18) -COR A (In the formula, R A is (a)C 1-6 Alkyl, (b) C 4-18 aryl, and (c) (C 4-18 Aryl)C 1-6 alkyl (e.g., selected from the group consisting of -Lk-Ar, where Lk is a divalent form of an optionally substituted alkyl group and Ar is an optionally substituted aryl); (19) -C(O)NR B R C (In the formula, R B and R C are each independently (a) hydrogen, (b) C 1-6 Alkyl, (c) C 4-18 aryl, and (d) (C 4-18 Aryl)C1-6 alkyl (e.g., selected from the group consisting of -Lk-Ar, where Lk is a divalent form of an optionally substituted alkyl group and Ar is an optionally substituted aryl); and (20) -NR G R H (In the formula, R G and R H are each independently (a) hydrogen, (b) an N-protecting group, or (c) C 1-6 Alkyl, (d) C 2-6 alkenyl (e.g., optionally substituted alkyl having one or more double bonds); (e) C 2-6 Alkynyl (e.g., optionally substituted alkyl having one or more triple bonds), (f) C 4-18 Aryl, (g) (C 4-18 Aryl)C 1-6 alkyl (e.g., Lk-Ar, where Lk is a divalent form of an optionally substituted alkyl group and Ar is an optionally substituted aryl); (h) C 3-8 cycloalkyl, and (i) (C 3-8 Cycloalkyl)C 1-6 The alkyl group may be substituted with one, two, three, or, in the case of alkyl groups of two or more carbons, four substituents independently selected from the group consisting of alkyl (e.g., -Lk-Cy, where Lk is a divalent form of an optionally substituted alkyl group and Cy is an optionally substituted cycloalkyl as described herein), and in one embodiment, two groups are not attached to the nitrogen atom via a carbonyl group. The alkyl group may 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 selected from the group consisting of C 1-2 , C 1-3 , C 1-6 , C 1-12 , C 1-16 , C 1-18 , C 1-20 , C 1-24、 C 2-3 , C 2-6 , C2-12 , C 2-16 , C 2-18 , C 2-20 , or C 2-24 It is an alkyl group.

[0074] "Alkylene" refers to the polyvalent (e.g., divalent) forms of the alkyl groups described herein. Representative alkylene groups include methylene, ethylene, propylene, butylene, and the like. In some embodiments, an alkylene group is a C 1-3 , C 1-6 , C 1-12 , C 1-16 , C 1-18 , C 1-20 , C 1-24 , C 2-3 , C 2-6 , C 2-12 , C 2-16 , C 2-18 , C 2-20 , or C 2-24 It is an alkylene group. The alkylene group may be branched or unbranched. The alkylene group may also be substituted or unsubstituted. For example, the alkylene group may be substituted with one or more of the substituents described herein for alkyl.

[0075] "Alkyleneoxy" means an alkylene group, as defined herein, attached to the parent molecular group through an oxygen atom.

[0076] "Alkylcarbonyl" means an alkyl group, as previously defined, appended to the parent molecular moiety through a carbonyl group. Representative non-limiting alkylcarbonyl groups include methylcarbonyl, ethylcarbonyl, and isopropylcarbonyl, among others.

[0077] "Alkynyl" refers to an optionally substituted C alkyl group having one or more triple bonds. 2-24It refers to an alkyl group. Alkynyl groups can be cyclic or acyclic and are exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can also be substituted or unsubstituted. For example, alkynyl groups can be substituted with one or more of the substituents described herein for alkyl. Non-limiting unsubstituted alkynyl groups include C 2-8 Alkynyl, C 2-6 Alkynyl, C 2-5 Alkynyl, C 2-4 Alkynyl, or C 2-3 Representative non-limiting alkynyl groups include ethynyl (-C≡CH), 1-propynyl (-C≡CCH), 2-propynyl or propargyl (-CHC≡CH), 1-butynyl (-C≡CCHCH), 2-butynyl (-CHC≡CCH), 3-butynyl (-CHCHC≡CH), and the like.

[0078] "Alkynylene" means a polyvalent (e.g., divalent) form of the alkynyl group, optionally substituted C 2-24 It is an alkyl group. An alkynylene group may be cyclic or acyclic. An alkynylene group may be substituted or unsubstituted. For example, an alkynylene group may be substituted with one or more substituents described herein for alkyl. Representative non-limiting alkynylene groups include -C≡C- or -C≡CCH2-.

[0079] "Amide" means -N(R N1 )C(O)-(wherein, R N1 means H, optionally substituted alkyl, or optionally substituted aryl).

[0080] "Amino" means -NR N1 R N2 (In the formula, R N1 and R N2are each independently H, optionally substituted alkyl, or optionally substituted acyl, or optionally substituted aryl, or R N1 and R N2 which together with the nitrogen atom to which they are attached form a heterocyclyl group as defined herein.

[0081] "Aminoalkyl" means an alkyl group, as defined herein, substituted with an amino group, as defined herein.

[0082] "Aminoaryl" means an aryl group, as defined herein, substituted with an amino group, as defined herein.

[0083] "Ammonium" refers to the protonated nitrogen atom N + Representative ammonium groups include -N + R N1 R N2 R N3 (In the formula, R N1 , R N2 , and R N3 are each independently H, optionally substituted alkyl, optionally substituted cycloalkyl, or optionally substituted aryl; or R N1 and R N2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl group or heterocycle; or R N1 and R N2 taken together form an optionally substituted alkylene or heteroalkylene (e.g., as described herein); or R N1 and R N2 and R N3 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl group or heterocycle, e.g., a heterocyclic cation).

[0084] "Aromatic," unless otherwise specified, refers to a cyclic, conjugated group or moiety of 5 to 15 ring atoms having a single ring (e.g., phenyl) or multiple fused rings, in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); i.e., at least one ring, and optionally multiple fused rings, have a contiguous, delocalized π-electron system. Typically, the number of out-of-plane π-electrons corresponds to Hückel's rule (4n+2). The point of attachment to the parent structure is typically through the aromatic portion of the fused ring system. Such aromatics may be unsubstituted or substituted with one or more groups, such as those described herein for alkyl or aryl groups. Other substituents may include aliphatic, haloaliphatic, halo, nitrate, cyano, sulfonate, sulfonyl, and the like.

[0085] "Aryl" includes, but is not limited to, phenyl, benzyl, anthracenyl, anthryl, benzocyclobutenyl, dibenzocyclooctenyl, biphenylyl, chrysenyl, dihydroindenyl, fluoranthenyl, indacenyl, indenyl, naphthyl, phenanthryl, phenoxybenzyl, picenyl, pyrenyl, terphenyl, and the like, including fused benzo-C such as indanyl, tetrahydronaphthyl, fluorenyl, and the like. 4-8 It refers to a group containing any carbon-based aromatic group, including a cycloalkyl radical (e.g., as defined herein). The term aryl also includes heteroaryl, which is defined as a group containing an aromatic group having 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. Similarly, the term non-heteroaryl, also included in the term aryl, defines a group containing an aromatic group that does not contain a heteroatom. An aryl group may be substituted or unsubstituted. An aryl group may be substituted with one, two, three, four, or five substituents, such as any of those described herein for alkyl.

[0086] "Arylalkoxy" means 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, where Ak is an optionally substituted alkylene, as defined herein, and Ar is an optionally substituted aryl, as defined herein.

[0087] "(Aryl)(alkyl)ene" refers to a divalent form comprising an arylene group, as described herein, linked to an alkylene or heteroalkylene group, as described herein. In some embodiments, the (aryl)(alkyl)ene group is -L-Ar- or -L-Ar-L- or -Ar-L-, where Ar is an arylene group and L is each independently an optionally substituted alkylene group or an optionally substituted heteroalkylene group.

[0088] "Arylalkylene" refers to an aryl group, as defined herein, attached to the parent molecular group via an alkylene group, as defined herein. In some embodiments, the arylalkylene group is -Ak-Ar, where Ak is an optionally substituted alkylene, as defined herein, 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 substituents described herein for aryl and / or alkyl. Representative unsubstituted arylalkylene groups are those of 7 to 16 carbons (C 7-16 aryl alkylene), and those having an aryl group having 4 to 18 carbons and an alkylene group having 1 to 6 carbons (i.e., (C 4-18 Aryl)C 1-6 alkylene).

[0089] "Arylene" refers to the polyvalent (e.g., divalent, trivalent, tetravalent, etc.) forms of the aryl groups described herein. Representative arylene groups include phenylene, naphthylene, biphenylene, triphenylene, diphenylether, acenaphthenylene, anthrylene, or phenanthrylene. In some embodiments, an arylene group is selected from the group consisting of C 4-18 , C 4-14 , C 4-12 , C 4-10 , C 6-18 , C 6-14 , C 6-12 , or C 6-10 It is an arylene group. The arylene group may be branched or unbranched. The arylene group may also be substituted or unsubstituted. For example, the arylene group may be substituted with one or more of the substituents described herein for aryl.

[0090] "Aryleneoxy" means an arylene group, as defined herein, appended to the parent molecular group through an oxygen atom.

[0091] "Aryloxy" means an aryl group, as defined herein, appended to the parent molecular group through an oxygen atom.

[0092] "Aryloyl" refers to an aryl group that is attached to the parent molecular group through a carbonyl group. In some embodiments, an unsubstituted aryloyl group is C 7-11 Aryloyl or C 5-19 In certain embodiments, an aryloyl group is —C(O)—Ar, where Ar is an aryl group as defined herein.

[0093] "Attaching," "attachment," or related forms of the term means any covalent or non-covalent interaction between two components. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions, halogen bonding, electrostatic interactions, pi-bonding interactions, hydrophobic interactions, inclusion complexes, clathration, van der Waals interactions, and combinations thereof.

[0094] "Azido" refers to -N3.

[0095] "Boranyl" refers to the radical -BR2, where each R can independently be H, halo, or optionally substituted alkyl.

[0096] "Borono" refers to the -BOH2 group.

[0097] "Branched alkenyl" refers to an isomer of a straight-chain alkenyl compound; one that has an alkyl group attached to the main carbon chain.

[0098] "Carbonyl" means the group -C(O)-, which may also be depicted as >C=O.

[0099] "Carboxyl" means a -CO2H group.

[0100] "Carboxylate anion" means -CO2 - means a group.

[0101] "Covalent bond" means a covalent interaction between two components. Non-limiting covalent bonds include single, double, triple, or spirocyclic bonds, where at least two molecular groups are bonded to the same carbon atom.

[0102] "Cyano" means -CN.

[0103] The term "cyclic group" is used herein to refer to either an aryl group, a non-aryl group (e.g., a cycloalkyl group or a heterocycloalkyl group), or both. A cyclic group has one or more ring systems that may 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] "Cycloalkyl," unless otherwise specified, means a monovalent saturated or unsaturated, non-aromatic or aromatic cyclic hydrocarbon group of 3 to 8 carbons, exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, and the like. Cycloalkyl groups can also be substituted or unsubstituted. For example, cycloalkyl groups can be substituted with one or more groups, including those described herein for alkyl.

[0105] "Dicarbonyl" means any moiety or compound containing two carbonyl groups, as defined herein. Non-limiting dicarbonyl moieties include 1,2-dicarbonyl (e.g., R C1 -C(O)-C(O)R C2 (In the formula, R C1 and R C2 are each independently an optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group); 1,3-dicarbonyl (e.g., R C1 -C(O)-C(R 1a R 2a )-C(O)R C2 (In the formula, R C1 and R C2 are each independently an optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group; R 1a and R 2aare each independently H or an optional substituent as defined herein for alkyl); and 1,4-dicarbonyl (e.g., R C1 -C(O)-C(R 1a R 2a )-C(R 3a R 4a )-C(O)R C2 (In the formula, R C1 and R C2 are each independently an optionally substituted alkyl, halo, optionally substituted alkoxy, hydroxyl, or a leaving group; R 1a , R 2a , R 3a , and R 4a are each independently H or an optional substituent provided for alkyl as defined herein.

[0106] By "electron-withdrawing moiety" is meant a moiety that can donate at least a portion of its electron density to a directly attached ring or functional group, eg, by resonance.

[0107] "Halo" means F, Cl, Br, or I.

[0108] "Halo-containing substituent" means a group that contains a halo, for example, a haloaliphatic or haloalkyl group.

[0109] "Haloaliphatic" means an aliphatic group, as defined herein, that is substituted with one or more halo.

[0110] "Haloalkenyl" means an alkenyl group, as defined herein, that is substituted with one or more halo.

[0111] "Haloalkynyl" means an alkynyl group, as defined herein, that is substituted with one or more halo.

[0112] "Haloalkyl" means an alkyl group, as defined herein, that is substituted with one or more halogens. Non-limiting unsubstituted haloalkyl groups include C 1-2 Haloalkyl, C 1-3 Haloalkyl, C 1-4 Haloalkyl, C 1-5 Haloalkyl, C 1-6 Haloalkyl, C 2-3 Haloalkyl, C 2-4 Haloalkyl, C 2-5 Haloalkyl, C 2-6 Haloalkyl, or C 3-6 Other non-limiting haloalkyl groups include -CX y H 3-y wherein y is 1, 2, or 3, and each X is independently halo (F, Cl, Br, or I); -CX z H 2-z CX y H 3-y wherein z is 0, 1, or 2, y is 0, 1, 2, or 3, and each X is independently halo (F, Cl, Br, or I), and at least one of z or y is not 0; -CHCX y H 3-y wherein y is 1, 2, or 3, and each X is independently halo (F, Cl, Br, or I); -CX z1 H 2-z1 CX z2 H 2-z2 CX y H 3-y wherein z1 and z2 are each independently 0, 1, or 2; y is 0, 1, 2, or 3; and X is each independently halo (F, Cl, Br, or I); and at least one of z1, z2, or y is not 0; and -CX z H 1-z [CX y1 H 3-y1 ][CX y2 H 3-y2wherein z is 0 or 1, y1 and y2 are each independently 0, 1, 2, or 3, and X is each independently halo (F, Cl, Br, or I), and at least one of z, y1, or y2 is not 0.

[0113] "Haloalkylene" means an alkylene group, as defined herein, that is substituted with one or more halo.

[0114] "Heteroaliphatic" means an aliphatic group, as defined herein, containing at least 1 heteroatom and up to 20 heteroatoms, e.g., 1 to 15 heteroatoms, or 1 to 5 heteroatoms, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and oxidized forms thereof in the group.

[0115] "Heteroalkyl" means an alkyl group, as defined herein, that contains one, two, three, or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorus, sulfur, selenium, or halo).

[0116] "Heteroalkylene" means an alkylene group, as defined herein, containing 1, 2, 3, or 4 non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorus, sulfur, selenium, or halo). A heteroalkylene group can be saturated or unsaturated (e.g., having one or more double or triple bonds). A heteroalkylene group can be substituted or unsubstituted. For example, a heteroalkylene group can be substituted with one or more substituents described herein for alkyl.

[0117] "Heteroaryl" means a subset of heterocyclyl groups, as defined herein, that are aromatic, i.e., contain 4n+2 pi electrons in a monocyclic or polycyclic ring system.

[0118] The term "heterocycloalkyl" refers to a cycloalkyl group, as defined above, in which at least one carbon atom and, if present, its associated hydrogen atom, is replaced by O, S, N, or NH. Heterocycloalkyl and heterocycloalkenyl groups can be substituted or unsubstituted. Cycloalkenyl and heterocycloalkenyl groups 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, azide, silyl, sulfonyl, sulfinyl, or thiol, as described herein.

[0119] "Heterocycle" means a compound having one or more heterocyclyl moieties. Non-limiting heterocycles include optionally substituted imidazole, optionally substituted triazole, 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 dioxin, 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 benzothiazole, optionally substituted benzothiadiazole, optionally substituted adenine, optionally substituted guanine, optionally substituted tetrahydroquinoline, optionally substituted dihydroquinoline, optionally substituted dihydroisoquinoline, optionally substituted quinoline quinolinone, optionally substituted quinolinone, optionally substituted isoquinolinone, 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 phenoxathiin, optionally substituted quinuclidine, optionally substituted azaadamantane, optionally substituted dihydroazepine,Optionally substituted azepines, optionally substituted diazepines, optionally substituted oxepanes, optionally substituted thiepines, optionally substituted thiazepines, optionally substituted azocanes, optionally substituted azocines, optionally substituted thiocanes, optionally substituted azonanes, optionally substituted azecines, etc. Optional substitutions include any of those described herein for aryl. Heterocycles can also include any of the cations and / or salts thereof (e.g., any of those 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] "Heterocyclyl," unless otherwise specified, refers to a 3-, 4-, 5-, 6-, or 7-membered ring containing 1, 2, 3, or 4 non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorus, sulfur, selenium, or halo). 3-membered rings have 0 to 1 double bond, 4- and 5-membered rings have 0 to 2 double bonds, and 6- and 7-membered rings have 0 to 3 double bonds. The term "heterocyclyl" also includes bicyclic, tricyclic, and tetracyclic groups, any of which are fused to 1, 2, or 3 rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, and another monocyclic heterocycle, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, and the like. Heterocycles include acridinyl, adenyl, alloxazinyl, azaadamantanyl, azabenzimidazolyl, azabicyclononyl, azacycloheptyl, azacyclooctyl, azacyclononyl, azahypoxanthinyl, azaindolyl, azecinyl, azepanyl, azepinyl, azetidinyl, azetyl, aziridinyl, azirinyl, azocanyl, azocinyl, azonanyl, benzimidazolyl, benzisothiazolyl, benzisoxazolyl, benzodiazepinyl, benzodiazocinyl, benzodihydrofuryl, benzodioxepinyl, benzodioxinyl, benzodioxolyl, benzodithiepinyl, benzodithiin ...thiazolyl, benzodihydrofuryl, benzisothiazolyl, benzodioxinyl, benzodioxolyl, benzodithiepinyl, benzodithiinyl, benzimidazolyl, benzisothiazolyl, benzodithiazolyl, benzodithiazolyl, benzodithiazolyl, benzodithiazolyl zodioxocinyl, 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, benzoxazosinyl, benzoxazolinonyl, benzoxazolinyl, benzoxazolyl, benzylsultamyl, benzylsaltimyl, bipyrazinyl, bipyridinyl, carbazolyl (e.g., 4H-carbazolyl), carbolinyl (e.g., β-carbolinyl), chromanonyl, chromanyl, chromenyl, cinnolinyl, coumarinyl, cytidinyl, cytosinyl, decahydroisoquinolinyl, decahydroquinolinyl, diazabicyclooctyl, diazetyl, diaziridinethionyl, diaziridinonyl, diazi Lysinyl, diazirinyl, dibenzisoquinolinyl, dibenzoacridinyl, dibenzocarbazolyl, dibenzofuranyl, dibenzophenazinyl, dibenzopyranonyl, dibenzopyronyl (xanthonyl), dibenzoquinoxalinyl, dibenzothiazepinyl, dibenzothiepinyl, dibenzothiophenyl, dibenzoxepinyl, dihydroazepinyl, dihydroazetyl, dihydrofuranyl, dihydrofuryl, dihydroisoquinolinyl, dihydropyranyl, dihydropyridinyl, dihydropyridyl, dihydroquinolinyl dihydrothienyl, dihydroindolyl, dioxanyl, dioxazinyl, dioxindolyl, dioxiranyl, dioxenyl, dioxinyl, dioxobenzofuranyl, dioxolyl, dioxotetrahydrofuranyl, dioxothiomorpholinyl, dithianyl, dithiazolyl, dithienyl, dithiinyl, furanyl, furazanyl, furoyl, furyl, guaninyl, homopiperazinyl, homopiperidinyl, hypoxanthinyl, hydantoinyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl (e.g., 1H-indazolyl), indo indolyl, indolinyl, indolizinyl, indolyl (e.g., 1H-indolyl or 3H-indolyl), isatinyl, isatyl, isobenzofuranyl, isochromanyl, isochromenyl, isoindazolyl, isoindolinyl, isoindolyl, isopyrazolonyl, isopyrazolyl, isoxazolidinyl, isoxazolyl, isoquinolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, morpholinyl, naphthoindazolyl, naphthoindolyl, naphthiridinyl, naphthopyranyl, naphthothiazolyl,Naphthothioxolyl, naphthotriazolyl, naphthoxindolyl, naphthyridinyl, octahydroisoquinolinyl, oxabicycloheptyl, oxauracil, oxadiazolyl, oxazinyl, oxaziridinyl, oxazolidinyl, oxazolidonyl, oxazolinyl, oxazolonyl, oxazolyl, oxepanyl, oxetanoyl, oxetanyl, oxetyl, oxtenayl, oxindolyl, oxiranyl, oxobenzisisothiazolyl, oxochromenyl, oxoisoquinolinyl nyl, 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, pyrronyl, pyrrolidinyl, pyrrolidonyl (e.g., 2-pyrrolidonyl), pyrrolinyl, pyrrolidizinyl, 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-1,2,5-thiadiazinyl or 2H,6H-1,5,2-dithiazinyl), thiadiazolyl, thianthrenyl, thianyl, thianaphthenyl, thiazepinyl, thiazinyl, thiazolidinedionyl, thiazolidinyl, thiazolyl, thienyl, thiepanyl, thiepinyl, thietanyl, thiethyl, thiiranyl, thiocanyl, thiochromanonyl,Examples of heterocyclyl groups include thiochromanyl, thiochromenyl, thiodiazinyl, thiadiazolyl, thioindoxyl, thiomorpholinyl, thiophenyl, thiopyranyl, thiopyronyl, thiotriazolyl, thiourazolyl, thioxanyl, thioxolyl, thymidinyl, thyminyl, triazinyl, triazolyl, trithianyl, urazinyl, urazolyl, uretidinyl, uretinyl, urisyl, uridinyl, xanthenyl, xanthinyl, xantthionyl, and the like, as well as modified forms thereof (e.g., containing one or more oxo and / or amino groups) and salts thereof. Heterocyclyl groups can be substituted or unsubstituted. For example, heterocyclyl groups can be substituted with one or more of the substituents described herein for aryl.

[0121] "Heterocyclyldiyl" refers to a divalent form of a heterocyclyl group described herein. In one example, a heterocyclyldiyl is formed by removing a hydrogen from a heterocyclyl group. Representative heterocyclyldiyl groups include piperzylidene, quinolinediyl, and the like. A heterocyclyldiyl group can also be substituted or unsubstituted. For example, a heterocyclyldiyl group can be substituted with one or more substituents described herein for heterocyclyl.

[0122] "Hydroxyalkyl" means an alkyl group, as defined herein, that is substituted with one or more hydroxyl groups.

[0123] "Hydroxyalkylene" means an alkylene group, as defined herein, that is substituted with one or more hydroxy groups.

[0124] "Hydroxyl" means --OH.

[0125] "Imino" means -NR-, where R can be H or optionally substituted alkyl.

[0126] "Isocyanate" means --NCO.

[0127] "Isothiocyanate" means -N=C=S.

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

[0129] "Nitro" refers to the -NO2 group.

[0130] "Oxo" means the group =O.

[0131] "Oxy" means --O--.

[0132] "Phosphate" refers to a group derived from phosphoric acid. An example of a phosphate is -OP(=O)(OR P1 )(OR P2 ) or -O-[P(=O)(OR P1 )-O] P3 -R P2 group (in the formula, R P1 and R P2 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, or optionally substituted arylalkylene, and P3 is an integer from 1 to 5. Further examples of phosphates include orthophosphoric acid, diphosphoric acid, tripolyphosphoric acid, tetrapolyphosphoric acid, trimetaphosphoric acid, and / or phosphoric anhydride, or combinations thereof.

[0133] "Phosphono" or "phosphonic acid" refers to the group -P(O)(OH).

[0134] "Salt" refers to an ionic form of a compound or structure (e.g., any formula, compound, or composition described herein), including cationic or anionic compounds that form electrically neutral compounds or structures. Salts are well known in the art. For example, non-toxic salts are described in Berge SM et al., "Pharmaceutical salts," J. Pharm. Sci. 1977 January; 66(1):1-19 and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Wiley-VCH, April 2011 (2nd rev. ed., eds. P.H. Stahl and C.G. Wermuth). 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 acid (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, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, hydroxyethanesulfonate, hydroxynaphthoate, iodide, lactate, lactobiolate, and the like. Examples of suitable salts include phosphate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl bromide, methyl nitrate, 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, and valerate salts. Representative cationic salts include metal salts, such as alkali metal or alkaline earth metal salts, such as barium, calcium (e.g., calcium edetate), lithium, magnesium, potassium, sodium, etc.; other metal salts, such as aluminum, bismuth, iron, and zinc salts; and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, pyridinium, etc. Other cationic salts include organic salts, such as chloroprocaine, choline, dibenzylethylenediamine, diethanolamine, ethylenediamine, methylglucamine, and procaine.Further salts include ammonium, sulfonium, sulfoxonium, phosphonium, iminium, imidazolium, benzimidazolium, amidinium, guanidinium, phosphazinium, phosphazenium, pyridinium, and the like, 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 ... tetrazolium, optionally substituted pyridinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted triazinium, optionally substituted tetrazinium, optionally substituted pyridinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted tetrazolium, optionally substituted pyridinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted tetrazolium, optionally substituted pyridinium, optionally substituted pyrimidinium, optionally substituted pyrazinium, optionally substituted tetrazolium, optionally substituted pyridinium, optionally substituted pyr optionally substituted quinolinium, optionally substituted quinolinium, optionally substituted isoquinolinium, optionally substituted quinolinium, optionally substituted dehydroquinolizinium, optionally substituted quinolinium, optionally substituted isoindolium, optionally substituted benzimidazolinium, and optionally substituted quinolinium.Still other salts include those containing anions such as halides (e.g., F. - , Cl - , Br - , or I - ), hydroxides (e.g., OH - ), borates (e.g., tetrafluoroborates (BF4 - ), carbonates (e.g., CO3 2- or HCO3 - ), or sulfates (e.g., SO4 2- ) may be included.

[0135] "Silyl" means -SiR 1 R 2 R 3 or -SiR 1 R 2 In some embodiments, R 1 , R 2 , and R 3 are each independently H, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aromatic, optionally substituted heteroaromatic, or optionally substituted amino. 1 , R 2 , and R 3 are each 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 (OR) b (NR2) cwherein each R is independently H, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aromatic, or optionally substituted heteroaromatic; a, b, and c are each ≧0; and a+b+c=3. In certain embodiments, each R is independently H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkyl-aryl, or optionally substituted aryl-alkyl.

[0136] "Spirocyclyl" also refers to alkylene diradicals in which both ends are bonded to the same carbon atom of a parent group to form a spirocyclyl group, and heteroalkylene diradicals in which both ends are bonded to the same atom. Non-limiting alkylene and heteroalkylene groups for use within a spirocyclyl group include C 2-12 , C 2-11 , C 2-10 , C 2-9 , C 2-8 , C 2-7 , C 2-6 , C 2-5 , C 2-4 , or C 2-3 Alkylene groups, as well as C groups with one or more heteroatoms 1-12 , C 1-11 , C 1-10 , C 1-9 , C 1-8 , C 1-7 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , or C 1-2 Heteroalkylene groups are included.

[0137] "Sulfate" refers to a group derived from sulfuric acid. An example of a sulfate is -OS(=O)2(OR S1 ) group (wherein, R S1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, or optionally substituted arylalkylene).

[0138] "Sulfo" or "sulfonic acid" refers to the group -S(O)2OH.

[0139] "Sulfonyl" means the group -S(O)- or -S(O)R, where R can be H, optionally substituted alkyl, or optionally substituted aryl. Non-limiting sulfonyl groups can include trifluoromethylsulfonyl groups (-SO-CF or Tf).

[0140] Use of the above terms is meant to encompass substituted and unsubstituted moieties. Substitution can be by one or more groups such as alcohol, ether, ester, amide, sulfone, sulfide, hydroxyl, nitro, cyano, carboxy, amine, heteroatom, lower alkyl, lower alkoxy, lower alkoxycarbonyl, alkoxyalkoxy, acyloxy, halogen, trifluoromethoxy, trifluoromethyl, alkyl, aralkyl, alkenyl, alkynyl, aryl, cyano, carboxy, carboalkoxy, carboxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, alkylheterocyclyl, heterocyclylalkyl, oxo, arylsulfonyl, and aralkylaminocarbonyl, or any of the substituents in the previous paragraph or any of these substituents are directly attached or attached by a suitable linker. Linkers are typically short chains of 1 to 3 atoms containing any combination of -C-, -C(O)-, -NH-, -S-, -S(O)-, -O-, -C(O)- or -S(O)O. The ring may be substituted multiple times.

[0141] The term "lower" modifying "alkyl," "alkenyl," "alkynyl," "alkoxy," or "alkoxycarbonyl" refers to a C1 to C6 unit for a particular functional group. For example, "lower alkyl" means a C1 to C6 alkyl.

[0142] "Substituted" means having one or more substituent moieties whose presence does not in itself interfere with the desired function or reactivity. Examples of substituents include alkyl, alkenyl, alkynyl, cycloalkyl (non-aromatic ring), Si(alkyl)3, Si(alkoxy)3, alkoxy, amino, alkylamino, alkenylamino, amido, amidine, guanidine, hydroxyl, thioether, alkylcarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, carbonate, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphate ester, phosphonato, cyano, halo, acylamino, imino, sulfhydryl, alkylthio, thiocarboxylate, dithiocarboxylate, sulfate, sulfato, sulfonate, sulfamoyl, sulfonamido, nitro, nitrile, azide, heterocyclyl, ether, ester, silicon-containing moiety, thioester, or combinations thereof. Substituents may themselves be substituted. For example, the amino substituent may itself be mono-substituted or di-substituted with further substituents independently defined above, such as alkyl, alkenyl, alkynyl, and cycloalkyl (non-aromatic rings).

[0143] By "sulfide" is meant a thioether -S-R', where R' can be, but is not limited to, an aliphatic group.

[0144] By "sulfhydryl" is meant thiol, i.e., --SH.

[0145] "Thiocyanato" means -SCN.

[0146] "Thioester" means --SC(O)R', where R' can be, but is not limited to, an aliphatic group.

[0147] "Unsubstituted" means that any open valence on the atom is occupied by a hydrogen. Also, if no occupant is specified for an open valence position for an atom, it is a hydrogen.

[0148] "Unsaturated" means a moiety that contains double or triple carbon-carbon bonds.

[0149] "Unsaturated substituent" means a double or triple bond containing aliphatic chains, cyclic groups, aryl groups, or heteroaryl groups.

[0150] Those skilled in the art will recognize that the definitions provided above are not intended to include impermissible substitution patterns (e.g., methyl substituted with five substituents, etc.). Such impermissible substitution patterns would be readily recognized by one of ordinary skill in the art. Any functional group disclosed herein and / or defined above may be substituted or unsubstituted, unless otherwise indicated herein.

[0151] As used herein, a mass tag includes any tag that contains an enriched heavy atom, such as an enriched metal isotope. A mass tag may include a polymer carrying an enriched metal isotope, and may optionally include a conjugated bioactive agent. A mass tag can be identified based on the atomic mass of its enriched metal isotope.

[0152] As used herein, mass cytometry is any method for detecting mass tags in a biological sample, e.g., simultaneously detecting multiple distinguishable mass tags with single-cell resolution. Mass cytometry includes suspension mass cytometry, imaging mass cytometry™ (IMC™), and secondary ion mass spectrometry (SIMS). Mass cytometry can atomize and ionize mass tags in a cellular sample by one or more of laser irradiation, ion beam irradiation, electron beam irradiation, and / or inductively coupled plasma (ICP). Mass cytometry can simultaneously detect distinct mass tags from a single cell, e.g., by time-of-flight mass spectrometry (TOF) or magnetic sector mass spectrometry (MS). As used herein, DBCO-NHS refers to (2,5-dioxopyrrolidin-1-yl)4-(2-azatricyclo[10.4.0.0].4,9 ]Hexadeca-1(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 (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies, e.g., bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The various classes of immunoglobulins have different, well-known subunit structures and three-dimensional configurations.

[0154] A "monoclonal antibody" refers to a homogeneous antibody population that is responsible for highly specific recognition and binding to a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to antibodies made in a 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 that has been engineered to contain minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and properties.

[0156] The term "human antibody" refers to 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 human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0157] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies from another (usually human) species 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 will readily recognize that a chemical antibody can be substituted for an antibody in any of the embodiments described herein.

[0159] The term "protein" refers to small proteins less than 100 amino acids in length and large biological molecules or macromolecules having one or more long chains of amino acid residues, including globular proteins, fibrous proteins, or membrane proteins, such as antibodies.

[0160] The term "chelator" refers to a moiety capable of coordinating (eg, stably coordinating) a metal atom.

[0161] Introduction and Background Traditional detection reagents for bioassays often consist of a binding moiety with specificity for the molecule of interest conjugated to an enzymatic or optical moiety. To date, these determinations are typically facilitated through the use of radioactive, fluorescent, or enzymatic tags. Among analytical methods of interest, flow cytometry provides a 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. Cell analysis is of particular interest. Modern tools typically employ multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for the simultaneous analysis of multiple labeled antibodies, allowing for more precise identification of target populations by phenotypic markers. In traditional flow cytometry, fluorescently labeled particles, such as live cells, fixed cells, and beads, are individually differentiated and separated based on their fluorescence and light scattering properties. After isolation, particle phenotypes can be further characterized.

[0162] Such conventional flow cytometry methods are limited by the number of simultaneous parameters that can be measured for a single particle, the overlap of fluorescence emissions during simultaneous measurements, and problems with background fluorescence or enzymatic activity. As the number of simultaneous parameters increases, this spectral overlap significantly complicates the analysis, affecting both the accuracy and sensitivity of the assay. In an alternative detection method, atomic mass spectrometry measurements have been used in combination with stable isotope tags of rare elements.

[0163] Existing elemental tag capture reagents for use in ICP-MS are based on chelating agents, such as maleimide-functionalized polymers of ethylenediaminetetraacetic acid (EDTA), tetraazacyclododecanetetraacetic acid (DOTA), or diethylenetriaminepentaacetic acid (DTPA), with an average length of 10–30 monomers. Such protocols allow for the conjugation of six or seven polymers to a typical antibody, thereby conjugating an average of 200 tagged 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 by the number of disulfide bonds that can be cleaved without disrupting immunoglobulin function. The number of metal chelating units that can be conjugated to a detection reagent is also limited because increasing the number can interfere with the detection reagent or induce nonspecific interactions, thereby interfering with the assay or causing high background.

[0164] Metalloprotein Conjugates Metalloprotein conjugates, in some embodiments, may be composed of a number of different metals, including mercury (Hg), lead (Pb), platinum (Pt), antimony (Sb), or arsenic (As). As used herein, the term "conjugate" refers to a metal-containing compound, e.g., a coordination complex. In some embodiments, the metal is a thiophilic metal. In some embodiments, the protein may be an antibody, and the metal and protein may be linked together by a variety of suitable conjugation means. For example, metals can be conjugated to proteins through covalent bonds (e.g., amine chemistry, thiol chemistry, phosphate chemistry), enzymatic reactions, redox reactions (e.g., using metal halides), and affinity intermediates (e.g., streptavidin or biotin), or a form of click chemistry (e.g., 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 to proceed rapidly and irreversibly, with high reaction specificity (sometimes both regiospecificity and stereospecificity) to give a single reaction product in high yield. The reactions typically produce few, if any, toxic by-products, are often suitable for performance under physiological conditions, i.e., in aqueous buffers at approximately neutral pH, are tolerant to typical buffer salts, and the resulting products are stable under physiological conditions. Even if these reactions do not strictly meet these criteria, they are characterized by their high yield and ability to be performed in convenient, non-toxic, or easily removed solvents. More importantly, the molecular reactions are bioorthogonal, meaning they proceed without interacting with any of the mechanisms typically found in biological systems.

[0166] In some embodiments, the metal and protein are directly bound to one another, such as when the protein is appropriately functionalized or derivatized to contain a moiety capable of chelating the metal. In other embodiments, the metal can be chelated by a chelator attached to the protein or derivatized protein via a linking moiety. These metalloprotein conjugates are useful in certain embodiments for mass cytometry applications.

[0167] Suitable conjugates include a metal atom, a sulfhydryl-containing moiety, and a protein, where the protein is conjugated to the sulfhydryl-containing moiety via a bond or via a linking moiety. The linking moiety has the formula (I): -L 1 -AL 2 - (I) (In the formula, L 1 and L 2 is a linking group). As used herein, a "linking group" refers to a compound that can link two moieties. In some embodiments, L 1is a first linking group, which may be an alkylcarbonyl, amide, amine, ether, or ester; L 1 may be covalently bound to proteins; L 2 is a second linking group containing a nitrogen-containing moiety, and L 2 may be covalently bound to the sulfhydryl-containing moiety; and A is a cyclic group; and a metal atom is chelated to the sulfhydryl-containing moiety.

[0168] Metalloprotein conjugates linked via linking moieties Metals can be indirectly bound to proteins, such as antibodies, via an intermediate binding moiety. The selection of an appropriate binding moiety depends on its ability to maximize metal-intermediate binding while minimizing cross-reactivity, and can include, but is not limited to, repeating polymers capable of forming bonds with metals or metal-binding agents. The synthetic route to a metal-antibody conjugate depends on the type of binding moiety utilized.

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

[0170] [ka]

[0171] An example of this preparation is illustrated in Scheme 1 below.

[0172] [ka]

[0173] Following synthesis and purification of DMPS-A, mercury chelation may be performed as the second step in the process for preparing metalloprotein conjugates. The metal chelator complex may be formed at neutral pH for stability purposes and to avoid the formation of insoluble hydroxides that can occur under alkaline conditions. In one embodiment, the complex is prepared by dissolving Hg(NO3)2 in a small amount of dilute nitric acid and adding 5 mM Hg(NO3)2. 2+ to 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0). DMPS-A from the first step can be added to the Hg-containing buffer at a 1:1 molar ratio (5 mM) and incubated at room temperature for 1 minute before use.

[0174] The DMPS-Hg complex prepared according to the second step may, in some embodiments, have a ratio of DMPS to Hg of 1:1, 2:1, or 4:1.

[0175] In the third step, the metal-loaded DMPS chelator can be covalently attached to a protein, such as an antibody, via a DBCO-azide click chemistry reaction, as shown in Scheme 2 below.

[0176] [ka]

[0177] In Scheme 2, the center structure labeled as an azido-residual DMPS peptide is an azido-residual metal-bearing DMPS chelator; whereas the structure on the right labeled as a DBCO-derivatized antibody is a metal-tagged antibody.

[0178] Other alkynes that can be utilized to derivatize antibodies include monofluorinated cyclooctyne, difluorocyclooctyne, dimethoxyazacyclooctyne, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, bicyclononyne, 2,3,6,7-tetramethoxydibenzocyclooctyne, sulfonylated dibenzocyclooctyne, carboxymethyl monobenzocyclooctyne, or pyrrolocyclooctyne.

[0179] In another embodiment, the metal chelating protein conjugate can be prepared using lipoic acid. Lipoic acid is a mercury chelator. In some embodiments, the lipoic acid-Hg complex can have a 1:1 configuration as seen below:

[0180] [ka]

[0181] Conjugate 1 can be conjugated to the antibody using a coupling agent capable of binding the metal-bearing chelator (Conjugate 1) to the antibody. Suitable coupling agents can be carbodiimides, such as dicyclohexylcarbodiimide or 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); or phosphonium salts, such as benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP). In some embodiments, the coupling agent is an amide coupling agent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (DMTMM).

[0182] An alternative method for forming metal-chelating protein conjugates using lipoic acid involves the use of functionalized polymers. For example, lipoic acid can be covalently attached to an azide-terminated poly(γ-benzyl L-glutamate (PBLG))-based polymer backbone. The polymer backbone can be prepared by aminolysis using ethylenediamine or 1,6-hexanediamine to introduce reactive amine groups. 50% of the polymer's repeating units are then functionalized with polyethylene glycol succinimidyl ester, e.g., PEG24-NHS ester, resulting in a functionalized polymer with good water solubility. Lipoic acid is then attached to the remaining polymer repeating units. The polymer is then loaded with a metal, and the polymeric azide end groups allow for conjugation to an antibody that has been treated with NHS-DBCO; thus, forming a metal-chelating protein conjugate.

[0183] In another embodiment, a method for preparing a metal-chelating protein conjugate, e.g., a mercury-antibody conjugate, includes conjugating an azide-containing chelator to an antibody prior to mercury chelation. This technique avoids potential cross-reactivity of azide with the chelator conjugate. In certain embodiments, the method includes the following steps: (1) incubation of the antibody with glutathione to cap free thiol groups, (2) conjugation of the DBCO-modified antibody with DMPS-A using the DBCO-azide click reaction, and (3) chelation of Hg using the chelator-antibody conjugate.

[0184] Directly linked metalloprotein conjugates In some embodiments, a metal chelating site or sites can be directly introduced into an antibody, allowing direct labeling of the antibody with a metal ion. Disulfide bonds (also called disulfide bridges) can be found, for example, in the hinge region of an antibody. The original disulfide bridge can be reduced to two sulfhydryls using a reducing agent, such as dithiothreitol (DTT) or tris-2-carboxyethylphosphine (TCEP). The proximity of two thiol groups can facilitate metal chelating.

[0185] In some embodiments, if the antibody does not contain native disulfide bonds, disulfide bonds can be added to the antibody.

[0186] Kits and Kit Components Aspects of the present application include manufacturing the kits discussed herein, or portions thereof. Aspects of the present application include using the kits described herein, for example, for mass cytometry or delivery of radioisotopes.

[0187] The kit, the components of the kit, and the steps of preparing the kit can include suitable storage media.For example, solvents and cosolubilizers can include, but are not limited to, water; sterile water for injection (SWFI); saline; alcohols, such as ethanol, benzyl alcohol, etc.; glycols and polyalcohols, such as propylene glycol, glycerin, etc.; esters of polyalcohols, such as diacetin, triacetin, etc.; polyglycols and polyethers, such as polyethylene glycol 400, propylene glycol methyl ether, etc.; dioxolanes, such as isopropylideneglycerol, etc.; dimethyl isosorbide; pyrrolidone derivatives, such as 2-pyrrolidone, N-methyl-2-pyrrolidone, polyvinylpyrrolidone (cosolubilizer only), etc.; polyoxyethylenated fatty alcohols; esters of polyoxyethylenated fatty acids; polysorbates, such as TWEEN, polyoxyethylenated derivatives of polypropylene glycol, such as PLURONICS.

[0188] Suitable stabilizers include, but are not limited to, one or more of monosaccharides (e.g., galactose, fructose, and fucose), disaccharides (e.g., lactose), polysaccharides (e.g., dextran), cyclic oligosaccharides (e.g., alpha-, beta-, and gamma-cyclodextrin), 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 solvents and / or stabilizers may be used in any step of the synthetic methods described above, or in the storage of any of the reagents described above (e.g., as provided in a kit).

[0189] In certain embodiments, the solution may be acidic. The acidic solution of the present application may comprise one or more strong acids, such as 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% (e.g., more than 0.05%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.5%, more than 1%, more than 2%, or more than 5%) and / or less than 10% (e.g., less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1%). For example, the acid may be present at 0.05% to 2%. The acidic solution may have a pH of 6 or less, 5 or less, 4.5 or less, or 4 or less. The lyophilized composition of the present application may have a water content (by weight) of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0190] At any step (e.g., when the conjugates discussed herein are provided in a kit), the conjugates can be lyophilized. For example, the conjugates can be lyophilized to a moisture content (e.g., by mass) of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Such lyophilization can allow for storage in the kit prior to use in mass cytometry and / or can allow flexibility in assay design if the lyophilization stabilizes the conjugate.

[0191] The kit may further comprise any additional components (eg, buffers, filters, etc.).

[0192] Alternatively, or in addition, the kit can include additional reagents for mass cytometry, such as buffers, standards, cell barcodes, and / or reagents containing heavy atoms of various masses (e.g., mass tags attached to or provided for attachment to bioactive agents).

[0193] In certain embodiments, the kit can include multiple antibodies (e.g., to various targets). Such a collection of antibodies can be provided together in a single panel. The panel can be provided in a solution or a lyophilized mixture containing less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% water by mass.

[0194] conclusion Although the above embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be made 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. Therefore, the present embodiments should be considered as illustrative and not restrictive, and the embodiments are not limited to the details given herein.

Claims

1. A conjugate comprising: metal atom; a sulfhydryl-containing moiety; and protein Including, The protein is conjugated to the sulfhydryl-containing moiety via a bond or via a linking moiety, the linking moiety having the formula (I): L １ -A-L ２ - (I) (In the formula, L １ is a first linking group comprising an alkylcarbonyl, an amide, an amine, an ether, or an ester; L １ is covalently bound to the protein; L ２ is a second linking group that includes a nitrogen-containing moiety, and L ２ is covalently attached to said sulfhydryl-containing moiety; and A is a cyclic group. The structure of the metal atom chelates to the sulfhydryl-containing moiety; Conjugates.

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

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

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

5. 2. 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 to the metal atom.

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

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

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

10. 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 an amino, an azide, or an amide.

13. 1. A method of forming a metal chelating protein conjugate for mass cytometry, comprising: reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelating agent; chelating a metal with the dithiol-containing sulfonyl azide chelator to form an azide-containing metal chelator complex; reacting the 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-chelating protein conjugate. A method comprising:

14. 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. 1. A method for forming a metal chelate chemistry antibody conjugate for mass cytometry, comprising: reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelating agent; 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, or As; reacting the 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 chelate chemical antibody conjugate. A method comprising:

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

18. 14. 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. 1. A method of forming a metal chelating protein conjugate for mass cytometry, comprising: Chelating a metal with lipoic acid to form a metal chelator complex; and covalently attaching said metal chelator complex to a protein using a coupling agent; A method comprising:

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

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

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

25. 25. The method of claim 24, wherein the coupling agent comprises 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride.

26. 1. A method of forming a metal chelating protein conjugate for mass cytometry, comprising: reacting the poly(amino acid) with ethylenediamine 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 attaching lipoic acid to the functionalized poly(amino acid) to form a lipoic acid-modified, functionalized poly(amino acid); chelating a metal to the lipoic acid-modified, functionalized poly(amino acid) to form a metal-chelating lipoic acid-modified, functionalized poly(amino acid); reacting the protein with a reagent to form an alkyne-containing protein derivative; and conjugating the metal-chelating lipoic acid-modified functionalized poly(amino acid) with the alkyne-containing protein derivative to form a metal-chelating protein conjugate. A method comprising:

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

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

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

30. The reagent is (2,5-dioxopyrrolidin-1-yl)4-(2-azatricyclo[10.4.0.0 ４，９ ]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

31. 1. A method of forming a metal chelating protein conjugate for mass cytometry, comprising: reacting a protein containing a thiol group with a reactant to form a modified protein; reacting a dithiol-containing sulfonic acid with sodium azide to form a dithiol-containing sulfonyl azide chelating agent; 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 said protein-chelator conjugate. A method comprising:

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

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

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

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

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

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

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

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

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

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

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

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

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

45. 41. The method of claim 40, wherein the step of introducing a sulfhydryl group comprises reacting an active site of the protein with the reagent.

46. 46. ​​The method of claim 45, wherein the active site comprises an amine.

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

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