Dithiol chelators for metal to antibody conjugation

Abstract

Provided are a metal-protein conjugate for use in mass spectrometry flow cytometry and a method for preparing the same. The metal-protein conjugate may have a metal that binds directly to the protein, or a metal that is attached to the protein through a linking moiety. The metal-protein conjugates include mercury chelated triazole-containing antibody complexes and methods of forming them from the derived antibodies and sulfonyl azide compounds.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of USSN 63 / 384,829, filed on November 23, 2022, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] In mass cytometry, cells are labeled with mass - tagged bioactive materials such as antibodies or oligonucleotides, and the mass tags can be detected by mass spectrometry with single - cell resolution.

[0004] These mass tags are typically lanthanide - chelate polymers loaded with enriched lanthanide isotopes for conjugation to antibodies. One drawback of conjugating lanthanides to antibodies is that proteins such as bovine serum albumin (BSA, commonly used in buffers for purified antibodies) can bind the lanthanides, resulting in failed antibody conjugation due to lanthanide adsorption.

[0005] 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 distinguishable mass - tagged bioactive materials is determined by the number of isotopes of different masses. Developing additional mass tags, particularly those conjugated with other metals such as mercury, would allow for an expansion in the number and types of targets detectable in mass cytometry applications and avoid unwanted interactions with other components of antibody formulations such as buffers or stabilizers (including glycerol).

[0006] The background description provided herein is to generally present the context of the present disclosure. To the extent described in this background art section, the work of the presently named inventors, and aspects that may not qualify as prior art at the time of filing, are neither expressly nor implicitly considered prior art to the present disclosure. Technical Field

[0007] The present disclosure relates to reagents and their use for elemental mass spectrometry (including imaging mass spectrometry) of biological samples. Summary of the Invention

[0008] The present disclosure provides metal - protein conjugates for use in mass cytometry and methods for their preparation. The metal - protein conjugates can have a metal directly bound to the protein or a metal attached to the protein through a linking moiety. The metal - protein conjugates include mercury - chelated triazole - containing antibody complexes and methods for forming them from a derived antibody and a sulfonyl azide.

[0009] Accordingly, in a first aspect, the present disclosure includes conjugates. In some embodiments, the conjugate includes a metal atom; a thiol-containing moiety; and a protein; wherein the protein is conjugated to the thiol-containing moiety either through a bond or through a linking moiety having the structure of formula (I):

[0010] -L 1 -A-L 2 -(I),

[0011] wherein L 1 is a first linking group including an alkylcarbonyl, amide, amine, ether, or ester, and L 1 is covalently bound to the protein; L 2 is a second linking group including a nitrogen-containing moiety, and L 2 is covalently bound to the thiol-containing moiety; and A is a cyclic group; and wherein the metal atom chelates the thiol-containing moiety.

[0012] In some embodiments, the thiol-containing moiety is a thiol, dithiol, cysteine, or N-acetylcysteine group.

[0013] In some embodiments, the dithiol is 1,2-dithiol, 1,3-dithiol, or 1,4-dithiol.

[0014] In some embodiments, the dithiol is lipoic acid.

[0015] In some embodiments, the metal atom is Hg, Pb, Pt, Sb, or As.

[0016] In some embodiments, the dithiol chelates the metal atom.

[0017] In some embodiments, two dithiols chelate one metal atom.

[0018] In some embodiments, four dithiols chelate one metal atom.

[0019] In some embodiments, the protein is an antibody.

[0020] In some embodiments, the protein is a chemical antibody.

[0021] In some embodiments, the chemical antibody is an aptamer, which may include one or more modifications.

[0022] In some embodiments, the antibody is a monoclonal antibody, bispecific antibody, multispecific antibody, chimeric antibody, human antibody, or humanized antibody.

[0023] In some embodiments, the antibody is a dibenzocyclooctyne-derived antibody.

[0024] In some embodiments, the nitrogen-containing moiety is an amino group, an azide group, or an amide group.

[0025] In a second aspect, the present disclosure includes 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.

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

[0027] In a third aspect, the present disclosure includes 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.

[0028] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0029] In some embodiments, the protein is an antibody.

[0030] In some embodiments, conjugating the azide-containing metal-chelator complex to the alkyne-containing protein derivative includes reacting the alkyne of the alkyne-containing protein derivative with the azide of the azide-containing metal-chelator complex to form a triazole.

[0031] In some embodiments, the dithiol-containing sulfonic acid is 2,3-dimercaptopropane-1-sulfonic acid.

[0032] In some embodiments, the reagent is DBCO-NHS.

[0033] In a fourth aspect, the present disclosure includes 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.

[0034] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0035] In some embodiments, the protein is an antibody.

[0036] In some embodiments, the protein is a chemical antibody.

[0037] In some embodiments, the chemical antibody is an aptamer, which may include one or more modifications.

[0038] In some embodiments, the coupling agent is an amide-containing coupling agent.

[0039] In some embodiments, the coupling agent is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride.

[0040] In a fifth aspect, the present disclosure includes a method of forming a metal-chelated protein conjugate for mass cytometry. In some embodiments, the method includes 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 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.

[0041] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0042] In some embodiments, the protein is an antibody.

[0043] In some embodiments, the protein is a chemical antibody.

[0044] In some embodiments, the chemical antibody, which may include one or more modifications.

[0045] In some embodiments, the poly(amino acid) is poly(γ-benzyl α,L-glutamate).

[0046] In some embodiments, the reagent is (2,5-dioxopyrrolidin-1-yl) 4-(2-azatricyclo[10.4.0.0 4,9 hexadec-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoate.

[0047] In a sixth aspect, the present disclosure includes a method of forming a metal-chelated protein conjugate for mass cytometry. In some embodiments, the method includes reacting a protein comprising 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.

[0048] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0049] In some embodiments, the protein is an antibody.

[0050] In some embodiments, the protein is a chemical antibody.

[0051] In some embodiments, the chemical antibody is an aptamer, which may include one or more modifications.

[0052] In some embodiments, the reactant is glutathione.

[0053] In some embodiments, the reagent is DBCO-NHS.

[0054] In a seventh aspect, the present disclosure includes 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 reducing agent to form a dithiol-containing protein; and complexing a metal to the dithiol-containing protein.

[0055] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0056] In some embodiments, the protein is an antibody.

[0057] In some embodiments, the protein is a chemical antibody.

[0058] In some embodiments, the chemical antibody is an aptamer, which may include one or more modifications.

[0059] In some embodiments, the reducing agent is dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, or tris-(2-carboxyethyl)phosphine.

[0060] In an eighth aspect, the present disclosure includes a method for forming a metal-chelating protein. In some embodiments, the method includes: introducing a thiol group into a protein by reacting the protein with a reagent to form a thiol-containing protein; and chelating a metal to the thiol-containing protein.

[0061] In some embodiments, the metal is Hg, Pb, Pt, Sb, or As.

[0062] In some embodiments, the protein is an antibody.

[0063] In some embodiments, the protein is a chemical antibody.

[0064] In some embodiments, the chemical antibody is an aptamer, which may include one or more modifications.

[0065] In some embodiments, introducing the thiol group includes reacting an active site on the protein with the reagent.

[0066] In some embodiments, the active site is an amine group.

[0067] In some embodiments, the reagent is 2-iminothiolane. Detailed Description

[0068] 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 so as not to unnecessarily obscure the disclosed embodiments. While the disclosed embodiments will be described in conjunction with specific embodiments, it should be understood that this is not intended to limit the disclosed embodiments.

[0069] Definitions

[0070] As used herein, the term "about" is understood to account for minor increases and / or decreases beyond the recited value, such that these variations do not significantly affect the desired function of the parameter beyond the recited value. In some instances, "about" includes + / - 10% of any recited value. As used herein, the term modifies any recited value, range of values, or endpoints of one or more ranges.

[0071] The terms "acyl" or "alkanoyl", which may be used interchangeably herein, represent an alkyl group as defined herein, or a hydrogen attached to a parent molecular group through a carbonyl as defined herein. Examples of such groups are formyl, acetyl, propionyl, butyryl, etc. The alkanoyl group may be substituted or unsubstituted. For example, the alkanoyl group may be substituted with one or more substituents as described herein for alkyl groups. In some embodiments, the unsubstituted acyl group is C 2-7 acyl or alkanoyl group. In certain embodiments, the alkanoyl group is -C(O)-Ak, where Ak is an alkyl group as defined herein.

[0072] "Aliphatic" means a hydrocarbon moiety having from at least one carbon atom to 50 carbon atoms (C 1-50 ), such as from 1 to 25 carbon atoms (C 1-25 ) or from 1 to 10 carbon atoms (C 1-10 ), and which includes saturated groups such as alkanes (or alkyls) and unsaturated groups such as alkenes (or alkenyls), alkynes (or alkynyls), and also includes their cyclic forms, and further includes straight-chain and branched-chain arrangements, and all stereoisomers and positional isomers. Such hydrocarbons may be unsubstituted or substituted with one or more groups, such as halogens or the groups described herein for alkyl groups.

[0073] "Alkenyl" means an optionally substituted C 2-24 alkyl group having one or more double bonds. The alkenyl group may be cyclic (e.g., C 3-24 cycloalkenyl) or acyclic. The alkenyl group may also be substituted or unsubstituted. For example, the alkenyl group may be substituted with one or more substituents as described herein for alkyl groups. Non-limiting unsubstituted alkenyl groups include C 2-8 alkenyl, C 2-6 alkenyl, C 2-5 alkenyl, C 2-4 alkenyl or C 2-3 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 (-CH=CHCH2CH3), 2-butenyl (-CH2CH=CHCH3), 3-butenyl (e.g., -CH2CH2CH=CH2) and 2-methyl-2-butenyl (e.g., =CH-CH=CHCH3), etc.

[0074] "Alkenylene" means a polyvalent (e.g., divalent) form of an alkenyl group, which is an optionally substituted C 2-24 alkyl group having one or more double bonds. The alkenylene group may be cyclic (e.g., C 3-24cycloalkenyl) or acyclic. The alkenylene group may be substituted or unsubstituted. For example, the alkenylene group may be substituted with one or more substituents as described herein for alkyl groups. Exemplary, non-limiting alkenylene groups include -CH=CH- or -CH=CHCH2-.

[0075] "Alkoxy" means -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 may be substituted or unsubstituted. For example, the alkoxy group may be substituted with one or more substituents as described herein for alkyl groups. Exemplary 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.

[0076] "Alkoxyalkyl" means an alkyl group as defined herein which is substituted with an alkoxy group as defined herein. Exemplary unsubstituted alkoxyalkyl groups include those having 2 to 12 carbons (C 2-12 alkoxyalkyl), and those having an alkyl group of 1 to 6 carbons and an alkoxy group of 1 to 6 carbons (i.e., C 1-6 alkoxy-C 1-6 alkyl).

[0077] "Alkyl" and the prefix "alk" mean 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), sec-butyl (s-Bu or sBu), tert-butyl (t-Bu or tBu), cyclobutyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl group may be cyclic (e.g., C 3-24 cycloalkyl) or acyclic. The alkyl group may be branched or unbranched. The alkyl group may also be substituted or unsubstituted. For example, the alkyl group may include haloalkyl, where the alkyl group is substituted with one or more halogen groups as described herein. In another example, the alkyl group may be substituted with one, two, three, or in the case of an alkyl group having two or more carbons, four substituents independently selected from the group consisting of: (1) C 1-6 alkoxy (e.g., -O-Ak, where Ak is an optionally substituted C 1-6(alkyl); (2) amino (e.g., -NR N1 R N2 , where R N1 and R N2 are each independently H or optionally substituted alkyl, or R N1 and R N2 together with the nitrogen atom to which they are attached form a heterocyclic group); (3) aryl; (4) arylalkoxy (e.g., -O-Lk-Ar, where Lk is a divalent form of optionally substituted alkyl and Ar is optionally substituted aryl); (5) aroyl (e.g., -C(O)-Ar, where Ar is optionally substituted aryl); (6) cyano (e.g., -CN); (7) carboxaldehyde (e.g., -C(O)H); (8) carboxyl (e.g., -CO2H); (9) C 3-8 cycloalkyl (e.g., a monovalent saturated or unsaturated non-aromatic cyclic C 3-8 hydrocarbyl group); (10) halogen (e.g., F, Cl, Br or I); (11) heterocyclic group (e.g., a 3-, 4-, 5-, 6- or 7-membered ring, containing one, two, three or four non-carbon heteroatoms such as nitrogen, oxygen, phosphorus, sulfur or halogen unless otherwise specified); (12) heterocyclic oxy (e.g., -O-Het, where Het is a heterocyclic group as described herein); (13) heterocyclic acyl (e.g., -C(O)-Het, where Het is a heterocyclic group as described herein); (14) hydroxy (e.g., -OH); (15) N-protected amino; (16) nitro (e.g., -NO2); (17) oxo (e.g., =O); (18) -CO2R A , where R A is selected from the group consisting of: (a) C 1-6 alkyl, (b) C 4-18 aryl and (c) (C 4-18 aryl)C 1-6 alkyl (e.g., -Lk-Ar, where Lk is a divalent form of optionally substituted alkyl group and Ar is optionally substituted aryl); (19) -C(O)NR B R C , where R B and R C are each independently selected from the group consisting of: (a) hydrogen, (b) C 1-6 alkyl, (c) C 4-18 aryl and (d) (C 4-18 aryl)C 1-6 alkyl (e.g., -Lk-Ar, where Lk is a divalent form of optionally substituted alkyl group and Ar is optionally substituted aryl); and (20) -NR G R H , where R G and RH Each independently selected from the group consisting of: (a) hydrogen, (b) an N-protecting group, (c) a C 1-6 alkyl group, (d) a C 2-6 alkenyl group (e.g., an optionally substituted alkyl group having one or more double bonds), (e) a C 2-6 alkynyl group (e.g., an optionally substituted alkyl group having one or more triple bonds), (f) a C 4-18 aryl group, (g) a (C 4-18 aryl)C 1-6 alkyl group (e.g., Lk-Ar, where Lk is a divalent form of an optionally substituted alkyl group and Ar is an optionally substituted aryl group), (h) a C 3-8 cycloalkyl group, and (i) a (C 3-8 cycloalkyl)C 1-6 alkyl group (e.g., -Lk-Cy, where Lk is a divalent form of an optionally substituted alkyl group and Cy is an optionally substituted cycloalkyl group as described herein), where in one embodiment, no two groups are bonded 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 halogens or alkoxy groups). In some embodiments, the unsubstituted alkyl group is a 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 -, C 2-12 -, C 2-16 -, C 2-18 -, C 2-20 - or C 2-24 alkyl group.

[0078] As described herein, "alkylene" means a polyvalent (e.g., divalent) form of an alkyl group. Exemplary alkylene groups include methylene, ethylene, propylene, butylene, etc. In some embodiments, the 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 2-24Alkylene group. The alkylene group can be branched or unbranched. The alkylene group can also be substituted or unsubstituted. For example, the alkylene group can be substituted by one or more substituents, as described herein for alkyl groups.

[0079] "Alkoxy" means an alkylene group as defined herein attached to the parent molecular group through an oxygen atom.

[0080] "Alkylcarbonyl" means an alkyl group as previously defined attached to the parent molecular moiety through a carbonyl group. Exemplary, non-limiting alkylcarbonyl groups include methylcarbonyl, ethylcarbonyl, and isopropylcarbonyl, etc.

[0081] "Alkynyl" means an optionally substituted C 2-24 alkyl group having one or more triple bonds. The alkynyl group can be cyclic or acyclic, and examples include ethynyl, 1-propynyl, etc. The alkynyl group can also be substituted or unsubstituted. For example, the alkynyl group can be substituted by one or more substituents, as described herein for alkyl groups. 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 alkynyl. Exemplary, non-limiting alkynyl groups include ethynyl (-C≡CH), 1-propynyl (-C≡CCH3), 2-propynyl or propargyl (-CH2C≡CH), 1-butynyl (-C≡CCH2CH3), 2-butynyl (-CH2C≡CCH3), 3-butynyl (-CH2CH2C≡CH), etc.

[0082] "Alkynylene" means the polyvalent (e.g., divalent) form of an alkynyl group, which is an optionally substituted C 2-24 alkyl group having one or more triple bonds. The alkynylene group can be cyclic or acyclic. The alkynylene group can be substituted or unsubstituted. For example, the alkynylene group can be substituted by one or more substituents, as described herein for alkyl groups. Exemplary, non-limiting alkynylene groups include -C≡C- or -C≡CCH2-.

[0083] "Amido" means -N(R N1 )C(O)-, where R N1 is H, an optionally substituted alkyl, or an optionally substituted aryl.

[0084] "Amino" means -NR N1 R N2 , where R N1 and R N2 are each independently H, an optionally substituted alkyl, or an optionally substituted acyl, or an optionally substituted aryl, or RN1 and R N2 together with the nitrogen atom to which it is attached form a heterocyclic group as defined herein.

[0085] "Aminoalkyl" means an alkyl group as defined herein which is substituted by an amino group as defined herein.

[0086] "Aminoaryl" means an aryl group as defined herein which is substituted by an amino group as defined herein.

[0087] "Ammonium" means a group containing a protonated nitrogen atom N + The exemplary ammonium groups include -N + R N1 R N2 R N3 wherein R N1 、R N2 and R N3 are each independently H, an optionally substituted alkyl, an optionally substituted cycloalkyl, or an optionally substituted aryl; or R N1 and R N2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic group or heterocycle; or R N1 and R N2 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 heterocyclic group or heterocycle, such as a heterocyclic cation.

[0088] Unless otherwise specified, "aromatic" means a cyclic conjugated group or moiety of 5 to 15 ring atoms, having a single ring (e.g., phenyl) or multiple fused rings, where at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridyl); that is, at least one ring and optionally multiple fused rings, having a continuous, delocalized π - electron system. Typically, the number of out - of - plane π - electrons corresponds to the Huckel rule (4n + 2). The point of attachment to the parent structure is typically through the aromatic portion of the fused - ring system. Such aromatic compounds can be unsubstituted or substituted by one or more groups (such as those described herein for alkyl or aryl groups). Other possible substituents can include aliphatic, haloaliphatic, halogen, nitrate, cyano, sulfonate, sulfonyl, or others.

[0089] "Aryl" means a group containing any carbon - based aromatic group, including but not limited to phenyl, benzyl, anthracenyl, anthryl, benzocyclobutenyl, benzocyclooctenyl, biphenyl, chrysenyl, indanyl, fluoranthenyl, indacenyl, indenyl, naphthyl, phenanthryl, phenoxybenzyl, picenyl, pyrenyl, terphenyl, etc., including fused benzo-C 4-8 cycloalkyl radicals (e.g., as defined herein), such as indanyl, tetrahydronaphthyl, fluorenyl, etc. The term aryl also includes heteroaryl, which is defined as a group containing an aromatic group with 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 within the term aryl) defines a group containing 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 of the substituents described herein for alkyl).

[0090] “Aryloxyalkyl” means an arylalkylene group as defined herein attached to the parent molecular group through an oxygen atom. In some embodiments, the aryloxyalkyl 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.

[0091] “(Aryl)(alkyl)subylene” means a divalent form comprising an arylene group attached to an alkylene or heteroalkylene group as described herein. In some embodiments, the (aryl)(alkyl)subylene group is -L-Ar- or -L-Ar-L- or -Ar-L-, where Ar is an arylene group, and each L is independently an optionally substituted alkylene group or an optionally substituted heteroalkylene group.

[0092] “Arylalkylene” means 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, 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 as described herein for aryl and / or alkyl. Exemplary unsubstituted arylalkylene groups have 7 to 16 carbons (C 7-16 arylalkylene), and those arylalkylene groups having an aryl group with 4 to 18 carbons and an alkylene group with 1 to 6 carbons (i.e., (C 4-18 aryl)C 1-6 alkylene).

[0093] As used herein, "arylene" means a polyvalent (e.g., divalent, trivalent, tetravalent, etc.) form of an aryl group. Exemplary arylene groups include phenylene, naphthylene, biphenylene, triphenylene, diphenyl ether, acenaphthylene, anthrylene, or phenanthrylene. In some embodiments, the arylene group is a 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 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 substituents as described herein for aryl groups.

[0094] "Aryloxy" means an arylene group as defined herein attached to a parent molecular group through an oxygen atom.

[0095] "Aryloxy" means an aryl group as defined herein attached to a parent molecular group through an oxygen atom.

[0096] "Aroyl" means an aryl group attached to a parent molecular group through a carbonyl group. In some embodiments, an unsubstituted aroyl group is a C 7-11 aroyl or C 5-19 aroyl group. In certain embodiments, the aroyl group is -C(O)-Ar, where Ar is an aryl group as defined herein.

[0097] "Attaching", "attachment", or related word forms mean any covalent or non-covalent bonding interaction between two components. Non-covalent bonding interactions include, but are not limited to, hydrogen bonding, ionic interactions, halogen bonding, electrostatic interactions, π-bond interactions, hydrophobic interactions, inclusion compounds, clathration, van der Waals interactions, and combinations thereof.

[0098] "Azido" means -N3.

[0099] "Boranyl" means a -BR2 group, where each R can independently be H, halogen, or an optionally substituted alkyl group.

[0100] "Dihydroxyboron" means a -BOH2 group.

[0101] "Branched alkenyl" means an isomer of a straight-chain alkenyl compound; it has an alkyl group bonded to the main carbon chain.

[0102] "Carbonyl" means a -C(O)- group, which can also be represented as >C=O.

[0103] "Carboxyl" means the -CO2H group.

[0104] "Carboxylate anion" means the -CO2 - group.

[0105] "Covalently bonded" means a covalent bonding interaction between two components. Non-limiting covalent bonds include single bonds, double bonds, triple bonds or spiro bonds, where at least two molecular groups are bonded to the same carbon atom.

[0106] "Cyano" means -CN.

[0107] As used herein, "cyclic group" refers to an aryl group, a non-aryl group (e.g., a cycloalkyl or heterocycloalkyl group), or both. The cyclic group has one or more ring systems that may or may not be substituted. The cyclic group may 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.

[0108] Unless otherwise specified, "cycloalkyl" means a monovalent saturated or unsaturated non-aromatic or aromatic cyclic hydrocarbon group having three to eight carbons, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, etc. The cycloalkyl group may also be substituted or unsubstituted. For example, the cycloalkyl group may be substituted with one or more groups (including those described herein for alkyl groups).

[0109] "Dicarbonyl" means any moiety or compound that includes two carbonyl groups, as defined herein. Non-limiting dicarbonyl moieties include 1,2-dicarbonyl (e.g., R C1 -C(O)-C(O)R C2 , where R C1 and R C2 are each independently an optionally substituted alkyl group, a halogen, an optionally substituted alkoxy group, a hydroxyl group or a leaving group); 1,3-dicarbonyl (e.g., R C1 -C(O)-C(R 1a R 2a )-C(O)R C2 , where R C1 and R C2 are each independently an optionally substituted alkyl group, a halogen, an optionally substituted alkoxy group, a hydroxyl group or a leaving group, and where R 1a and R 2a are each independently H or an optionally substituted group provided for the alkyl group, as defined herein); and 1,4-dicarbonyl (e.g., R C1 -C(O)-C(R 1a R 2a )-C(R 3aR 4a )-C(O)R C2 wherein R C1 and R C2 are each independently an optionally substituted alkyl group, a halogen, an optionally substituted alkoxy group, a hydroxyl group or a leaving group, and wherein R 1a , R 2a , R 3a and R 4a are each independently H or an optionally substituted group provided for the alkyl group, as defined herein).

[0110] "Electron-withdrawing moiety" means a moiety capable of contributing at least a portion of its electron density, such as by resonance, to the ring or functional group to which it is directly attached.

[0111] "Halogen" means F, Cl, Br or I.

[0112] "Halogen-containing substituent" means a group containing a halogen, such as a haloaliphatic or haloalkyl group.

[0113] "Haloaliphatic" means an aliphatic group substituted with one or more halogens as defined herein.

[0114] "Haloalkenyl" means an alkenyl group substituted with one or more halogens as defined herein.

[0115] "Haloalkynyl" means an alkynyl group substituted with one or more halogens as defined herein.

[0116] "Haloalkyl" means an alkyl group substituted with one or more halogens as defined herein. 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 haloalkyl. Other non-limiting haloalkyl groups include -CX y H 3-y , where y is 1, 2 or 3, and wherein each X is independently a halogen (F, Cl, Br or I); -CX z H 2-z CX y H 3-y, where z is 0, 1, or 2, where y is 0, 1, 2, or 3, and where each X is independently a halogen (F, Cl, Br, or I), with at least one of z or y not being 0; -CH2CX y H 3-y , where y is 1, 2, or 3, and where each X is independently a halogen (F, Cl, Br, or I); -CX z1 H 2-z1 CX z2 H 2-z2 CX y H 3-y , where z1 and z2 are each independently 0, 1, or 2, where y is 0, 1, 2, or 3, and where each X is independently a halogen (F, Cl, Br, or I), with at least one of z1, z2, or y not being 0; and -CX z H 1-z [CX y1 H 3-y1 [CX y2 H 3-y2 , where z is 0 or 1, where y1 and y2 are each independently 0, 1, 2, or 3, and where each X is independently a halogen (F, Cl, Br, or I), with at least one of z, y1, or y2 not being 0.

[0117] "Halogenoalkylene" means an alkylene group substituted with one or more halogens as defined herein.

[0118] "Heteroaliphatic" means an aliphatic group as defined herein that includes at least one heteroatom to 20 heteroatoms, such as 1 to 15 heteroatoms, or 1 to 5 heteroatoms within the group, said heteroatoms being selected from, but not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and their oxidized forms.

[0119] "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 halogen).

[0120] "Heteroalkylene" means an alkylene 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 halogen). The heteroalkylene group can be saturated or unsaturated (e.g., having one or more double or triple bonds). The heteroalkylene group can be substituted or unsubstituted. For example, the heteroalkylene group can be substituted with one or more substituents as described herein for alkyl.

[0121] "Heteroaryl" means a subset of the heterocyclic groups as defined herein that are aromatic, i.e., that contain 4n + 2 π electrons within a monocyclic or polycyclic system.

[0122] The term "heterocycloalkyl" is a cycloalkyl group as defined above in which at least one carbon atom and its attached hydrogen atom(s), if any, are replaced by O, S, N or NH. Heterocycloalkyl groups and heterocycloalkenyl groups may be substituted or unsubstituted. As described herein, cycloalkenyl groups and heterocycloalkenyl groups may be substituted by one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, sulfonic acid, sulfinic acid, fluoric acid, phosphonic acid, ester, ether, halide, hydroxy, ketone, nitro, cyano, azide, silyl, sulfonyl, sulfinyl or thiol.

[0123] "Heterocycle" means a compound having one or more heterocyclic 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 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 substituents include any of the substituents described herein for aryl. The heterocycle may also include cations and / or salts of any of these heterocycles (e.g., any such substances 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).

[0124] "Heterocyclic group" means a 3-, 4-, 5-, 6- or 7-membered ring, containing one, two, three or four non-carbon heteroatoms (e.g., independently selected from the group consisting of nitrogen, oxygen, phosphorus, sulfur, selenium or halogen), unless otherwise specified. The 3-membered ring has zero to one double bond, the 4- and 5-membered rings have zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds. The term "heterocyclic group" also includes bicyclic, tricyclic and tetracyclic groups, wherein any of the above heterocycles 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, a cyclopentene ring and another monocyclic heterocycle, such as indolyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, benzofuranyl, benzothienyl, etc. Heterocycles 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, benzodioxocinyl, 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., β-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, dioxinyldioxobenzofuranyl, dioxolyl, dioxotetrahydrofuranyl, dioxothiomorpholinyl, dithianyl, dithiazolyl, dithienyl, dithiinyl, furanyl, furazanyl, furoyl, furyl, guaninyl, homopiperazinyl, homopiperidinyl, hypoxanthinyl, hydantoinyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl (e.g., 1H-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, naphthotriazolyl, 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, pyridopyrazinylpyridopyrimidinyl, 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-1,2,5-thiadiazinyl or 2H,6H-1,5,2-dithiadiazinyl), thiadiazolyl, thianthrenyl, thianyl, thianaphthenyl, thiazepinyl, thiazinyl, thiazolidinedionyl, thiazolidinyl, thiazolylThienyl, 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, etc., and their modified forms (e.g., including one or more oxo groups and / or amino groups) and their salts. The heterocyclic group can be substituted or unsubstituted. For example, the heterocyclic group can be substituted by one or more substituents as described herein for aryl groups.

[0125] As used herein, "heterocyclic diyl" means the divalent form of a heterocyclic group. In one example, the heterocyclic diyl is formed by removing hydrogen from the heterocyclic group. Exemplary heterocyclic diyl groups include piperdylidene, quinoline diyl, etc. The heterocyclic diyl group can also be substituted or unsubstituted. For example, the heterocyclic diyl group can be substituted by one or more substituents as described herein for heterocyclic groups.

[0126] "Hydroxyalkyl" means an alkyl group substituted by one or more hydroxyl groups as defined herein.

[0127] "Hydroxyalkylene" means an alkylene group substituted by one or more hydroxyl groups as defined herein.

[0128] "Hydroxy" means -OH.

[0129] "Imino" means -NR-, where R can be H or an optionally substituted alkyl group.

[0130] "Isocyanate" means -NCO.

[0131] "Isothiocyanate" means -N=C=S.

[0132] "Leaving group" means an atom (or group of atoms) having electron-withdrawing ability that can be replaced as a stable species and take away the bonding electrons, or an atom (or 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 trifluoromethanesulfonate (-OTf), methanesulfonate (-OM), toluenesulfonate (-OT), p-bromobenzenesulfonate (-OB), acetate, Cl, Br, and I.

[0133] "Nitro" means the -NO2 group.

[0134] "Oxo" means the =O group.

[0135] "Oxy" means -O-.

[0136] "Phosphate" means a group derived from phosphoric acid. An example of a phosphate includes -O-P(=O)(OR P1 )(OR P2 ) or -O-[P(=O)(OR P1 )-O] P3 -R P2 groups, where R P1 and R P2 are each independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted aryl group, or an optionally substituted arylalkylene group, and where P3 is an integer from 1 to 5. Other examples of phosphates include orthophosphoric acid, pyrophosphoric acid, tripolyphosphoric acid, tetrapolyphosphoric acid, trimetaphosphoric acid, and / or phosphoric anhydride, or combinations thereof.

[0137] "Phosphonyl" or "phosphonic acid" means the -P(O)(OH)2 group.

[0138] "Salt" means the ionic form of a compound or structure (e.g., any of the formulas, compounds, or compositions described herein), which includes a cationic or anionic 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. Sci. January 1977; 66(1):1-19; and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Wiley-VCH, April 2011 (2nd revised edition, edited by 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 present invention, or separately by reacting a free basic group with a suitable organic acid (thereby producing an anionic salt) or by reacting an acidic 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, dodecylsulfate, edetate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide,hydroxyethanesulfonate, hydroxynaphthoate, iodide, lactate, lactobionate, laurate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, 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, etc. Representative cationic salts include metal salts such as alkali metal or alkaline earth metal salts, e.g., barium, calcium (e.g., calcium edetate), lithium, magnesium, potassium, sodium, etc.; other metal salts such as aluminum, bismuth, iron, and zinc; 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. 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. Other additional salts may include anions such as halides (e.g., F, - , Cl - , Br - or I - ), hydroxides (e.g., OH - ), borates (e.g., tetrafluoroborate (BF4 - ), carbonates (e.g., CO3 2- or HCO3 - ), or sulfates (e.g., SO4 2- ).

[0139] "Silyl" means -SiR 1 R 2 R 3 or -SiR 1 R 2 - group. 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. In certain embodiments, R 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) c , where each R is independently H, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aromatic, or optionally substituted heteroaromatic; each of a, b, and c is ≥ 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.

[0140] "Spiro group" means an alkylene diradical whose two ends are bonded to the same carbon atom of the parent group to form a spiro group, and also means a heteroalkylene diradical whose two ends are bonded to the same atom. Non-limiting alkylene and heteroalkylene groups for use within the spiro 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 an alkylene group, and a C having 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 a heteroalkylene group.

[0141] "Sulfate" means a group derived from sulfuric acid. An example of a sulfate includes the -O-S(=O)2(OR S1 ) group, where R S1 is H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted aryl, or an optionally substituted arylalkylene.

[0142] "Sulfo" or "sulfonic acid" means the -S(O)2OH group.

[0143] "Sulfonyl" means the -S(O)2- or -S(O)2R group, where R can be H, an optionally substituted alkyl, or an optionally substituted aryl. Non-limiting sulfonyl groups can include the trifluoromethylsulfonyl group (-SO2-CF3 or Tf).

[0144] The use of the above terms means including substituted and unsubstituted moieties. Substitution can be carried out by one or more groups such as alcohol, ether, ester, amide, sulfone, sulfide, hydroxyl, nitro, cyano, carboxyl, amine, heteroatom, lower alkyl, lower alkoxy, lower alkoxycarbonyl, alkoxyalkoxy, acyloxy, halogen, trifluoromethoxy, trifluoromethyl, alkyl, arylalkyl, alkenyl, alkynyl, aryl, cyano, carboxyl, carbonylalkoxy, carboxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclic group, alkylheterocyclic group, heterocycloalkyl, oxo, arylsulfonyl, and arylalkylaminocarbonyl, or any of the substituents in the preceding paragraph, or those substituents directly attached or attached through a suitable linker. The linker is typically a short chain of 1-3 atoms containing any combination of -C-, -C(O)-, -NH-, -S-, -S(O)-, -O-, -C(O)-, or -S(O)O. The ring can be substituted multiple times.

[0145] The term "lower" modifying "alkyl", "alkenyl", "alkynyl", "alkoxy", or "alkoxycarbonyl" refers to a C1-C6 unit of a specific functionality. For example, "lower alkyl" means C1-C6 alkyl.

[0146] "Substituted" means having one or more substituent moieties whose presence does not interfere with the desired function or reactivity. Examples of substituents are alkyl, alkenyl, alkynyl, cycloalkyl (non-aromatic ring), Si(alkyl)3, Si(alkoxy)3, alkoxy, amino, alkylamino, alkenylamino, amide, amidine, guanidine, hydroxy, thioether, alkylcarbonyl, alkylcarbonyloxy, alkoxycarbonyloxy, carbonate, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, cyano, halogen, acylamino, imino, mercapto, alkylthio, thiocarboxylate, dithiocarboxylate, sulfate, sulfate group, sulfonate, sulfamoyl, sulfonamide, nitro, nitrile, azide, heterocyclic group, ether, ester, silicon-containing moiety, thioester or a combination thereof. The substituent itself may be substituted. For example, the amino substituent itself may be mono-substituted or independently di-substituted by other substituents defined above such as alkyl, alkenyl, alkynyl and cycloalkyl (non-aromatic ring), etc.

[0147] "Sulfide" means a thioether -S-R', where R' may be, but is not limited to, an aliphatic group.

[0148] "Mercapto" means a thiol, i.e., -SH.

[0149] "Thiocyanate group" means -SCN.

[0150] "Thioester" means -SC(O)R', where R' may be, but is not limited to, an aliphatic group.

[0151] "Unsubstituted" means any open valence of an atom occupied by hydrogen. Additionally, if the occupant of the open valence position on the atom is not specified, it is hydrogen.

[0152] "Unsaturated" means a moiety containing a double carbon-carbon bond or a triple carbon-carbon bond.

[0153] "Unsaturated substituent" means an aliphatic chain, cyclic, aryl or heteroaryl group containing a double bond or a triple bond.

[0154] One of ordinary skill in the art will recognize that the definitions provided above are not intended to include non-permissible substitution patterns (e.g., a methyl group substituted by five substituents, etc.). One of ordinary skill in the art can readily identify such non-permissible substitution patterns. Unless otherwise specified herein, any functional group disclosed herein and / or defined above may be substituted or unsubstituted.

[0155] As used herein, a mass tag includes any tag containing enriched heavy atoms (such as enriched metal isotopes). The mass tag may include a polymer loaded with an enriched metal isotope and may optionally include a conjugated bioactive material. Mass tags can be distinguished based on the atomic mass of their enriched metal isotopes.

[0156] As used herein, mass cytometry is any method for detecting mass tags in a biological sample, such as simultaneously detecting multiple distinguishable mass tags at single-cell resolution. Mass cytometry includes suspension mass cytometry and imaging mass cytometry TM (IMC TM ) and secondary ion mass cytometry (SIMS). Mass cytometry can atomize and ionize the mass tags of a cell sample by one or more of laser radiation, ion beam radiation, electron beam radiation, and / or inductively coupled plasma (ICP). Mass cytometry can simultaneously detect different 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-1-yl) 4-(2-azatricyclo[10.4.0.0 4,9 hexadec-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutyrate.

[0157] 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, multispecific antibodies (such as bispecific antibodies produced from at least two intact antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antigen-determining portions of antibodies, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Based on the characteristics of their heavy-chain constant domains, which are designated α, δ, ε, γ, and μ, respectively, antibodies can 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). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.

[0158] "Monoclonal antibody" refers to a population of homologous antibodies that involve highly specific recognition and binding to a single antigen determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies against different antigen determinants. The term "monoclonal antibody" encompasses 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 containing antibody portions, and any other modified immunoglobulin molecule containing an antigen recognition site. In addition, "monoclonal antibody" refers to such antibodies prepared in any number of ways, including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0159] 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, a humanized antibody is a human immunoglobulin in which the residues from the complementarity determining regions (CDRs) are replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and capacity.

[0160] 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 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 one human heavy chain and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0161] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences in an antibody derived from another (usually human) species to avoid eliciting an immune response in that species.

[0162] As used herein, the term "chemical antibody" encompasses aptamers (e.g., protein aptamers or nucleic acid aptamers). It will be readily understood by those skilled in the art that in any of the embodiments described herein, a chemical antibody can replace an antibody.

[0163] The term "protein" refers to small proteins less than 100 amino acids in length and large biological or macromolecules having one or more long chains of amino acid residues, including globular, fibrous, or membrane proteins, such as antibodies.

[0164] The term "chelator" refers to a moiety that can coordinate (e.g., stably coordinate) a metal atom.

[0165] Introduction and Background

[0166] Conventional detection reagents for biometric assays typically consist of a binding moiety specific for a molecule of interest, conjugated to a moiety with enzymatic or optical properties. To date, these assays have generally been facilitated by the use of radioactive, fluorescent, or enzyme tags. In the assay methods of interest, flow cytometry provides a means for simultaneous multi-parameter analysis of the physical and / or chemical characteristics of up to thousands of particles per second and is commonly used in research and clinical diagnostic applications, including both particle analysis and particle sorting. The analysis of cells is of particular interest. Modern instruments typically have multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for the simultaneous analysis of multiple labeled antibodies and enables more precise identification of target populations by their phenotypic markers. In conventional flow cytometry, fluorescently labeled particles, such as live cells, fixed cells, beads, etc., are individually distinguished and separated based on their fluorescence and light scattering characteristics. The phenotype of the particles can be further studied after they are separated.

[0167] Such conventional flow cytometry methods are limited by the number of simultaneous parameters that can be measured on a single particle, and there are problems of fluorescence emission overlap during simultaneous measurements; as well as background fluorescence or enzyme activity. As the number of simultaneous parameters increases, this spectral overlap severely interferes with the analysis, thus affecting both the accuracy and sensitivity of the assay. In alternative detection methods, atomic mass spectrometry measurements have been used in combination with stable isotope tags of rare elements.

[0168] Existing elemental labeling capture reagents for use in ICP-MS are based on chelating agents, such as ethylenediaminetetraacetic acid (EDTA), tetraazacyclododecane-tetraacetic acid (DOTA), or diethylenetriaminepentaacetic acid (DTPA), for example, a maleimide-functionalized polymer of DOTA with an average length between 10 and 30 monomers. Such a scheme allows for conjugation with a typical antibody of 6 or 7 polymers, thus conjugating an average of 200 labeled isotope atoms per antibody. The sensitivity of this method is directly related to the number of elemental isotope tags in each detection reagent molecule. The number of polymers that can be attached is limited to the number of disulfide bonds that can be cleaved on the immunoglobulin without disrupting its function. The number of metal chelating units that can be conjugated to the detection reagent is also limited because an increased number may interfere with the detection reagent or induce non-specific interactions and thus interfere with or induce high background in the assay.

[0169] Metal-protein conjugates

[0170] In some embodiments, the metal-protein conjugate can be composed of a variety 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, such as a coordination complex. In some embodiments, the metal is a thiophilic metal. In some embodiments, the protein can be an antibody, and the metal and the protein can be linked together by various suitable conjugation methods. For example, the metal can be conjugated to the protein by covalent bonding (e.g., amine chemistry, thiol chemistry, phosphate chemistry), enzymatic reactions, redox reactions (such as with metal halides), and affinity intermediates (e.g., streptavidin or biotin), or click chemistry forms (such as strain-promoted click chemistry or metal-catalyzed click chemistry).

[0171] As used herein, "click chemistry", "click chemical reaction", or "click reaction" refers to a reaction designed to have a high thermodynamic driving force that drives the reaction rapidly and irreversibly to a high yield of a single reaction product, with high reaction specificity (in some cases, with both regioselectivity and stereoselectivity). The reaction typically produces few (if any) toxic by-products, and many reactions can be carried out under physiological conditions, that is, in an aqueous buffer, at an approximately neutral pH, and tolerant of typical buffer salts, while the resulting product is also stable under physiological conditions. The characteristics of reactions that do not strictly meet these criteria are still characterized by their high yields, simplicity, and ability to be carried out in mild or easily removable solvents. More importantly, the molecular reactions are bioorthogonal, that is, they proceed without interacting with any functions common in biological systems.

[0172] In some embodiments, the metal and the protein are directly linked together, for example, when the protein is appropriately functionalized or derivatized to include moieties that can chelate the metal. In other embodiments, the metal can be chelated by a chelator attached to the protein or the derivatized protein through a linking moiety. In certain embodiments, these metal-protein conjugates can be used in mass cytometry applications.

[0173] Suitable conjugates include a metal atom, a thiol-containing moiety, and a protein, wherein the protein is conjugated to the thiol-containing moiety by a bond or through a linking moiety. The linking moiety contains a structure of formula (I):

[0174] -L 1 -A-L 2 -(I),

[0175] wherein L 1 and L 2is a linking group. As used herein, "linking group" refers to a compound that can bind to two moieties. In some embodiments, L 1 is a first linking group, which can be an alkylcarbonyl, amide, amine, ether, or ester, and L 1 can covalently bind to a protein; L 2 is a second linking group that includes a nitrogen-containing moiety, and L 2 can covalently bind to a thiol-containing moiety; and A is a cyclic group; and wherein the metal atom chelates the thiol-containing moiety.

[0176] Metal-protein conjugate attached through a linking moiety

[0177] The metal can be indirectly linked to a protein, such as an antibody, through an intermediate linking moiety. The choice of a suitable linking moiety depends on its ability to maximize metal-intermediate bonding while minimizing cross-reactivity, which can include, but is not limited to, polymeric repeating moieties that can form bonds with the metal or metal binding agent. The synthetic route of the metal-antibody conjugate will depend on the type of linking moiety to be used.

[0178] In one embodiment, the method for preparing the 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).

[0179]

[0180] An example of this preparation is shown in Scheme 1 below.

[0181]

[0182] After the synthesis and purification of DMPS-A, mercury chelation can be carried out as the second step of the method for preparing the metal-protein conjugate. For stability purposes and to avoid the formation of insoluble hydroxides that may occur under basic conditions, the metal-chelator complex can be formed at neutral pH. In one embodiment, the complex can be formed by dissolving Hg(NO3)2 in a small amount of dilute nitric acid, and 5 mM Hg 2+ can be added to 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0). The DMPS-A from the first step can be added to the Hg-containing buffer in a 1:1 molar ratio (5 mM), and incubated at room temperature for 1 minute before use.

[0183] In some embodiments, the DMPS-Hg complex prepared according to the second step can have a ratio of DMPS to Hg of 1:1, 2:1, or 4:1.

[0184] In the third step, the metal-loaded DMPS chelator can be covalently conjugated to a protein (such as an antibody) via a DBCO-azide click chemical reaction, as shown in Scheme 2 below.

[0185]

[0186] In Scheme 2, the intermediate structure labeled as azide-residual DMPS peptide is the azide-residual metal-loaded DMPS chelator; while the structure labeled as DBCO-derived antibody on the right is the metal-labeled antibody.

[0187] Other alkynes that can be used to derivatize antibodies include monofluorocyclooctyne, difluorocyclooctyne, dimethoxyazacyclooctyne, dibenzoazacyclooctyne, dibenzocyclooctyne, bisarylazacyclooctyne, bicyclononyne, 2,3,6,7-tetramethoxydibenzocyclooctyne, sulfonated dibenzocyclooctyne, carboxymethylmonobenzocyclooctyne, or pyrrolocyclooctyne.

[0188] In another embodiment, the metal-chelated protein conjugate can be prepared with lipoic acid. Lipoic acid is a mercury chelator. In some embodiments, the lipoic acid-Hg complex can have a 1:1 configuration, as shown below:

[0189]

[0190] Complex 1

[0191] The complex 1 can be attached to the antibody using a coupling agent that is capable of linking the metal-loaded chelator (complex 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)-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).

[0192] Alternative methods for forming metal chelated protein conjugates with lipoic acid involve 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 ammonolysis with ethylenediamine or 1,6-hexanediamine to introduce reactive amine groups. Subsequently, 50% of the polymer repeat units are functionalized with polyethylene glycol succinimidyl esters such as PEG24-NHS ester to yield a functionalized polymer with good water solubility. Then lipoic acid is attached to the remaining polymer repeat units. The polymer will then be loaded with metal, and the polymeric azide end groups will allow attachment to an antibody that has been treated with NHS-DBCO; thereby forming a metal chelated protein conjugate.

[0193] In another embodiment, a method for preparing a metal chelated protein conjugate such as a mercury-antibody conjugate involves conjugating an azide-containing chelator to an antibody prior to chelating mercury. This technique can avoid cross-reactivity of azide that may occur in chelator complexes. In certain embodiments, the method comprises the steps of: (1) incubating the antibody with glutathione to block free thiol groups, (2) conjugating a DBCO-modified antibody with DMPS-A using a DBCO-azide click reaction, and (3) chelating Hg using a chelator-antibody complex.

[0194] Directly bound metal-protein conjugates

[0195] In some embodiments, one or more metal chelation sites can be introduced directly onto the antibody, allowing direct labeling of the antibody with metal ions. Disulfide bonds (also known as disulfide bridges) can be found, for example, in the hinge region of an antibody. Using a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), native disulfide bridges can be reduced to two thiol groups. The proximity of the two thiol groups facilitates metal chelation.

[0196] In some embodiments, when the antibody does not include native disulfide bonds, they can be added to the antibody.

[0197] Kits and kit components

[0198] Aspects of the present application include the preparation of a kit or a portion thereof as discussed herein. Aspects of the present application include the use of the kits described herein, such as for mass cytometry or delivery of radioisotopes.

[0199] Kits, kit components, and steps for preparing kits can include a suitable storage medium. For example, solvents and cosolvents can include, but are not limited to, water; sterile water for injection (SWFI); saline; alcohols such as ethanol, benzyl alcohol, etc.; diols and polyols such as propylene glycol, glycerol, etc.; esters of polyols such as diacetin, triacetin, etc.; polyethylene glycols and polyethers such as polyethylene glycol 400, propylene glycol methyl ether, etc.; dioxolanes such as isopropylidene glycerol, etc.; dimethyl isosorbide; pyrrolidone derivatives such as 2-pyrrolidone, N-methyl-2-pyrrolidone, polyvinylpyrrolidone (only as cosolvent), etc.; polyoxyethylated fatty alcohols; esters of polyoxyethylated fatty acids; polysorbates such as TWEEN, polyoxyethylene derivatives of polypropylene glycol such as PLURONICS.

[0200] Suitable stabilizers 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., α-, β-, γ-cyclodextrin), aliphatic polyols (e.g., mannitol, sorbitol, and thioglycerol), cyclic polyols (e.g., inositol), organic solvents (e.g., ethanol and glycerol), and / or aprotic solvents (pyridine, ethyl acetate, DMF, HMPA, and DMSO). The above solvents and / or stabilizers can be used in any step of the above synthesis method or for storing any of the above reagents (e.g., provided in a kit).

[0201] In some aspects, the solution can be acidic. The acidic solutions of the present application can include strong acids such as one or more of nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, hydrochloric acid, and chloric acid. The acid can be present in 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 can be present at 0.05% to 2%. The pH of the acidic solution can be equal to or lower than 6, equal to or lower than 5, equal to or lower than 4.5, or equal to or lower than 4. The lyophilized compositions of the present application can have a water content of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% (by weight).

[0202] At any step (e.g., when conjugates discussed herein are provided in a kit), the conjugates can be lyophilized. For example, the conjugates can be lyophilized with a water content of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% (e.g., by mass). Such lyophilization can allow storage in a kit before use in mass cytometry, and / or when lyophilization stabilizes the conjugates, it can allow flexibility in assay design.

[0203] The kit may further include any additional components (e.g., buffers, filters, etc.).

[0204] Alternatively or in addition, the kit may include additional reagents for mass cytometry, such as buffers, standards, cell barcodes, and / or reagents comprising heavy atoms of different masses (e.g., mass tags attached to or provided for attachment to bioactive materials).

[0205] In some aspects, the kit may include multiple antibodies (e.g., against different targets). Such a collection of antibodies may be provided together in a single panel. The panel may be provided in solution or as a lyophilized mixture that contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by mass of water.

[0206] Conclusion

[0207] Although the foregoing embodiments have been described in detail for purposes of clear 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 devices of the embodiments of the present invention. Accordingly, the embodiments of the present invention are considered illustrative rather than restrictive, and the embodiments are not limited to the details given herein.

Claims

1. A conjugate comprising: a metal atom; a thiol - containing moiety; and a protein; wherein the protein is conjugated to the thiol - containing moiety either through a bond or through a linking moiety, the linking moiety comprising a structure of formula (I): -L 1 -A-L 2 -(I), wherein L 1 is a first linking group comprising an alkylcarbonyl, an amide, an amine, an ether or an ester, and 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 bound to the thiol-containing moiety; and A is a cyclic group; and wherein the metal atom chelates the thiol - containing moiety.

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

3. The conjugate according to claim 2, wherein the dithiol comprises 1,2 - dithiol, 1,3 - dithiol, or 1,4 - dithiol.

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

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

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

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

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

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

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

11. The conjugate according to claim 9, wherein the antibody comprises a dibenzocyclooctyne - derived antibody.

12. The conjugate according to claim 1, wherein the nitrogen - containing moiety comprises an amino group, azide group, or amide group.

13. A method for forming a metal - chelated protein conjugate for mass cytometry, the method 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 according to claim 13, wherein the metal comprises Hg, Pb, Pt, Sb, or As.

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

16. A method for forming a metal - chelated chemical antibody conjugate for mass cytometry, the method 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, 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.

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

18. The method according to claim 13, wherein 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.

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

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

21. A method of forming a metal-chelated protein conjugate for mass cytometry, the method 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 according to claim 21, wherein the metal comprises Hg, Pb, Pt, Sb or As.

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

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

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

26. A method of forming a metal-chelated protein conjugate for mass cytometry, the method comprising: 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 succinimide 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 according to claim 26, wherein the metal comprises Hg, Pb, Pt, Sb or As.

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

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

30. The method according to claim 26, wherein the reagent is (2,5-dioxopyrrolidin-1-yl) 4-(2-azatricyclo[10.4.0.0 4,9 hexadec-1(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, the method comprising: reacting a protein comprising 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.

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

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

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

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

36. A method for forming a metal-chelated protein, the method 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 according to claim 36, wherein the metal comprises Hg, Pb, Pt, Sb or As.

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

39. The method according to claim 36, wherein the reducing agent comprises dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine or tris-(2-carboxyethyl)phosphine.

40. A method for forming a metal-chelated protein, the method comprising: Introducing a thiol 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.

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

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

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

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

45. The method according to claim 40, wherein introducing the thiol group comprises reacting an active site on the protein with the reagent.

46. The method according to claim 45, wherein the active site comprises an amine.

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

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