CN117813339A - Metal-containing polymers for mass flow cytometry - Google Patents

Metal-containing polymers for mass flow cytometry Download PDF

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Publication number
CN117813339A
CN117813339A CN202280052397.1A CN202280052397A CN117813339A CN 117813339 A CN117813339 A CN 117813339A CN 202280052397 A CN202280052397 A CN 202280052397A CN 117813339 A CN117813339 A CN 117813339A
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China
Prior art keywords
compound
polymer
soft metal
group
independently
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CN202280052397.1A
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Chinese (zh)
Inventor
M·A·温尼克
E·C·N·王
Y·张
D·马约尼斯
P·刘
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Sibaituo Canada Inc
University of Toronto
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Sibaituo Canada Inc
University of Toronto
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Priority claimed from PCT/CA2022/051073 external-priority patent/WO2023279211A1/en
Publication of CN117813339A publication Critical patent/CN117813339A/en
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Abstract

The present disclosure relates to compounds of formula I, elemental tags comprising soft metals, and methods and kits thereof for performing mass flow cytometry.

Description

Metal-containing polymers for mass flow cytometry
Cross Reference to Related Applications
The present disclosure claims priority from U.S. patent application Ser. No. 63/219,787, filed on 7.8 of 2021, and U.S. patent application Ser. No. 63/359,182, filed on 7.7 of 2022, the contents of which are incorporated herein by reference in their entireties.
Technical Field
The present disclosure relates to metal-containing polymers, particularly soft metal-containing polymers as elemental tags for mass flow cytometry.
Background
Metal-containing polymers are one of the important classes of polymers developed in the 20 th century. 1,2 By introducing different metal units into conventional organic polymers, functional polymers with novel magnetic, optical, electronic, catalytic and bioactive properties can be obtained and are widely used in various fields such as sensing, catalysis, bioimaging, drug delivery, anticancer drugs and biocides. 3-7 Recently, a new single cell proteomics technique, called mass spectrometry flow cytometry, was developed to address the limitations of multiplexing capability of conventional flow cytometry due to spectral overlap. 8 One feature of this technique is the use of metal-labeled antibodies in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-MS) detection. By labeling antibodies with isotopes of different heavy metal ions, one can examine the expression of multiple biomarkers in a single cell by monitoring signals in different mass channels simultaneously. The development of mass flow cytometry provides new opportunities for metal-containing polymers as elemental mass tags for high-parameter single-cell analysis.
Over the last 15 years, several types of metal chelating polymers have been developed with suspended aminocarboxylate chelators such as diethylenetriamine pentaacetic acid (DTPA) or 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) for use in mass flow cytometry. 9-13 These chelating agents are particularly effective in binding hard metal ions such as lanthanoids, yttrium and bismuth. Using these polymeric tags, researchers can measure more than 40 different biomarkers/cells. These chelating agents are less effective at binding ions of soft metals such as Pd or Pt. One study explored a series of metal chelating polymers with various aminocarboxylate side groups as carriers for platinum or palladium ions in mass flow cytometry applications. 14 Although metal-containing polymers can be prepared, they do not stain target biomarkers and appear to lose heavy metals through interaction with cell-associated soft ligands (possibly thiols). However, newer metals are introduced into mass flow cytometry and increasedThe need to add the number of mass spectrometry channels that can be used is still growing and in this way extends the capabilities of mass flow cytometry.
Disclosure of Invention
Described herein are metal chelating polymers having pendant heterocyclic groups, such as lutidine amine (DPA) and imidazole, which are suitable for binding soft metal ions including Re, hg, or Ag. These polymers have proven useful in mass flow cytometry applications. As shown in the embodiments herein, the metal-labeled antibodies provide accurate quantification for single cell immunophenotyping assays and can be used in combination with commercial reagents for mass flow cytometry immunoassays. Polymers containing DPA chelating groups were used in 4-plex assays for PBMCs and were demonstrated to be able to quantify cell populations. DPA is an effective metal chelator for many different polarizable heavy metal ions. Thus, these results introduce new mass units for mass flow cytometry.
Due to d of Tc (I) and Re (I) 6 The resulting chelate exhibits great stability to ligand substitution and decomposition in low spintronic configurations. 17,19,20 It is understood that stability can be observed in other soft metals like electronic configurations. Since each soft metal element has a variety of naturally occurring isotopes, soft metal chelating polymers provide new mass channels for mass spectrometry applications.
The metal-containing polymer to be used in mass cytometry applications may have one or more of the following characteristics: first, the polymer may have a relatively narrow chain length distribution such that each labeled antibody carries a similar number of metal ions. Second, metals may be combined in such a way that little or no exchange occurs during storage of the application (e.g., in lyophilized form) and/or during use in aqueous solution (mass cytometry experiments may be performed within these hours). Third, the polymer may contain functional groups for antibody conjugation. Finally, the polymer may be water-soluble, as the bioassay is performed in an aqueous medium. Satisfying the above-mentioned various features simultaneously represents an integrated challenge.
In one aspect, the present disclosure includes compounds of formula I
Wherein the method comprises the steps of
A is a polymer backbone, optionally, the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof;
each B is independently a nitrogen-containing 5-to 7-membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof;
l is absent or a linker;
each L 2 Independently absent or a linker;
each R 2 Independently a first modifying group selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof;
x is a functional group selected from esters, ethers, and amides;
each L 1 Independently absent or a linker;
each R 1 Is independently H, C to C8 alkyl, C3-C8 cycloalkyl, OH, C1 to C10 alkoxy, C1 to C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof;
n is an integer between 0 and 50;
m is an integer between 0 and 40;
p is an integer between 0 and 30; and is also provided with
q is an integer greater than 0.
In another aspect, the present disclosure includes a compound of formula I or a derivative or salt thereof, wherein the compound of formula I is chelated to one or more metals M, and wherein the compound has the structure of formula II
In another aspect, the present disclosure includes a composition comprising one or more compounds of formula I and one or more metals M.
In another aspect, the disclosure includes a compound having formula I or II for use in mass flow cytometry.
In another aspect, the present disclosure includes an elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups, wherein at least one chelating group is chelated to a soft metal atom of a soft metal, the soft metal being a monoisotope.
In another aspect, the disclosure includes a kit comprising
An isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal; and
an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, the chelating groups comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group comprises or is capable of binding at least one soft metal atom of an isotopic composition;
wherein the kit does not comprise any radioactive soft metals.
In another aspect, the present disclosure includes a method comprising:
Providing an isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
providing an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group is capable of binding at least one of the soft metal atoms of the isotopic composition; and
binding the soft metal atoms of the isotope composition to one or more chelating groups of the elemental tag;
wherein the soft metal atoms are non-radioactive.
In another aspect, the present disclosure includes a method for analyzing an analyte in a biological sample, comprising:
(i) Incubating an element-labeled affinity reagent with an analyte, the element-labeled affinity reagent comprising an affinity reagent labeled with an element tag comprising a linear or branched polymer having a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, the element tag further comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
wherein:
each chelating group of the elemental tag includes or is capable of binding at least one of the soft metal atoms,
The soft metal atoms are non-radioactive and
the affinity reagent specifically binds to the analyte,
(ii) Separating unbound elemental-labeled affinity reagent from bound elemental-labeled affinity reagent; and
(iii) Elemental tags bound to affinity reagents attached to the analyte are analyzed by mass spectrometry atomic spectrometry.
Drawings
Exemplary embodiments of the present disclosure will be further described with reference to the accompanying drawings, wherein:
FIG. 1 is a compound I-1 1 H-NMR (600 MHz) spectrum.
FIG. 2 (a) is an aromatic region of a Re-loaded polymer 1 H-NMR (600 MHz) spectrum. FIG. 2 (b) is the FTIR spectra of Re salt, compound I-1 and Re-supporting Compound II-1.
FIGS. 3 (a) - (e) are human PBMC at different titers 170 Er-CD3 compared to 187 Biaxial scattergram of Re-CD 20. FIG. 3 (f) is the human PMBC at optimal titer 170 Er-CD3 compared to 147 Biaxial scattergrams of Sm-CD 20.
FIG. 4 (a) is a RAFT reaction mixture 1 H-NMR (600 MHz) spectrum, and fig. 4 (b) is GPC trace of poly (PFPA) synthesized by RAFT polymerization of PFPA monomers.
FIG. 5 is a poly (PFPA) 1 H-NMR (600 MHz) spectrum and poly (PFPA) 19 F-NMR (564 MHz) spectrum.
FIG. 6 is a graph of compounds 1-2 (top), compounds 1-3 (middle) and compounds 1-4 (bottom) 1 H-NMR (600 MHz) spectrum.
FIG. 7 is a series of 19 F-NMR (564 MHz) spectra depicting the ammonolysis of poly PFPA with lysine based rhenium chelators.
FIG. 8 is a schematic representation of Polymer 2-2 1 H-NMR (600 MHz) spectrum and Polymer 2-2 19 F-NMR (564 MHz) spectrum.
FIG. 9 is a UV-vis spectrum of Polymer 2-2 and DDMAT CTA.
FIG. 10 is a block diagram of PEGylated Polymer 2-3 by Polymer 2-2 1 H-NMR (600 MHz) spectrum.
FIG. 11 (a) is Bis-Mal-PEG 6 A kind of electronic device 1 H-NMR (600 MHz) spectrum. FIG. 11 (b) is Bis-Mal-PEG 6 And rhenium salt mixtures 1 H-NMR (600 MHz) spectrum.
FIG. 12 is an image of lyophilized rhenium-loaded polymer compound II-1.
FIG. 13 (a) is the UV-vis spectrum of Re-loaded polymer compound II-1 in PBS. FIG. 13 (b) is a FPLC chromatogram of a pure CD20 antibody. Fig. 13 (c) is an FPLC chromatogram of an antibody-polymer conjugate.
FIG. 14 (a) is a polymer 3-2/I-5 1 H-NMR (600 MHz) spectrum, and FIG. 14 (b) is the compound I-1 1 H-NMR (600 MHz) spectrum.
FIG. 15 is an FTIR spectrum of Polymer 3-2/I-5.
FIG. 16 is a Pt-loaded polymer 4-1/II-2 1 H-NMR (600 MHz) spectrum.
FIG. 17 is a graph of human PBMC at varying titers 170 Er-CD3 compared to 195 Pt-CD20, T-lymphocyte and B-lymphocyte 170 Er-CD3 compared to 147 A series of biaxial scattergrams of Sm-CD 20.
FIG. 18 is a schematic illustration of polymers 2-3 1 H-NMR (600 MHz) spectrum.
FIG. 19 is a schematic diagram of Hg-supporting Polymer 5-1/II-3 1 H-NMR (600 MHz) spectrum.
FIG. 20 is a schematic diagram of Ag-supporting Polymer 6-1/II-4 1 H-NMR (600 MHz) spectrum.
FIG. 21 is a graph of (a) Pt-loaded polymer 14-3/II-6 (where the arrow shows the change in chemical shift of the pyridyl proton after metallization with Pt) and (b) Hg-loaded polymer 14-2/II-5 (where the arrow shows the change in chemical shift of the pyridyl proton after metallization with Hg) 1 H-NMR (600 MHz) spectrum.
FIG. 22 is a photograph of Compound 11-4 1 H-NMR (600 MHz) spectrum.
FIG. 23 is a diagram of Compound I-11 1 H-NMR (600 MHz) spectrum.
FIG. 24 is a diagram of Compound I-12 1 H-NMR (600 MHz) spectrum.
Figure 25 is a mass cytometry immunoassay result of identification of cd20+ B cells from PBMCs with rhenium-labeled zwitterionic solubility modifiers containing polymers of the present disclosure conjugated to CD20 antibodies at different concentrations of the polymer conjugates. Maxpar TM 147 Sm-CD20 conjugate was used as positive control.
Figure 26 is a mass cytometry immunoassay result of identification of cd8+ T cells from PBMCs with rhenium-labeled zwitterionic solubility modifiers containing polymers of the present disclosure conjugated to CD8a antibodies at different concentrations of the polymer conjugates. Maxpar TM 146 Nd-CD8a conjugate was used as positive control.
FIG. 27 is a graph showing the use of rhenium-labeled zwitterionic solubility modifiers obtained from non-T/B cells (CD 3-CD 20-) within PBMC 187 Re signal 147 A plot of signal distribution histograms of Sm signals containing polymers of the present disclosure conjugated to CD20 antibodies at different concentrations of polymer conjugate. Maxpar TM 147 Sm-CD20 conjugate was used as a control.
FIG. 28 is a graph showing the use of rhenium-labeled zwitterionic solubility modifiers obtained from B cells (CD 3-CD20+) in PBMC 187 Re signal 146 Graph of signal distribution histogram of Nd signal, the zwitterionic solubilityThe modifier contains polymers of the present disclosure conjugated to CD8a antibodies at different concentrations of the polymer conjugate. Maxpar TM 146 Nd-CD8a conjugates were used as controls.
Fig. 29 is a graph showing the results of non-specific binding assays for both PEG-modified rhenium polymers (group a, polymer concentrations of 1, 2, and 5 ug/mL) and zwitterionic-modified rhenium polymers (group B, polymer concentrations of 1, 2, and 5 ug/mL).
FIG. 30 is a photograph of Compound 16-3 1 H-NMR (600 MHz) spectrum.
Fig. 31 is a series of graphs showing the results of non-specific binding assays for glutathione-modified polymers of the present disclosure compared to non-glutathione-modified polymers of the present disclosure. Maxpar TM Used as positive control.
Detailed Description
I. Definition of the definition
The definitions and embodiments described in this and other sections are intended to apply to all embodiments and aspects of the disclosure as understood by those skilled in the art as appropriate, unless otherwise indicated.
The term "compound of the present disclosure" or "compound of the present disclosure" and the like as used herein refers to a compound having formula I or II, and salts, solvates and/or derivatives thereof.
The term "and/or" as used herein means that the listed items are present or used, either alone or in combination. Indeed, this term means "at least one" or "one or more" used or present in the listed items. The term "and/or" with respect to pharmaceutically acceptable salts and/or solvates thereof means that the compounds of the present disclosure exist as a combination of individual salts and hydrates, e.g., solvates of salts of the compounds of the present disclosure.
As used in this disclosure, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, embodiments that include "a compound" are understood to exhibit certain aspects with one compound or two or more additional compounds.
In embodiments that include an "additional" or "second" component (such as an additional or second compound), the second component as used herein is chemically different from the other component or the first component. For example, when the second combination and the first component may have the same chelating agent, the metal chelated to the second component may be different than the metal chelated to the first component. The "third" component is different from the other components, the first component, and the second component, and similarly, the further enumerated or "additional" components are different.
As used in this disclosure and in the claims, the terms "comprise" (and any form of comprising), such as "comprises" and "comprising)", "having" (and any form of having), such as "having" and "having", "including)", "and any form of including, such as" including "and" comprising "," or "containing", "and any form of containing" such as "containing" and "containing" are inclusive or open-ended, and do not exclude additional unrecited elements or method steps.
The term "consisting of … …" and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but also exclude the presence of other unstated features, elements, components, groups, integers, and/or steps.
The term "consisting essentially of … …" as used herein is intended to specify the presence of stated features, elements, components, groups, integers, and/or steps, and those that do not materially affect one or more of the basic and novel characteristics of such features, elements, components, groups, integers, and/or steps.
The term "suitable" as used herein means that the selection of a particular compound or condition will depend on the particular synthetic manipulation to be performed, the nature of the molecule or molecules to be converted, and/or the particular use of the compound, but such selection will be well within the skill of the person trained in the art.
In embodiments of the present disclosure, the compounds described herein may have at least one asymmetric center. Where the compounds have more than one asymmetric center, they may exist as diastereomers. It is understood that all such isomers and mixtures thereof in any ratio are encompassed within the scope of the present disclosure. It is further understood that while the stereochemistry of a compound may be as shown in any given compound listed herein, such compounds may also contain an amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of a compound of the disclosure having alternative stereochemistry. Any optical isomer (as an isolated, pure or partially purified optical isomer or racemic mixture thereof) is intended to be included within the scope of the present disclosure.
The compounds of the present disclosure may also exist in different tautomeric forms, and any tautomeric forms, and mixtures thereof, that are intended for formation of the compounds are included within the scope of the present disclosure.
The present specification refers to a number of chemical terms and abbreviations used by those skilled in the art. However, for clarity and consistency, definitions of selected terms are provided.
The terms "about," "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least or up to + -5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context otherwise suggests to one of ordinary skill in the art.
The term "alkyl", as used herein, whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The possible number of carbon atoms in the mentioned alkyl groups is indicated by the prefix "Cn1-n 2". For example, the term C1-10 alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
The term "alkylene", whether used alone or as part of another group, means a saturated alkylene group, straight or branched, i.e., a saturated carbon chain containing substituents at both ends thereof. The possible number of carbon atoms in the alkylene groups mentioned is indicated by the prefix "Cn1-n 2". For example, the term C2-6 alkylene means an alkylene having 2, 3, 4, 5 or 6 carbon atoms.
The term "available" as in "available hydrogen atoms" or "available atoms" refers to atoms that will be known to those skilled in the art to be capable of being replaced by substituents.
The term "amine" or "amino", as used herein, whether used alone or as part of another group, refers to a group having the general formula NR 'R ", wherein R' and R" are each independently selected from hydrogen or C1-6 alkyl.
The term "cycloalkyl", as used herein, whether used alone or as part of another group, means a saturated carbocyclic group containing one or more rings. The possible number of carbon atoms in the cycloalkyl radicals mentioned is indicated by the numerical prefix "Cn1-n 2". For example, the term C3-10 cycloalkyl means cycloalkyl having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
The term "aryl", as used herein, whether used alone or as part of another group, refers to a carbocyclic group containing at least one aromatic ring. In embodiments of the present disclosure, aryl contains 6, 9, or 10 carbon atoms, such as phenyl, indanyl, or naphthyl.
The terms "heterocycle", "heterocyclic", and the like, as used herein, whether used alone or as part of another group, refer to a cyclic group containing at least one aromatic or non-aromatic ring in which one or more atoms are heteroatoms selected from O, S and N. Heterocyclic groups are saturated or unsaturated (i.e., contain one or more double bonds). When the heterocyclic group contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more, suitably 1 to 5 ring atoms are replaced by heteroatoms as defined above.
The terms "heteroaryl", "heteroaromatic", and the like, as used herein, whether used alone or as part of another group, refer to a cyclic group containing at least one heteroaromatic ring in which one or more atoms is a heteroatom selected from O, S and N. When heteroaryl contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more, suitably 1 to 5, ring atoms are replaced by heteroatoms as defined above.
All cyclic groups (including aryl and cyclic groups) contain one or more than one ring (i.e., are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spiro, or linked by a bond.
The first ring and the second ring being "fused" means that the first ring and the second ring share two adjacent atoms therebetween.
By "bridging" a first ring with a second ring is meant that the first ring and the second ring share two non-adjacent atoms therebetween.
The first ring and the second ring are "spiro-fused" means that the first ring and the second ring share one atom therebetween.
The term "halogen" as used herein refers to a halogen atom and includes fluoro, chloro, bromo and iodo.
The term "optionally substituted" refers to a group, structure, or molecule that is unsubstituted or substituted with one or more substituents.
The term "atm" as used herein refers to atmospheric pressure.
The term "MS" as used herein refers to mass spectrometry.
The term "aq." as used herein refers to aqueous.
The term "protecting group" or "PG" or the like as used herein refers to a chemical moiety that protects or masks the reactive portion of a molecule to prevent side reactions in these reactive portions of the molecule when manipulating or reacting different portions of the molecule. After the completion of the procedure or reaction, the protecting groups are removed without degrading or decomposing the remainder of the molecule. Suitable protecting groups can be selected by those skilled in the art. Many conventional protecting groups are known in the art, for example as described in the following documents: "Protective Groups in Organic Chemistry" McOmie, J.F.W. editions, plenum Press,1973,in Greene,T.W. And Wuts, P.G.M. "," Protective Groups in Organic Synthesis ", john Wiley & Sons, 3 rd edition, 1999 and Kocienski, P.protective Groups, 3 rd edition, 2003,Georg Thieme Verlag (The America).
The term "inert organic solvent" as used herein refers to a solvent that is generally considered to not react with functional groups present in the compounds to be combined together in any given reaction, so that it does not interfere with or inhibit the desired synthetic transformations. The organic solvent is generally non-polar and dissolves the compounds insoluble in the aqueous solution.
The term "cell" as used herein refers to a single cell or a plurality of cells, and includes cells in a cell culture or optionally in a subject.
The term "solvate" as used herein means a compound or a salt or derivative of a compound in which a molecule of a suitable solvent is incorporated into the crystal lattice. Examples of suitable solvents may include ethanol, water, and the like. When water is the solvent, the molecule is referred to as a "hydrate".
The term "antibody" as used herein is intended to include any and all antibodies and fragments thereof, including monoclonal, polyclonal and chimeric antibodies and binding fragments thereof. Antibodies can be derived from recombinant sources and/or produced in transgenic animals. Antibodies can be fragmented using conventional techniques. For example, F (ab') 2 fragments can be produced by treating antibodies with pepsin. The resulting F (ab ') 2 fragments may be treated to reduce disulfide bridges, thereby producing Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, fab 'and F (ab') 2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques. Antibody fragments as used herein means binding fragments
The term "oligonucleotide" as used herein refers to a nucleic acid comprising: nucleotide or nucleoside monomer sequences consisting of naturally and non-naturally occurring bases, sugars and inter-sugar (backbone) linkages, and include single-and double-stranded molecular RNAs and DNAs. Oligonucleotides can be long (e.g., greater than 1000 monomers and up to 10K monomers), medium-sized (e.g., between 200 and 1000 nucleotides and inclusive), or short, e.g., less than 200 monomers, 100 monomers, 50 monomers, including non-naturally occurring monomers. The term "oligonucleotide" includes, for example, single stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA reverse transcribed from RNA), messenger RNA (mRNA), "antisense oligonucleotide" and "miRNA" and oligonucleotide analogs such as "morpholino oligonucleotides", "phosphorothioate oligonucleotides" or any oligonucleotides known to those skilled in the art or analogs thereof.
The terms "elemental tag," "tag," and the like as used herein refer to a chemical moiety that includes an element or elements having one or more isotopes (such as soft metals) attached to a supporting molecular structure, or capable of binding the one or more elements or one or more isotopes. The elemental tag may also include means for attaching the elemental tag to a molecule of interest or target molecule (e.g., a biomolecule such as an analyte). Different element tags may be distinguished based on the element composition of the tag. An elemental tag may contain multiple copies of a given isotope, and may have reproducible copy numbers of each isotope in each tag. An elemental tag is functionally distinguishable from many other elemental tags in the same sample because its elemental or isotopic composition is different from the other tags.
The term "ICP-MS" as used herein refers to inductively coupled plasma mass spectrometry, an elemental analyzer based on sensitive mass spectrometry. The different ICP-MS configurations are primarily distinguished by the mass selection technique employed and may be, for example, quadrupole or time of flight (ICP-TOF) or magnetic sector (high resolution ICP-MS). There are many commercially available ICP-MS models with a wide variety of configurations, functions and modifications.
The term "polymer" as used herein refers to a substance consisting of molecules characterized by multiple repetitions of one or more atoms or groups of atoms (building blocks) that are linked to each other in an amount sufficient to provide a set of properties that do not change significantly with the addition or removal of one or several building blocks. (IUPAC definition, see E.S.White, J.Chem.Inf.Comput.Sci.1997,37, 171-192). A polymer molecule can be considered to be its backbone, a connecting chain of atoms spanning the length of the molecule, and a pendant group attached to the backbone portion of each constituent unit. The pendant groups are typically chemically and functionally different from the backbone chain. Side groups having a high affinity for metal ions may act as chelating groups or ligands for these ions. In some cases, the polymer may have from about 10 to about 300 units.
The term "copolymer" as used herein refers to a polymer composed of two or more chemically distinct constituent units. A "linear polymer" is a polymer characterized by a linear sequence of constituent units. A "block copolymer" is a linear polymer having a sequence of constituent units of a common type linked to a sequence of constituent units of a different type. A "branched polymer" is a polymer in which additional polymer chains (branches) extend from the backbone of the polymer. One typically refers to the longest linear sequence as the "backbone". Branched polymers in which the chemical composition of the constituent units of the branches is different from the chemical composition of the constituent units of the main chain are referred to as "graft copolymers".
The term "star polymer" as used herein refers to a polymer having multiple linear polymer chains derived from a common constituent unit or core. The term "hyperbranched polymer" as used herein refers to a multi-branched polymer in which the backbone atoms are arranged in a tree. These polymers are associated with "dendrimers" that have three unique structural features: an initiator core, an inner layer (layer generation) composed of repeating units attached radially to the initiator core, and an outer surface attached to the terminal functional groups of the outermost layer generation. "dendrimers" differ from hyperbranched polymers in that they have very good symmetry, highly branched and maximized (telechelic) terminal functionality.
The term "metal-tagged polymer" (also referred to as "polymeric metal tag carrier", or "metal-polymer conjugate", or "chelate-derived polymer") and the like as used herein refers to various element tags that consist of a polymer backbone with at least one pendant chelating group to which a metal atom is attached. These metal-tagged polymers may be, but are not limited to, linear, star-shaped, branched or hyperbranched homopolymers or copolymers, block or graft copolymers.
The term "metal binding pendant group" as used herein is a pendant group on a polymer that is capable of binding a metal or metal isotope. It may also be referred to as a chelator.
The term "chelation" as used herein refers to the process of combining a ligand, chelant, chelator, or chelating agent with a metal ion to form a metal complex, chelate. With simple monodentate ligands such as H 2 O or NH 3 In contrast, polydentate chelators form multiple bonds with metal ions.
The term "metal" as used herein refers to an element having one of the following atomic numbers: 3. 4, 11-13, 19-33, 37-52, 55-84, 87-102.
The term "soft metal" as used herein refers to a metal that is considered to be soft according to Pearson's lewis hard acid base theory.
It is contemplated that when referring to a monoisotope, it basically refers to a monoisotope of a metal. For example, a monoisotope may contain trace amounts of other isotopes of a metal and/or trace amounts of another metal. For example, substantially monoisotopic may mean an isotopic purity of any of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%, or an isotopic purity of 100%. For example, a monoisotope may constitute about 95% or more than 95% of the isotope and about 5% or less than 5% of the other isotopes. In some embodiments, a monoisotope may constitute about 97% or more than 97% of the isotope and about 3% or less than 3% of the other isotopes. In some embodiments, a monoisotope may constitute about 98% or more than 98% of the isotope and about 2% or less than 2% of the other isotopes. In some embodiments, a monoisotope may constitute about 99% or more than 99% of the isotope and about 1% or less than 1% of the other isotopes. In some embodiments, a monoisotope may constitute about 99.5% or more than 99.5% of the isotope and about 0.5% or less than 0.5% of other isotopes. In some embodiments, a monoisotope may constitute about 99.9% or more than 99.9% of the isotope and about 0.1% or less than 0.1% of other isotopes. In some embodiments, the monoisotope constitutes 100% of the isotope.
The terms Mn, mw and PDI (polydispersity index): mw/Mn is used to represent the number and average molecular weight, respectively, and the polydispersity index describes the molecular weight distribution.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and fractions thereof are considered to be modified by the term "about".
Furthermore, the definitions and embodiments described in the specific section are intended to apply to other embodiments described herein as understood by those skilled in the art as appropriate. For example, in the following paragraphs, various aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Sub-ranges, such as each 0.1 increment therebetween, are also contemplated for the ranges described herein. For example, if the range is 0ppm to about 5ppm, 0.1ppm to about 5ppm, 0ppm to about 4.9ppm, 0.1ppm to about 4.9ppm, etc. are also contemplated.
Compounds, compositions and kits
In one aspect, the present disclosure includes compounds of formula I
Wherein the method comprises the steps of
A is a polymer backbone, optionally, the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof;
each B is independently a nitrogen-containing 5-to 7-membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof;
l is absent or a linker;
each L 2 Independently absent or a linker;
each R 2 Independently a first modifying group selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof;
x is a functional group selected from esters, ethers, and amides;
each L 1 Independently absent or a linker;
each R 1 Is independently H, C to C8 alkyl, C1 to C8 alkyl, C2 to C8 alkenyl, C3-C8 cycloalkyl, OH, C1 to C10 alkoxy, C1 to C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof;
n is an integer between 0 and 50;
m is an integer between 0 and 40;
p is an integer between 0 and 30; and is also provided with
q is an integer greater than 0.
In another aspect, the present disclosure includes a compound of formula I or a derivative or salt thereof, wherein the compound of formula I is chelated to one or more metals M, and wherein the compound has the structure of formula II
In another aspect, the present disclosure includes a composition comprising one or more compounds of formula I or one or more compounds of formula II, and a solvent.
In another aspect, the disclosure includes a compound having formula I or II for use in mass flow cytometry.
In some embodiments, the polymer backbone a may comprise 10 to 300 monomer units.
In some embodiments, n is an integer between 0 and 20, between 1 and 10, or between 0 and 7. In some embodiments, m is an integer between 0 and 30, between 0 and 20, between 0 and 10, or between 0 and 4. In some embodiments, p is an integer between 0 and 20, between 0 and 10, between 0 and 5, or between 0 and 3. In some embodiments, q is an integer greater than 0. For example, q is an integer between 2 and 300, between 2 and 200, between 2 and 150, between 2 and 100, between 4 and 80, between 4 and 60, between 4 and 20, between 4 and 12, or between 10 and 60. For example, q is an integer of at least 2, at least 4, or at least 10. For example, q is an integer up to 300, up to 250, up to 200, up to 150, up to 100, up to 80, up to 60, up to 20, up to 12, or up to 10. In some embodiments, q may be greater than 1 but less than 20 to avoid steric hindrance and/or reduce background, such as when used to stain tissue for imaging mass flow cytometry or to label intracellular targets in suspension mass flow cytometry. In some embodiments, n is an integer between 0 and 7; m is an integer between 0 and 4; p is an integer between 0 and 3; and q is an integer greater than 0. For example, n is 2, 3, 4 or 5. In some embodiments, m is 0, 1, or 2. In some embodiments, p is 1 or 2.
As used herein, the term "modifying group," such as in a first modifying group or a second modifying group, refers to a group, moiety, structure, and/or substituent that, when attached to a chemical entity or chemical structure, such as a polymer, modifies, alters, modulates, or alters the functionality and/or characteristics of the chemical entity or chemical structure, such as a polymer. For example, a modifying group may modify, change, modulate, or alter the solubility, reactivity, and/or hydrophobicity of a chemical entity, or the affinity of the chemical entity for another chemical entity. In some embodiments, the modifying group may be a solubility modifier and/or a reactive functional group.
As used herein, "solubility modifier" refers to a chemical entity or chemical entity when attached to itGroups, moieties, structures and/or substituents that modify, change, regulate or alter the solubility of a chemical entity or chemical structure in water when the structure is a chemical structure, such as an oligomer or a polymer. For example, the solubility modifier may include a water-soluble polymer, such as polyethylene glycol (PEG), a zwitterionic polymer, or a charged polymer. The zwitterionic polymers may include poly (sulfobetaine methacrylate) (PSBMA) and poly (carboxybetaine methacrylate) (PCBMA). In some embodiments, each R 2 The solubility modifier of the first modification group and the solubility modifier of the second modification group each independently comprise polyethylene glycol (PEG), a sugar, an oligosaccharide, or a zwitterionic polymer, such as poly (carboxybetaine) methacrylate or poly (sulfobetaine) methacrylate (PBSMA). The solubility modifier can increase the solubility of a polymer (e.g., a metal-loaded polymer as described herein), such as can increase by a factor of two in a solution (e.g., an aqueous solution, such as a buffer solution having a pH of between 6 and 8 or 6 and 8), as compared to the absence of the solubility modifier. In certain aspects, the solubility modifier can increase the solubility of a polymer (e.g., a metal-loaded polymer as described herein) compared to the absence of the solubility modifier. In some embodiments, the oligomer may have up to 10 monomer units. In some embodiments, the solubility modifier may include a polymer having from about 10 to about 5000 units. For example, the solubility modifier may be a PEG group. For example, the PEG group may have about 10 to about 350 units, about 10 to about 300 units, about 10 to about 250 units, about 10 to 200 units, about 10 to 150 units, or about 110 units of ethylene glycol. For example, the PEG group may have at least 10, at least 20, or at least 30 units of ethylene glycol. For example, the PEG group may have up to 300, up to 250, up to 200, up to 150, up to 100, or up to 50 units of ethylene glycol. For example, the PEG groups can have Mn of about 5000g/mol to about 10000 g/mol.
In certain aspects, solubility modifiers can also reduce non-specific binding of the disclosed compounds to targets in a sample. In some embodiments, certain solubility modifiers have been demonstrated to be more effective in reducing non-specific binding. For example, zwitterionic solubility modifiers have been demonstrated to exhibit less non-specific binding than PEG solubility modifiers.
In some embodiments, at least a portion or all of the metal M may be coordinated with one or more solubility modifiers. For example, the solubility modifier may be a ligand for metal M. In some embodiments, the solubility modifier may include a thiol small molecule. For example, the thiol small molecule may be selected from glutathione, cysteine, thioglycollic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaptopropanol, dimercaptosuccinic acid, 2, 3-dimercapto-1-propanesulfonate, and combinations thereof. In some embodiments, the solubility modifier is glutathione. It is understood that the solubility modifier may coordinate to the metal M by a ligand exchange reaction.
As used herein, a "reactive functional group" refers to a group of atoms or a single atom that interacts or reacts with another group of atoms or a single atom to form a chemical interaction between the two groups of atoms or atoms. For example, attaching one or more reactive functional groups to a chemical entity or chemical structure, such as a polymer, modifies or alters the reactivity of the chemical entity or chemical structure, such as a polymer, to allow the chemical entity or chemical structure to interact or react with an atomic group on another chemical entity or chemical structure, such as a biomolecule. It will be appreciated that in some cases, a given reactive functional group may interact or react with a particular functional group to form a chemical interaction. For example, it is known that azides can undergo click chemistry reactions, while maleimides can react with thiols. In some embodiments, the chemical interaction is covalent or ionic. For example, the chemical interactions are covalent. In some embodiments, the reactive functional groups are for attachment to one or more biomolecules. In some embodiments, each R 2 The reactive functional groups of the first modification group and the reactive functional groups of the second modification group of (a) are each independently selected from carboxylic acid, N-hydroxysuccinimide ester, tetrafluorophenyl ester, pentafluorophenylA base ester, maleimide, thiol, azide, dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO), tetrazine, furan, hydrazide, or aldehyde.
It will be appreciated that the reactive functional groups may be reversibly protected or terminated with suitable protecting groups until the reactive functional groups are required for further reaction. For example, the thiol may be capped with a thiol capping group such as maleimide or other groups known in the art. For example, thiol-containing polymers may be temporarily protected or may be temporarily present as disulfide dimers, which may be reduced using known methods (e.g., DTT reduction) to reveal thiol groups as desired. Thus, it is contemplated that reactive functional groups as described herein also include protected forms of reactive functional groups.
As used herein, when a metal is non-radioactive, it means that the metal is substantially non-radioactive. For example, the radioactive metal may have a decay rate suitable for radiation detection assays, while the non-radioactive metal may have a decay rate unsuitable for radiation detection assays, or lower than the detection limit of common radiation detection assays used in the radiolabeling field. In some embodiments, the non-radioactive metal may have a half-life of greater than about 150,000 years, greater than 200,000 years, or greater than about 210,000 years. For example, it is known that, 99 Tc isotopes have a half-life of 210,000 years and are therefore considered non-radioactive for the purposes of this disclosure.
In some embodiments, each B is independently a 5 or 6 membered heterocycle. For example, each B may independently be substituted or unsubstituted tetrahydropyrrole. In some embodiments, each B is independently a nitrogen-containing 5-or 6-membered heteroaryl, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and wherein optionally one or more B coordinates with a soft metal, and/or is conjugated to one or more biomolecules.
For example, each B is independently pyridine or imidazole, optionally substituted with one or more polar functional groups selected from C1 to C5 alkyl, C2 to C5 alkenyl, COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and wherein optionally one or more B coordinates to a soft metal, and/or is conjugated to one or more biomolecules.
It is contemplated that two B attached to the same nitrogen are not necessarily the same chelating group. However, for ease of synthesis, two B attached to the same nitrogen may be identical.
In some embodiments, one or more B coordinates with a soft metal. It is contemplated that not all of B of the compound of formula I may be necessarily coordinated to the metal. For example, in some embodiments, about 30% to about 95%, about 40% to about 90%, about 50% to about 85% of B coordinates with the metal. In some embodiments, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, or at least or about 85% of B coordinates to the metal. In some embodiments, up to 95%, up to 90%, up to 85%, up to 80%, up to 75%, up to 70%, or up to 65% of B coordinates to the metal. In some embodiments, about 75% to about 80% of B coordinates to the metal. In some embodiments, all B coordinates to the metal. Without wishing to be bound by theory, it is understood that two B attached to the same nitrogen atom may chelate to the same metal atom in a bidentate fashion.
In some embodiments, B is optionally substituted pyridine.
In some embodiments, B is substituted or unsubstituted imidazole. Optionally, B is a 2-substituted or 4-substituted imidazole. Suitable imidazole-based chelators include those described in Maresca et al, bioconjugate chem, 2010,21,1032, the contents of which are incorporated by reference in their entirety.
In some embodiments, R 1 Is a biological molecule. For example, R 1 May be an antibody. In some embodiments, R 1 Is an affinity reagent.
In some embodiments, R 2 Is a biological molecule. For example, R 2 May be an antibody. In some embodiments, R 2 Is an affinity reagent.
In some embodiments, X is an amide. For example, X is-C (O) NR 4 -or-NR 4 C (O) -, wherein R 4 Is H or C1 to C4 alkyl.
In some embodiments, X is-C (O) NR 4 -, and the compound has the structure of formula Ia
In some embodiments, X is-NR 4 C (O) -, and the compound has the structure of formula Ib
In some embodiments, X is-C (O) NR 4 -, and the compound has the structure of formula Ic
Wherein each R is 3 Independently selected from H, C to C5 alkyl, C2 to C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, or polyether.
In some embodiments, X is-C (O) NR 4 -, and the compound has the structure of formula Id or Ie
In some embodiments, X is-NR 4 C (O) -, and the compound has the structure of formula If
Wherein each R is 3 Independently selected from H, C to C5 alkylC2 to C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether or polyether.
In some embodiments, X is-NR 4 C (O) -, and the compound has the structure of formula Ig or Ih
In some embodiments, R 4 Is H. In some embodiments, R 4 Is a C1 to C3 alkyl group.
In some embodiments, the compound of formula I has the structure of formula Ii
Wherein A is 1 Is a monomer of a, and r is from about 3 to about 300, from about 3 to about 250, from about 3 to about 200, from about 3 to about 150, from about 3 to about 100, from about 3 to about 50, from about 6 to about 30, or from about 10 to about 25. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10. Optionally, the polymer backbone a is a linear polymer or copolymer.
In some embodiments, the compound of formula I has the structure of formula Ij
Wherein A is 1 And A 2 Each is a monomer of a, and r is from about 3 to about 300, from about 3 to about 250, from about 3 to about 200, from about 3 to about 150, from about 3 to about 100, from about 3 to about 50, from about 6 to about 30, or from about 10 to about 25, and the polymer backbone a is a linear copolymer. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10。
In some embodiments, each R 3 Independently selected from H, - (CH) 2 ) 1-3 COOH、-(CH 2 ) 1-3 O(CH 2 ) 1-2 CH 3 、-(CH 2 ) 2-4 OH、-(CH 2 ) 2-5 P(O)(OCH 2 CH 3 ) 2 or-CH 2 CH(OMe) 2 。
In some embodiments, a is selected from the group consisting of polyacrylates, polyacrylamides, polyethers, polyamino acids, polyvinylamines, poly (2-oxazolines), polyethylene glycols, polysaccharides, dendrimers, copolymers thereof, or combinations thereof.
For example, a may be a polyamino acid. For example, the polyamino acid may be optionally substituted polyglutamic acid, polyaspartic acid, polylysine, poly (2, 4-dimethylaminobutyric acid) (polyDab), poly (2, 4-diaminopimelic acid) (polyDap), derivatives thereof, or combinations thereof.
It is contemplated that the polymer backbone a of the compounds of the present disclosure may be a copolymer. For example, it may be a copolymer comprising PEG.
In some embodiments, the polymer backbone is a linear polymer. For example, the compound of formula I may have the structure shown below.
In some embodiments, the polymer backbone is a branched polymer, such as a hyperbranched polymer or a grafted polymer. Exemplary representatives of the compounds of formula I include the structures shown below.
Each a (e.g. a 1 、a 2 、……、a r ) Is a monomer unit of a polymer backbone. The polymer backbone of the compound of formula I may be a homopolymer or a copolymer. The copolymer may comprise a graft copolymer or a block copolymer. It is understood that each monomer unit (e.g., a 1 、a 2 、……、a r ) May be the same, for example in a homopolymer, or different, for example in a copolymer. For example, a 2 Can be equal to a 1 The same monomer, or a different monomer. Similarly, a 3 Can be equal to a 2 And/or a 1 The same monomer, or a different monomer.
It is contemplated that at least one monomer unit a is attached to the structureThe latter can chelate to metals, such as soft metals. In some embodiments, each monomer unit of the polymer backbone is attached toIn some other embodiments, some, but not all, monomer units of the polymer backbone are attached to +.>
In some embodiments, the first modifying group R 2 May be present at the ends of the polymer backbone. For example, each end of the polymer backbone may be attached by an optional linker L 2 With a first modifying group R 2 Functionalization, each first modification group R 2 And each joint L 2 Are defined independently herein. In some embodiments, some, but not all, of the ends of the polymer backbone may be joined by optional linkers L 2 With a first modifying group R 2 Functionalization, each first modification group R 2 And each joint L 2 Are defined independently herein.
In some embodiments, the polymer backbone may be a copolymer of monomers comprising different pendant groups. For example, it is contemplated that in the polyacrylamide backbone, the acrylamide monomer may be attached to a chelating agent side group or a modifying group, such as a solubility modifying group or a reactive functional group. Exemplary polymer compounds of the present disclosure having a copolymer backbone are shown below.
In some embodiments, the Degree of Polymerization (DP) may be about 1 to 1000 (1 to 2000 backbone atoms). As will be appreciated by those skilled in the art, larger polymers with the same functionality are within the scope of the invention and are possible. Typically, the degree of polymerization will be between 10 and 250. The polymers can be synthesized by pathways that result in relatively narrow polydispersities. The polymer may be synthesized by Atom Transfer Radical Polymerization (ATRP), reversible addition-fragmentation (RAFT) polymerization or ring-opening polymerization, which will result in Mw/Mn values in the range of 1.1 to 1.2. An alternative strategy involves anionic polymerization, wherein polymers with Mw/Mn of about 1.02 to 1.05 can be obtained. Thus, the polymer may have a polydispersity index of 1.02 to 1.5 (such as 1.02 to 1.2, 1.02 to 1.05, or 1.2 to 1.5). These methods allow control of the end groups by selection of the initiator or terminator. This allows the synthesis of polymers to which the linker can be attached. Strategies may be employed to prepare polymers containing functional pendant groups in the repeat units to which ligand transition metal units (e.g., soft metal units) may be attached in a later step. This embodiment has several advantages. It avoids complications that may arise from carrying out the polymerization of ligand-containing monomers. In addition, the polymer backbone is a known backbone that can be adapted to most, if not all, soft metal containing polymers. Thus, the polymers may have a common average chain length and chain length distribution.
In some embodiments, each linker independently comprises or is independently selected from C3-C8 alkylamine, C3-C8 alkylene, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, 5-or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C (O) O, amide, amine, thioether, maleimide-thiol conjugate, polyethylene glycol (PEG), or mixtures thereof, optionally, the amine, alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl are each independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
In some embodiments, each L 2 Independently comprising or independently selected from a C3-C8 alkylene, a C3-C8 alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, optionally, each of said alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
In some embodiments, each L 1 Independently comprising or independently selected from a C3-C8 alkylene, a C3-C8 alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, optionally, each of said alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
In some embodiments, L is absent or is a C3-C8 alkylamine.
In some embodiments, it is understood that the linker may include a functional group that attaches the linker to the remainder of the compound.
Biomolecules may be classified as proteins, oligonucleotides, lipids, carbohydrates or small molecules or combinations thereof. Alternatively or additionally, biomolecules may be classified by their function. The biomolecules are not particularly limited and different functionalities may be used to conjugate the biomolecules to the compounds of the present disclosure. For example, the oligonucleotide can be a single stranded DNA molecule, optionally, a cDNA that hybridizes under stringent conditions to a target nucleic acid analyte (e.g., a sample nucleic acid biomolecule), or the oligonucleotide can be an aptamer. For example, the biomolecule may be an oligonucleotide that specifically hybridizes to a target oligonucleotide (e.g., to a sample oligonucleotide), such as a target mRNA endogenous to the sample. Hybridization may be hybridization of sequences of more than 8, more than 10, more than 15 or more than 20 nucleotides.
In certain aspects, biomolecules may be classified by their function. For example, the biomolecule may be an affinity reagent, an antigen (e.g., an analyte specifically bound by the affinity reagent), or an enzyme substrate. The affinity reagent may be an antibody (e.g., or fragment thereof), an aptamer, a receptor (e.g., or portion thereof), or any other biomolecule that specifically binds to a target (e.g., avidin, such as streptavidin, that specifically binds biotin). For example, elemental tags may be associated with antibodies, which may be used to detect and/or analyze a sample for the presence of its target antigen, such as the presence of cytokines, viral proteins, cancer biomarkers, and the like. In certain methods and kits, an element tag can be functionalized with avidin for attachment of another biomolecule functionalized with biotin (e.g., to allow the compounds of the present disclosure to be suitable for any of a variety of different assays). An antigen may be a protein (or peptide sequence thereof) comprising an epitope that is specifically bound by an affinity reagent, such as an antibody. For example, compounds of the present disclosure may be attached to a viral antigen (such as a viral protein sequence) and may be used to detect the presence of antibodies in a sample that specifically bind to the viral antigen, as further described herein. The enzyme substrate may be any substrate that is acted upon by a particular enzyme, such as an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. For example, the substrate may be a protein (e.g., or a peptide sequence thereof) that is a substrate for an enzyme, such as a protease, phosphatase, kinase, methyltransferase, demethylase. Non-protein substrates include, for example, double-stranded oligonucleotides comprising restriction sequences cleavable by restriction enzymes or sites for DNA repair (e.g., nicks), oligonucleotide sequences comprising sequences targeted by DNA methyltransferases, or any non-protein substrate known to those of skill in the art. For example, a compound of the present disclosure may be attached to a substrate and exposed to a sample comprising an enzyme that modifies the substrate, and the modification of the substrate (or lack thereof) may be detected (e.g., as further described herein).
In some embodiments, the one or more biomolecules are each independently selected from a small molecule, a polypeptide, an oligonucleotide, a lipid, a carbohydrate, or a mixture thereof.
In some embodiments, the one or more biomolecules are each independently an affinity reagent, optionally wherein the affinity reagent is an antibody.
In some embodiments, the affinity reagent is or includes an antibody or binding fragment thereof. The antibody may be, for example, a biotinylated antibody or binding fragment, and may be added directly or indirectly to a compound of the disclosure.
In some embodiments, the compound of formula I is selected from
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Wherein R is from about 3 to about 300, from about 3 to about 250, from about 3 to about 200, from about 3 to about 150, from about 3 to about 100, from about 3 to about 50, from about 6 to about 30, or from about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined herein. In some embodimentsIn this case, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30 or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.
In some embodiments, the compound of formula I is selected from
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Wherein s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30, and R is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.
In some embodiments, the compound of formula I is selected from
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Wherein s is about 1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30, and R is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.
In some embodiments, the compound of formula I is selected from
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Wherein s is about1 to about 50, about 2 to about 40, about 5 to about 30, about 10 to about 30, about 5 to about 35, or about 20 to about 30, and R is about 3 to about 300, about 3 to about 250, about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 6 to about 30, or about 10 to about 25, and wherein R is 3 As defined herein. In some embodiments, r is at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 30, or at most 25. In some embodiments, r is at least 3, at least 6, or at least 10.
In some embodiments, the compound of formula I is selected from
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In some embodiments, the compound of formula II is selected from
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Wherein R is H,Or other suitable thiol capping groups known in the art. In some embodiments, R represents a second compound having formula II that forms a dimer through disulfide bonding.
It will be appreciated that bis-heterocyclic chelating agents such as DPA, bis ((1-methyl-imidazol-4-yl-) methyl) amine, bis ((1-methyl-imidazol-2-yl-) methyl) amine, bis ((1H-imidazol-4-yl-) methyl) amine and bis ((1H-imidazol-2-yl-) methyl) amine may stably chelate metals, particularly soft metals. Polymeric compounds of the present disclosure comprising one or more pendent groups (including bis-heterocyclic chelators such as DPA, bis ((1-methyl-imidazol-4-yl-) methyl) amine, bis ((1-methyl-imidazol-2-yl-) methyl) amine, bis ((1H-imidazol-4-yl-) methyl) amine, and bis ((1H-imidazol-2-yl-) methyl) amine) can chelate metals, including soft metals. When chelated to metals, the compounds of the present disclosure introduce new mass channels for applications, such as mass flow cytometry represented by stable isotopes of metals.
The examples herein show exemplary chelates formed using the polymer compounds of the present disclosure with metals including Re, pt, hg, and Ag. It is known that chelating agents such as DPA can form stable chelates with other soft metals in non-polymeric environments. Thus, polymer compounds of the present disclosure with chelating agents such as DPA, bis ((1-methyl-imidazol-4-yl-) methyl) amine, bis ((1-methyl-imidazol-2-yl-) methyl) amine, bis ((1H-imidazol-4-yl-) methyl) amine, and bis ((1H-imidazol-2-yl-) methyl) amine) can also be chelated to other soft metals.
For example, seubert et al [ "Chimeric GNA/DNA metal-mediated base pairs", chem. Commun.,2011,47,11041-11043] reported computational analysis of Au (III) chelated to DPA. Messori et al [ J.Med. Chem.2000,43,3541-3548] report a series of Au (III) complexes with diamines and triamines.
Molybdenum complexes with DPA [ Mo (dipic) (CO) ] 3 ]Has been described by van Staveren et al, labelling of Leu5]Enkephalin with organometallic Mo complexes by solid-phase synthesis, chem.Commun.,2002, 1406-1407.
Minard et al [ In situ generation of water-stable and-soluble rutheniumcomplexes of pyridine-based Chemical-ligands and their use for the hydrodeoxygenation of biomass-related substrates in aqueous acidic medium, journal of Molecular Catalysis A:chemical 422 (2016) 175-187 ]Report [ Ru III (DMF) 6 ](OTf) 3 May be reacted with lutidine amine in aqueous solution, which reduces Ru (III) to Ru (II). [ Ru (2, 2' -dimethylpyridine amine) (OH) 2 ) 3 ](OTf) 2 Is stable to 150 ℃.
The crystal structure of Rh, pd and Pt was prepared with tris (2-pyridylmethyl) amine by London et al [ Rhodium, palladium and platinum complexes of tris (pyridylalky) amine and tris (benzozolylethyl) amine N4-tripodal ligands, dalton Trans.,2006,3785-3797, DOI:10.1039/b602556k ].
Song et al [ Cadmedium (II) complexes containing N-substituted N, N-di (2-picolyl) amine: the formation of monomeric versus dimeric complexes is affected by the N' -substitution group on the amine moiety, J.organometallicchem.783 (2015) 55e63, doi.org/10.1016/j.joganche.2015.02.011]It was confirmed that N-alkyl dimethyl pyridine amine derivatives tended to react with Cd 2+ A dimeric complex is formed.
Thus, it is contemplated that the polymer compounds of the present disclosure comprising heterocyclic chelators, such as DPA and bis ((1H-imidazol-2-yl-) methyl) amine, can be used to chelate many different soft metals, and that stable isotopes based on metals open many new mass channels for mass flow cytometry applications. A non-limiting summary is shown in table 1 below.
Table 1 exemplary metals and corresponding mass channels
In some embodiments, M is a soft metal. For example, M may be selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or mixtures thereof.
In some embodiments, M is non-radioactive. It is contemplated that the compounds of the present disclosure may be used in radiation detection assays. Thus, M may be radioactive when the compound is used in a radiation detection assay.
In some embodiments, M is isotopically enriched. For example, M does not include naturally occurring isotopic mixtures.
In another aspect, the present disclosure includes a compound of formula II as defined herein for use in mass flow cytometry.
In another aspect, the present disclosure includes an elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups, wherein at least one chelating group is chelated to a soft metal atom of a soft metal, the soft metal being a monoisotope.
In some embodiments, the element tag is a compound of the present disclosure.
In another aspect, the disclosure includes a kit comprising
An isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal; and
An elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, the chelating groups comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group comprises or is capable of binding at least one soft metal atom of an isotopic composition.
In some embodiments, the kit does not include any radioactive soft metals.
In some embodiments, the isotopic composition does not comprise a natural mixture of isotopes.
It is contemplated that the elemental tag may be functionalized to bind biomolecules. For example, an elemental tag may be covalently attached to a biomolecule.
In some embodiments, the kit further comprises a biomolecule.
For example, the biomolecule may be an oligonucleotide. For example, the biomolecule may be an antibody or other affinity reagent.
In some cases, each chelating group includes at least one soft metal atom of the isotopic composition.
In some embodiments, the isotopic composition is a soft metal solution provided separately from the elemental tag, and wherein each chelating group is capable of binding at least one soft metal atom of the isotopic composition.
In some embodiments, the kit further comprises an additional isotopic composition. For example, the further isotopic composition comprises a plurality of further soft metal atoms of a further monoisotope of a soft metal, the further monoisotope being different from the monoisotope of the soft metal of the isotopic composition.
In some embodiments, the kit further comprises an additional element tag comprising an additional linear or branched polymer comprising a plurality of additional chelating groups.
In some aspects, each chelating group of the linear or branched polymer of the element tag comprises at least one soft metal atom of the isotopic composition, and wherein each further chelating group of the further linear or branched polymer of the further element tag comprises at least one further soft metal atom of the further isotopic composition.
In some embodiments, each elemental tag is covalently bound to a different antibody.
In one embodiment, each chelating group is capable of binding at least one soft metal atom of the isotopic composition and is selected from the group consisting of lutidine amine or bis ((1H-imidazol-2-yl) methyl) amine, wherein each imidazole is optionally substituted with one or more polar functional groups selected from the group consisting of C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof.
In some embodiments, the kit further comprises reagents for covalently attaching an elemental tag to an antibody.
In some embodiments, each element tag is independently a compound of formula I as described herein or a compound of formula II as described herein.
The kits described in the above embodiments can have any of the additional aspects described herein, such as an elemental tag comprising one or more solubility modifiers (e.g., on the same side group as the chelating group).
III methods and uses
In another aspect, the present disclosure includes a method comprising:
providing an isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
providing an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group is capable of binding at least one of the soft metal atoms of the isotopic composition; and
the soft metal atoms of the isotope composition are bound to one or more chelating groups of the elemental tag.
In some embodiments, the soft metal atoms are non-radioactive.
In some embodiments, the isotopic composition does not comprise a natural mixture of isotopes.
In some embodiments, the method may further comprise providing an additional isotopic composition, wherein the additional isotopic composition comprises a plurality of additional soft metal atoms of an additional monoisotope of a non-radioactive soft metal, the additional monoisotope being different from the monoisotope of the non-radioactive soft metal of the isotopic composition.
For example, the method further comprises providing an additional element tag comprising an additional linear or branched polymer comprising a plurality of chelating groups.
In some embodiments, each chelating group of the linear or branched polymer of the element tag comprises at least one soft metal atom of the isotopic composition, and wherein each further chelating group of the further linear or branched polymer of the further element tag comprises at least one further soft metal atom of the further isotopic composition.
In some embodiments, the method further comprises:
Providing a biomolecule; and
the biomolecules are covalently bound to the elemental tags.
In another aspect, the present disclosure includes a method for analyzing an analyte in a biological sample, comprising:
(i) Incubating an element-labeled affinity reagent with an analyte, the element-labeled affinity reagent comprising an affinity reagent labeled with an element tag comprising a linear or branched polymer having a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, the element tag further comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
wherein:
each chelating group of the elemental tag includes or is capable of binding at least one of the soft metal atoms, an
The affinity reagent specifically binds to the analyte,
(ii) Separating unbound elemental-labeled affinity reagent from bound elemental-labeled affinity reagent; and
(iii) Elemental tags bound to affinity reagents attached to the analyte are analyzed by mass spectrometry atomic spectrometry.
In some embodiments, the soft metal atoms are non-radioactive.
In some embodiments, the soft metal does not comprise a natural mixture of isotopes.
In some embodiments, incubating the element-labeled affinity reagent with the analyte comprises:
incubating two or more different elemental-labeled affinity reagents with two or more analytes, wherein the elemental-labeled affinity reagents specifically bind to the two or more analytes to produce two or more differently labeled analytes, wherein analyzing the elemental tags bound to the affinity reagents comprises analyzing the different elemental tags bound to the two or more analytes by mass spectrometry.
In some cases, the affinity reagent is further labeled with a fluorescent label.
In some embodiments, the mass atomic spectrum is ICP-MS. In one embodiment, mass spectrometry atomic spectroscopy is performed by a mass spectrometer based flow cytometer.
In some embodiments, the affinity reagent is an antibody.
In some embodiments, the affinity reagent specifically binds biotin.
In some embodiments, the affinity reagent is an oligonucleotide.
In some embodiments, the element-labeled affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells. In some embodiments, the element-labeled affinity reagent is configured to bind with an analyte in a biological sample, and the soft metal is a non-naturally occurring element in the biological sample.
In some embodiments, the soft metal is selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or a mixture thereof.
In some embodiments, the element tag is a compound of formula I as described herein or a compound of formula II as described herein.
The methods of the above embodiments can have any of the additional aspects described herein, such as an elemental tag comprising one or more solubility modifiers (e.g., on the same side group as the chelating group).
The present disclosure also provides the following embodiments:
embodiment 1A compound of formula I
Wherein the method comprises the steps of
A is a polymer backbone, optionally, the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof;
each B is independently a nitrogen-containing 5-to 7-membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof; and is also provided with
L is absent or a linker;
each L 2 Independently absent or a linker;
each R 2 Independently a first modifying group selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof;
X is a functional group selected from esters, ethers, and amides;
each L 1 Independently absent or a linker;
each R 1 Is independently H, C to C8 alkyl, C2 to C8 alkenyl, C3-C8 cycloalkyl, OH, C1 to C10 alkoxy, C1 to C10 alkylamine, solubility modifier, reactive functional groups, biomolecules, and combinations thereof;
n is an integer from 0 to 7;
m is an integer from 0 to 4;
p is an integer from 0 to 3; and is also provided with
q is an integer greater than 0.
Embodiment 2. The compound of embodiment 1 wherein each B is independently a nitrogen-containing 5-or 6-membered heteroaryl, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and wherein optionally one or more B coordinates to a soft metal, and/or is conjugated to one or more biomolecules.
Embodiment 3. The compound of embodiment 1 or 2, wherein each B is independently pyridine or imidazole, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and wherein optionally one or more B coordinates with a soft metal, and/or is conjugated to one or more biomolecules.
Embodiment 4. The compound of any one of embodiments 1 to 3, wherein one or more B coordinates with a soft metal.
Embodiment 5. The compound of any one of embodiments 1 to 4 wherein R 1 Or R is 2 Is said biomolecule, optionally said biomolecule is an affinity agent, such as an antibody.
Embodiment 6. The compound of any one of embodiments 1 to 5, wherein X is an amide.
Embodiment 7A compound according to embodiment 6 wherein X is-C (O) NR 4 -or-NR 4 C (O) -, wherein R 4 Is H or C1 to C4 alkyl.
Embodiment 8 the compound of any one of embodiments 1 through 7 wherein X is-C (O) NR 4 -, and the compound has the structure of formula Ia
Embodiment 9. The compound of any one of embodiments 1 to 7 wherein X is-NR 4 C (O) -, and the compound has the structure of formula Ib
Embodiment 10. The compound of any one of embodiments 1 through 7 wherein X is-C (O) NR 4 -, and the compound has the structure of formula Ic
Wherein each R is 3 Independently selected from H, C to C5 alkyl, C2 to C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, or polyether.
Embodiment 11 the compound of embodiment 10 wherein the compound has the structure of formula Id or Ie
Embodiment 12 the compound of any one of embodiments 1 through 7 wherein X is-NR 4 C (O) -, and the compound has the structure of formula If
Wherein each R is 3 Independently selected from H, C to C5 alkyl, C2 to C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, or polyether.
Embodiment 13 the compound of embodiment 12 wherein the compound has the structure of formula Ig or Ih
Embodiment 14. The compound of any one of embodiments 10 to 13 wherein each R 3 Independently selected from H, - (CH) 2 ) 1-3 COOH、-(CH 2 ) 1-3 O(CH 2 ) 1-2 CH 3 、-(CH 2 ) 2-4 OH、-(CH 2 ) 2-5 P(O)(OCH 2 CH 3 ) 2 or-CH 2 CH(OMe) 2 。
Embodiment 15. The compound of any one of embodiments 1 to 14, wherein n is 2, 3, 4, or 5.
Embodiment 16. The compound of any one of embodiments 1 to 15, wherein m is 0, 1 or 2.
Embodiment 17 the compound of any one of embodiments 1 to 16, wherein p is 1 or 2.
Embodiment 18. The compound of any of embodiments 1 to 17 wherein a is selected from the group consisting of polyacrylates, polyacrylamides, polyethers, polyamino acids, polyvinylamines, poly (2-oxazolines), polyethylene glycols, polysaccharides, dendrimers, copolymers thereof, or combinations thereof.
Embodiment 19. The compound of embodiment 18 wherein a is a polyamino acid.
Embodiment 20. The compound of embodiment 18 or 19, wherein the polyamino acid is polyglutamic acid, polyaspartic acid, polylysine, poly (2, 4-dimethylaminobutyric acid) (polyDab), poly (2, 4-diaminopimelic acid) (polyDap), derivatives thereof, or combinations thereof.
Embodiment 21. The compound of any one of embodiments 1 to 20, wherein each linker independently comprises or is independently selected from the group consisting of C3-C8 alkylamine, C3-C8 alkylene, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, 5-or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C (O) O, amide, amine, thioether, maleimide-thiol conjugate, polyethylene glycol (PEG), or mixtures thereof, optionally, the amine, alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl are each independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
Embodiment 22. The compound of any one of embodiments 1 to 21 wherein each L 2 Independently comprising or independently selected from a C3-C8 alkylene, a C3-C8 alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, optionally, each of said alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
Embodiment 23 the compound of any one of embodiments 1 to 22 wherein each L 1 Independently comprising or independently selected from a C3-C8 alkylene, a C3-C8 alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, optionally wherein the alkylene and alkyl groups are each independently unsubstituted or taken from one or more ofSubstitution of substituents: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
Embodiment 24. The compound of any one of embodiments 1 to 23, wherein L is absent or is a C3-C8 alkylamine.
Embodiment 25 the compound of any one of embodiments 1 to 24 wherein each R 2 The solubility modifier of the first modification group and the solubility modifier of the second modification group each independently comprise polyethylene glycol (PEG), a sugar, an oligosaccharide, or a zwitterionic polymer, such as poly (carboxybetaine) methacrylate or poly (sulfobetaine) methacrylate (PBSMA).
Embodiment 26. The compound of any one of embodiments 1 to 25, wherein the reactive functional group is for attachment to one or more biomolecules.
Embodiment 27 the compound of any one of embodiments 1 through 26 wherein each R 2 Each independently selected from carboxylic acid, maleimide, thiol, azide, dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO), tetrazine, furan, or aldehyde.
Embodiment 28 the compound of any one of embodiments 1 to 27 wherein the one or more biomolecules are each independently selected from a small molecule, a polypeptide, an oligonucleotide, a lipid, a carbohydrate, or a mixture thereof.
Embodiment 29. The compound of embodiment 28, wherein each of the one or more biomolecules is independently an affinity reagent, optionally wherein the affinity reagent is an antibody.
Embodiment 30. The compound of embodiment 1 wherein the compound is selected from
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Wherein R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined in any one of embodiments 10 to 14.
Embodiment 31 the compound of embodiment 1 wherein the compound is selected from
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Wherein s is from about 1 to about 50, from about 2 to about 40, from about 5 to about 30, from about 10 to about 30, from about 5 to about 35, or from about 20 to about 30, and R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined in any one of embodiments 10 to 14.
Embodiment 32. The compound of embodiment 1 wherein the compound is selected from
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Wherein s is from about 1 to about 50, from about 2 to about 40, from about 5 to about 30, from about 10 to about 30, from about 5 to about 35, or from about 20 to about 30, and R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined in any one of embodiments 10 to 14.
Embodiment 33 the compound of embodiment 1 wherein the compound is selected from
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Wherein s is from about 1 to about 50, from about 2 to about 40, from about 5 to about 30, from about 10 to about 30, from about 5 to about 35, or from about 20 to about 30, and R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 3 As defined in any one of embodiments 10 to 14.
Embodiment 34. A compound of formula I as defined in any one of embodiments 1 to 33, or a derivative or salt thereof, wherein the compound of formula I is chelated to one or more metals M, and wherein the compound has the structure of formula II
Embodiment 35. The compound of embodiment 34 wherein M is a soft metal.
Embodiment 36 the compound of embodiment 34 or 35 wherein M is selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or a mixture thereof.
Embodiment 37 the compound of any one of embodiments 34 to 36, or the composition of any one of embodiments 35 to 37, wherein M is non-radioactive.
Embodiment 38 the compound of any one of embodiments 34 to 37, or the composition of any one of embodiments 35 to 38, wherein M is isotopically enriched.
Embodiment 39 a composition comprising one or more compounds of formula I as defined in any one of embodiments 1 to 33, each independently, or one or more compounds of formula II as defined in any one of embodiments 34 to 38, each independently, and a solvent.
Embodiment 40 a compound of formula I as defined in any one of embodiments 1 to 33 or a compound of formula II as defined in any one of embodiments 34 to 38 for use in mass spectrometry flow cytometry.
Embodiment 41. An elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups, wherein each chelating group is capable of binding a soft metal, the soft metal being a monoisotope, and wherein at least one chelating group is chelated to a soft metal atom of the soft metal.
Embodiment 42A kit comprising
An isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal; and
an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, the chelating groups comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group of the elemental tag comprises or is capable of binding at least one soft metal atom of the isotopic composition;
optionally, wherein the kit does not comprise any radioactive soft metals.
Embodiment 43 the kit of embodiment 42, wherein the isotopic composition does not comprise a natural mixture of isotopes.
Embodiment 44 the kit of embodiment 42 or 43, wherein the element tag is functionalized to bind a biomolecule.
Embodiment 45 the kit of embodiment 42 or 43, wherein the elemental tag is covalently attached to a biomolecule.
Embodiment 46 the kit of any one of embodiments 42 to 44, further comprising a biomolecule.
Embodiment 47. The kit of any one of embodiments 43 to 46, wherein the biomolecule is an oligonucleotide.
Embodiment 48 the kit of any one of embodiments 43 to 46, wherein the biomolecule is an antibody.
Embodiment 49 the kit of any one of embodiments 42 to 48, wherein each chelating group comprises at least one soft metal atom of the isotopic composition.
Embodiment 50. The kit of any one of embodiments 42 to 48, wherein the isotopic composition is a soft metal solution provided separately from the elemental tag, and wherein each chelating group is capable of binding at least one soft metal atom of the isotopic composition.
Embodiment 51 the kit of any one of embodiments 42 to 50 further comprising an additional isotopic composition, wherein the additional isotopic composition comprises a plurality of additional soft metal atoms of an additional monoisotope of a soft metal that is different from the monoisotope of the soft metal of the isotopic composition.
Embodiment 52 the kit of embodiment 51 further comprising an additional element tag comprising an additional linear or branched polymer comprising a plurality of additional chelating groups.
Embodiment 53. The kit of embodiment 52, wherein each chelating group of the linear or branched polymer of the elemental tag comprises at least one soft metal atom of the isotopic composition, and wherein each additional chelating group of the additional linear or branched polymer of the additional elemental tag comprises at least one additional soft metal atom of the additional isotopic composition.
Embodiment 54 the kit of any one of embodiments 42 to 53, wherein each elemental tag is covalently bound to a different antibody.
Embodiment 55. The kit of any one of embodiments 42 to 54, wherein each chelating group is capable of binding at least one soft metal atom of the isotopic composition, and each chelating group is selected from the group consisting of lutidine amine or bis ((1H-imidazol-2-yl) methyl) amine, wherein each imidazole is optionally substituted with one or more polar functional groups selected from the group consisting of C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof.
Embodiment 56 the kit of any one of embodiments 42 to 55, further comprising reagents for covalently attaching the elemental tag to an antibody.
Embodiment 57 the kit according to any one of embodiments 42 to 56, wherein each element tag is independently a compound of formula I as defined in any one of embodiments 1 to 33 or a compound of formula II as defined in any one of embodiments 34 to 38.
Embodiment 58. A method comprising:
providing an isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
providing an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group is capable of binding at least one of the soft metal atoms of the isotopic composition; and
binding the soft metal atom of the isotopic composition to the one or more chelating groups of the elemental tag;
wherein the soft metal atoms are non-radioactive.
Embodiment 59. The method of embodiment 58, wherein the isotopic composition does not comprise a natural mixture of isotopes.
Embodiment 60 the method of embodiment 59, further comprising providing an additional isotopic composition, wherein said additional isotopic composition comprises a plurality of additional soft metal atoms of an additional monoisotope of a non-radioactive soft metal, said additional monoisotope being different from said monoisotope of said non-radioactive soft metal of said isotopic composition.
Embodiment 61 the method of any one of embodiments 58-60, further comprising providing an additional element tag comprising an additional linear or branched polymer comprising a plurality of chelating groups.
Embodiment 62. The method of any of embodiments 58 to 61, wherein each chelating group of the linear or branched polymer of the elemental tag comprises at least one soft metal atom of the isotopic composition, and wherein each additional chelating group of the additional linear or branched polymer of the additional elemental tag comprises at least one additional soft metal atom of the additional isotopic composition.
Embodiment 63 the method of any one of embodiments 58 to 62, further comprising:
Providing a biomolecule; and
covalently binding said biomolecule to said elemental tag.
Embodiment 64 a method for analyzing an analyte in a biological sample, comprising:
(i) Incubating an element-labeled affinity reagent with the analyte, the element-labeled affinity reagent comprising an affinity reagent labeled with an element tag comprising a linear or branched polymer having a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, the element tag further comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
wherein:
each chelating group of the elemental tag comprises or is capable of binding at least one of the soft metal atoms,
the soft metal atoms are non-radioactive and
the affinity reagent specifically binds to the analyte,
(ii) Separating unbound elemental-labeled affinity reagent from bound elemental-labeled affinity reagent; and
(iii) Elemental tags bound to affinity reagents attached to the analyte are analyzed by mass spectrometry atomic spectrometry.
Embodiment 65. The method of embodiment 64, wherein the soft metal does not comprise a natural mixture of isotopes.
Embodiment 66. The method of embodiment 64 or 65, wherein incubating the element-labeled affinity reagent with the analyte comprises:
incubating two or more different elemental-labeled affinity reagents with two or more analytes, wherein the elemental-labeled affinity reagents specifically bind to the two or more analytes to produce two or more differently labeled analytes, wherein analyzing the elemental tags bound to the affinity reagents comprises analyzing the different elemental tags bound to the two or more analytes by mass spectrometry.
Embodiment 67. The method of any one of embodiments 64 to 66, wherein the affinity reagent is further labeled with a fluorescent label.
Embodiment 68. The method of any one of embodiments 64 to 67, wherein the mass spectrometry atomic spectrum is ICP-MS.
Embodiment 69. The method of any one of embodiments 64 to 67, wherein the mass spectrometry atomic spectroscopy is performed by a mass spectrometer-based flow cytometer.
Embodiment 70. The method of any one of embodiments 64 to 69, wherein the affinity reagent is an antibody.
Embodiment 71 the method of any one of embodiments 64-70, wherein the affinity reagent specifically binds biotin.
Embodiment 72. The method of any one of embodiments 64 to 69, wherein the affinity reagent is an oligonucleotide.
Embodiment 73 the method of any one of embodiments 64-72, wherein the element-labeled affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells.
Embodiment 74 the method of any one of embodiments 64 to 73 wherein the soft metal is selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or a mixture thereof.
Embodiment 75 the method of any one of embodiments 64 to 74 wherein said soft metal is a non-naturally occurring element in said biological sample.
Embodiment 76 the method of any one of embodiments 54 to 75 wherein the element tag is or comprises a compound of formula I as defined in any one of embodiments 1 to 33 or a compound of formula II as defined in embodiment 34.
The foregoing disclosure generally describes the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These embodiments are described for illustrative purposes only and are not intended to limit the scope of the present application. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms are employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Examples
While the present disclosure has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the disclosure is not limited to the disclosed embodiments. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
Example 1
Preparation of chelating Polymer Compound I-1
Polymers having a lutidine amine (DPA) chelator attached to a pendant group were synthesized, wherein the pendant group can chelate rhenium, which expands the number of mass channels available for mass flow cytometry. The synthesis of metal chelator polymers and materials used are described.
Exemplary chelating polymer compounds of the present disclosure, I-1, were prepared according to scheme 2. Exemplary activated ester polymer 2-1 was reacted with exemplary Lys-DPA chelator 1-4. The resulting polymer 2-2 was then attached to a modifying group comprising PEG and maleimide to obtain compound I-1. Chelating agents 1-4 were synthesized according to scheme 1. It will be appreciated that other compounds of the present disclosure can be prepared with appropriate modifications using similar methods, techniques and principles as described below.
Material
Triethylamine (TEA, catalog No. 471283), acryloyl chloride (catalog No. 549797), 2- (dodecylthiocarbonylthio) -2-methylpropanoic acid (DDMAT), catalog No. 723010), 2' -azobis (2-methylpropanoic acid) (AIBN, catalog No. 44109), N epsilon-Boc-L-lysine (catalog No. 359661), sodium triacetoxyborohydride (starb, catalog No. 316393), pyridine-2-carbaldehyde (catalog No. P62003), HCl (4M in dioxane, catalog No. 345547), tris (2-carboxyethyl) phosphine hydrochloride solution (TCEP, catalog No. 646547) were obtained from Sigma Aldrich corporation (Sigma Aldrich). Pentafluorophenol was purchased from Matrix Scientific company (catalog No. 006058). mPEG (polyethylene glycol) 6 -NH 2 (catalog number 281204) is available from ChemPep corporation. Bis-Mal-PEG 6 (catalog number BP-22152) is available from BroadPharm. 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM, catalog number D461245) was purchased from Toronto Research Chemicals company. All organic solvents (anhydrous) were obtained from commercial sources and used without further purification.
Synthesis of lysine-based rhenium chelators
As shown in scheme 1, chelators 1-4 were prepared in three steps by (i) direct reductive alkylation of Boc protected lysine precursors, 20 (ii) Deprotection by Boc of hydrochloric acid (HCl in dioxane) followed by (iii) conversion of amine hydrochloride to the free base with NaOH. By passing through 1 H-NMR characterizes the reaction intermediates and the resulting products to confirm their structure (FIG. 6).
Scheme 1 Synthesis of chelators 1-4
To a mixture of N ε -Boc-L-lysine 1-1 (2 g) and STAB (4.5 g) in dichloroethane (45 mL) was added at 0deg.C under nitrogenPyridine-2-carbaldehyde (1.85 g, dissolved in 5mL of dichloroethane). The suspension was stirred at room temperature for about 5h and turned into a uniform bright yellow color. The reaction mixture was decomposed with deionized water (30 mL) and diluted with dichloroethane (100 mL). The separated organic layer was washed with deionized water (30 mL. Times.4), and dried over Na 2 SO 4 Dried and concentrated by rotary evaporation to give t-butoxycarbonyl (Boc) protected lysine-based chelator 1-2 (2.5 g). 1 H NMR(600MHz,CDCl 3 )δ8.53(ddd,J＝5.1,1.8,0.9Hz,2H),7.65(td,J＝7.7,1.8Hz,2H),7.32(dt,J＝7.9,1.1Hz,2H),7.20(ddd,J＝7.6,5.0,1.2Hz,2H),4.70(s,1H),4.10(d,J＝3.8Hz,4H),3.47(dd,J＝7.8,6.5Hz,1H),3.16-3.07(m,2H),2.04-1.92(m,1H),1.88-1.72(m,1H),1.52-1.45(m,4H),1.43(s,9H)。
To remove the Boc group, a solution of hydrogen chloride (4 m in dioxane, 7 mL) was slowly added to a solution of the above compound 1-2 (1.5 g) in DCM (5 mL) at 0 ℃. The resulting solution was stirred at room temperature for 24h. A yellow precipitate formed during the reaction. The solvent was discarded and the precipitate was dissolved in methanol (5 mL) and precipitated into diethyl ether (15 mL). The precipitate was collected by centrifugation (2700 Xg, 10 min). This dissolution-precipitation cycle was repeated once more. Finally, the precipitate was dried in a vacuum oven at room temperature for 24h to obtain Boc-deprotected lysine-based DPA chelator 1-3 (1.3 g) as an amine salt. 1 H NMR(600MHz,D 2 O)δ8.68(ddd,J＝6.0,1.6,0.7Hz,2H),8.49(td,J＝7.9,1.6Hz,2H),8.05(dt,J＝8.0,1.0Hz,2H),7.92(ddd,J＝7.5,5.9,1.3Hz,2H),4.42(q,J＝16.5Hz,4H),3.57-3.51(m,1H),2.96(t,J＝7.7Hz,2H),1.92(ddd,J＝9.9,8.4,5.8Hz,1H),1.89-1.82(m,1H),1.69-1.61(m,2H),1.52-1.44(m,2H)。
To convert the amine salt to the free base, a concentrated NaOH solution (5M) was slowly added to the amine salt solution (0.5 g, dissolved in 2mL of water) at 0 ℃ until the pH reached about 13. The alkalized solution was then lyophilized to obtain a light brown solid. Dichloromethane (5 mL) was added to dissolve the free base. Undissolved solids were removed by centrifugation (16000 Xg, 10 min). The supernatant was collected, concentrated, and dried under vacuum at room temperature for 24h to obtain the final lysine-based DPA chelator 1-4 (0.3 g) as the free base. 1 H NMR(500MHz,D 2 O)δ8.24(ddd,J＝5.0,1.8,0.9Hz,2H),7.60(td,J＝7.7,1.8Hz,2H),7.37(dt,J＝7.9,1.2Hz,2H),7.14(ddd,J＝7.6,5.0,1.2Hz,2H),3.95(d,J＝14.6Hz,2H),3.79(d,J＝14.6Hz,2H),3.21(dd,J＝8.5,6.3Hz,1H),2.59(t,J＝6.8Hz,2H),1.75-1.64(m,2H),1.45-1.26(m,4H)。
Synthesis of pentafluorophenyl acrylate (PFPA) monomer
To prepare activated ester polymer 2-1, pentafluorophenyl acrylate (PFPA) monomer was first synthesized by slowly adding triethylamine (18.3 mL) to a solution of pentafluorophenol (20 g in 130mL of methylene chloride) under nitrogen at 0 ℃ followed by the addition of 10.6mL of acryloyl chloride. The reaction mixture was stirred at 0 ℃ for 2h and then at room temperature overnight. The salts were removed by filtration. The solution was concentrated by rotary evaporation and then purified using silica gel column chromatography using hexane as eluent. 1 H NMR(500MHz,CDCl 3 )δ6.71(dd,J＝17.3,1.0Hz,1H),6.37(dd,J＝17.3,10.6Hz,1H),6.17(dd,J＝10.5,0.9Hz,1H)。 19 F NMR(564MHz,CDCl 3 )δ-153.17--153.27(m),-158.74(t,J＝21.5Hz),-163.11(td,J＝23.0,22.5,5.4Hz)。
RAFT polymerization of PFPA monomers
The synthesis of activated ester polymer 2-1, poly (pentafluorophenyl acrylate) (PPFPA), was achieved by reversible addition-fragmentation chain transfer (RAFT) polymerization. 21,22 In 1, 4-dioxane (6.0 mL), polymerization was performed at 70 ℃ in a Schlenk flask equipped with a stirring bar using 2- (dodecylthiocarbonylthio) -2-methylpropanoic acid (DDMAT, 0.28 mmol) as Chain Transfer Agent (CTA) and 2,2' -azobis (2-methylpropanenitrile) (AIBN, 0.028 mmol) as thermal initiator. The solution was degassed by three freeze-pump-thaw cycles, after which the flask was sealed and placed in a preheated oil bath (70 ℃) for 9 hours. After polymerization, the solution was cooled to room temperature with cold water and exposed to air. The polymer was precipitated into an excess of cold hexane (30 mL). The polymer obtained was dissolved in chloroform (5 mL) and precipitated again in hexane (30 mL). This dissolution-precipitation process was repeated 3 times. After drying overnight at room temperature under vacuum, the final polymer was obtained as a yellow powder, poly (PFPA) 2-1 。
The molar ratio of monomer to CTA to initiator ([ M) was chosen to be 30:1:0.1]:[CTA]:[I]) To tailor the molecular weight of the resulting polymer 2-1, its apparent number average molecular weight (M n GPC ) 9,400g/mol and a relatively narrow molecular weight distribution is obtained at a conversion of about 73%(FIG. 4).
Using 1 H NMR spectra, the Degree of Polymerization (DP) was determined by comparing the integral of PPFPA backbone peak (δ=3.11 ppm) with the integral of methyl units at the dodecyl chain end (δ=0.88 ppm) (fig. 5S 2). The DP of the corresponding polymer 2-1 was about 20. Polymer 2-1 19 The F NMR spectra showed three broad peaks at-153.2, -156.8 and-162.3 ppm, with an integration ratio of 2:1:2, corresponding to pentafluorophenyl groups along the polymer backbone (FIG. 5).
Ammonolysis of poly (PFPA) with lysine-based rhenium chelators
Next, polymer 2-1 was treated with a slight excess of lysine-based chelating agent 1-4 (1.6 equivalents relative to PFPA repeat units) at room temperature to obtain polymer 2-2 (scheme 2, step a). Chelating agents 1-4 were prepared in three steps as shown in scheme 1 above. By passing through 19 F NMR spectroscopy monitored the ammonolysis reaction. Over time, the broad signal corresponding to PFPA units along the scaffold disappeared, while the sharp signal corresponding to released pentafluorophenol appears (fig. 7). After stirring overnight (13 h) at room temperature, 100 μl of ethanolamine was added to the reaction mixture, and the reaction mixture was stirred for 3h. The polymer was precipitated in diethyl ether (50 mL) and then dissolved in H 2 O. Excess chelator was removed using a spin filter (Amicon, ultra-15,3 kDa), using H 2 O was washed twice, washed three times with PBS buffer and used with H 2 O was washed three times. After freeze-drying, polymer 2-2 (polyDPA) was obtained as a light brown powder.
Scheme 2 synthesis of rhenium chelating polymers. (a) 1) lysine-based rhenium chelator 1-4, RT,13h, DMF; 2) Ethanolamine, RT,3h; (b) 1) DMTMM, RT,5min,PB buffer (0.2M, pH 8.0); 2) mPEG (polyethylene glycol) 6 -NH 2 ，RT，15h；(c)1)TCEP(50mM)，RT，1h，H 2 O；2)Bis-Mal-PEG 6 RT,90min, DMF/PB buffer (0.2M, pH 7.0).
After purification, polymer 2-2 1 H NMR shows a set of resonances corresponding to the introduction of chelators 1-4, whereas 19 The F spectrum shows no signal (fig. 8). These NMR studies showed that PFPA units were fully modified as chelators 1-4 were introduced near quantitatively along the polymer backbone. In addition, the UV-vis spectrum of polymer 2-2 in methanol showed negligible absorption at 309nm, indicating cleavage of the trithiocarbonate group during ammonolysis (FIG. 9). However, a sharp absorption peak at 262nm was observed, corresponding to the absorption of the 2-pyridyl group of the chelating agent.
The carboxylic acid (COOH) moiety of 1-4 can be used for two purposes. First, it is expected that carboxylate ions will increase the water solubility of the metal-loaded polymer. Second, such metal-polymer complexes may have a net positive charge. Each pendant positively charged polymer can interact non-specifically with cells that typically have a negatively charged outer membrane. The carboxylate provides a potential counterion such that each pendant group is zwitterionic. Rhenium-loaded polymer 2-2 was found to have low solubility in water or in PBS buffer. To further increase the water solubility, polymer 2-2 was treated with short methoxypolyethylene glycol (mPEG) 6 -NH 2 ) Modification as shown in scheme 2, step b. The goal here is not only to increase the water solubility of the polymer, but also to provide a PEG corona to protect the positively charged complex from interacting with the cell.
Polymer 2-2 and mPEG 6 -NH 2 Polyethylene glycol of (a)
First, in PB buffer (4 mL,0.2M, pH 8.0), excess (4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine hydrochloride) (DMTMM, 1mL H was used 2 288mg in O, about 8 molar equivalents per carboxyl group) of treated Polymer 2-2 (50.3 mg) (scheme 2Step b). The reaction mixture was stirred at room temperature for 5min to activate the carboxylic acid functionality. Subsequently, an excess of mPEG was added rapidly 6 -NH 2 (about 7 molar equivalents per carboxyl group) and the reaction solution was stirred at room temperature overnight (15 h). A PEGylated version of Polymer 2-2, referred to herein as Polymer 2-3, is then obtained. Polymer 2-3 was purified using spin filters (Amicon, ultra-15, 10 kDa) with H 2 O was washed three times, twice with PBS buffer and with H 2 O was washed three times. After freeze drying, the final polymer was obtained as a light brown solid, polyDPA-mPEG 6 2-3。
Polymers 2 to 3 1 H NMR spectra confirmed PEGylation with new peaks at 3.32ppm and 3.59ppm, corresponding to mPEG 6 Methoxy and ethylene glycol repeat units of (a) (fig. 10). Rhenium loading experiments demonstrated that metal loaded polymers 2-3 were soluble in both water and PBS buffer.
Attachment of maleimide functional groups to polymers 2-3
The polymer 2-3 was further modified to mount maleimide functional groups (scheme 2, step c). First, tris (2-carboxyethyl) phosphine (TCEP) is used to reduce any disulfide bonds that may have formed in the previous step. To Polymer 2-3 (10 mg) at H 2 TCEP (80. Mu.L, 0.5M) was added to the solution in O (1 mL). The concentration of TCEP in the reaction solution was about 50mM. The reaction mixture was stirred at room temperature for 1h. The polymer was then washed twice with acetic acid solution (approximately 5mM, pH 3.5) using a spin filter (Amicon, ultra-15, 10 kDa) to remove excess TCEP.
The newly reduced thiol group is then reacted with an excess of bismaleimide (Bis-Mal-PEG 6 ) To give maleimide-functionalized compound I-1. Next, the concentrated polymer solution (approximately 300. Mu.L) was transferred to a 2-dram glass vial, 0.5mL of PB buffer (0.2M, pH 7.0) was added, followed by Bis-Mal-PEG6 (22 mg in 120. Mu.L DMF). The reaction solution was stirred at room temperature for 90min. Compound I-1 was then purified using a spin filter (Amicon, ultra-15, 10 kDa), using H 2 O was washed twice, washed once with PB buffer (0.2M, pH 7.0) and usedH 2 O was washed three times. After washing, the concentrated polymer solution was centrifuged at 12000×g for 10min to remove undissolved solids. The supernatant was taken and lyophilized to obtain the final polymer: polyDPA-mPEG as a light brown solid 6 Mal Compound I-1.
Compound I-1 1 The H NMR spectrum showed a new peak at 6.89ppm corresponding to maleimide (FIG. 1). The integral ratio of this peak to the chelator proton at 8.27ppm was 1:20, indicating that maleimide functionality was almost quantitatively incorporated into the polymer.
Rhenium loading of Compound I-1
By combining Compound I-1 (2.1 mg in 2mL of anhydrous MeOH) with a slight excess of rhenium salt [ NEt 4 ] 2 [Re(CO) 3 Br 3 ] 24 (3 mg, 1.2-fold each chelator, dissolved in 0.5mL anhydrous MeOH) was incubated in a 2-dram glass vial to achieve metal loading. The reaction mixture was incubated at 37℃for 2h without stirring. After incubation, the solution is poured into a container pre-filled with H 2 O (12 mL) in an Amicon spin filter (Ultra-15, 10 kDa). By H 2 O the polymer was washed three times. After washing, the polymer solution was lyophilized to obtain a rhenium-loaded polymer.
FIG. 2 (a) shows Re-supporting Compound I-1 (Compound II-1) in an aromatic region 1 Part of H NMR. All pyridine-matrix sub-signals are transferred to the low field due to the electron withdrawing induced effect of Re (I) compared to the unsupported polymer. Importantly, maleimide groups remain present under these reaction conditions (fig. 11). As shown in fig. 2 (b), successful loading was further confirmed by Fourier Transform Infrared (FTIR) spectroscopy. Rhenium salts at 1848 and 1998cm -1 Two strong carbonyl absorptions are shown. Compound I-1 at 1650 and 1095cm -1 The absorption bands are shown corresponding to c=o extension of the amide group in PEG and vibration of the ether C-O-C bond, respectively. 25 After loading, two new absorption bands corresponding to carbonyl groups appear at 1908 and 2030cm -1 Thereby indicating fac- [ Re (CO) 3 ] + The presence of a core. Rhenium-loaded polymers can be lyophilized for long-term storage and prior to bioconjugationRedissolved in buffer (lyophilized samples are shown in fig. 12).
Example 2
Re-loaded polymer compound II-1 was labeled with primary antibodies and used in mass cytometry immunoassays.
Antibody labelling of Re-loaded polymers
To evaluate the performance of exemplary elemental tag compound II-1 in mass cytometry immunoassays, the standard Maxpar was followed TM Antibody labelling protocol, primary antibody CD20 was labelled with a polymer label. Briefly, antibodies were partially reduced by TCEP, washed in a spin filter, then mixed with excess polymer, and the mixture incubated for 1h at 37 ℃. The antibody-polymer conjugate was purified by flash protein liquid chromatography to remove excess unconjugated polymer (fig. 13).
Antibody titration experiments
An antibody titration experiment was then performed with human Peripheral Blood Mononuclear Cells (PBMC) to evaluate the performance of the purified conjugates. In these experiments, human PBMC were treated with the 4-plex antibody panel (Fluidigm Maxpar TM Reagent) staining, including 154 Sm-CD45、 160 Gd-CD14、 170 Er-CD3 187/185 Re-CD20. Separate dye sets (wherein 187/185 Re-CD20 is replaced with 147 Sm-CD 20) was used as a positive control. Titrating at concentrations of 0.1, 0.3, 0.5, 1 and 2.5 μg/mL 187/185 Re-CD20 conjugates. As shown in the figure 3 of the drawings, 187/185 Re-CD20 allow CD20 + The B cell subpopulation was significantly separated from the remaining cell subpopulations in the PBMCs. In addition, by using 0.3. Mu.g/mL 187/185 Re-CD20 (FIG. 3 (b)) or optimal titre 147 Sm-CD20 (FIG. 3 (f)) achieved a highly comparable percentage of the major cell subsets within PBMC. These results indicate that rhenium-labeled antibodies provide accurate quantification for single cell immunophenotyping experiments and can be used in combination with commercial reagents for mass flow cytometry immunoassays.
Example 3
Chelating agents with one or more platinum atoms chelated by DPA
Polymers having a lutidine amine (DPA) chelating agent attached to a pendant group are synthesized, wherein the pendant group can chelate platinum useful in mass flow cytometry. Exemplary metal chelator polymers synthesized with platinum and materials used are described.
Material
Polymer 2-2 as in example 1 was used to prepare Polymer 3-2 (scheme 3). azido-PEG 6 -NH 2 (catalog number 76172) Potassium chloroplatinite (K) 2 PtCl 4 Catalog number 520853) is available from sigma aldrich.
Polymer 2 with azide-PEG 6 -NH 2 Polyethylene glycol of (a)
The resulting polymer 3-2 was characterized by 1H-NMR (FIG. 14). FTIR further confirmed the successful introduction of azide groups (fig. 15). To a solution of the blocked polymer 3-1 (20 mg in 2mL PB buffer, 0.2M, pH 8.0) was added DMTMM solution (0.5 mL H) 2 124mg in O, about 8 times each COOH). The solution was stirred at room temperature for 5min to activate COOH groups. After 5min, add the mixture containing mPEG 6 -NH 2 (54 mg, 3.5 equivalents per COOH) and azide-PEG 6 -NH 2 (64 mg, 3.5 equivalents per COOH) of PEG mixture. The reaction mixture was stirred at room temperature overnight. The resulting polymer 3-2 was purified using a spin filter (Amicon, ultra-5, 10 kDa) with H 2 O was washed three times, washed twice with PB buffer (0.2M, pH 7.6) and with H 2 O was washed three times. The polymer solution was then lyophilized overnight to give the final product 3-2.
Scheme 3 Synthesis of clickable chelating polymers
Platinum loading of Polymer 3-2 platinum loading on Polymer 3-2 was performed according to scheme 4). By using 1 H-NMR confirmed the successful chelation of Pt (FIG. 16). To a solution of polymer 3-2 (5.8 mg in 5.5mL dry MeOH) in a 2-dram (about 7 mL) glass vial was added K 2 PtCl 4 (200. Mu.L, 50mM in DMSO, 1.2 equivalents of each chelator). The vials were wrapped with aluminum foil to avoid exposure to sunlight. The reaction mixture was incubated in an oil bath at 45℃for 2h without stirring. After incubation, the solution is poured into a container pre-filled with H 2 O (10 mL) in an Amicon spin filter (Ultra-15, 10 kDa). The resulting solution of Polymer 4-1 (Compound II-2) was washed three times with NaCl solution (20 mM) and with H 2 O is washed once. After washing, the polymer solution was lyophilized to obtain Pt-loaded polymer 4-1.
Scheme 4 Synthesis of azido functionalized Pt chelating Polymer 4-1/II-2
Example 4
Pt-loaded polymers were labeled with primary antibodies and used for mass flow cytometry immunoassays.
Antibody labelling of Pt-loaded polymers
To evaluate the performance of the polymeric element tag 4-1/II-2 in mass cytometry immunoassays, the primary antibody CD20 was labeled with the polymeric element tag 4-1/II-2. To a solution of Dibenzocyclooctyne (DBCO) -modified CD20 Ab (160 μg,1.6 mg/mL) was added a solution of Pt-loaded polymer 4-1 (50 μ L H) 2 0.27mg in O, about 13-fold molar excess to Ab). The reaction mixture was vortexed at room temperature for 2h and then at 4 ℃ overnight. The conjugate was purified by washing five times with PBS using a spin filter (Amicon, ultra-0.5, 100 kDa).
Antibody titration experiments
Antibody titration experiments were performed as follows. FIG. 17 shows that CD20-PolyPt was effective in isolating B cells from T cells at titers of 0.5 or 1.0 μg/mL.
By combining differentMCP-Ab conjugates nat Pt-Ab conjugate cocktail antibody staining mixtures were prepared(70. Mu.L). For use of nat Pt-CD20 was subjected to an immunoassay to prepare four antibody staining mixtures. A mixture contains only ∈>The MCP-Ab conjugate (i.e., 154 Sm-CD45、 160 Gd-CD14、 170 Er-CD3 147 Sm-CD 20) and used as positive control. The other three mixtures consist of->The MCP-Ab conjugate (i.e., 154 Sm-CD45、 160 Gd-CD14 170 Er-CD 3) nat Both Pt-CD20 conjugates consisted of, with titers of 0.5, 1.0 and 2.5 μg/mL, respectively, in each mixture nat The concentration of Pt-CD20 conjugate was varied.
For the staining procedure, PBMC suspensions (30. Mu.LApproximately 300 ten thousand cells in cell staining buffer, fc blocked) were added to the antibody mixture (70 μl). The mixture was gently vortexed and incubated at room temperature for 30min. After incubation, cells were washed twice with cell staining buffer and then fixed with 1.6% formaldehyde/PBS solution for 10min at room temperature. The immobilized cells were pelleted and a cell intercalation solution (Ir intercalator, 1mL, final concentration: 125 nM) was added. The cells were then incubated overnight at 4 ℃. After incubation, the cells were washed twice with cell staining buffer and with +. >The cell harvesting solution was washed twice. Resuspending the pelleted cells in the presence of EQ TM The four element calibration beads were taken up in cell collection solution (100 tens of thousands of cells/mL) and analyzed by mass cytometry.
Uptake of CD20-PolyPt conjugate by monocytes was also observed.
Example 5
Chelating agents with one or more mercury atoms chelated by DPA
Polymers having a lutidine amine (DPA) chelating agent attached to a pendant group are synthesized, wherein the pendant group can sequester mercury useful in mass flow cytometry. The synthesis of exemplary mercury-sequestering agent polymers and materials used are described.
Material
PolyDPA polymer (2-3) was prepared as described in example 1. Mercury acetate (catalog No. 176109) and methanol (catalog No. 322415) were purchased from sigma aldrich corporation.
Hg loading to polyDPA (Polymer 2-3)
To a solution of Polymer 2-3 in a 2dram glass vial (2.5 mg polymer in 2mL methanol) was added a solution of Hg salt (1.5 mg Hg (OAc) in 0.5mL methanol) 2 1.4-fold excess of DPA chelator) followed by gentle swirling. The resulting solution was incubated at 40℃for 2h without stirring (scheme 5). After incubation, the solution was poured into a spin filter (Amicon Ultra4, 10 kDa) pre-filled with water (1.5 mL). The resulting solution of polymer 5-1/II-3 was washed three times with water (2700 g,20 min) and then lyophilized overnight to obtain the final product. For NMR measurements, polymer 5-1/II-3 was redissolved in D 2 O. By using 1 H-NMR confirmed successful mercury sequestration (FIGS. 18 and 19).
Scheme 5 Synthesis of Hg-supporting Polymer 5-1
Example 6
Chelating agents with one or more silver atoms chelated by DPA
Polymers having a lutidine amine (DPA) chelator attached to a pendant group were synthesized, where the pendant group can chelate silver that can be used in mass flow cytometry. The synthesis of exemplary silver chelator polymers and materials used are described.
Material
Polymers 2-3 were prepared as described in example 1. Silver perchlorate (catalog number 226548) and methanol (catalog number 322415) were purchased from sigma aldrich corporation.
Ag loading to polyDPA (Polymer 2-3)
To a solution of polymer 2-3 (2.5 mg polymer in 2mL methanol) in a 2Dram glass vial was added a solution of Ag salt (1.0 mg AgClO in 0.5mL methanol) 4 1.2-fold excess of DPA chelator) followed by gentle swirling. The resulting solution was incubated at 40℃for 2h without stirring (scheme 6). After incubation, the solution was poured into a spin filter (Amicon Ultra4, 10 kDa) pre-filled with water (1.5 mL). The resulting solution of polymer 6-1/II-4 was washed three times with water (2700 g,20 min) and then lyophilized overnight to obtain the final product. For NMR measurements, polymer 6-1/II-4 was redissolved in D 2 O. By using 1 H-NMR confirmed successful chelation of silver (FIGS. 18 and 20).
Scheme 6 Synthesis of Ag-supporting Polymer
Instrument for measuring and controlling the intensity of light
On an Agilent DD2 500MHz spectrometer or an Agilent DD2 600MHz spectrometer 1 HNMR and 19 f NMR experiment.
UV-vis measurements were performed on an Agilent Cary 300UV-vis spectrophotometer.
PerkinElmer Spectrum Two with ATR attachment TM FT-IR measurements were performed on an infrared spectrometer. At 500-4000cm -1 Within 1cm -1 All spectra were collected at resolution.
GPC measurements were performed on Waters 515HPLC equipped with a Viscotek VE 3580 Refractive Index (RI) detector. Tetrahydrofuran (THF) containing 2.5g/L tetra-n-butylammonium bromide (TBAB) was used as eluent (35 ℃, flow = 0.6 mL/min). The system was calibrated with PMMA standards.
FPLC experiments were performed on the AKTA pure 25L system. To purify the polymer-antibody conjugate, superdex was used TM 200 10/300GL column.
In fluid Canada (Markham, ON) of OntarioHelios TM Mass flow cytometry experiments were performed systematically. The data is obtained in FCS3.0 file format and processed by FlowJo software.
Example 7
Preparation of zwitterionic poly (sulfobetaine methacrylate) PSBMA as solubility modifier
Material
4-cyano-4- (phenylthioformyl thio) pentanoic acid (CTA reagent), ethyl 2- (N-3-sulfopropyl-N, N-dimethylammonium) methacrylate (SBMA), 4' -azobis (4-cyanopentanoic acid) (ACVA), 2-Trifluoroethanol (TFE) were obtained from Sigma Aldrich company.
Experimental details
CTA (14.51 mg,5.19×10 -2 mmol,1.0 eq.), SBMA (501 mg,1.78mmol,35 eq.) and ACVA (1.692 mg, 5.13x10) -3 mmol,0.1 eq) was dissolved in TFE (2.63 mL). The polymerization solution was subjected to 3 freeze-pump-thaw cycles. The polymerization solution was heated in a preheated oil bath at 70℃for 8h. The polymerization was quenched by freezing the solution via plunging the Schlenk flask into liquid nitrogen. After thawing the polymerization solution, it was stored at 4 ℃ overnight. One part of crude polymerization solution 1 H-NMR analysis indicated a monomer conversion of about 70%. Excess solvent was evaporated in vacuo using a rotary evaporator. The crude polymer mixture was redissolved in TFE (about 1 mL), precipitated in methanol (about 13 mL), and centrifuged at 2700rcf for 5min: this procedure was repeated 3 times. The resulting polymer was then dried in vacuo overnight to obtain the desired polymer. The solid product had a distinct isolated pink and white solid.
Scheme 7 preparation of Poly (sulfobetaine methacrylate) PSBMA (Polymer 7-1)
PSBMA such as polymer 7-1 can optionally be attached to a polymeric chelator such as compound 2-2 through a diamine linker, as shown in example 8. For example, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine hydrochloride (DMTMM) or other similar chemicals may be used as coupling agents.
Scheme 8 attachment of PSBMA to Polymer 2-2
EXAMPLE 8 preparation of imidazole-based chelating Polymer
It will be appreciated that imidazole-based chelators may be prepared using a method similar to that used to prepare DPA-based chelators in example 1. An exemplary synthesis is shown in scheme 9. Other examples of lysine-imidazole chelators include those described in Maresca et al, bioconjugate chem.,2010,21,1032-1042, the contents of which are incorporated herein by reference in their entirety.
Scheme 9 preparation of lysine-imidazole chelators
N epsilon-protected lysine 9-1 may be functionalized with two imidazole groups at the alpha-amino group by reductive amination and deprotection to give lysine-imidazole chelator 9-4. Once obtained, lysine-imidazole chelators can be incorporated into a polymer scaffold by a procedure similar to scheme 2. An exemplary method is shown in scheme 10.
Scheme 10 preparation of Polymer 10-3 containing imidazole chelator
The activated ester polymer 10-1 can be coupled to a lysine-imidazole chelator such as compound 9-4 to obtain a chelate polymer 10-2/compound I-7. Optionally, a modifying group such as a solubility modifier (e.g., PEG) to obtain polymer 10-3/compound I-8. In addition, reactive functional groups (e.g., maleimide) may be attached to polymer 10-3 to obtain polymer 10-4/compound I-9.
Example 9 preparation of DPA chelator-containing Polymer with zwitterionic solubility modifier
Two exemplary zwitterionic sulfobetaine solubility modifiers were prepared and attached to a polymer containing a DPA chelator, such as compound 12-1.
Based on the previously reported synthetic procedure, 3- ((3-aminopropyl) -dimethylammonium) propane-1-sulfonate 11-4 was prepared as shown in scheme 11. (26)
Scheme 11 preparation of 3- ((3-aminopropyl) -dimethylammonium) propane-1-sulfonate
Compound 11-2:
di-tert-butyl dicarbonate (15.6 g,71.5 mmol) is added to a solution of 3- (dimethylamino) -1-propylamine (4.9 g,6mL,47.7 mmol) in 50mL1, 4-dioxane. The solution was stirred at 0 ℃ for 2h and further stirred at room temperature overnight for 18h. After 18h, the solvent was removed in vacuo and 50mL MilliQ water was added to the crude product. The product was extracted three times with ethyl acetate (30 mL). Ethyl acetate was dried to give a yellowish transparent oil as compound 11-2. The product was used in the subsequent synthesis step without further purification.
Compound 11-3:
compound 11-2 (1 equivalent, 2.5g,0.012 mol) was dissolved in 15mL anhydrous DMF. 1, 3-propane sultone (1.4 eq, 2.113g,0.017 mol) was added to a solution of compound 11-2 and stirred at room temperature for 3 days. The crude product was dried in vacuo to remove DMF until a slightly yellowish viscous oil remained. The viscous oil was washed with diethyl ether (30 mL) followed by a second wash with ethyl acetate (30 mL) to remove the remaining 1, 3-propane sultone. The oil was freeze-dried to give a white solid, compound 11-3.
Compound 11-4:
compound 11-3 (4 g,0.012 mol) was dissolved in 50mL DCM and cooled to 0deg.C to give a slightly cloudy, slightly white solution. After cooling to 0 ℃, 4M HCl in dioxane (5 mL) was added to the solution and stirred for 1h. After 1h, the solution became clear and a solid white massive precipitate formed. The solvent was removed in vacuo to dryness and precipitated with DCM/isopropanol/MeOH (10:5:1 v/v ratio). The precipitated product was a freeze-dried viscous white block. The final product compound 11-4 was obtained as a white solid and stored in a vacuum sealed bag in a refrigerator. Compound 11-4 1 The H NMR spectrum is shown in fig. 22.
Compound 12-2/I-11
The sulfobetaine 11-4 was then attached to the DPA-containing polymer chelator 12-1 as shown in scheme 12 to obtain compound 12-2/polymer I-11.
Scheme 12 preparation of sulfobetaine containing Polymer I-11
Polymer 12-1 containing DPA chelator was dissolved in 0.4mL of 0.2M sodium phosphate buffer pH 8. DMTMM (28.8 mg,0.104mmol,7.3 eq/side group) was dissolved in 0.1mL MilliQ water and added to the polymer solution and allowed to pre-react for 5min. After 5min, sulfobetaine 11-4 (14.58 mg,0.065mmol,5 eq/side group) was added to the DPA chelator-containing polymer 12-1 solution and the reaction mixture was stirred overnight at room temperature for 20 hours. After 20 hours, the reaction mixture was purified using a 3k MWCO Amicon Ultra 4mL spin filter, washed 5 times with MilliQ water. The retentate was removed and freeze-dried to give product 12-2. The 1H NMR spectrum of compound 12-2/I-11 is shown in FIG. 23.
Compound I-12
PBSMA7-1 was prepared as shown in scheme 7 in example 7. An ethylenediamine linker was then attached to 7-1 and the resulting compound 13-1 was attached to DPA chelator-containing polymer 12-1 to produce polymer I-12 as shown in scheme 13. The 1H NMR spectrum of compound I-12 is shown in FIG. 24.
Scheme 13 preparation of sulfobetaine containing Polymer I-12
Polymer compound 12-1 containing DPA-chelator (Dp=20, M n =6000 g/mol,0.0005mmol,3 mg) in 0.1ml of 0.2m sodium phosphate buffer, ph 8. A solution of DMTMM (7.3 eq/side group, 20.20mg,0.073 mmol) in 0.1mL MilliQ water was added to the polymer solution containing DPA-chelator and allowed to pre-react for 10min. Then, compound 13-1 (M n =5600 g/mol,112mg,0.02mmol,2 eq/side chain amine) was added to the pre-reacted DPA-chelator-containing polymer, compound 12-1 solution and stirred overnight at room temperature for about 20h. The crude product was purified 2 times with MilliQ water, 2 times with 20mM NaCl solution, 3 times with MilliQ water using an Amicon Ultra-4mL 10kDa MWCO rotary filter, and freeze-dried to yield a polymer, compound 13-2/I-12.
Alternatively, compound 13-1 can be prepared according to scheme 13 a.
Scheme 13a-13-1 preparation
4-cyano-4- (phenylthioformyl-thio) pentanoic acid 13a-1 (500 mg,1.79mmol,1 eq.), EDC (555.8 mg,3.58mmol,2 eq.) and NHS (412 mg,3.58mmol,2 eq.) were dissolved in a minimum amount of acetonitrile and vortexed for 15min. Next, N-Boc-ethylenediamine 13a-2 (430 mg,2.685mmol,1.5 eq.) was added and stirred overnight. After 24h, the reaction was dried via compressed air and then purified via flash column chromatography on silica gel. The solvent system was a gradient elution of 100% DCM to 1:1 DCM/EtOAc. The purified product was dried in vacuo to give a red-pink solid, N-boc CTA13a-3, as the desired product.
N-boc CTA13a-3 (45 mg,0.1067 mmol), SBMA monomer 13a-5 (1.4 g,2.252 mmol) and 4,4' -azobis (4-cyanovaleric acid) initiator 13a-4 (3.038 mg,0.0108 mmol) were dissolved in 4mL 2, 2-trifluoroethanol and the solution was bubbled with nitrogen for 20min. After bubbling the solution with nitrogen for 20min, the reaction was stirred at 70 ℃ for 6 hours. After 6H, the reaction was exposed to air, a small portion of the crude product was taken for H NMR, and trifluoroacetic acid (1.7 mL) was added to the remaining crude product and stirred overnight at room temperature to hydrolyze the Boc group. After 21h, the sample was precipitated with 7:3 diethyl ether/methanol and centrifuged at 2.7k rcf for 15min to precipitate. The supernatant was discarded. The precipitate was redissolved once with 2, 2-trifluoroethanol and reprecipitated with 7:3 diethyl ether/methanol and centrifuged again under the same conditions. This procedure was repeated twice. The sample was dried in vacuo to give compound 13-1.
EXAMPLE 10 preparation of Metal-chelated DPA chelator-containing Polymer Using various solubility modifiers
Three polymers containing DPA chelating agents were prepared based on the methods described in examples 3 and 9 (scheme 14). Using K respectively 2 PtCl 4 Or HgCl 2 Each polymer was metallized with platinum or mercury. Platinum metallization was performed in methanol at 45 ℃ for 2 hours. Mercury metallization was performed in methanol at room temperature for 1 hour. Successful metallization was assessed by proton NMR by changes in pyridine matrix chemical shifts. NMR spectra of compounds 14-2/II-7 and 14-3/II-8 are shown in FIG. 21.
Scheme 14-chelator polymers
Polymer 12-1 containing DPA-chelator (Dp=20, M n =6000 g/mol,0.0005mmol,3 mg) in 0.1ml of 0.2m sodium phosphate buffer, ph 8. A solution of DMTMM (7.3 eq/side group, 20.20mg,0.073 mmol) in 0.1mL MilliQ water was added to a polymer solution containing DPA-chelatorAnd allowed to pre-react for 10min. Then, poly (SBMA) 13-1 (M n =5600 g/mol,112mg,0.02mmol,2 eq/side chain amine) was added to the pre-reacted DPA-chelator-containing polymer, compound 12-1 solution and stirred overnight at room temperature for about 20h. The crude product was purified 2 times with MilliQ water, 2 times with 20mM NaCl solution, 3 times with MilliQ water using an Amicon Ultra-4mL 10kDa MWCO rotary filter, and freeze-dried to yield a polymer, compound 13-2/I-12.
Scheme 15-preparation of metallized Polymer
Compound 13-2/I-12 (11.6 mg dissolved in methanol/2, 2-trifluoroethanol (1:1 by volume). HgCl 2 To a solution of compound 13-2/I-12 (2 mg/mL,7mM,360 mL) and stirred at room temperature. A red precipitate formed within 10 minutes and the reaction was stirred at room temperature for 1h, and a red precipitate precipitated via centrifugation at 10k rcf for 10 minutes. The supernatant was discarded and the precipitate was further washed 2 times with MeOH. The red precipitate was freeze-dried to give compound 14-6/II-11. (method adaptation from 27)
Compound 13-2/I-12 (20 mg dissolved in aqueous MeOH (1:1 by volume). Will K 2 PtCl 4 To compound 13-2/I-12 (50 mM,360 mL) and then heated at 45℃for 2h, to give a yellow solution. The yellow solution was spin-filtered with Amicon Ultra-15ml 10k MWCO spin filter, washed 2 times with MilliQ water, 3 times with 20mM NaCl solution, and 1 time with MilliQ water. The sample was then freeze-dried to give compound 14-7/II-12.
The metallization reaction can also be accomplished with 2, 2-trifluoroethanol and MeOH/2, 2-trifluoroethanol as solvent systems.
Example 11 Mass Spectrometry flow cytometry testing of rhenium-containing antibody conjugated polymers with zwitterionic solubility modifiers
Rhenium chelating polymers of the present disclosure comprising zwitterionic solubility modifiers were conjugated to antibodies and non-specific binding was assessed using mass flow cytometry.
Antibody conjugation
To evaluate the performance of rhenium-tagged polymers with zwitterionic solubility modifiers in mass flow cytometry immunoassays, the modified Maxpar was followed TM Antibody labelling protocol, two primary antibodies, CD20 and CD8a, were labelled with a polymer label. Briefly, antibodies were partially reduced by TCEP, washed in a spin filter (30 kDa), and then mixed with an excess (20-fold) of DBCO-PEG 4-maleimide. The mixture was incubated at 37℃for 30min. DBCO modified Abs were purified using spin filters (30 kDa) and then mixed with the desired polymer mass tags. The mixture was incubated at 37℃for 90min. Excess polymer was removed by spin-on filter (100 kDa).
Mass flow cytometry titration experiments
Antibody titration experiments were then performed with human Peripheral Blood Mononuclear Cells (PBMC) to evaluate the performance of the conjugates. In these experiments, human PBMC were treated with the 7-plex antibody panel (Fluidigm Maxpar TM Reagent) staining, including 145 Nd-CD4、 146 Nd-CD8a、 165 Ho-CD16、 154 Sm-CD45、 160 Gd-CD14、 170 Er-CD3 147 Sm-CD20. For titration of rhenium conjugates, a separate staining set was used, in which 147 Sm-CD20 146 Nd-CD8a quilt 187/185 Re-CD20 187/185 Re-CD8a substitution. Titrating at concentrations of 0.25, 0.5, 1 and 2.0 μg/mL 187/185 Re-CD20/CD8a conjugates. As shown in figure 25 of the drawings, 187/185 Re-CD20 allow CD20 + The B cell subpopulation was significantly separated from the remaining cell subpopulations in the PBMCs. In a similar manner to that described above, 187/185 Re-CD8a also allows for CD8 to be quantified at all titers + The T cell subpopulation was significantly separated from the remaining cell subpopulations in PBMCs (fig. 26). Importantly, both conjugates showed minimal non-specific binding to the other cell populations, as shown in figures 27 and 28. Rhenium-labeled CD20 conjugates showed minimal non-specific binding to non-T/B cells. (FIG. 27) rhenium-labeled CD8a conjugates showed minimal non-specificity with B cellsAnd (3) sex binding. (FIG. 28)
Comparison with PEG-modified Re-containing Polymer
To evaluate the non-specific binding properties of zwitterionic solubility modifiers compared to other solubility modifiers, PEG-modified polymers were synthesized and sequestered to Re metal based on the method described in example 1. For mass flow cytometry experiments, two polymers were mixed with antibody staining mixtures of different concentrations (1, 2 and 5 ug/mL) and the resulting antibody mixtures were used to stain PBMCs according to the conventional staining protocol as described above. It was observed that this PEG modified polymer showed non-specific binding to the major cell subsets within PBMC at 1 ug/mL. (see FIG. 29)
The polymers of the present disclosure modified with the PEG solubility modifier exhibit higher non-specific binding to PBMCs than polymers modified with the zwitterionic solubility modifier. (see FIG. 29) the polymer modified with the zwitterionic solubility modifier showed minimal non-specific binding to the major subset of PBMC at 5 ug/mL. The zwitterionic modified rhenium polymer showed relatively less non-specific binding to PBMCs than the PEG modified rhenium polymer.
EXAMPLE 12 preparation of H-Dap lutidine chelator
Chelating agents based on H-Dap lutidine were prepared as shown in scheme 16.
Scheme 16-H-Dap dimethyl pyridinamine chelating agentIs prepared from
H-Dap (Boc) -OMe HCl 16-1 (0.5 g,1.86mmol,1 eq.) was dissolved in about 30mL dry acetonitrile and stirred with N 2 (g) Bubbling for 30 minutes. Next, 2-chloromethylpyridine hydrochloride (2.2 eq., 671.21mg,4.092 mmol), K were added successively 2 CO 3 (3.2 eq, 5.95mmol,822.6 mg) and the reaction stirred at room temperature for 2h. After stirring for 2 hours, potassium iodide (1 equivalent679.27mg,4.092 mmol) and the reaction was heated and stirred at reflux (about 85 ℃). The reaction was then allowed to react overnight (about 18 h). The sample was dried in vacuo to remove acetonitrile. The dried crude product was redissolved in DCM. The DCM organic layer was washed with water 3X 100 mL. The organic layer was collected and dried in vacuo to give the product as a brown solid, H-Dap Boc-OMe lutidine amine 16-2.
H-Dap Boc-OMe lutidine amine 16-2 (185 mg) was dissolved in a 1:1 mixture of 40mL MillIQ water and concentrated HCl (to make about 6M HCl). The sample was then refluxed overnight at 110℃and the reaction started at 11:40 am. After 24h, the reaction mixture was dried in vacuo. The samples were redissolved in approximately 20mL MilliQ water and basified with 1M NaOH to neutralize the acid (checked with pH paper). The samples were freeze dried overnight and then redissolved with DCM. The DCM layer was then centrifuged at 2.7k rcf for 15min to remove any salts from the neutralization. Product 16-3 was dried and collected as a dark brown solid. The 1H NMR spectrum of compound 16-3 is shown in FIG. 30.
Chelating agent 16-3 is used to prepare the polymers of the present disclosure as described herein.
EXAMPLE 13 addition of modifier to Metal-containing Polymer by ligand exchange
Modifiers such as solubility modifiers have been attached to metal-containing polymers via metal centers by ligand exchange with small molecules such as glutathione. (schemes 17 and 18)
Scheme 17 ligand exchange for Hg containing Polymer
Scheme 18 ligand exchange for Pt-containing polymers
To a solution (3 mg in 250 uL) containing a mercury-containing polymer or a platinum-containing polymer, a glutathione solution (325 mM,50 uL) was added, and the reaction was gently vortexed at room temperature for 15 minutes. The sample was then filtered 8 times with MilliQ water at 2.7k rcf on Amicon, ultra-0.5,3 kDa. The sample was freeze-dried to give a polymer.
Other examples of small molecule thiols containing one or more thiol functional groups for use in the ligand exchange reactions described herein include, but are not limited to, cysteine, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaptopropanol, dimercaptosuccinic acid, 2, 3-dimercapto-1-propanesulfonate.
It was observed that the polymer containing glutathione ligand was more soluble than the original polymer with halide ligand. For example, for Hg polymers, any undissolved material was observed in MilliQ water. These precipitates were filtered out using a 0.2um nylon syringe filter. Glutathione addition allowed redissolution of the pellet, which was then purified by spin filtration using an Amicon, ultra-0.5,3kda spin filter. 1 H NMR showed that the redissolved (in water) material was a DPA-sulfobetaine modified polymer. Therefore, ligand exchange with a solubility modifier installed at the metal center can increase solubility.
Several glutathione-modified Pt-containing polymers of the present disclosure were prepared and compared to their counterparts without glutathione ligand as solubility modifiers:
TABLE 2 Metal-containing Polymer with or without glutathione ligand
The polymer was conjugated to an antibody and non-specific binding was assessed using mass cytometry methods based on those described in example 11. The results are shown in fig. 31. As shown by the mass cytometry results, glutathione-modified polymers exhibited lower non-specific binding to all polymers tested.
While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. When a term in the present application is found to have a different definition in the literature incorporated herein by reference, the definition provided herein is used as the definition of the term.
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Claims (30)

1. A compound of formula I
Wherein the method comprises the steps of
A is a polymer backbone, optionally, the polymer is a linear polymer, a branched polymer, a hyperbranched polymer, a copolymer, or a combination thereof;
Each B is independently a nitrogen-containing 5-to 7-membered heterocycle optionally substituted with one or more polar functional groups selected from C1-C6COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof; and is also provided with
L is absent or a linker;
each L 2 Independently absent or a linker;
each R 2 Independently a first modifying group selected from a solubility modifier, a reactive functional group, a biomolecule, or a combination thereof;
x is a functional group selected from esters, ethers, and amides;
each L 1 Independently absent or a linker;
each R 1 Is independently H, C to C8 alkyl, C2 to C8 alkenyl, C3-C8 cycloalkyl, OH, C1 to C10 alkoxy, C1 to C10 alkylamine, solubility modifier, reactive functional group, biomolecule, and combinations thereof;
n is an integer from 0 to 7;
m is an integer from 0 to 4;
p is an integer from 0 to 3; and is also provided with
q is an integer greater than 0.
2. The compound of claim 1, wherein each B is independently a nitrogen-containing 5-or 6-membered heteroaryl, optionally wherein each B is independently pyridine or imidazole, optionally substituted with one or more polar functional groups selected from COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, polyether, or a combination thereof, and wherein optionally one or more B coordinates with a soft metal, and/or is conjugated to one or more biomolecules.
3. The compound of claim 1 or 2, wherein one or more B coordinates with a soft metal, optionally selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or mixtures thereof.
4. A compound according to any one of claims 1 to 3 wherein R 1 Or R is 2 Is said biomolecule, optionally said biomolecule is an affinity agent, such as an antibody.
5. The compound of any one of claims 1 to 4, wherein X is-C (O) NR 4 -or-NR 4 C (O) -, wherein R 4 Is H or C1 to C4 alkyl.
6. The compound of any one of claims 1 to 5, wherein X is-C (O) NR 4 -, and the compound has the structure of formula Ia
Or wherein X is-NR 4 C (O) -, and the compound has the structure of formula Ib
7. The compound of any one of claims 1 to 5, wherein X is-C (O) NR 4 -, and the compound has the structure of formula Ic
Alternatively, wherein X is-NR 4 C (O) -, and the compound has the structure of formula If
Or wherein the compound has the structure of formula Id or Ie
Or wherein the compound has the structure of formula Ig or Ih
Wherein each R is 3 Independently selected from H, C to C5 alkyl, C2 to C5 alkenyl, C1-C6 COOH, C1-C6 alkoxy, C1-C6 alkylphosphonate, alkyl ether, or polyether.
8. The compound of claim 7 or 8, wherein each R 3 Independently selected from H, - (CH) 2 ) 1-3 COOH、-(CH 2 ) 1-3 O(CH 2 ) 1-2 CH 3 、-(CH 2 ) 2-4 OH、-(CH 2 ) 2-5 P(O)(OCH 2 CH 3 ) 2 Or-CH 2 CH(OMe) 2 And/or wherein n is 2, 3, 4 or 5, wherein m is 0, 1 or 2, or wherein p is 1 or 2.
9. The compound of any one of claims 1 to 8, wherein a is selected from the group consisting of polyacrylates, polyacrylamides, polyethers, polyamino acids, polyvinylamines, poly (2-oxazolines), polyethylene glycols, polysaccharides, dendrimers, copolymers thereof, or combinations thereof.
10. The compound of any one of claims 1 to 9, wherein each linker independently comprises or is independently selected from C3-C8 alkylamine, C3-C8 alkylene, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, 5-or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C (O) O, amide, amine, thioether, maleimide-thiol conjugate, polyethylene glycol (PEG), or mixtures thereof, optionally, the amine, alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl are each independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
11. The compound of any one of claims 1 to 10, wherein each L 1 And/or L 2 Independently comprising or independently selected from a C3-C8 alkylene, a C3-C8 alkylamine, an ester, an amine, an amide, a thioether, a maleimide-thiol conjugate, PEG, or a mixture thereof, optionally, each of said alkylene and alkyl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
12. The compound of any one of claims 1 to 11, wherein each R 2 The solubility modifier of the first modification group and the solubility modifier of the second modification group each independently comprise polyethylene glycol (PEG), a sugar, an oligosaccharide, or a zwitterionic polymer, such as poly (carboxybetaine) methacrylate or poly (sulfobetaine) methacrylate (PBSMA).
13. The compound of any one of claims 1 to 12, wherein the reactive functional group is for attachment to one or more biomolecules, optionally the one or more biomolecules are each independently selected from a small molecule, a polypeptide, an oligonucleotide, a lipid, a carbohydrate, an affinity reagent, optionally an antibody or a mixture thereof.
14. The compound of claim 1, wherein the compound is selected from the group consisting of
Or wherein the compound is selected from
Wherein s is from about 1 to about 50, from about 2 to about 40, from about 5 to about 30, from about 10 to about 30, from about 5 to about 35, or from about 20 to about 30, and R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 1 、R 2 、L 1 、L 2 And R is 3 Each as defined in any one of claims 7 to 8.
15. The compound of claim 1, wherein the compound is selected from the group consisting of
/>
/>
/>
/>
/>
/>
Wherein s is from about 1 to about 50, from about 2 to about 40, from about 5 to about 30, from about 10 to about 30, from about 5 to about 35, or from about 20 to about 30, and R is from about 3 to about 200, from about 6 to about 30, or from about 10 to about 25, and wherein R is 3 As defined in any one of claims 7 to 8.
16. A compound of formula I as defined in any one of claims 1 to 15, or a derivative or salt thereof, wherein the compound of formula I is chelated to one or more metals M, and wherein the compound has the structure of formula II
17. A compound of formula I as defined in any one of claims 1 to 15 or a compound of formula II as defined in claim 16 for use in mass flow cytometry.
18. An elemental tag comprising a linear or branched polymer comprising a plurality of chelating groups, wherein each chelating group is capable of binding a soft metal, the soft metal being a monoisotope, and wherein at least one chelating group is chelated to a soft metal atom of the soft metal.
19. A kit comprises
An isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal; and
an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, the chelating groups comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group of the elemental tag comprises or is capable of binding at least one soft metal atom of the isotopic composition;
optionally, wherein the kit does not comprise any radioactive soft metals.
20. The kit of claim 19, wherein the elemental tag is functionalized to bind and/or attach to a biomolecule.
21. The kit of claim 19 or 20, wherein the isotopic composition is a soft metal solution provided separately from the elemental tag, and wherein each chelating group is capable of binding at least one soft metal atom of the isotopic composition.
22. The kit of any one of claims 19 to 21, further comprising a biomolecule, optionally an oligonucleotide or antibody and/or further comprising a further isotopic composition, wherein the further isotopic composition comprises a plurality of further soft metal atoms of a further monoisotope of a soft metal, the further monoisotope being different from the monoisotope of the soft metal of the isotopic composition.
23. The kit of any one of claims 19 to 22, wherein each element tag is independently a compound of formula I as defined in any one of claims 1 to 15 or a compound of formula II as defined in claim 16.
24. A method, the method comprising:
providing an isotopic composition comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
providing an elemental tag comprising a linear or branched polymer, the polymer comprising a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, wherein each chelating group is capable of binding at least one of the soft metal atoms of the isotopic composition; and
binding the soft metal atom of the isotopic composition to the one or more chelating groups of the elemental tag;
wherein the soft metal atoms are non-radioactive.
25. The method of claim 24, wherein the isotopic composition does not comprise a natural mixture of isotopes, optionally wherein the method further comprises providing a further isotopic composition, wherein the further isotopic composition comprises a plurality of further soft metal atoms of a further monoisotope of a non-radioactive soft metal, the further monoisotope being different from the monoisotope of the non-radioactive soft metal of the isotopic composition.
26. A method for analyzing an analyte in a biological sample, the method comprising:
(i) Incubating an element-labeled affinity reagent with the analyte, the element-labeled affinity reagent comprising an affinity reagent labeled with an element tag comprising a linear or branched polymer having a plurality of chelating groups, each chelating group independently comprising two nitrogen-containing 5-or 6-membered heterocycles, the element tag further comprising a plurality of soft metal atoms of a monoisotope of a soft metal;
wherein:
each chelating group of the elemental tag comprises or is capable of binding at least one of the soft metal atoms,
the soft metal atoms are non-radioactive and
the affinity reagent specifically binds the analyte,
(ii) Separating unbound elemental-labeled affinity reagent from bound elemental-labeled affinity reagent; and
(iii) Analyzing the elemental signature bound to the affinity reagent attached to the analyte by mass spectrometry atomic spectrometry.
27. The method of claim 26, wherein incubating the element-labeled affinity reagent with the analyte comprises:
Incubating two or more different elemental-labeled affinity reagents with two or more analytes, wherein the elemental-labeled affinity reagents specifically bind to the two or more analytes to produce two or more differently labeled analytes, wherein analyzing the elemental tags bound to the affinity reagents comprises analyzing the different elemental tags bound to the two or more analytes by mass spectrometry.
28. The method of claim 26 or 27, wherein the element-labeled affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells.
29. The method of any one of claims 26 to 28, wherein the soft metal is selected from Re, pt, pd, nb, tc, hg, ag, au, mo, ru, rh, cd, W, os or a mixture thereof.
30. The method of any one of claims 24 to 74, wherein the element tag is or comprises a compound of formula I as defined in any one of claims 1 to 15 or a compound of formula II as defined in claim 16.
CN202280052397.1A 2021-07-08 2022-07-08 Metal-containing polymers for mass flow cytometry Pending CN117813339A (en)

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US63/219,787 2021-07-08
US202263359182P 2022-07-07 2022-07-07
US63/359,182 2022-07-07
PCT/CA2022/051073 WO2023279211A1 (en) 2021-07-08 2022-07-08 Metal-containing polymers for mass cytometry

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