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WO2024039743A1 - Compositions et procédés de marquage sélectif d'amides primaires avec des composés iodovinyliques - Google Patents

Compositions et procédés de marquage sélectif d'amides primaires avec des composés iodovinyliques Download PDF

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WO2024039743A1
WO2024039743A1 PCT/US2023/030385 US2023030385W WO2024039743A1 WO 2024039743 A1 WO2024039743 A1 WO 2024039743A1 US 2023030385 W US2023030385 W US 2023030385W WO 2024039743 A1 WO2024039743 A1 WO 2024039743A1
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compound
certain embodiments
iodovinyl
amide
methods
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Monika Raj
Kuei TANG
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Emory University
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Emory University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid

Definitions

  • Amino acid sequencing of naturally occurring proteins and antibodies may be accomplished by reconstructing segments of degraded and labeled protein fragments including the utilization of unique amino acid markers and size exclusion chromatography. Mass spectrometry can be helpful to identify sequences of smaller segments, but the technique becomes inherently less reliable as the protein segments become larger. Thus, there is a need to identify improved methods for identifying and sequencing proteins using selective amino acid labels.
  • compositions and methods for selective labeling of compounds containing primary amides relate to compositions and methods for selective labeling of compounds containing primary amides.
  • the primary amide is present in peptides containing the amino acids asparagine and/or glutamine.
  • the primary amide is present on aliphatic or aromatic compounds or nucleic acids.
  • this disclosure relates to methods of forming N-vinyl amide labeled peptides or compounds, comprising contacting a compound containing a primary amide group with iodovinyl compounds disclosed herein.
  • this disclosure relates to methods of labeling a primary amide compound comprising contacting a primary amide compound with an iodovinyl compound, and a copper salt such that the primary amide compound reacts with the iodovinyl compound providing a labeled compound with a N-vinyl amide compound formed in place of the iodo group on the iodovinyl compound.
  • the copper salt is copper iodine.
  • the reaction further comprises a copper chelating ligand.
  • the copper chelating ligand is N,N'-dimethylethylenediamine.
  • the reaction further comprises a cesium salt. In certain embodiments, the cesium salt is cesium carbonate.
  • contacting a primary amide compound with an iodovinyl compound is in an aqueous pH buffered solution.
  • the aqueous pH buffered solution contains is a phosphate buffer.
  • the iodovinyl compound comprises a label.
  • the label is an aromatic molecule, a fluorescent dye, a ligand, biotin, a receptor, an antibody, or an antigen.
  • this disclosure relates to compounds, compositions, and materials comprising or coated with compounds disclosed herein.
  • Figure 1 illustrates a method of labeling a primary amide compound comprising contacting a primary amide compound with an iodovinyl compound and copper salt such that the primary amide compound reacts with the iodovinyl compound providing a labeled compound with a N- vinyl amide compound formed in place of the iodo group.
  • Benzene acts as a label as benzene is aromatic thus detectable using ultraviolet light.
  • the dipeptide asparagine-phenylalanine has a terminal amine on the N-terminal asparagine and a methyl ester on the C-terminal phenylalanine.
  • the amino acid side chain of asparagine contains a primary amide which is substituted with a vinyl group in place of the iodo group providing a N-vinyl amide.
  • Figure 2 illustrates a method wherein an iodovinyl compound is used to label a peptide with a primary amide providing a N-vinyl amide compound with an alkynyl group that allows for reaction with solid surface coated with an azide providing a heterocyclic linking group attached the solid surface due to an azide-alkyne cycloaddition reaction.
  • Embodiment indicates that it is an example and not necessarily limited to such example.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods.
  • protein refers to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a protein molecule.
  • An “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein.
  • terms such as “peptide” or “protein” are not meant to limit be limited to natural amino acids.
  • the term includes naturally and non-naturally derived material optionally having naturally or non- naturally occurring amino acids and modifications such as, substitutions, glycosylations, and addition of hydrophilic or lipophilic moieties.
  • the protein/peptide/polypeptide comprises more than three, four, five, six, seven, eight, nine, or ten amino acids.
  • nucleobase polymer refers to a polymer comprising nitrogen containing aromatic or heterocyclic bases that bind to naturally occurring nucleic acids through hydrogen bonding otherwise known as base pairing.
  • a typical nucleobase polymer is a nucleic acid, RNA, DNA, or chemically modified form thereof.
  • a nucleobase polymer may contain DNA or RNA or a combination of DNA or RNA nucleotides or may be single or double stranded or both, e g., they may contain overhangs, hairpins, bends, etc.
  • Nucleobase polymers may contain naturally occurring or synthetically modified bases and backbones.
  • conjugation refers to linking molecular entities through covalent bonds, or by other specific binding interactions, such as due to hydrogen bonding and other van der Walls forces.
  • the force to break a covalent bond is high, e.g., about 1500 pN for a carbon-to- carbon bond.
  • the force to break a combination of strong protein interactions is typically a magnitude less, e g., biotin to streptavidin is about 150 pN.
  • conjugation must be strong enough to bind molecular entities in order to implement the intended results.
  • a "linking group” refers to any variety of molecular arrangements that can be used to bridge or conjugate molecular moieties together.
  • linking groups include bridging alkyl groups, alkoxyalkyl, and aromatic groups.
  • specific binding agent refers to a molecule, such as a proteinaceous molecule, that binds a target molecule with a greater affinity than other random molecules or proteins.
  • specific binding agents include antibodies that bind an epitope of an antigen or a receptor which binds a ligand.
  • Specifically binds refers to the ability of a specific binding agent (such as a ligand, receptor, enzyme, antibody or binding region/fragment thereof) to recognize and bind a target molecule or polypeptide, such that its affinity (as determined by, e.g., affinity ELISA or other assays) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity of the same for any other or other random molecule or polypeptide.
  • a specific binding agent such as a ligand, receptor, enzyme, antibody or binding region/fragment thereof
  • ligand refers to any organic molecule, i.e., substantially comprised of carbon, hydrogen, and oxygen, that specifically binds to a “receptor.”
  • Receptors are organic molecules typically found on the surface of a cell. Through binding a ligand to a receptor, the cell has a signal of the extra cellular environment which may cause changes inside the cell.
  • a ligand is usually used to refer to the smaller of the binding partners from a size standpoint, and a receptor is usually used to refer to a molecule that spatially surrounds the ligand or portion thereof.
  • the terms can be used interchangeably as they generally refer to molecules that are specific binding partners.
  • a glycan may be expressed on a cell surface glycoprotein and a lectin protein may bind the glycan.
  • a lectin protein may bind the glycan.
  • the glycan is typically smaller and surrounded by the lectin protein during binding, it may be considered a ligand even though it is a receptor of the lectin binding signal on the cell surface.
  • An antibody may be a receptor, and the epitope may be considered the ligand.
  • a ligand is contemplated to be a compound that has a molecular weight of less than 500 or 1,000.
  • a receptor is contemplated to be a protein-based compound that has a molecular weight of greater than 1,000, 2,000 or 5,000. In any of the embodiments disclosed herein, the position of a ligand and a receptor may be switched.
  • label refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • a peptide "label” refers to incorporation of a heterologous polypeptide in the peptide, wherein the heterologous sequence can be identified by a specific binding agent, antibody, or bind to a metal such as nickel/ nitrilotriacetic acid, e.g., a poly-histidine sequence.
  • Specific binding agents and metals can be conjugated to solid surfaces to facilitate purification methods.
  • a label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • marked avidin for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35 S or 131 I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. Tn some embodiments, labels may be attached by spacer arms of various lengths to reduce potential steric hindrance.
  • fluorescent labels such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphor
  • an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule and an antigen to the antibody.
  • the immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for elimination or degradation. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody.
  • antibody is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, and binding fragments, such as single chain binding fragments. These antibodies may have chemical modifications.
  • monoclonal antibodies refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen.
  • the term “monoclonal” is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.
  • Hydrophilic polymers contain polar or charged functional groups, rendering them soluble in water.
  • Examples include polyethylene glycol, polylactides, polyglycolide, poly(e- caprolactone), poly(2-methoxyethyl acrylate), poly(tetrahydrofurfuryl acrylate), poly(2- methacryloyloxyethyl phosphorylcholine), poly(p-dioxanone), poly(serine methacrylate), poly[oligo(ethylene glycol) vinyl ether], poly ⁇ [2-(methacryloyloxy)ethyl], copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(alpha-hydroxy acid), and poly(vinyl alcohol).
  • PEG polyethylene glycol
  • the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue.
  • the derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur atom, replacing an amino group with a hydroxyl group, replacing a nitrogen with a protonated carbon (CH) in an aromatic ring, replacing a bridging amino group (-NH-) with an oxy group (-O-), or vice versa.
  • the derivative may be a prodrug.
  • a derivative may be a polypeptide variant.
  • Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.
  • R a and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • the carboxylic acid group on the C-terminus of a peptide may be converted by synthetic means to contain a primary amide referred to as a "C-terminal" amide.
  • a “bridging amide” refers to an amide that is not a primary amide, i.e., because the amide contains a substitution of one of the hydrogens connected to the amide nitrogen. Such is the case when amides link amino acids together in a peptide chain.
  • alkyl means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms.
  • Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl” or “alkynyl", respectively).
  • Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2- butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.
  • Haloalkyl refers to an alkyl group wherein one or more or all of the hydrogens are substituted with a halogen(s), e g., -CH2CH2CI or -CF3.
  • Alkylthio refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a sulfur bridge.
  • An example of an alkylthio is methylthio, (e.g., -S-CH3).
  • Alkoxy refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge.
  • alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy.
  • Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t- butoxy.
  • Alkylamino refers an alkyl group as defined above with the indicated number of carbon atoms attached through an amino bridge.
  • An example of an alkylamino is methylamino, (e.g., - NH-CH3).
  • Alkylthio refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a sulfur bridge.
  • An example of an alkylthio is methylthio, (e g., -S-CH3).
  • Alkoxy refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t- butoxy.
  • Alkylamino refers an alkyl group as defined above with the indicated number of carbon atoms attached through an amino bridge.
  • An example of an alkylamino is methylamino, (e.g., - NH-CHs).
  • Aryl means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl.
  • Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.
  • Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or “carbocyclyl” groups.
  • Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like.
  • heterocycle or “heterocyclyl” refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom.
  • the mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings.
  • Heterocycle includes heterocarbocycles, heteroaryls, and the like.
  • heteroaryl or “heteroaromatic” refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems.
  • Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.
  • heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl” includes N-alkylated derivatives such as a 1-methylimidazol- 5-yl substituent.
  • this disclosure relates to methods of forming N-vinyl amide labeled compounds, comprising contacting a compound containing a primary amide group with iodovinyl compounds.
  • this disclosure relates to methods of labeling a primary amide compound comprising contacting a primary amide compound with an iodovinyl compound, and copper salt such that the primary amide compound reacts with the iodovinyl compound providing a labeled compound with a N-vinyl amide compound formed in place of the iodo group.
  • the copper salt is a copper iodine.
  • the reaction further comprises a copper chelating ligand.
  • the copper chelating ligand is N,N'-dimethylethylenediamine.
  • the copper chelating ligand is a bidentate ligand of nitrogen, oxygen (carboxylate), or a combination thereof.
  • reaction further comprises a cesium salt.
  • cesium salt is cesium carbonate, cesium chloride, or cesium sulphate.
  • contacting a primary amide compound with an iodovinyl compound is in an aqueous pH buffered solution.
  • the aqueous pH buffered solution contains is a phosphate buffer, sulfate buffer, or citrate buffer.
  • reactions are in an aqueous solution with a pH of between 6.5 to 8.5. In certain embodiments, for any of the methods disclosed herein, reactions are in an aqueous solution with a pH of between 7.0 to 8.5. In certain embodiments, for any of the methods disclosed herein reactions are in an aqueous solution with a pH of between 7.0 to 8.0.
  • the primary amide compound is a peptide comprising an asparagine or glutamine or C-terminal amide of a peptide and methods provide a labeled N-vinyl (amide) asparagine, labeled N-vinyl (amide) glutamine, and/or a labeled N-vinyl C-terminal peptide.
  • the iodovinyl compound comprises a label.
  • the label is an aromatic molecule, a fluorescent dye, a ligand, biotin, a receptor, an antibody, or an antigen.
  • this disclosure relates to compounds, compositions, and materials comprising or coated with compounds disclosed herein.
  • this disclosure relates to methods and compositions for labeling, isolating, detecting, measuring, and purifying compounds containing primary amides made by processes disclosed herein.
  • the primary amide is asparagine, glutamine amino acid, or a C-terminal amide of a peptide.
  • this disclosure relates to methods of labeling, isolating, detecting, measuring, purifying, quantifying, and amino acid sequencing compounds/peptides comprising an asparagine, glutamine amino acid, or a C-terminal amide of a peptide containing the same from a sample optionally utilizing solid supports.
  • the sample is from a human subject.
  • the sample is a blood sample, urine sample, stool sample, saliva sample, or sputum sample.
  • the peptide or compound for labeling having a primary amide is asparagine, glutamine, synthetic amino acid, C-terminal amide of a peptide, a nucleobase, nucleic acid, DNA, RNA, nucleobase polymer, alkyl, aryl, carbocyclyl, or heteroaryl optionally substituted with one or more, the same or different substituents such as a halogen, hydroxy, amino, thiol, alkyl, alkoxy, alkylamino, alkylthio, dialkylamino, acetamido, formyl, alkanoyl, carboxyl, carbonyl ester, carbamoyl, aryl, carbocyclyl or heterocyclyl, or N-substituted carbamoyl group, which is optionally further substituted or conjugated to a label or a solid support through a linking group.
  • substituents such as a halogen, hydroxy, amino, thiol,
  • the methods further comprise determining the molecular weight or exact mass of the peptide or N-vinyl amide compound.
  • reactions are at about room temperature, e.g., between 10 and 30 degrees Celsius. In certain embodiments, for any of the methods disclosed herein reactions are at between about 5 and 40 degrees Celsius. In certain embodiments, for any of the methods disclosed herein reactions are at between about 35 and 55 degrees Celsius.
  • this disclosure relates to methods of contacting a compound or protein containing a primary amide, asparagine, or glutamine wherein the compound or protein containing a primary amide, asparagine or glutamine is in a sample such as a biological sample, (e.g., cell, tissue, etc.) or environmental sample.
  • Biological samples may be obtained from animals (including humans) and encompass fluids, blood, solids, tissues, and gases.
  • Environmental samples include environmental material such as surface matter, soil, water, and industrial samples.
  • the conditions for providing a N-vinyl amide labeled peptide or compound does not result in the labeling of bridging amides and/or terminal amines, including primary, secondary, nor tertiary amines, in the peptide compound or sample. In certain embodiments, for any of the methods disclosed herein, the conditions for providing a N-vinyl amide labeled peptide or compound does not result in the labeling of bridging amides in the peptide compound or sample.
  • the compound having a N-vinyl amide labeled peptide or compound is a benzene para substituted with an alkynyl group.
  • the compound having a N-vinyl amide labeled peptide or compound is further substituted with a label.
  • the label is biotin, aromatic molecule, a fluorescent dye, a second alkynyl group, ligand, receptor, antibody, or antigen.
  • the methods further comprise contacting the alkynyl labeled compound with a solid surface conjugated to a triazene under conditions such that a triazole compound is conjugated to the solid surface.
  • methods further comprise contacting the triazole compound conjugated to the solid surface with an acid solution such that the purified compound is cleaved from the solid surface providing purified compound.
  • the label is a ligand providing ligand labeled compound or peptide.
  • the method further comprises contacting the ligand labeled or peptide with a solid surface conjugated to a receptor under conditions such that the ligand labeled compound or peptide is conjugated to the solid surface.
  • the label is biotin providing biotin labeled compound or peptide.
  • the method further comprises contacting the biotin labeled compound or peptide with a solid surface conjugated to avidin or streptavidin under conditions such that the biotin labeled compound or peptide is conjugated to the solid surface.
  • this disclosure relates to methods of forming N-vinyl amide labeled compounds comprising contacting a compound or peptide containing a primary amide with a compound comprising an aromatic group with iodovinyl group substituted with an alkynyl group under conditions such that a N-vinyl amide labeled compound or peptide is formed at the amide group.
  • the aromatic group is benzene substitute with an iodovinyl group.
  • the N-vinyl amide labeled compound is an aliphatic amide, aromatic amide, phenyl amide, benzyl amide, asparagine, glutamine, peptide, other compound containing the same.
  • the iodovinyl compound comprising an aromatic group has the following formula, or derivatives thereof, wherein R is optionally substituted to a label or conjugated to a solid support through a linking group.
  • the linking group is a hydrophilic polymer.
  • R is alkyl, aryl, carbocyclyl, or heterocyclyl, wherein R is optionally substituted with one or more substituents, or conjugated to a label or a solid support through a linking group.
  • R 1 is hydrogen, alkyl, halogen, haloalkyl, alkoxy, alkylthio, dialkylamino, acetamido, nitrile, nitro, formyl, carboxyl, carbonyl ester, carbamoyl, or N-substituted carbamoyl group, wherein R 1 is optionally substituted.
  • R 2 is hydrogen, alkyl, halogen, haloalkyl, alkoxy, alkylthio, dialkylamino, acetamido, nitrile, nitro, formyl, carboxyl, carbonyl ester, carbamoyl, or N-substituted carbamoyl group, wherein R 2 is optionally substituted.
  • R, R 1 or R 2 are individually and independently at each occurrence hydrogen, alkyl, halogen, haloalkyl, alkoxy, alkylthio, dialkylamino, acetamido, nitrile, nitro, formyl, carboxyl, carbonyl ester, carbamoyl, or N-substituted carbamoyl group, wherein R, R 1 or R 2 are optionally substituted e.g., conjugated to a label or a solid support through a linking group, or R 1 or R, or R 1 or R 2 , and the attached atoms together form an aromatic or nonaromatic ring optionally substituted e.g., conjugated to a label or a solid support through a linking group.
  • this disclosure relates to methods of purifying a compound comprising a primary amide in a sample comprising contacting the sample with a compound with an iodovinyl group substituted with an alkynyl group (e.g., 2-ethynylaniline) with a copper salt, providing a N-vinyl amide labeled compound at the iodovinyl group.
  • a compound with an iodovinyl group substituted with an alkynyl group e.g., 2-ethynylaniline
  • the N-vinyl amide compound substituted with an alkynyl group is formed by the process of contacting an iodovinyl compound a copper salt, a cesium salt, and a phosphate salt at pH of between 6.5 to 8.5.
  • this disclosure relates to methods of purifying a compound comprising a primary amide in a sample comprising contacting the sample with an iodovinyl compound, a copper salt, cesium salt, and a phosphate salt at pH of about 7 providing a compound with a N-vinyl bridging amide.
  • the N-vinyl amide (aromatic or phenyl) compound comprises a meta or para substituted group comprising an alkynyl group (e.g., N-(prop-2-yn-l-yl)benzamide) and the method further comprises contacting the N-vinyl amide labeled compound comprising an alkynyl group with a solid support conjugated to a triazene group under conditions such that the meta or para alkynyl group reacts with the triazene group on the solid support to form a triazole linkage providing a solid support conjugated to the N-vinyl labeled compound; washing the solid support to remove unreacted material from the sample providing a purified solid support conjugated to the N-vinyl labeled compound; and contacting the purified solid support conjugated to the N-vinyl amide labeled compound to conditions providing a composition with a purified compound comprising a N-vinyl amide released from the solid support.
  • an alkynyl group
  • the conditions are acid conditions, e.g., exposure to trifluoroacetic acid.
  • the methods further comprise determining the molecular weight or exact mass of the N-vinyl amide labeled compound or peptide comprising an asparagine or glutamine amino acid.
  • the methods further comprise separating the composition with a purified N-vinyl amide into two or more compounds with a N-vinyl amide. In certain embodiments, separating is by chromatography.
  • the methods further comprise separating a purified, cleaved, or isolated peptide composition or N-vinyl amide compound composition into two or more peptides or labels compounds.
  • separating is by chromatography, e.g., adsorption and/or size-based separations by chromatography or filtration through a gel.
  • methods comprise obtaining a sample from a subject comprising a protein, contacting the sample comprising a protein in a sample with a protease providing a cleaved protein, contacting the cleaved protein with a labeled iodovinyl compound providing a N- vinyl amide labeled cleaved protein in the sample, and purifying the N-vinyl amide labeled cleaved protein, e.g., by chromatography.
  • methods comprise obtaining a sample comprising a protein from a subject, contacting the sample comprising a protein in a sample with a labeled iodovinyl compound providing a N-vinyl amide labeled protein in the sample, contacting N-vinyl amide labeled protein with a protease providing a cleaved N-vinyl amide labeled protein, and purifying the cleaved N-vinyl amide labeled protein by chromatography.
  • this disclosure relates to methods of purifying a compound with a primary amide in a sample comprising, contacting the sample with a solid support conjugated to a compound comprising an iodovinyl such that the compounds with a primary amide in the sample reacts with the iodovinyl providing a N-vinyl amide compound immobilized to the solid support; washing the solid support to remove unreacted material from the sample providing a purified N- vinyl compound immobilized to the sold support.
  • the method further comprises exposing the purified compound immobilized to the solid support to conditions releasing a purified N-vinyl amide compound.
  • this disclosure relates to a labeled compound, peptide, or solid support such as a particle conjugated with a compound having the following formula: or derivative or salt thereof wherein,
  • W is a solid support such as a particle or a label
  • X is a peptide
  • Y is O, NH, or S
  • Z is a linking group
  • Ris alkyl, aryl, carbocyclyl, or heterocyclyl optionally substituted with one or more substituents, or conjugated to a label or a solid support through a linking group;
  • R 1 is hydrogen, alkyl, halogen, haloalkyl, alkoxy, alkylthio, dialkylamino, acetamido, nitrile, nitro, formyl, carboxyl, carbonyl ester, carbamoyl, or N-substituted carbamoyl group, wherein R 1 is optionally substituted with one or more substituents, or conjugated to a label or a solid support through a linking group;
  • R 2 is individually and independently at each occurrence hydrogen, alkyl, halogen, haloalkyl, alkoxy, alkylthio, dialkylamino, acetamido, nitrile, nitro, formyl, carboxyl, carbonyl ester, carbamoyl, or N-substituted carbamoyl group, wherein R 2 is optionally substituted with one or more substituents, -Z-W, or -Z-Y-W, or two R 2 s and the attached atoms come together to form an aromatic or non-aromatic ring that is optionally substituted with one or more substituents or -Z-W, or -Z-Y-W.
  • this disclosure relates to a compound or material comprising or coated with or conjugate to chemical arrangements disclosed herein.
  • the material, solid support, or particle further comprises or is coated with or conjugated to a hydrophilic polymer.
  • the solid support is a magnetic material, e.g., magnetic bead
  • methods of purifying entail capturing the magnetic bead with a magnetic field thereby separating the magnetic bead and contents thereof from a solution or mixture.
  • iodovinyl compounds can be accomplished by halide-exchange reactions of vinyl bromides with potassium iodide in the presence of a copper catalyst. See, e.g., Feng et al. Synthesis, 2017, 49, 2727-2732.
  • CeHi4 alkyl group could be any R group (aliphatic or aromatic, alkyne and others).

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  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des compositions et des procédés de marquage sélectif de composés contenant des amides primaires. Dans certains modes de réalisation, l'amide primaire est présent dans des peptides contenant les acides aminés asparagine et/ou glutamine. Dans certains modes de réalisation, l'amide primaire est présent sur des composés aliphatiques ou aromatiques ou des acides nucléiques. Dans certains modes de réalisation, la présente divulgation concerne des procédés de formation de peptides ou de composés marqués par N-vinylamide, comprenant la mise en contact d'un composé contenant un groupe amide primaire avec des composés iodovinyliques divulgués dans les présentes.
PCT/US2023/030385 2022-08-17 2023-08-16 Compositions et procédés de marquage sélectif d'amides primaires avec des composés iodovinyliques Ceased WO2024039743A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXIS COSTE, FRANÇOIS COUTY, GWILHERM EVANO: "Synthesis of Ynamides by Copper-Mediated Coupling of 1,1-Dibromo-1- alkenes with Nitrogen Nucleophiles. Preparation of 4-Methyl-N-(2-phenylethynyl)-N- (phenylmethyl)benzenesulfonamide", XP093142544, [retrieved on 20240318] *
BAQI YOUNIS: "Recent Advances in Microwave-Assisted Copper-Catalyzed Cross-Coupling Reactions", CATALYSTS, M D P I AG, CH, vol. 11, no. 1, CH , pages 46, XP093142538, ISSN: 2073-4344, DOI: 10.3390/catal11010046 *
GILLE FRANZISKA, KIRSCHNING ANDREAS: "Studies on the synthesis of peptides containing dehydrovaline and dehydroisoleucine based on copper-mediated enamide formation", BEILSTEIN JOURNAL OF ORGANIC CHEMISTRY, BIOMED CENTRAL, LONDON, GB, vol. 12, 22 March 2016 (2016-03-22), GB , pages 564 - 570, XP093142540, ISSN: 1860-5397, DOI: 10.3762/bjoc.12.55 *
RICARD SIMON, GAGNON ALEXANDRE, DAOUST BENOIT: "Copper‐Catalyzed β‐Iodovinylation of Carbamates: Expedient Access to Highly Functionalized Vinyl‐Carbamates", CHEMISTRYSELECT, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 3, no. 17, 8 May 2018 (2018-05-08), DE , pages 4923 - 4929, XP093142541, ISSN: 2365-6549, DOI: 10.1002/slct.201800824 *
WANG LEIMING, LEI XINSHENG, WANG QUANRUI, LI YINGXIA: "Copper-catalyzed cross-coupling of amino acid-derived amides with (Z)-vinyl iodides: Unexpected solvent effect and preparation of plocabulin", TETRAHEDRON, ELSEVIER SIENCE PUBLISHERS, AMSTERDAM, NL, vol. 83, 1 March 2021 (2021-03-01), AMSTERDAM, NL , pages 131953, XP093142537, ISSN: 0040-4020, DOI: 10.1016/j.tet.2021.131953 *

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