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WO2016149899A1 - Réactif, activateur et précurseur orthogonaux biologiques, et kit de réactif orthogonal biologique - Google Patents

Réactif, activateur et précurseur orthogonaux biologiques, et kit de réactif orthogonal biologique Download PDF

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Publication number
WO2016149899A1
WO2016149899A1 PCT/CN2015/074897 CN2015074897W WO2016149899A1 WO 2016149899 A1 WO2016149899 A1 WO 2016149899A1 CN 2015074897 W CN2015074897 W CN 2015074897W WO 2016149899 A1 WO2016149899 A1 WO 2016149899A1
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bioorthogonal
group
reagent
precursor
activator
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Chinese (zh)
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洪梅
赵劲
段平平
陈颖
陈波
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Peking University Shenzhen Graduate School
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Peking University Shenzhen Graduate School
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Priority to PCT/CN2015/074897 priority Critical patent/WO2016149899A1/fr
Priority to CN201580002757.7A priority patent/CN106170304A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates

Definitions

  • the invention belongs to the technical field of bioorthogonal reactions, in particular to bioorthogonal reagents, bioorthogonal activators, bioorthogonal precursors and bioorthogonal kits.
  • Macromolecular fluorescent probes such as the Green Fluorescent Protein encoded by the Green Fluorescent Protein and its variants of Fluorescent Proteins, have been widely used to label and track in vivo proteins with great success and won the promise in 2008. Bell Chemistry Prize.
  • gene-encoding probes are not suitable for monitoring non-protein biomolecules, including polysaccharides, lipids, nucleic acids, etc., and have large steric hindrance, and have greater flexibility for labeling sites and fluorescence detection. limit.
  • organic small molecule optical probes have advantages in flexibility and spatial size, are suitable for any sample including the human body, and are relatively inexpensive and easy to handle, providing a high signal-to-noise ratio through chemical design and regulation. And target applicability.
  • fluorescent small molecule probe can realize real-time visual tracer of active small molecules in cells and even living organisms. It has non-destructive, real-time, high sensitivity, easy operation, low background and signal in researching signal transduction and cell physiology and pathology. Can adjust and other advantages. Fluorescent small molecule probes are the most widely used detection tools in life systems. They have been widely used in the analysis and detection of gas molecules such as nitric oxide, carbon monoxide and hydrogen sulfide in living systems, as well as the detection of metal ions in the body. Organic small molecule fluorescent probes are also increasingly valued for the labeling and coloration of biomolecules such as proteins, free radicals, peptides, and enzymes. The use of bioorthogonal chemical reactions to specifically label in vivo biomacromolecules as an emerging chemical biology method is gaining more and more attention and interest.
  • Bioorthogonal reactions refer to a class of chemical reactions that can be carried out in living cells or tissues without interfering with the biochemical reaction of the organism. It introduces a first bioorthogonal group of a specific tag (Tagging) in a biomolecule, such as a target protein, by an activator, which is also called a chemical reporter (Chemical Reporter), and is complementary to a second organism containing or modifying A cross-linking agent, also known as a triggering group (Trigger), performs a bioorthogonal reaction to achieve specific labeling of the target biomolecule.
  • Tagging a specific tag
  • Trigger a triggering group
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a bioorthogonal precursor and a bioorthogonal reagent or bioorthogonal activator based on the bioorthogonal precursor to overcome the present
  • Another object of the present invention is to provide a bioorthogonal kit containing the bioorthogonal reagent or bioorthogonal precursor of the present invention to improve the efficiency of existing bioorthogonal reactions.
  • a bioorthogonal precursor comprising a multifunctional functional group at least as a localization group of a coupling reaction and a bioorthogonal group, wherein the multifunctional functional group is a hydroxylamine group.
  • bioorthogonal precursor molecular structure of the present invention has the following structural formula (A):
  • R 1 and R 2 are the same or different and are H, a linear or branched alkyl group, a linear or branched acyl group or an alkoxycarbonyl group, and R 3 represents Any of H, an alkyl group, an alkenyl group, an aryl group and a heteroaryl group, and the structure represented by the arc is a chain structure or a cyclic structure.
  • P 1 is an optical probe molecule fragment or a structural group.
  • bioorthogonal activator which is a molecular structural formula (A) or a derivative thereof according to the present invention, wherein the bioorthogonal activator is capable of activating a biomolecule by a dehydrogenation coupling reaction under biological conditions.
  • the molecular structure of the activated molecule is as follows (C):
  • P 2 is represented as a biomolecule fragment or a member group.
  • a bioorthogonal kit for biomarkers comprising a bioorthogonal reagent comprising a second bioorthogonal group and a bioorthogonal reaction with the bioorthogonal reagent and capable of direct or indirect Generating an activator of a first bioorthogonal group, such as the bioorthogonal reagent of the present invention as described above; or the activator is a bioorthogonal precursor of the invention as described above or an organism of the invention as described above Orthogonal activator.
  • the above-described bioorthogonal precursor of the present invention uses a hydroxylamine group as a functional group having a chelate catalyst to activate a substrate action and a bioorthogonal property, and thus, the bioorthogonal precursor of the present invention activates carbon-hydrogen /
  • the coupling group of the coupling reaction and the bioorthogonal group are combined into one, and a bio-orthogonal "trigger" group can be selected on the small molecule optical probe, or a specific organism can be selectively introduced into the target biomolecule. Submit the "Chemical Report" group.
  • the above-described bioorthogonal precursor structural formula of the present invention is designed as described above for the structural formula (A), that is, the ortho position of the hydroxylamine group-bonded carbon is a carbon-carbon double bond, which is effective in combination with biomolecules.
  • Alkenyl carbon undergoes atomic economic dehydrogenation coupling, and the resulting modified biomolecule is labeled with a diene structure; or can be atomically and economically dehydrogenically coupled with an alkenyl carbon in a small molecule optical probe to form a double A bioorthogonal reagent for an alkenyl group.
  • bioorthogonal reagent and bioorthogonal activator of the present invention have a strong substrate suitability, mildness and biocompatible reaction due to the bioorthogonal precursor group of the present invention.
  • Conditions can be used as tools to modify exogenous fluorescent probes, or to modify bulk biomolecules, to easily trigger biological "click" reactions to achieve bioorthogonal labeling.
  • bio-orthogonal kit for biomarker can realize bioorthogonal labeling quickly and efficiently because it contains the bioorthogonal reagent or bioorthogonal precursor of the present invention as described above.
  • route a represents a chemical reporter group after bio-orthogonal precursor carbon-hydrogen activation in the embodiment of the present invention
  • route b represents a route diagram for providing a trigger group and a bioorthogonal reaction coupling after carbon-hydrogen activation of the bioorthogonal precursor of the present invention
  • Figure 2 is a graph showing the relationship between fluorescence intensity and time for surface labeling of HeLa cells using the Bio-orthogonal kit of Example 28 of the present invention.
  • Bioorthogonal reaction refers to a type of chemical reaction that can be carried out in living cells or tissues without interfering with the biochemical reaction of the organism. It introduces a first bioorthogonal group of a specific tag (Tagging) in a biomolecule, such as a target protein, by an activator, which is also called a chemical reporter (Chemical Reporter), and is complementary to a second organism containing or modifying A cross-linking agent, also known as a triggering group (Trigger), performs a bioorthogonal reaction to achieve specific labeling of the target biomolecule.
  • Tagging a specific tag
  • Trigger a triggering group
  • Positioning group means a specific "inert" carbon-hydrogen bond used to activate the substrate, with a sufficiently high concentration of transition metal catalyst around it to achieve the desired reaction kinetics, and to coordinate the transition metal with a specific carbon-hydrogen The bonds are linked to achieve a precise regioselective group in multiple carbon-hydrogen bonds.
  • Bioorthogonal group Represents an interactive group that can clicks on each other in living systems and other complex biological environments.
  • the bioorthogonal groups are orthogonal, characterized in that the reactive components are compatible with each other and do not undergo cross-reaction, selectively bioorthogonal coupling without interfering with natural biomolecules or biological processes. Ideally, their mutually orthogonal reactions can be used in tandem dual biomolecule labeling.
  • the first bioorthogonal group in the interactive bioorthogonal group is mounted on the biomolecule as a chemical reporter group
  • the second bioorthogonal group is mounted on the probe molecule as a trigger base. group.
  • Carbon-hydrogen activation refers to a carbon-hydrogen bond cleavage process coordinated by an organometallic compound, wherein the bond cleavage is accomplished by coordination of carbon-hydrogen bonds to the transition metal inner electron cloud without the pre-functionalization step of the substrate. .
  • the Directing Group is very important. It acts as a group on the substrate to coordinate the transition metal to a specific carbon-hydrogen bond, thereby achieving multiple carbon-hydrogen The precise regioselectivity of the bond; and due to its coordination, a sufficiently high concentration of transition metal catalyst around the activated "inert" carbon-hydrogen bond to achieve the desired reaction kinetics.
  • the carbon-hydrogen bond activated substrate can be cross-coupled with another coupled substrate.
  • embodiments of the invention are based on the recognition that when the localization group of the carbon-hydrogen activation/coupling reaction is retained after the coupling reaction and acts as a bioorthogonal group, the carbon-hydrogen activation /Coupling reaction Strong substrate suitability, mild and biocompatible reaction conditions can be used as a tool to modify exogenous small molecule optical probes, or to modify bulk biomolecules, to easily trigger biological "click" reactions to achieve bioorthogonality mark.
  • embodiments of the present invention provide a bioorthogonal precursor comprising a multifunctional functional group capable of at least a positioning group and a bioorthogonal group as a coupling reaction, wherein the multifunctional functional group
  • the group is a hydroxylamine group.
  • the above-described bioorthogonal precursor of the present invention uses a hydroxylamine group as a functional group, which has an active substrate action and a bioorthogonal property. Therefore, the bioorthogonal precursor of the present invention activates carbon-hydrogen.
  • the coupling group of the coupling reaction and the bioorthogonal group are combined into one, and a bio-orthogonal "trigger" group can be selected on the small molecule optical probe, or a specific organism can be selectively introduced into the target biomolecule.
  • a bio-orthogonal "trigger” group can be selected on the small molecule optical probe, or a specific organism can be selectively introduced into the target biomolecule.
  • Submit the "Chemical Report” group Submit the "Chemical Report” group. Among them, the route in which the carbon-hydrogen activation and the bioorthogonal reaction are combined is shown in FIG.
  • the above bioorthogonal precursor is a general-purpose reagent for providing bioorthogonal groups.
  • the universal reagent can modify the biomolecule to provide a first bioorthogonal chemical reporter group, or the modified probe molecule to provide a second bioorthogonal triggering group, ie, when the bioorthogonal precursor of the embodiment of the invention is an activator, Can be introduced into the target biomolecule as a first bioorthogonal group, ie, a "chemical reporter group”; when the bioorthogonal precursor of the embodiment of the invention is a bioorthogonal reagent precursor, it can be introduced into small molecule optics As a second bioorthogonal group in the probe, ie, a "trigger group", It meets the needs of the chemical biology community to expand bio-orthogonal kits and orthogonal methods, and provides new tools for modifying biomolecules and studying molecular networks and functions in biological systems.
  • bioorthogonal precursor molecular structure of the embodiment of the present invention has the following general formula (A):
  • R 1 and R 2 are the same or different and are H, a linear or branched alkyl group, a linear or branched acyl group or an alkoxycarbonyl group
  • R 3 represents Any of H, an alkyl group, an alkenyl group, an aryl group and a heteroaryl group, and the structure represented by the arc is a chain structure or a cyclic structure. Therefore, when the ortho position of the hydroxylamine group-bonded carbon in the bioorthogonal precursor represented by the formula A is a carbon-carbon double bond, it can be efficiently atomically and economically dehydrogenated with the alkenyl carbon in the target biomolecule.
  • the generated modified target biomolecule is labeled with a diene structure, or can be efficiently dehydrogenically coupled with the alkenyl carbon in the small molecule optical probe to form a bioorthogonal reagent containing a dienyl group.
  • At least one of R 1 and R 2 in formula A is not represented by H, in which case the hydroxylamine group in the bioorthogonal precursor of the above-described embodiments of the invention is R 1 and/or R 2 protection.
  • the bioorthogonal precursor to which the hydroxylamine group is protected is preferred in the practice of the invention, and the reaction conditions are controlled such that it is completely preserved as a bioorthogonal group in the above carbon-hydrogen activation/coupling reaction.
  • R 1 , R 2 in the bioorthogonal precursor represented by Formula A represents a linear or branched acyl group
  • the acyl group is selected from the group consisting of an acetyl group, a benzoyl group, and a benzyl group.
  • R 3 in the bioorthogonal precursor represented by Formula A has a positive influence on the optical property regulation, the adjustment of the solubility property, and the conformational adjustment of the modified biomolecule of the bioorthogonal reagent constructed by the bioorthogonal precursor of the present invention.
  • the alkenyl group is selected from the group consisting of ethenyl, propenyl, isopentenyl, octenyl, and twelfth Any of an alkenyl group and a cyclopentenyl group; or
  • R 3 represents an aryl group
  • the aryl group is selected from a benzene ring and a derivative thereof;
  • R 3 represents a heteroaryl group
  • the heteroaryl group is selected from any one of a pyrrolyl group, a furyl group, an imidazolyl group, a pyridyl group, a thiazolyl group, a pyrimidinyl group, a thienyl group, and a pyrazolyl group.
  • the chain structure represented by the arc in the above molecular structure formula (A) is selected from a chain structure containing a double bond.
  • the cyclic structure shown by the arc is selected from the group consisting of cyclopropene, cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetrahydropyridine, dihydrofuran, and dithiazine.
  • cyclopropene cyclobutene
  • cyclopentene cyclohexene
  • cyclooctene tetrahydropyridine
  • dihydrofuran and dithiazine.
  • hydroxylamine functional group in the bioorthogonal precursor of the embodiment of the invention described above is coated with a hydrazide, an ester group or It is a group replacement with a positioning effect, which is also within the scope of the embodiments of the present invention.
  • the embodiment of the invention further provides a bioorthogonal reagent.
  • the bioorthogonal reagent is formed by reacting an optical probe molecule or group with the bioorthogonal precursor described above by carbon-hydrogen activation/coupling, that is, the bioorthogonal reagent of the embodiment of the present invention contains In the text of the invention, the bioorthogonal precursor group is used.
  • the bioorthogonal reagent of the embodiment of the invention can modify the exogenous small molecule optical probe, that is, provide the second bioorthogonal group (trigger group), and simultaneously regulate Properties of small molecule optical probes such as sensitivity, selectivity, detection limits, optical properties, and biocompatibility.
  • bioorthogonal reagent of the present invention utilizes the bioorthogonal precursors of the embodiments of the invention described above to multi-conjugate the existing probes, incorporate polycyclic and heterocyclic systems, and increase the length of the conjugated chain.
  • the bioorthogonal reagents of the embodiments of the present invention are effective in meeting the needs for imaging in vivo in animals and humans.
  • the bioorthogonal reagent of the present invention is formed by the dehydrogenation coupling reaction of the bioorthogonal precursor of the embodiment of the present invention represented by the above formula (A).
  • the structural formula is as follows (B):
  • P 1 is an optical probe molecule fragment or a structural group. among them,
  • P 1 in the formula B is coupled to the biological positive by a coupling reaction of an unsaturated bond with a bioorthogonal precursor represented by the above formula (A) And a ketone carbonyl group, wherein the unsaturated bond is a part of the optical probe molecule fragment or a structural group.
  • P 1 is attached to the organism by a double bond such as a carbon-carbon double bond in a coupling reaction with a bioorthogonal precursor of the molecular structure formula (A) described above.
  • a bioorthogonal precursor of the molecular structure formula (A) described above in the case of orthogonal precursors, has the following general formula (B 1 ):
  • P 1 ' is an optical probe molecule fragment or a structural group.
  • the formula B 1 hydroxylamine group attached ortho to the carbon-containing diene group, and therefore, the diene group may be used as a second bio-orthogonal group (group trigger). That is, the biological reagent orthogonal molecular structure shown in Formula 1 B except hydroxylamine groups as the second group of bio-orthogonal, may also be provided as a second group diene bio-orthogonal group.
  • the bioorthogonal reagent represented by the above formula B has a molecular structure of the following formula (B 2 ), and the bioorthogonal reagent represented by the above formula B 1 has a molecular structural formula of The following (B 3 ):
  • optical probe molecule fragments or structural groups represented by P 1 , P 1 ' in the molecular structure of each of the above bioorthogonal reagents may be the same or different one or more optical probe molecule fragments or structural groups,
  • the optical probe molecule fragment of P 1 , P 1 ' is a small molecule fluorescent probe fragment or a colorimetric probe fragment.
  • the optical probe molecule fragment of P 1 , P 1 ' may be pyrene, quinine sulfate, boron fluoride-boron-dipyrromethene , BODIPY), fluorescein, rhodamine, or any of rhodamine.
  • the structural group described by P 1 , P 1 ' may be any of an alkene, an alkyne, a diene, a thioketone, a sulfinyl group, a nitrosobenzene, an azide, and the like.
  • the above bioorthogonal reagent can be prepared by reacting according to the route b in FIG. 1, and the bioorthogonal precursor and the optical probe molecule of the embodiment of the invention described above are subjected to carbon-hydrogen catalysis under the catalysis of a metal catalyst.
  • the reaction temperature is preferably between 0 and 100 °C.
  • the metal catalyst may be selected from a palladium catalyst, a rhodium catalyst, a rhodium catalyst, a copper catalyst, a nickel catalyst, etc., preferably a palladium catalyst, more preferably an organically coordinated palladium catalyst, such as bis(2-amino).
  • bioorthogonal reagent shown above because the molecular structure of formula B-containing bis alkenyl group, therefore, a dehydrogenation coupling reaction in which the two carbon alkenyl group, a small molecule generated optical probe bioorthogonal
  • the reagent contains a diene structure and can be used as a bioorthogonal triggering group to activate and recognize a biomolecular target of a corresponding chemical reporter group in a physiological environment.
  • the reaction solvent for the carbon-hydrogen activation/dehydrogenation coupling is preferably a polar solvent, including but not limited to 1,2-xylene, chlorobenzene, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane. Hexacyclohexane, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, tert-butanol, tert-amyl alcohol, water or a mixed solvent thereof, preferably an aqueous solvent, more preferably dimethyl A sulfoxide/water mixed solvent.
  • a polar solvent including but not limited to 1,2-xylene, chlorobenzene, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane. Hexacyclohexane, dimethyl sulfoxide, N,N-dimethylformamide, aceton
  • the hydrogen removed by the carbon-hydrogen activation/dehydrogenation coupling reaction can be removed as hydrogen or captured by an oxidizing agent, so the oxidizing agent can be an oxidizing agent well known to organic chemists such as phenylhydrazine, silver carbonate, oxygen, air, and Sulfate, high iodine reagent, peroxide, etc., are preferably air.
  • organic chemists such as phenylhydrazine, silver carbonate, oxygen, air, and Sulfate, high iodine reagent, peroxide, etc., are preferably air.
  • the hydroxylamine functional group in the bioorthogonal reagent molecule is protected, that is, if R 1 and R 2 in the bioorthogonal reagent molecule represented by the molecular structural formula B and B 1 are not hydrogen
  • a deprotection reaction can be carried out to release the hydroxylamine functional group.
  • the hydroxylamine deprotection may be a method well known to an organic chemist, for example, using a mixed solvent of tetrahydrofuran and water at room temperature under the action of a copper salt and an acid.
  • the present invention also provides a bioorthogonal activator.
  • the bioorthogonal activator of the present invention is a bioorthogonal precursor as described above, which can undergo a carbon-hydrogen activation/coupling reaction with a biomolecule, that is, the bioorthogonal activator of the embodiment of the present invention contains
  • the bioorthogonal precursor group therefore, the bioorthogonal activator of the present invention contains a hydroxylamine functional group to provide a first bioorthogonal group, ie, a "chemical reporter group", which can be used as Marking biomolecules such as proteins to biocompatiblely install bio-orthogonal reaction “handles” to study the structure, function and interaction of target biomolecules, and to detect their expression, transport and Positioning and other changes, as well as obtaining information about the physiological metabolic processes of biological tissues.
  • the bioorthogonal activating agent of the embodiment of the present invention is a bioorthogonal precursor of the embodiment of the present invention represented by the above formula (A), which is modified by a dehydrogenation coupling reaction.
  • the molecular structure of the biomolecule is as follows (C):
  • P 2 is represented as a target biomolecule fragment or a member group.
  • P 2 in the formula C is linked to the biodenormalized precursor represented by the above formula (A) by an unsaturated bond to the On the bioorthogonal precursor, wherein the unsaturated bond is an unsaturated bond composed of C, O, S, N, or P, wherein the unsaturated bond is a part of the biomolecule.
  • P 2 is attached to the organism by a dehydrogenation coupling reaction of a double bond composed of a carbon atom with a bioorthogonal precursor represented by the above formula (A)
  • a bioorthogonal precursor represented by the above formula (A)
  • the molecular structure of the modified bioorthogonal activator is as follows (C 1 ):
  • P 2 ' is represented as a biomolecule fragment or a member group.
  • the diene group as a first group of orthogonal bio (chemical reporter group). That is, the bioorthogonal activator of the molecular structure formula (A) can form a dienyl group as the first bioorthogonal group in addition to the hydroxylamine group as the first bioorthogonal group.
  • C 1 formula when the same representation of R 1 and R 2 in the biomolecules after the orthogonal activation shown by the above C, C 1 formula is H, at this time, the above-described embodiment of the present invention as the first bioorthogonal group The hydroxylamine group of the group is not protected.
  • the bio-orthogonal activator represented by the above formula C its molecular structure represented by the following general formula (C 2), bio-orthogonal activator shown above C 1 through the molecular structure of formula The formula is as follows (C 3 ):
  • P 2 is represented as a biomolecule fragment or a member group. among them,
  • P 2 , P 2 ' may be one or more biomolecule fragments or member groups, and in some embodiments, the biomolecules indicated by P 2 , P 2 '
  • the fragment or member group can be any of a protein, nucleic acid or polysaccharide fragment or group.
  • P 2 ' is a protein fragment such as metalloporphyrins and its constructed heme, tryptophan;
  • P 2 , P In the case of 2 ' nucleic acid fragment, when a nucleic acid fragment such as cytosine, thymine, P 2 or P 2 'is an unsaturated polysaccharide fragment, an unsaturated polysaccharide fragment such as heparin disaccharides, It does not contain any of unsaturated fat and the like.
  • P 2 ' is part P 2, i.e., P 2' together constitute a carbon-carbon double P 2;
  • P 2 necessarily contains P 2 ', P 2 i.e. the portion connected to the activator of the present invention Not necessarily carbon-carbon double bonds, but they represent the same type of substance.
  • the above bio-orthogonal activation can be carried out according to the route a in FIG. 1 for the bio-orthogonal reaction "handle", and the above-described embodiments of the present invention are bioorthogonal precursors and biomolecules in metal catalyzed conditions.
  • the carbon-hydrogen activation/dehydrogenation coupling reaction is carried out.
  • the conditions of the carbon-hydrogen activation/dehydrogenation coupling are mild and biocompatible, provided that a metal catalyst is added, wherein, in one embodiment, the metal catalyst is a palladium catalyst, a rhodium catalyst, a rhodium catalyst, a copper catalyst, or a nickel catalyst.
  • a palladium catalyst is used, more preferably an organically coordinated palladium catalyst such as bis(2-amino-4,6-dihydroxypyrimidine)palladium acetate.
  • the hydrogen removed by the carbon-hydrogen activation/dehydrogenation coupling reaction carbon-hydrogen activation/dehydrogenation coupling reaction may be removed as hydrogen or captured with an oxidizing agent, preferably under oxidizing conditions, in one embodiment.
  • the oxidizing agent may be an oxidizing agent well known to organic chemists such as phenylhydrazine, silver carbonate, silver fluoride, oxygen, air, sodium persulfate, a high iodine reagent, a peroxide, etc., preferably air.
  • the reaction solvent for the carbon-hydrogen activation/dehydrogenation coupling is preferably water or a mixed solvent, including but not limited to water, ethanol/water, tetrahydrofuran/water, dimethyl sulfoxide/water, acetonitrile/water, ethylene glycol/ Water, preferably an aqueous mixed solvent, more preferably dimethyl sulfoxide/water solvent.
  • the hydroxylamine functional group in each of the bioorthogonal activator molecules is protected, that is, R 1 and R 2 in each of the above bioorthogonal activator molecules are not hydrogen, after the dehydrogenation coupling reaction
  • the deprotection reaction is carried out to release the hydroxylamine functional group, and as a bio-orthogonal reporter group, the biomolecule target can be recognized under the activation of the exogenous trigger group in a physiological environment.
  • the hydroxylamine deprotection may be a method well known to an organic chemist, for example, using a mixed solvent of tetrahydrofuran and water at room temperature under the action of a copper salt and an acid.
  • the above-described bioorthogonal reagent and bioorthogonal activator of the present invention contain the bioorthogonal precursor group and the carbon-hydrogen activation localization group of the embodiment of the present invention described above, and therefore, the carbon- Hydrogen Activation/Coupling Reactions Strong substrate suitability, mild and biocompatible reaction conditions can be used as tools to modify exogenous small molecule optical probes, or to modify bulk biomolecules, to easily trigger biological "click" reactions to achieve biological Orthogonal marking.
  • bio-orthogonal kit includes a bioorthogonal reagent containing a second bioorthogonal group (trigger group) and a bioorthogonal reaction with the bioorthogonal reagent and capable of directly or indirectly generating a first bioorthogonality Activator of the group (chemical reporter group).
  • the bio-orthogonal reagent is selected from the bio-orthogonal reagent of the embodiment of the invention described above, or the activator is selected from the bio-orthogonal precursor of the embodiment of the invention described above.
  • the activator selected is capable of bioorthogonal reaction with the bioorthogonal reagent, such as but not only for the ruthenium reaction or Diels-Alder's cycloaddition reaction; when the activator is selected from the bioorthogonal precursors described above, the selected bioorthogonal reagent is capable of bioorthogonal reaction with the bioorthogonal activator For example, but not only for the condensed synthesis reaction or the Diels-Alder cycle addition reaction.
  • the activator in the bioorthogonal kit of the present invention (referred to as kit 1) is selected from the bioorthogonal precursors described above, when the bioorthogonal activator and target organism After the carbon-hydrogen activation/dehydrogenation coupling reaction of the molecular or structural group, the activated biomolecule of the embodiment of the invention as described above is produced, as described in the above structural formula C, in which case P 2 is the A biomolecule fragment or member group formed by dehydrogenation of a target biomolecule or structural group.
  • the activated biomolecule formed by the carbon-hydrogen activation/dehydrogenation coupling reaction between the bioorthogonal precursor and the target biomolecule or structural group utilizes the hydroxylamine group contained as the first bioorthogonal group.
  • Group (chemical reporter group) the bio-orthogonal reagent in the kit 1 uses a non-biological probe molecule, and the non-biological probe molecule provides a second bioorthogonal group (trigger group) It is an aldehyde carbonyl group or a ketone carbonyl group, that is, a bio-orthogonal reagent in the kit 1 is a small molecule optical probe containing a carbonyl group.
  • the hydroxylamine group provided by the activator in the kit 1 is used as the first bioorthogonal group, the hydroxylamine group (unprotected) has a strong ⁇ -effect, which can be provided by the bioorthogonal reagent.
  • the aldehyde carbonyl or ketone carbonyl undergoes a condensation bioorthogonal reaction (ie, condensed into a hydrazine reaction) to release its optical imaging function.
  • the catalyst used for the condensed synthesis reaction may be an acid or an aniline.
  • the hydroxylamine group in the bioorthogonal precursor as the activator in the above kit 1 is protected during the carbon-hydrogen activation/dehydrogenation coupling reaction with the biomolecule or structural group to be tested, if
  • the hydroxylamine group in the bioorthogonal precursor is an unprotected hydroxylamine group, ie, as described above, in the general formula A, R 1 and R 2 are the same H, then the carbon-hydrogen activation/dehydrogenation coupling
  • the hydroxylamine group should be protected prior to the reaction.
  • the bioorthogonal activator formed after the carbon-hydrogen activation/dehydrogenation coupling reaction between the bioorthogonal precursor and the biomolecule or structural group to be tested is performed before the condensation reaction with the bioorthogonal reagent
  • the deprotected treatment of the protected hydroxylamine group, such that the deprotected hydroxylamine group as the first orthogonal group, is as described above and will not be described herein.
  • the target biomolecule that undergoes a carbon-hydrogen activation/dehydrogenation coupling reaction with the activator in the kit 1 described above is, but not limited to, a protein, a nucleic acid, or a polysaccharide, preferably an unsaturated bond.
  • Biomolecules such as heme containing metalloporphyrins and their symbiotic hemoglobin, myoglobin, unsaturated fat, heparin disaccharides, etc. .
  • the small molecular optical probe bioorthogonal reagent containing the second bioorthogonal trigger group in the above kit 1 may be a fluorescent probe containing an aldehyde carbonyl group or a ketone carbonyl group, such as fluorescein or fluorine.
  • fluorescein or fluorine such as fluorescein or fluorine.
  • BODIPY Boron-dipyrromethene
  • phenyl substituted pyrazoline phenyl pyrazoline
  • coumarin-like core structure and its derivatives such as fluorescein represented by the following structural formula.
  • Bodipy bodipy hydrocarbons such as represented by the following structural formula D 2
  • phenyl substituted pyrazolines as shown in the following structural formula D 3
  • phenyl substituted pyrazoline coumarin core structure as shown in the following structural formula D 4.
  • kit 1 for a chemical tool for binding a substance in a physiological environment, or the bioorthogonal precursor of the embodiment of the invention described above and a trigger group aldehyde or ketone carbonyl
  • a ruthenium reaction as a chemical tool for binding substances in a physiological environment.
  • the activator in the bioorthogonal kit of the present invention is selected from the bioorthogonal precursors of the embodiments of the invention described above, and the bioorthogonal front
  • the first bioorthogonal group produced by the body and the coupled modification target biomolecule is a dienyl group, that is, using the bioorthogonal precursor described above, preferably the above molecular structure formula A
  • the carbon-hydrogen activation/coupling reaction site occurring between the bio-orthogonal precursor and the modified target biomolecule is on the unsaturated bond of the biomolecule, preferably on the carbon-carbon double bond, and is catalyzed by the metal.
  • the alkenyl carbon in the target biomolecule and the bioorthogonal precursor of the embodiment of the invention described above are preferably the same in the bioorthogonal precursor represented by the above molecular structure formula A
  • the adjacent alkenyl carbon undergoes atomic economic dehydrogenation coupling, and the resulting modified biomolecule is labeled with a diene structure, and the diene structural group is used as a bioorthogonal chemical reporter group, so that it can be exogenous in a physiological environment.
  • Recognition of biomolecular targets by triggering group activation are preferably the same in the bioorthogonal precursor represented by the above molecular structure formula A
  • kits 2 a carbon-hydrogen activation/coupling reaction site occurring between the bioorthogonal precursor represented by the above formula M of the molecular structure and the modified target biomolecule containing a carbon-carbon double bond biomolecules carbon - carbon double bond, as described above to generate the molecular structure of formula C 1, C 3 bioorthogonal activator containing group represented diene.
  • the di-alkenyl group produced by the carbon-hydrogen activation/dehydrogenation coupling reaction between the bioorthogonal precursor and the target biomolecule or structural group containing an unsaturated bond, particularly a carbon-carbon double bond is at least a first bioorthogonal group (chemical reporter group), on the basis of which the bioorthogonal reagent in the kit 2 is selected from a dienophile as a non-biological probe containing a second bioorthogonal group molecule.
  • the chemically reported dienyl group provided by the activator that produces the dienyl group in kit 2 induces Diels- between the dienophile group as a triggering group in the bioorthogonal reagent.
  • the Diels-Alder's cycloaddition reaction acts as a bioorthogonal tool to activate a bioorthogonal reagent that turns fluorescence on or off in a physiological environment, resulting in the release of fluorescent imaging functions at the biological target.
  • the DAc reaction occurs between the electron-rich 1,3-diene and the electron-deficient dienophile, and a six-membered carbon ring is formed by [4 ⁇ +2 ⁇ ] cycloaddition.
  • Diels-Alder's cycloaddition reaction has high selectivity and compatibility with biomacromolecules, and can be used for polypeptide sequences, small molecules, deoxyribonucleic acid (DNA), protein modification and conjugation. And the presence of water has a significant acceleration on the reaction, and the reaction rate can be accelerated by several orders of magnitude relative to the pure organic phase. Since the dienophile is not present in any biomolecule, the DAc reaction can achieve the desired chemoselectivity as a bioorthogonal reaction without a protecting group.
  • the existing DAc reaction as a bioorthogonal reaction technique can only install a first bioorthogonal chemical reporter group by reacting with a diene-containing activator, which leads to limited selection of existing activators, and is difficult to obtain.
  • Large, long chain length, and installed chemical reporter group diene are far from the target of biomolecules, reducing the targeting, selectivity and credibility of bioorthogonal labeled biomolecules, and significantly changed The size of the biomolecule.
  • the above kit 2 overcomes the technical defect that both carbon-carbon double bonds in the diene must be obtained from the activator, and directly dehydrogenates and couples using the unsaturated bond present in the target biomolecule.
  • An bioorthogonal precursor preferably having a molecular structure of the general formula A bioorthogonal precursor as an activator, produces at least two conjugated double bonds on-line, the conjugated double bond being modified at a distance from the biomolecule target Producing a modified biomolecule containing a dienyl group as shown by the above molecular formulas C 1 and C 3 , which greatly improves the targeting, selectivity and credibility of the DAc reaction as a bioorthogonal labeled biomolecule To avoid significant changes in the size of the target biomolecule.
  • the hydroxylamine group in the bioorthogonal precursor as the activator in the above kit 2 may be a protected or unprotected hydroxylamine group. Then it may also be protected or unprotected during the carbon-hydrogen activation/dehydrogenation coupling reaction with the biomolecule or structural group to be tested, also in the bioorthogonal precursor and the biomolecule or structural group to be tested.
  • the bioorthogonal activator formed after the carbon-hydrogen activation/dehydrogenation coupling reaction of the group may also be protected or unprotected before the DAc reaction with the bioorthogonal reagent.
  • the second bioorthogonal triggering group contained in the bioorthogonal reagent of the dienophile in the kit 2 is a dienyl group such as an alkene, an alkyne, a dilute, a thioketone, Sulfonyl, nitrosobenzene, azide, and the like.
  • the bioorthogonal reagent is selected from the following structural formulas, but not limited to vinyl pyrene, quinidine, vinylbenzyl bromide complex (vinylbenzyl BODIPY), snail [ Isobenzofuran-1 (3H), 9'-[9H] xanthen]-3-1,5-vinyl-3',6'-dihydroxy (vinyl fluorescein), maleimide-containing rodane Mother nucleus and its derivatives such as maleimide-containing rhodamine B.
  • the kit 2 contains a di-alkenyl group-containing small molecule optical probe bioorthogonal reagent and a dienyl group-containing activator-modified biomolecule between Diels-Alder
  • the cycloaddition reaction is carried out as a bioorthogonal reaction, and the reaction conditions are carried out at room temperature in the aqueous phase, preferably the reaction is carried out without adding any metal catalyst.
  • the bioorthogonal reagent in the bio-orthogonal kit (referred to as the kit 3) of the embodiment of the present invention is selected from the bio-orthogonal reagent of the embodiment of the invention described above, preferably as the molecular structure is a bioorthogonal reagent of the formula B, B 2 , wherein the activator in the kit 3 is capable of directly or indirectly producing the first bioorthogonal group as an aldehyde carbonyl group. Or an activator of a ketone carbonyl group.
  • the bioorthogonal reagent in the kit 3 utilizes the hydroxylamine group contained as the second bioorthogonal group (trigger group), and on the basis of this, the activator utilization in the kit 3
  • the induced aldehyde carbonyl or ketone carbonyl provides a first bioorthogonal group (chemical reporter group). Therefore, based on the setting of the bioorthogonal reagent and the activator in the kit 3, the reaction principle between the bioorthogonal reagent and the activator is similar to that of the kit 1 described above, and the hydroxylamine group is also utilized.
  • the protected hydroxylamine group should be deprotected before decomposing with the activator to cause deprotection.
  • the hydroxylamine group serves as the first bioorthogonal group, and the deprotection method is as described above, and will not be described herein. If the hydroxylamine group in the bioorthogonal reagent is not protected, such as the bioorthogonal reagent shown in the above formula B2, it can be directly condensed with the activated biomolecule or fragment.
  • the aldehyde carbonyl or ketone carbonyl-containing activator of kit 3 may be activated DMSO, hypervalent iodine compounds, collins reagent, Any of piperidine oxynitride (TEMPO), tetra-n-propylammonium perruthenate/N-methylmorpholine-N-oxide (TPAP/NMO).
  • DMSO dimethyl methacrylate
  • TEMPO piperidine oxynitride
  • TPAP/NMO tetra-n-propylammonium perruthenate/N-methylmorpholine-N-oxide
  • kits of the present invention orthogonal to the biological agent, such as selection of the molecular structure of the above Formula B 1, B 3 shown in EXAMPLES Bioorthogonal reagents.
  • the activator in the kit 4 contains or induces the production of a dienophile under the premise of the bioorthogonal reagent.
  • the bioorthogonal reagent contains a dienyl group, and then the bioorthogonal reagent uses at least the dienyl group contained as the second bioorthogonal group (trigger group), then Based on this, the dienyl group contained in the activator in the kit 4 serves as a first bioorthogonal group (chemical reporter group). Therefore, based on the setting of the bioorthogonal reagent and the activator in the kit 4, the reaction principle between the bioorthogonal reagent and the activator is similar to that of the kit 2 described above, and is also utilized as a chemical report.
  • a Diels-Alder cycloaddition reaction occurs between the dienyl group of the group and the dienophile group as a triggering group in the activator.
  • the Diels-Alder's cycloaddition reaction acts as a bioorthogonal tool to activate a bioorthogonal reagent that turns fluorescence on or off in a physiological environment, resulting in the release of fluorescent imaging functions at the biological target.
  • the specific DAc is as described above and will not be described here.
  • the hydroxylamine group contained in the bioorthogonal reagent in the above kit 4 may be a protected or unprotected hydroxylamine group.
  • the hydroxylamine group contained in the activator may also be protected or unprotected prior to the DAc reaction with the bioorthogonal reagent.
  • the dienophile as the activator in the kit 4 may be a maleimide derivative, a maleic anhydride derivative, an acrylate derivative, or the like. Any one of a benzoquinone derivative, a dimethyl azodicarboxylate derivative, and a dioxotriazole derivative.
  • bio-orthogonal kit for biomarkers of the above-described embodiments of the present invention can realize bioorthogonal labeling quickly and efficiently due to the above-described bioorthogonal reagent or bioorthogonal precursor of the present invention.
  • Epoxybutene (1 eq), allylamine (2 eq) and 0.4 mL of water were added to the sealed tube, and then heated to 100 ° C for 6 h. After completion of the reaction, it was directly dried to give a crude 1-(allylamino)butyl-3-en-2-ol, which was directly used for the next reaction.
  • the bioorthogonal precursor N-(cyclopentyl-2-en-1-yloxy)acetamide (16.9 mg, 0.1 mmol) shown in Example 4 was added to a 15 ml sealed tube, bis(2- Amino-4,6-dihydroxypyrimidine) palladium acetate sodium salt (2.8 mg, 0.005 mmol). Allyl methyl ester (25.8 mg, 0.3 mmol) and dimethyl sulfoxide/water (1.0 ml, 20% DMSO) were added to the sealed tube, and the mixture was stirred at room temperature for about 6 hours after the dropwise addition. After the reaction was completed, it was filtered and concentrated to give a crude material. The crude product was purified by column chromatography to give a product.
  • a dehydrogenation coupling reaction of a bioorthogonal activator with a biomolecule to mount a chemical reporter group to a biomolecule :
  • the bioorthogonal activator N-(cyclohexyl-2-en-1-yloxy)acetamide (3.7 mg, 0.024 mmol) shown in Example 1 was added to the sealed tube, and the heme of the hemin was added.
  • High-resolution mass spectrometry (HRMS) analysis was carried out to obtain a molecular weight of 2, which was 797.2905, which was consistent with the calculation of the molecular weight of [C 44 H 47 FeN 5 O 6 , M-Cl - ] + 797.2876, and the molecular weight of 3 was 950.3702.
  • the calculated molecular weight of [C 44 H 47 FeN 5 O 6 , M-Cl - ] + is 950.3666, which further confirms the molecular structure of the product after modification of the biomolecule by the activator, and confirms that the reaction can be selectively carried out. Single target activation of biomolecules.
  • a dehydrogenation coupling reaction of a bioorthogonal activator with a biomolecule to mount a chemical reporter group to a biomolecule :
  • the bioorthogonal activator N-(cyclohexyl-2-en-1-yloxy)acetamide shown in Example 1 was added to the sealed tube (11.6 Mg, 0.075 mmol), dimethyl ester compound 1 (20.4 mg, 0.03 mmol), bis(2-amino-4,6-dihydroxypyrimidine) palladium acetate sodium acetate (0.9 mg, 5 mol) %) and dimethyl sulfoxide/water (0.5 ml, 20% DMSO), and after stirring, stirred at room temperature for about 14 hours. The remaining steps are the same as in the embodiment 24.
  • the product 2 (3 mg) and 3 (10.6 mg) were obtained in a total yield of 48%, and the molar ratio of the product 2 to the product 3 was 1:3. This result further confirmed the molecular structure of the product after modification of the biomolecule by the activator. It was also confirmed that the reaction can selectively perform multi-target activation of biomolecules.
  • the bioorthogonal precursor N-(cyclohexyl-2-en-1-yloxy)acetamide (18.6 mg, 0.12 mmol) and the alkenylated boron fluoride shown in Example 1 were added to the sealed tube.
  • NMR nuclear magnetic resonance spectrum
  • the bioorthogonal reagent-modified boron fluoride complexed dipyrromethene fluorescent molecule (25 mg, 0.05 mmol) obtained in Example 26 was dissolved in THF/H 2 O (0.2 mL / 0.2 mL), and then CuCl was added thereto. 2 (6.6 mg, 0.05 mmol) and HCl (5M, 20 ⁇ L). Stir at room temperature for about 36 hours. It was neutralized by the addition of saturated sodium hydrogencarbonate, extracted three times with dichloromethane, and the organic layer was collected and then dried over MgSO 4 . Finally, 0.3 g of silica gel was added and concentrated to give a crude product. The crude product was purified by column chromatography to obtain a boron fluoride complexed dipyrromethene-based fluorescent molecule (9.2 mg) containing a deprotected hydroxylamine structure in a yield of 40%.
  • High-resolution mass spectrometry (HRMS) analysis was carried out to obtain a molecular weight of 462.2492, which was consistent with the calculation of the molecular weight of [C 29 H 33 BF 2 N 3 O 2 , M+H] + 462.2528, which further confirmed the modified boron fluoride.
  • the hydroxylamine group of the complexed dipyrromethene-based fluorescent molecule has been deprotected, and the resulting unprotected hydroxylamine group serves as the first bioorthogonal chemical reporter group.
  • a bioorthogonal kit comprising a bioorthogonal reagent for modifying a boron fluoride complexed dipyrromethene fluorescent molecule and an activator for activating cells as described in Example 27, and a method of using the same.
  • Kit composition bioorthogonal reagent, here modified boron fluoride complexed dipyrromethene fluorescent molecule or derivative thereof as described in Example 27; activator, here is periodic acid for biomolecule labeling Salt or its analogues.
  • the activator activates the surface of the biomolecule to form a chemical reporter group, and the activated biomolecule is further subjected to a bioorthogonal condensation reaction with the bioorthogonal reagent.
  • Periodate sialic acid oxidation of cell surface Hela adherent cells were first cultured in culture dishes for 48 hours, washed three times with phosphate buffered saline (pH 7.4), and then stored in ice bath at 1 mM high. Sodium iodate in phosphate buffered saline (pH 7.4) was used for half an hour at room temperature as a control test. The oxidation reaction was quenched with 1 mM glycerol and then washed three times with a phosphate buffered saline solution (pH 7.4).
  • An aniline-catalyzed condensation reaction to form hydrazine Hela cells were immersed in a phosphate buffered saline solution (pH 6.7) containing 5% fetal calf serum, 100 ⁇ M modified boron fluoride complexed dipyrromethene fluorescent molecule as described in Example 36, and 10 mM aniline. The cells were placed under dark and ice bath conditions, gently agitated by a shaker, then trypsinized with cells, centrifuged, and washed three times with phosphate buffered saline (pH 7.4).
  • the cells were placed in RIPA lysate containing HaltTM protease inhibitor (Thermo) to a concentration of 1 x 10 7 cells/mL. The lysate was then subjected to 10% polyacrylamide gel electrophoresis to detect the corresponding protein. In situ fluorescence was detected directly by Typhoon FLA 9500.
  • HaltTM protease inhibitor Thermo

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Abstract

L'invention concerne un précurseur orthogonal biologique, un réactif orthogonal biologique, un activateur orthogonal biologique, et un kit de réactif orthogonal biologique à utiliser comme biomarqueur. Le précurseur orthogonal biologique de la présente invention contient un groupe fonctionnel hydroxylamine qui a au moins de multiples fonctions, peut être utilisé comme groupe de positionnement dans une réaction de couplage, et est utilisé comme groupe orthogonal biologique. Le kit de réactif orthogonal biologique de la présente invention comprend le réactif orthogonal biologique comprenant un second groupe orthogonal biologique, de la présente invention, ou comprend un activateur orthogonal biologique capable de générer directement ou indirectement un premier groupe orthogonal biologique, de la présente invention.
PCT/CN2015/074897 2015-03-23 2015-03-23 Réactif, activateur et précurseur orthogonaux biologiques, et kit de réactif orthogonal biologique Ceased WO2016149899A1 (fr)

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