WO2016149899A1 - 生物正交试剂、活化剂和前体及生物正交试剂盒 - Google Patents

生物正交试剂、活化剂和前体及生物正交试剂盒 Download PDF

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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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French (fr)
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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 patent/WO2016149899A1/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

本发明公开了一种生物正交前体、生物正交试剂和生物正交活化剂以及用于生物标记的生物正交试剂盒。本发明生物正交前体含有至少多功能的羟胺官能基团可作为偶联反应的定位基团并作为生物正交基团。本发明生物正交试剂盒包括含有第二生物正交基团的本发明生物正交试剂或包括能直接或间接产生第一生物正交基团的本发明正交活化剂。

Description

生物正交试剂、活化剂和前体及生物正交试剂盒 技术领域
本发明属于生物正交反应技术领域，具体的说是涉及生物正交试剂、生物正交活化剂、生物正交前体和生物正交试剂盒。
背景技术
在医学成像领域，使用小分子光学探针来可视化探测活细胞和生物体的生物事件已经成为强有力的工具。由于我们的身体和细胞的生物学事件通常不能提供足够用肉眼或仪器检测的视觉或数据信息使我们能够理解生物事件，如细胞信号传导和蛋白表达，因而采用外源性的小分子光学探针来特异性地检测体内特定分子或靶点，对于无论是基础生物学研究，还是疾病的诊断和治疗都有非常重要的意义。荧光实时成像方法无需依靠特殊或昂贵的仪器，灵敏度高，有足够的空间和时间分辨率来分析动态细胞信号传导过程，因而在化学生物学领域正得到广泛使用。大分子荧光探针，如绿色荧光蛋白基因编码的荧光蛋白(Green Fluorescent Protein)和其变种的荧光蛋白(Fluorescent Proteins)被广泛应用于标记和跟踪体内蛋白，取得巨大成功，并于2008年获得诺贝尔化学奖。然而此类基因编码探针不适合于监测非蛋白质的生物分子，包括多糖、脂质、核酸等，同时其具有较大的空间位阻，对标记位点及荧光检测的灵活性有较大的限制。相比而言，有机小分子光学探针在灵活性和空间尺寸上都有优势，适用于任何样品包括人体，而且相对便宜和容易处理，通过化学设计和调控，能够提供较高的信噪比和靶点适用性。
荧光小分子探针的成像技术能够实现对细胞甚至活体中活性小分子的实时可视化示踪，在研究信号转导、细胞生理病理功能方面具有无损、实时、灵敏度高、操作简便、背景低、信号可调控等优势。荧光小分子探针是生命体系中使用最为广泛的检测工具，已被广泛用于生命体系中气体分子如一氧化氮、一氧化碳及硫化氢等信号传导分子的分析检测，以及体内金属离子的探测。有机小分子荧光探针对生物分子如蛋白质、自由基、多肽、酶的标记和着色方向也正被逐渐重视。其中利用生物正交化学反应对体内生物大分子进行特异标记作为一种新兴的化学生物学方法，正日益得到更广泛的关注和兴趣。
生物正交反应统指一类能够在活体细胞或组织中不干扰生物自身生化反应条件下进行的化学反应。它通过活化剂在生物分子内如目标蛋白中引入特定标签(Tagging)的第一生物正交基团，又称为化学报告基团(Chemical Reporter)，与含有或修饰有互补的第二生物正交基团，又称为触发基团(Trigger)的探针进行生物正交反应，从而实现对目标生物分子的特异标记。这两种正交基团被称为生物正交反应对(Bioorthogonal Pair)，在生理条件下相互间发生“点击”反应，对周围分子保持惰性。然而到目前为止只有少数的几个反应可以满足生物正交的条件，已成为生物正交反应发展和技术应用的瓶颈。同时，现有生物正交反应的步骤复杂，使得生物正交反应效率低。
技术问题
本发明的目的在于克服现有技术的上述不足，提供一种生物正交前体、以及以所述生物正交前体为基础基团的生物正交试剂或生物正交活化剂，以克服现有生物正交反应发展和技术应用的瓶颈技术问题。
本发明的另一目的在于提供一种含有本发明生物正交试剂或生物正交前体的生物正交试剂盒，以提高现有生物正交反应效率。
发明内容
为了实现上述发明目的，本发明的技术方案如下：
一种生物正交前体，其含有至少可作为偶联反应的定位基团和生物正交基团的多功能官能基团，其中，所述多功能官能基团为羟胺基团。
进一步地，上述本发明生物正交前体分子结构通式为下述(A)：
Figure PCTCN2015074897-appb-000001
所述A式中，R1、R2相同或不相同的为H、含有直链或支链的烷基、含有直链或支链的酰基、烷氧羰基中的任一种，R3表示H、烷基、烯基、芳基、杂芳基中的任一种，弧形所表示的结构为链状结构或环状结构。
以及，一种生物正交试剂，由上述本发明分子结构通式(A)所示的生物正交前体经脱氢偶联反应而成，其分子结构通式为下述(B)：
Figure PCTCN2015074897-appb-000002
所述B式中，P1为光学探针分子片段或结构基团。
以及，一种生物正交活化剂，其是上述本发明分子结构通式(A)或其衍生物，所示的生物正交活化剂可在生物条件下经脱氢偶联反应活化生物分子，其活化后的分子结构通式为下述(C)：
Figure PCTCN2015074897-appb-000003
所述C式中，P2表示为生物分子片段或构件基团。
以及，一种用于生物标记的生物正交试剂盒，其包括含有第二生物正交基团的生物正交试剂和能与所述生物正交试剂发生生物正交反应的且能直接或间接产生第一生物正交基团的活化剂，所述生物正交试剂为如上述本发明生物正交试剂；或所述活化剂为如上述本发明生物正交前体或为如上述本发明生物正交活化剂。
因此，上述本发明生物正交前体选用羟胺基团作为功能基团，其具有螯合催化剂以活化底物作用和可生物正交性能，因此，本发明生物正交前体将碳-氢活化/偶联反应的定位基团与生物正交基团合二为一，可选择在小分子光学探针上引入生物正交“触发”基团，或者可选择在目标生物分子中引入特定生物正交“化学报告”基团。
优选地，将上述本发明生物正交前体结构式设计如上文所述的结构通式(A)，即羟胺基团连接碳的邻位为碳-碳双键，其能有效与生物分子中的烯基碳发生原子经济地脱氢偶联，生成的修饰生物分子被标记上双烯结构；或能有效与小分子光学探针中的烯基碳发生原子经济地脱氢偶联，生成含双烯基团的生物正交试剂。
上述本发明生物正交试剂和生物正交活化剂由于含有本发明生物正交前体基团，因此，所述碳-氢活化/偶联反应强大的底物适用性、温和及生物兼容的反应条件可以作为修饰外源荧光探针，或修饰本体生物分子的工具，可轻松触发生物“点击”反应来实现生物正交标记。
有益效果
上述用于生物标记的生物正交试剂盒由于含有上文所述的本发明生物正交试剂或生物正交前体，因此，其能快速高效地实现生物正交标记。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为本发明实施例生物正交前体碳-氢活化和生物正交反应糅合的路线图；其中，路线a表示本发明实施例生物正交前体碳-氢活化后提供化学报告基团和生物正交反应糅合的路线图；路线b表示本发明实施例生物正交前体碳-氢活化后提供触发基团和生物正交反应糅合的路线图；
图2为利用本发明实施例28生物正交试剂盒用于Hela细胞表面标记的荧光强度与时间关系图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
除非另外定义，否则下文对于本发明实施例技术方案的阐述内容中使用的技术和科学术语具有与本发明领域的技术人员理解相同的含义。为了有利于本领域技术人员的理解，与本发明实施例技术方案相关的定义列举如下：
生物正交反应：统指一类能够在活体细胞或组织中不干扰生物自身生化反应条件下进行的化学反应。它通过活化剂在生物分子内如目标蛋白中引入特定标签(Tagging)的第一生物正交基团，又称为化学报告基团(Chemical Reporter)，与含有或修饰有互补的第二生物正交基团，又称为触发基团(Trigger)的探针进行生物正交反应，从而实现对目标生物分子的特异标记。这两种正交基团被称为生物正交反应对(Bioorthogonal Pair)，在生理条件下相互间发生“点击”反应，对周围分子保持惰性。
定位基团：表示用于活化底物中的特定“惰性”碳-氢键，使其周围有足够高浓度的过渡金属催化剂来实现理想的反应动力学，并协调过渡金属与特定的碳-氢键相连从而实现在多个碳-氢键中精准区域选择性的基团。
生物正交基团：表示在生命系统和其它复杂的生物环境中可以互相进行点击化学反应(click reactions)的交互基团。所述生物正交基团具有正交性，其特征是反应组分是彼此相容并且不发生交叉反应，选择性地生物正交偶联而不干扰天然生物分子或生物过程。理想地，其相互正交的反应可以在串列式双生物分子标记中使用。本发明实施例中，所述交互生物正交基团中的第一生物正交基团安装在生物分子上作为化学报告基团，第二生物正交基团安装在探针分子上作为触发基团。
碳-氢活化：指由有机金属化合物协调的碳-氢键断裂过程，其中所述键断裂是通过碳-氢键配位到过渡金属内层电子云来完成，无需底物的预官能化步骤。在碳-氢活化反应中，定位基团(Directing Group)是非常重要的，它作为底物上的基团参与协调过渡金属与特定的碳-氢键相连，从而实现了在多个碳-氢键中精准的区域选择性；同时由于它的配位作用，使得被活化的特定“惰性”碳-氢键周围有足够高浓度的过渡金属催化剂来实现理想的反应动力学。所述碳-氢键被活化的底物可以与另一个偶联底物实现交叉偶联反应。
另外，在广义上，本发明实施例是基于以下认识：当碳-氢活化/偶联反应的定位基团在偶联反应后被保留并作为生物正交基团后，所述碳-氢活化/偶联反应强大的底物适用性、温和及生物兼容的反应条件可以作为修饰外源小分子光学探针，或修饰本体生物分子的工具，可轻松触发生物“点击”反应来实现生物正交标记。
基于此，本发明实施例提供了一种生物正交前体，其含有至少可作为偶联反应的定位基团和生物正交基团的多功能官能基团，其中，所述多功能官能基团为羟胺基团。这样，上述本发明实施例生物正交前体选用羟胺基团作为功能基团，其具有活化底物作用和可生物正交性能，因此，本发明实施例生物正交前体将碳-氢活化/偶联反应的定位基团与生物正交基团合二为一，可选择在小分子光学探针上引入生物正交“触发”基团，或者可选择在目标生物分子中引入特定生物正交“化学报告”基团。其中，其碳-氢活化与生物正交反应糅合的路线如图1中所示。
由图1所示的路线a和b清晰表明，上述生物正交前体作为提供生物正交基团的通用试剂。该通用试剂可以修饰生物分子提供第一生物正交化学报告基团，或者修饰探针分子提供第二生物正交触发基团，即当本发明实施例生物正交前体为活化剂时，其可被引入目标生物分子中作为第一生物正交基团，即“化学报告基团”；当本发明实施例生物正交前体为生物正交试剂前体时，其可被引入小分子光学探针中作为第二生物正交基团，即“触发基团”，能 够满足化学生物学界对于拓展生物正交试剂盒和正交方法的需求，为修饰生物分子、研究生物体系中分子网络和功能提供了新的工具。
在一实施例中，上述本发明实施例生物正交前体分子结构通式为下述(A)：
所述A式中，R1、R2相同或不相同的为H、含有直链或支链的烷基、含有直链或支链的酰基、烷氧羰基中的任一种，R3表示H、烷基、烯基、芳基、杂芳基中的任一种，弧形所表示的结构为链状结构或环状结构。因此，该式A所示的生物正交前体中羟胺基团连接碳的邻位为碳-碳双键时，其能有效与目标生物分子中的烯基碳发生原子经济地脱氢偶联，生成的修饰目标生物分子被标记上双烯结构，或能有效与小分子光学探针中的烯基碳发生原子经济地脱氢偶联，生成含双烯基团的生物正交试剂。
另外，基于式A所示的生物正交前体中R1、R2所表示的基团，在一实施例中，式A中的R1、R2相同的表示为H时，此时，上述本发明实施例生物正交试剂中羟胺基团没有被保护。
在另一实施例中，式A中的R1、R2中的至少一个不表示为H时，此时，上述本发明实施例生物正交前体中羟胺基团则被R1和/或R2保护。该羟胺基团被保护的生物正交前体是本发明实施例优选，控制反应条件可使其在上述碳-氢活化/偶联反应中被完整地保存下来作为生物正交基团。
进一步地，在一些实施例中，当式A所示的生物正交前体中R1、R2表示含有直链或支链的酰基时，所述酰基选自乙酰基、苯甲酰基、苄酰基、特戊酰基、十二酰基、三氟乙酰基中的任一种或两种；所述R1、R2表示烷氧羰基时，所述烷氧羰基选自叔丁氧羰基、苯氧羰基、苄氧羰基、烯丙氧羰基、乙氧羰基、三氯乙氧羰基、芴甲氧羰基中的任一种或两种。
式A所示的生物正交前体中R3对本发明实施例生物正交前体构建的生物正交试剂的光学性能调控和溶解性能的调节、以及修饰生物分子的构象调整都有积极的影响。因此，在一些实施例中，当式A所示的生物正交前体中R3表示烯基时，所述烯基选自乙烯基、丙烯基、异戊烯基、辛烯基、十二烯基、环戊烯基中的任一种；或
所述R3表示芳基时，所述芳基选自苯环及其衍生物；或
所述R3表示杂芳基时，所述杂芳基选自吡咯基、呋喃基、咪唑基、吡啶基、噻唑基、嘧啶基、噻吩基、吡唑基中的任一种。
在一些实施例中，上述分子结构通式(A)中弧形所示的链状结构选自含双键的链状结构。
在另一些实施例中，弧形所示的环状结构选自环丙烯、环丁烯、环戊烯、环己烯、环辛烯、四氢吡啶、二氢呋喃、二噻嗪中的任一种或其衍生物。
另外，如果上文所述的本发明实施例生物正交前体中的羟胺功能基团被酰肼、酯基或其 它有定位效果的基团替换，也在本发明实施例公开的范围之内。
相应地，在上文本发明实施例生物正交前体的基础上，本发明实施例还提供了一种生物正交试剂。该生物正交试剂为光学探针分子或基团与上文所述的生物正交前体经碳-氢活化/偶联反应而成，也即是说，本发明实施例生物正交试剂含有上文本发明实施例生物正交前体基团，因此，本发明实施例生物正交试剂能修饰外源小分子光学探针，即提供第二生物正交基团(触发基团)，同时调控小分子光学探针的性质，如灵敏度、选择性、检测限、光学性质、以及生物相容性。
尽管小分子荧光探针被发现已经有上百年的时间了，但对小分子荧光染料的结构修饰才刚刚起步。由于生物组织在红色至近红外(Near-infrared,NIR)这一波长范围内透明，设计和合成出在红色至近红外波段内发射明亮且耐光的荧光染料尤其重要，能够用于动物和人类体内成像的需求。而本发明实施例生物正交试剂利用上文所述的本发明实施例生物正交前体对现有探针进行多共轭化修饰，并入多环和杂环体系，增加共轭链长度，从而使得本发明实施例生物正交试剂能有效满足用于动物和人类体内成像的需求。
在一实施例中，本发明实施例生物正交试剂由上文所述的分子结构通式(A)所示的本发明实施例生物正交前体经脱氢偶联反应而成，其分子结构通式为下述(B)：
Figure PCTCN2015074897-appb-000005
所述B式中，P1为光学探针分子片段或结构基团。其中，
在进一步实施例中，所述B式中的P1是通过不饱和键与上文所述的分子结构通式(A)所示的生物正交前体发生偶联反应连接至所述生物正交前体上的，其中，所述不饱和键为双键、三键、醛羰基、或酮羰基，且所述不饱和键为所述光学探针分子片段或结构基团的一部分。
因此，在进一步实施例中，P1是通过双键如碳-碳双键与上文所述的分子结构通式(A)所示的生物正交前体发生偶联反应连接至所述生物正交前体时，所述生物正交试剂分子结构通式为下述(B1)：
Figure PCTCN2015074897-appb-000006
所述B1式中，P1’为光学探针分子片段或结构基团。因此，在该B1式中，羟胺基团连接碳的邻位含有双烯基团，因此，该双烯基团可以作为第二生物正交基团(触发基团)。也即是，该分子结构通式B1所示的生物正交试剂除了羟胺基团作为第二生物正交基团之外，还 可以提供双烯基团作为第二生物正交基团。
另外，所述B、B1式中，R1、R2和R3如同上文生物正交前体中所述，为了节约篇幅，在此不再赘述。因此，
在进一步实施例中，当上述B、B1式所示的生物正交试剂中的R1、R2相同的表示为H时，此时，上述本发明实施例生物正交前体中羟胺基团没有被保护。此时，在一具体实施例中，上述B式所示的生物正交试剂其分子结构通式为下述(B2)，上述B1式所示的生物正交试剂其分子结构通式为下述(B3)：
Figure PCTCN2015074897-appb-000007
对于上述各生物正交试剂分子结构中的P1、P1’所示的光学探针分子片段或结构基团可以是相同或不相同的一个或多个光学探针分子片段或结构基团，在一些实施例中，P1、P1’所述的光学探针分子片段为小分子荧光探针片段或比色探针片段。在进一步实施例中，该P1、P1’所述的光学探针分子片段可以是芘(pyrene)类、硫酸奎宁(quinine sulfate)类、氟化硼络合二吡咯甲川(boron-dipyrromethene,BODIPY)类、荧光素(fluorescein)类、罗丹明(rhodamine)类中的任一种。
在另一些实施例中，P1、P1’所述的结构基团可以是烯、炔、联烯、硫酮、亚磺酰、亚硝苯、叠氮等中的任一种。
另外，上述生物正交试剂可以按照如图1中的路线b进行反应制备，将上文所述的本发明实施例生物正交前体与光学探针分子在金属催化剂的催化下进行碳-氢活化/脱氢偶联反应，反应温度优选在0-100℃之间。其中，在一实施例中，金属催化剂可以选用钯催化剂、钌催化剂、铑催化剂、铜催化剂、镍催化剂等，优选用钯催化剂，更优选用有机配位的钯催化剂，例如双(2-胺基-4,6-二羟基嘧啶)合醋酸钯(Pd(OAc)2(ADHP)2)。另外，上述分子结构通式B1所示的生物正交试剂由于含有双烯基团，因此，其脱氢偶联反应发生在两个烯基碳上，产生的小分子光学探针生物正交试剂含双烯结构，可作为生物正交触发基团的用途，使其在生理环境中能够激活相应化学报告基团的生物分子靶点并被识别。
该碳-氢活化/脱氢偶联的反应溶剂优选为极性溶剂，包含但不限于1,2-二甲苯、氯苯、二氯甲烷、二氯乙烷、四氢呋喃、1,4-二氧六环、二甲基亚砜、N,N-二甲基甲酰胺、乙腈、甲醇、叔丁醇、叔戊醇，水或其混合溶剂，优选为有水溶剂，更为优选地为二甲基亚砜/水混合溶剂。
该碳-氢活化/脱氢偶联反应脱除的氢可以作为氢气被移出，或用氧化剂捕捉，所以氧化剂可以是有机化学家所熟知的氧化剂，例如苯醌、碳酸银、氧气、空气、过硫酸盐、高碘试剂、过氧化物等，优选地为空气。
另外，当生物正交试剂分子中的羟胺功能基团被保护，也即是如分子结构式B、B1所示的生物正交试剂分子中的R1、R2表示不为氢时，待脱氢偶联反应后可以进行脱保护反应，使得羟胺功能基团得以释放。其中，所述羟胺脱保护可以是有机化学家所熟知的方法，例如在室温条件下采用四氢呋喃与水的混合溶剂，在铜盐和酸的共同作用下进行。当上述生物正交试剂为B1式所述的含有双烯基团时，R1、R2可以被保护或不被保护。
相应地，在上文本发明实施例生物正交前体的基础上，本发明实施例同时还提供了一种生物正交活化剂。本发明实施例生物正交活化剂为上文所述的生物正交前体，可与生物分子发生碳-氢活化/偶联反应，也即是说，本发明实施例生物正交活化剂含有上文本发明实施例生物正交前体基团，因此，本发明实施例生物正交活化剂其含有的羟胺功能基团提供第一生物正交基团，即“化学报告基团”，能作为标记如蛋白质等目标生物分子，实现生物相容性地安装带有生物正交反应“把手”的方法，从而对目标生物分子的结构、功能和相互作用进行研究，实时检测观察其表达、运输和定位等变化过程，以及获得关于生物组织生理代谢过程的相关信息。
在一实施例中，本发明实施例生物正交活化剂为上文所述的分子结构通式(A)所示的本发明实施例生物正交前体，其经脱氢偶联反应修饰的生物分子结构通式为下述(C)：
Figure PCTCN2015074897-appb-000008
所述C式中，P2表示为目标生物分子片段或构件基团。
在一实施例中，所述C式中的P2是通过不饱和键与上文所述的分子结构通式(A)所示的生物正交前体发生脱氢偶联反应连接至所述生物正交前体上的，其中，所述不饱和键由C、O、S、N、或P构成的不饱和键，其中所述不饱和键为所述生物分子的一部分。
因此，在进一步实施例中，P2是通过碳原子构成的双键与上文所述的分子结构通式(A)所示的生物正交前体发生脱氢偶联反应连接至所述生物正交活化剂上时，得到生物正交活化剂修饰后的分子结构通式为下述(C1)：
Figure PCTCN2015074897-appb-000009
所述C1式中，P2’表示为生物分子片段或构件基团。因此，在该C1式中，羟胺基团连接碳的邻位含有双烯基团，因此，该双烯基团可以作为第一生物正交基团(化学报告基团)。也即是，该分子结构通式(A)所示的生物正交活化剂除了羟胺基团作为第一生物正交基团 之外，还可以生成双烯基团作为第一生物正交基团。
另外，所述C、C1式中，R1、R2和R3如同上文生物正交前体中所述，为了节约篇幅，在此不再赘述。因此，
在进一步实施例中，当上述C、C1式所示正交活化后的生物分子中R1、R2相同的表示为H时，此时，上述本发明实施例作为第一生物正交基团的羟胺基团没有被保护。此时，在一具体实施例中，上述C式所示的生物正交活化剂其分子结构通式为下述(C2)，上述C1式所示的生物正交活化剂其分子结构通式为下述(C3)：
Figure PCTCN2015074897-appb-000010
所述C式中，P2表示为生物分子片段或构件基团。其中，
对于上述各生物正交活化剂分子结构中的P2、P2’可以是一个或多个生物分子片段或构件基团，在一些实施例中，该P2、P2’所示的生物分子片段或构件基团可以是蛋白质、核酸或多糖中的任一种片段或基团。
在一些具体实施例中，该P2、P2’所示蛋白质片段时，蛋白片段如金属卟啉(metalloporphyrins)及其构建的血红素(heme)、色氨酸(tryptophan)；P2、P2’所示核酸片段时，核酸片段如胞嘧啶(cytosine)、胸腺嘧啶(thymine)；P2、P2’所示不饱和多糖片段时，不饱和多糖片段如肝素双糖(heparin disaccharides)、不包含脂肪(unsaturated fat)等中的任一种。另外，从分子结构式可知，P2’是P2的一部分，即P2’加上碳碳双键构成P2；P2不一定包含P2’，即P2与本发明活化剂相连的部分不一定是碳碳双键，但它们表示的是同一类物质。
另外，上述生物正交活化可以按照如图1中的路线a进行生物正交反应“把手”的安装，将上文所述的本发明实施例生物正交前体与生物分子在金属催化的条件下进行碳-氢活化/脱氢偶联反应。该碳-氢活化/脱氢偶联的条件温和且生物兼容，条件是加入金属催化剂，其中，在一实施例中，该是金属催化剂选用钯催化剂、钌催化剂、铑催化剂、铜催化剂、镍催化剂等，优选用钯催化剂、更优选用有机配位的钯催化剂，例如双(2-胺基-4,6-二羟基嘧啶)合醋酸钯。同样，该碳-氢活化/脱氢偶联反应碳-氢活化/脱氢偶联反应脱除的氢可以作为氢气被移出，或用氧化剂捕捉，优选是在氧化剂条件下进行，在一实施例中，所以氧化剂可以是有机化学家所熟知的氧化剂，例如苯醌、碳酸银、氟化银、氧气、空气、过硫酸钠、高碘试剂、过氧化物等，优选为空气。
该碳-氢活化/脱氢偶联的反应溶剂优选为水或混合溶剂，包含但不限于水、乙醇/水、四氢呋喃/水、二甲基亚砜/水、乙腈/水、乙二醇/水，优选为含水混合溶剂，更为优选地为二甲基亚砜/水溶剂。
当上述各生物正交活化剂分子中的羟胺功能基团被保护，也即是上述各生物正交活化剂 分子中的R1、R2表示不为氢时，待脱氢偶联反应后可以进行脱保护反应，使得羟胺功能基团得以释放，作为释放生物正交报告基团，使其在生理环境中能够在外源触发基团激活下实现生物分子靶点的识别。其中，所述羟胺脱保护可以是有机化学家所熟知的方法，例如在室温条件下采用四氢呋喃与水的混合溶剂，在铜盐和酸的共同作用下进行。当上述生物正交活化剂为C1式所述的含有双烯基团时，R1、R2可以被保护或不被保护。
因此，上述本发明实施例生物正交试剂和生物正交活化剂由于含有上文所述的本发明实施例生物正交前体基团兼碳-氢活化定位基团，因此，所述碳-氢活化/偶联反应强大的底物适用性、温和及生物兼容的反应条件可以作为修饰外源小分子光学探针，或修饰本体生物分子的工具，可轻松触发生物“点击”反应来实现生物正交标记。
相应地，在上文本发明实施例生物正交前体、生物正交试剂的基础上，本发明实施例还提供了用于生物标记的生物正交试剂盒。该生物正交试剂盒包括含有第二生物正交基团(触发基团)的生物正交试剂和能与所述生物正交试剂发生生物正交反应且能直接或间接产生第一生物正交基团(化学报告基团)的活化剂。其中，所述生物正交试剂选用上文所述的本发明实施例生物正交试剂，或所述活化剂选用上文所述的本发明实施例生物正交前体。
应当理解的是，当生物正交试剂选用上文所述的生物正交试剂时，选用的活化剂是能够与该生物正交试剂进行生物正交反应的，如但不仅为缩合成肟反应或狄尔斯-阿尔德尔环加成反应；当所述活化剂选用上文所述的生物正交前体时，选用的生物正交试剂是能够与该生物正交活化剂进行生物正交反应的，如但不仅为缩合成肟反应或狄尔斯-阿尔德尔环加成反应。
因此，在一实施例中，本发明实施例生物正交试剂盒(记为试剂盒1)中的活化剂选用上文所述的生物正交前体，当该生物正交活化剂与目标生物分子或结构基团发生碳-氢活化/脱氢偶联反应后，生成如上文所述的本发明实施例的活化生物分子，如上文结构通式C所述，此时，P2即是该目标生物分子或结构基团经脱氢后形成的生物分子片段或构件基团。
此时，该生物正交前体与目标生物分子或结构基团发生碳-氢活化/脱氢偶联反应后所形成的活化生物分子是利用所含的羟胺基团作为第一生物正交基团(化学报告基团)，那么在此基础上，该试剂盒1中的生物正交试剂选用非生物探针分子，且该非生物探针分子提供第二生物正交基团(触发基团)为醛羰基或酮羰基，也即是试剂盒1中的生物正交试剂选用含羰基的小分子光学探针。
这是因为，试剂盒1中的活化剂提供的羟胺基团作为第一生物正交基团时，该羟胺基团(未被保护)具有强α-效应，其能与生物正交试剂提供的醛羰基或酮羰基发生缩合生物正交反应(即缩合成肟反应)使其光学成像功能释放。其中，缩合成肟反应所用的催化剂可以是酸或苯胺。
因此，上述试剂盒1中作为活化剂的生物正交前体中羟胺基团在与待测生物分子或结构基团发生碳-氢活化/脱氢偶联反应过程中是被保护的，如果该生物正交前体中的羟胺基团为非保护的羟胺基团，即如上文所述的分子结构通式A中R1、R2相同为H，那么在碳-氢活化/脱氢偶联反应之前，应该对羟胺基团进行保护处理。在生物正交前体与待测生物分子或结构基团发生碳-氢活化/脱氢偶联反应之后所生成的生物正交活化剂在与生物正交试剂进行缩 合成肟反应之前，应该将被保护的羟胺基团进行脱保护处理，使得脱保护的羟胺基团作为第一正交基团的脱保护方法如上文所述，在此不再赘述。
另外，在一些具体实施例中，与上述试剂盒1中活化剂发生碳-氢活化/脱氢偶联反应的目标生物分子为但不限于蛋白质、核酸、或多糖，优选地为含不饱和键的生物分子，如含金属卟啉(metalloporphyrins)的血红素(heme)及其共生的血红蛋白(hemoglobin)、肌红蛋白(myoglobin)，不包含脂肪(unsaturated fat)，肝素双糖(heparin disaccharides)等。在上述试剂盒1中所述含第二生物正交触发基团的小分子光学探针生物正交试剂可以是如下含醛羰基或酮羰基的荧光探针，如荧光素类(fluorescein)、氟硼荧类(boron-dipyrromethene,BODIPY)、苯基取代吡唑啉(phenyl pyrazoline)类、香豆素(coumarin)类母核结构及其衍生物。在具体实施例中，荧光素类如下述结构式D1所示的荧光素，氟硼荧类如下述结构式D2所示的氟硼荧，苯基取代吡唑啉类如下述结构式D3所示的苯基取代吡唑啉，香豆素母核结构如下述结构式D4所示。
Figure PCTCN2015074897-appb-000011
基于上述试剂盒1的构成，该试剂盒1用于在生理环境中结合物质的化学工具的用途，或上文所述的本发明实施例生物正交前体与触发基团醛或酮羰基的缩合成肟反应作为用于在生理环境中结合物质的化学工具的用途。
在另一实施例中，本发明实施例生物正交试剂盒(记为试剂盒2)中的活化剂选用上文所述的本发明实施例生物正交前体，且所述生物正交前体与偶联修饰目标生物分子产生的所述第一生物正交基团为双烯基团，也即是利用上文所述的生物正交前体优选的是上文分子结构通式A所示的生物正交前体与修饰目标生物分子之间发生的碳-氢活化/偶联反应位点是生物分子的不饱和键上，优选地发生在碳-碳双键上，利用金属催化配位作用，将目标生物分子中的烯基碳与上文所述的本发明实施例生物正交前体优选的是上文分子结构通式A所示的生物正交前体中与定位基相邻的烯基碳发生原子经济地脱氢偶联，生成的修饰生物分子被标记上双烯结构，将双烯结构基团作为生物正交化学报告基团，使其在生理环境中能够在外源触发基团激活作用下实现生物分子靶点的识别。
具体地，在试剂盒2中，如上文分子结构通式A所示的生物正交前体与含有碳-碳双键的修饰目标生物分子之间发生的碳-氢活化/偶联反应位点是生物分子的碳-碳双键上，生成如上文分子结构通式C1、C3所示的含有双烯基团的生物正交活化剂。此时，该生物正交前体与含有不饱和键特别是碳-碳双键的目标生物分子或结构基团发生碳-氢活化/脱氢偶联反应后所产生的双烯基团至少作为第一生物正交基团(化学报告基团)，那么在此基础上，该试剂盒2中的生物正交试剂选用亲双烯体，作为含第二生物正交基团的非生物探针分子。
这是因为，试剂盒2中的产生双烯基团的活化剂提供的化学报告双烯基团与生物正交试剂中作为触发基团的亲二烯体基团之间诱导发生狄尔斯-阿尔德尔环加成反应(Diels–Alder Cycloaddition,DAc)。所述狄尔斯-阿尔德尔环加成反应作为生物正交工具来活化生物正交试剂，在生理环境中开启或关闭荧光，导致在生物靶点的荧光成像功能的释放。
该DAc反应发生在富电子的1,3-二烯和缺电子亲二烯体之间，通过[4π+2π]环加成生成六元碳环。狄尔斯-阿尔德尔环加成反应反应选择性高，与生物大分子兼容性好，可被用于多肽序列、小分子、脱氧核糖核酸(Deoxyribonucleic Acid,DNA)、蛋白质的修饰和缀合，并且水的存在对反应有明显的加速作用，反应速度相对于纯有机相可以加速几个数量级。由于亲二烯体不存在于任何生物分子中，DAc反应作为生物正交反应无需保护基就可以达到理想的化学选择性。
但是现有利用DAc反应作为生物正交反应技术只能通过与含双烯的活化剂反应安装第一生物正交的化学报告基团，这导致了现有活化剂选择受限、不易得、分子大、链长较长，而且安装的化学报告基团双烯与生物分子的靶点间隔远，降低了生物正交反应标记生物分子的靶向性、选择性和可信度，并且显著改变了生物分子的尺寸。而上述试剂盒2克服了双烯中的两个碳-碳双键都必须从活化剂中得来的技术缺陷，利用目标生物分子中大量存在的不饱和键，直接脱氢偶联本发明实施例生物正交前体，优选如上文分子结构通式A生物正交前体作为活化剂，在线产生至少两个共轭双键，所述共轭双键零距离地修饰在生物分子靶点上，生成如上文分子结构通式C1、C3所示的含有双烯基团的修饰生物分子，大大提高了DAc反应作为生物正交技术标记生物分子的靶向性、选择性和可信度，避免显著改变目标生物分子的大小。
因此，上述试剂盒2中作为活化剂的生物正交前体中羟胺基团可以是被保护或非保护的羟胺基团。那么其在与待测生物分子或结构基团发生碳-氢活化/脱氢偶联反应过程中也可以是被保护或非保护的，同样在生物正交前体与待测生物分子或结构基团发生碳-氢活化/脱氢偶联反应之后所生成的生物正交活化剂在与生物正交试剂进行DAc反应之前，所含的羟胺基也可以是被保护或非保护的。
另外，在一些具体实施例中，上述试剂盒2中亲双烯体的生物正交试剂所含第二生物正交触发基团为亲双烯基团如烯、炔、联稀、硫酮、亚磺酰、亚硝苯、叠氮等。具体的，该生物正交试剂选用如下结构式所示的但不仅仅限于乙烯芘(vinyl pyrene)、奎尼丁(quinidine)、乙烯苄基氟化硼络合二吡咯甲川(vinylbenzyl BODIPY)、螺[异苯并呋喃-1(3H),9'-[9H]呫吨]-3-1,5-乙烯基-3',6'-二羟基(vinyl fluorescein)、含马来酰亚胺的罗丹明B(maleimide-containing rhodamine B)等母核及其衍生物。
Figure PCTCN2015074897-appb-000012
当然，本领域的技术人员应清楚地知道试剂盒2中亲双烯体的上述生物正交试剂所述结构的拓展将远远超过本示例所给出的母核结构及其衍生物。
基于上述试剂盒2的构成，该试剂盒2含亲双烯基团的小分子光学探针生物正交试剂和含有双烯基团的活化剂修饰的生物分子之间的狄尔斯-阿尔德尔环加成反应作为生物正交反应，反应条件是在水相室温下进行，优选地所述反应不添加任何金属催化剂。
在再一实施例中，本发明实施例生物正交试剂盒(记为试剂盒3)中的生物正交试剂选用上文所述的本发明实施例生物正交试剂，优选的如分子结构通式B、B2所示生物正交试剂，在此生物正交试剂的前提下，该试剂盒3中的所述活化剂为能直接或间接产生所述第一生物正交基团为醛羰基或酮羰基的活化剂。因此，该试剂盒3中的生物正交试剂是利用所含的羟胺基团作为第二生物正交基团(触发基团)，那么在此基础上，该试剂盒3中的活化剂利用所诱导的醛羰基或酮羰基提供第一生物正交基团(化学报告基团)。因此，基于试剂盒3中的生物正交试剂与活化剂的设定，生物正交试剂与活化剂之间的反应原理与上文所述的试剂盒1的反应原理类似，也是利用羟胺基团(未被保护)具有强α-效应，其能与活化剂提供的醛羰基或酮羰基发生缩合生物正交反应(即缩合成肟反应)使其荧光成像功能的释放。具体的缩合成肟反应如同上文所述，在此不赘述。
那么同样，上述试剂盒3中，如果生物正交试剂中的羟胺基团被保护，则其与活化剂进行缩合成肟反应之前，应该将被保护的羟胺基团进行脱保护处理，使得脱保护的羟胺基团作为第一生物正交基团，脱保护方法如上文所述，在此不再赘述。如果生物正交试剂中的羟胺基团没有被保护，如上述分子结构通式B2所示生物正交试剂，则其与活化生物分子或片段直接可进行缩合成肟反应。
在一些具体实施例中，试剂盒3的含有醛羰基或酮羰基的活化剂可以选用活化二甲亚砜(activated DMSO)、高碘试剂(hypervalent iodine compounds)、柯林斯试剂(collins reagent)、四甲基哌啶氮氧化物(TEMPO)、四正丙基过钌酸铵/N-甲基吗啉-N-氧化物(TPAP/NMO)中的任一种。
在又一实施例中，本发明实施例生物正交试剂盒(记为试剂盒4)中的生物正交试剂选用上文所述的如分子结构通式B1、B3所示的本发明实施例生物正交试剂。在此生物正交试剂的前提下，该试剂盒4中的所述活化剂含有或诱导产生亲双烯体。因此，该试剂盒4中，生物正交试剂含有双烯基团，那么该生物正交试剂至少是利用所含的双烯基团作为第二生物正交基团(触发基团)，那么在此基础上，该试剂盒4中的活化剂所含的亲双烯基团作为第一生物正交基团(化学报告基团)。因此，基于试剂盒4中的生物正交试剂与活化剂的设定，生物正交试剂与活化剂之间的反应原理与上文所述的试剂盒2的反应原理类似，也是利用作为化学报告基团的双烯基团与活化剂中作为触发基团的亲二烯体基团之间发生狄尔斯-阿尔德尔环合加成反应。所述狄尔斯-阿尔德尔环加成反应作为生物正交工具来活化生物正交试剂，在生理环境中开启或关闭荧光，导致在生物靶点的荧光成像功能的释放。具体DAc如同上文所述，在此不赘述。
那么同样，上述试剂盒4中生物正交试剂所含的羟胺基团可以是被保护或非保护的羟胺基团。活化剂在与生物正交试剂进行DAc反应之前，所含的羟胺基也可以是被保护或非保护的。
在一些具体实施例中，试剂盒4中作为活化剂的亲双烯体可以选用马来酰亚胺(maleimide)衍生物、马来酸酐(maleic anhydride)衍生物、丙烯酸酯(acrylate)衍生物、苯醌(benzoquinone)衍生物、偶氮二甲酯(dimethyl azodicarboxylate)衍生物、二氧三唑(dioxotriazole)衍生物等中的任一种。
因此，上述本发明实施例用于生物标记的生物正交试剂盒由于含有上文所述的本发明生物正交试剂或生物正交前体，能快速高效地实现生物正交标记。
现结合实施例对本发明进行进一步详细说明。以下实施例仅仅用以进一步说明本发明的生物正交前体、生物正交试剂、生物正交活化剂、用于生物标记的生物正交试剂盒等方面，并不用于限定本发明。
实施例1
一种生物正交前体试剂N-(环己基-2-烯-1-基氧基)乙酰胺的制备：
将N-羟基邻苯二甲酰亚胺(1.1eq)，三苯基膦(1.5eq)溶于干燥的四氢呋喃中，然后向其中加入2-环己烯-1-醇(1eq)。将反应体系冷却至0℃，然后逐滴加入偶氮二甲酸二乙酯(1.5eq)。反应混合物恢复至室温，过夜搅拌后，将溶液浓缩，过柱得到2-(环己基-2-烯-1-氧基)异吲哚啉酮-1,3-二酮。
将2-(环己基-2-烯-1-基氧基)异吲哚啉酮-1,3-二酮(1.0eq)溶解在含有10％甲醇的氯仿(0.1M)中，然后向其中加入单水合肼(3.0eq)，混合物室温搅拌过夜，然后过滤，滤液浓缩，过柱；
将过柱获得的产物(1.0eq)溶解在乙醚(0.2M)中，冰浴条件下缓慢加入醋酸酐(2.0eq)，然后恢复至室温，搅拌3小时。将混合物浓缩，过柱得到相应的N-(环己-2-烯-1-基氧基)乙酰胺产物。
N-(环己基-2-烯-1-基氧基)乙酰胺制备的化学反应式如下：
Figure PCTCN2015074897-appb-000013
将制备的产物N-(环己基-2-烯-1-基氧基)乙酰胺进行核磁共振谱图(NMR)分析，其结果为：1H NMR(500MHz,CDCl3)δ8.58(s,1H),5.98(d,J＝17.8,1H),5.80(s,1H),4.31(d,J＝81.4,1H),2.18–1.96(m,3H),1.84(m,4H),1.79–1.48(m,2H)；13C NMR(126MHz,CDCl3)δ168.33,133.57,125.02,78.58,26.89,25.19,19.74,18.63。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为178.0838，与计算[C8H13NO2Na]+的分子量178.0844相符，该结果进一步证实了产物N-(环己基-2-烯-1-基氧基)乙酰胺分子结构正如上述分子结构。
实施例2
一种生物正交前体试剂苄基-5-(乙酰氨基)-5,6-二氢吡啶-1(2H)-羧酸的制备：
在封管中加入环氧丁烯(1eq)，烯丙胺(2eq)和0.4mL水，然后加热到100℃，反应6h。待反应结束后，直接旋干，得到粗产物1-(烯丙基氨基)丁基-3-烯-2-醇，直接用于下一步反应。
将上述产物溶解在二氯甲烷中，然后加入三乙胺(1.2eq),冷却至0℃，缓慢滴加氯甲酸苄酯(1eq)，恢复至室温，混合物在室温下进行反应。待原料转化完全，向体系中加入一点甲醇，然后旋干，过柱，得到苄基烯丙基(2-羟基丁基-3-烯-1-炔)氨基甲酸酯。
在室温下将上述产物溶解在二氯甲烷中，然后加入催化量的格拉布催化剂，保持反应体系浓度为0.01M，反应约10h,得到产物苄基-5-羟基-5,6-二氢吡啶-1(2H)-羧酸。
苄基-5-羟基-5,6-二氢吡啶-1(2H)-羧酸制备的化学反应式如下：
Figure PCTCN2015074897-appb-000014
由苄基-5-羟基-5,6-二氢吡啶-1(2H)-羧酸到苄基-5-(乙酰氨基)-5,6-二氢吡啶-1(2H)-羧酸的反应过程与实施例1类似，在此不再赘述，其反应化学式如下：
Figure PCTCN2015074897-appb-000015
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(500MHz,CDCl3)δ8.39(s,1H),7.29(d,J＝28.5Hz,5H),5.97(d,J＝24.0Hz,2H),5.20(dd,J＝40.2,12.8Hz,1H),5.08(d,J＝12.3Hz,1H),4.53–3.94(m,3H),3.74(d,J＝18.9Hz,1H),3.24(dd,J＝96.4,13.3Hz,1H),2.18–1.53(m,3H)；13C NMR(126MHz,CDCl3)δ168.12,156.31,136.32,131.01,128.59(2C),128.21(2C),127.86,123.19,74.47,67.60,44.02,43.03,19.74。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为313.1158，与计算[C15H18N2O4Na]+的分子量313.1165相符，该结果进一步证实了产物分子结构正如上述分子结构。
实施例3
一种生物正交前体试剂N-(环庚基-2-烯-1-基氧基)乙酰胺的制备:
将N-羟基邻苯二甲酰亚胺(1.1eq)，三苯基膦(1.5eq)溶于干燥的四氢呋喃中，然后向其中加入环庚-2-烯醇(1eq)。其余步骤与实施例1相同。
将制备的产物进行核磁共振谱图NMR分析，其结果为：1H NMR(400MHz,CDCl3)δ8.54(dd,J＝45.7,45.2Hz,1H),6.19–5.56(m,2H),4.86–4.23(m,1H),2.25–1.85(m,7H),1.73–1.49(m,4H)；13C NMR(126MHz,MeOD)δ168.64,133.27,130.94,84.78,30.99,27.98,26.37,26.26,18.05。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为192.0999，与计算[C9H15NO2Na]+的分子量192.0995相符，该结果进一步证实了产物分子结构为N-(环庚基-2-烯-1-基氧基)乙酰胺。
实施例4
一种生物正交前体试剂N-(环戊基-2-烯-1-基氧基)乙酰胺的制备:
将N-羟基邻苯二甲酰亚胺(1.1eq)，三苯基膦(1.5eq)溶于干燥的四氢呋喃中，然后向其中加入环戊-2-烯醇(1eq)。其余步骤与实施例1相同。
将制备的产物进行核磁共振谱图NMR分析，其结果为：1H NMR(500MHz,MeOD)δ6.18–6.14(m,1H),5.88–5.85(m,1H),5.01–4.97(m,1H),3.31(dt,J＝3.2,1.6Hz,1H),2.54–2.44(m,1H),2.34–2.26(m,1H),2.11(dddd,J＝14.2,8.8,7.6,5.2Hz,1H),1.95(ddt,J＝15.5,13.8,5.2Hz,1H),1.86(s,3H)；13C NMR(126MHz,MeOD)δ168.87,138.14,128.38,90.79,30.60,27.78,18.00。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为164.0687，与计算[C7H11NO2Na]+的分子量164.0682相符，该结果进一步证实了产物分子结构为N-(环戊基-2-烯-1-基氧基)乙酰胺。
实施例5
一种生物正交前体试剂与三氟甲基苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入3-三氟甲基苯乙烯(51.6mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率86％。
N-(环己基-2-烯-1-基氧基)乙酰胺与3-三氟甲基苯乙烯的脱氢偶联反应方程式如下：
Figure PCTCN2015074897-appb-000016
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.73(s,2H),7.53–7.41(m,3H),6.83(d,J＝16.3Hz,1H),6.37–6.15(m,1H),4.72(s,1H),2.29(m,1H),2.24(m,1H),2.22–2.10(m,1H),1.92(s,3H),1.88–1.76(m,1H),1.61(m,1H),1.56–1.42(m,1H)；13C NMR(75MHz,MeOD)δ168.86,139.33,136.63,133.31,131.39,130.52,129.11,128.88,126.18,125.76,122.88,122.51,76.00,25.94,25.22,18.27,16.24。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为348.1182，与计算[C17H18NO2F3Na,M+Na]+的分 子量348.1187相符，该结果进一步证实了产物分子结构为(E)-N-2-(3-三氟甲基苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例6
一种生物正交前体试剂与氟苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入3-氟苯乙烯(36.6mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率74％。
N-(环己基-2-烯-1-基氧基)乙酰胺与3-氟苯乙烯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000017
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ7.40–7.25(m,3H),7.20(d,J＝10.1Hz,1H),6.90(dt,J＝8.7,3.5Hz,1H),6.77(d,J＝16.3Hz,1H),6.19(m,1H),4.71(s,1H),2.37–2.12(m,3H),1.92(s,3H),1.89–1.77(m,1H),1.62(m,1H),1.49(m,1H)；13C NMR(101MHz,MeOD)δ168.86,163.23,140.89,136.06,133.36,130.82,129.71,126.15,121.97,113.04,111.97,76.10,25.91,25.28,18.26,16.27。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为298.1215，与计算[C16H18NO2FNa,M+Na]+的分子量298.1219相符，该结果进一步证实了产物分子结构为(E)-N-2-(3-氟苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例7
一种生物正交前体试剂与氯苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入4-氯苯乙烯(41.6mg，0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率85％。
N-(环己基-2-烯-1-基氧基)乙酰胺与4-氯苯乙烯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000018
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.44(d,J＝8.5Hz,2H),7.29(m,3H),6.73(d,J＝16.3Hz,1H),6.21–6.07(m,1H),4.70(s,1H),2.39–2.12(m,3H),1.91(s,3H),1.88–1.74(m,1H),1.61(m,1H),1.49(m,1H)；13C NMR(75MHz,MeOD)δ168.80,136.99,135.69,133.41,132.00,130.21,128.19,127.32,125.95,76.08,25.91,25.26,18.30,16.28。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为314.0917， 与计算[C16H18NO2ClNa,M+Na]+的分子量314.0924相符，该结果进一步证实了产物分子结构为(E)-N-2-(4-氯苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例8
一种生物正交前体试剂与甲氧基苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入4-甲氧基苯乙烯(40.3mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率66％。
N-(环己基-2-烯-1-基氧基)乙酰胺与4-甲氧基苯乙烯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000019
将制备的产物I-l进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ7.40(d,J＝8.7Hz,2H),7.22(d,J＝16.3Hz,1H),6.85(d,J＝8.8Hz,2H),6.60(d,J＝16.4Hz,1H),6.19–6.03(m,1H),4.70(s,1H),3.77(s,3H),2.34–2.25(m,1H),2.23(m,1H),2.20–2.13(m,1H),1.91(s,3H),1.86(m,1H),1.64–1.56(m,1H),1.48(m,1H)；13C NMR(101MHz,MeOD)δ168.78,159.04,133.74,133.59,130.88,127.40,127.11,126.75,113.59,76.27,54.31,25.83,25.38,2479,18.29,16.39。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为310.1413，与计算[C17H21NO3Na,M+Na]+的分子量310.1419相符，该结果进一步证实了产物分子结构为(E)-N-2-(4-甲氧基苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例9
一种生物正交前体试剂与叔丁基苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入4-叔丁基苯乙烯(48.1mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率77％。
N-(环己基-2-烯-1-基氧基)乙酰胺与4-叔丁基苯乙烯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000020
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.41(d,J＝8.5Hz,2H),7.33(d,J＝8.6Hz,2H),7.25(dd,J＝16.4,4.5Hz,1H),6.71(d,J＝16.3Hz,1H),6.20–6.08(m,1H),4.72(s,1H),2.37–2.12(m,3H),1.91(s,3H),1.90–1.79(m,1H),1.61(m,1H),1.56–1.42(m,1H),1.31(s,9H)；13C NMR(75MHz,MeOD)δ168.79,149.72,135.30,134.44,133.56,128.66,126.96,125.70,124.96,76.17,33.94,30.36,25.84,25.34,18.26, 16.34。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为336.1933，与计算[C20H27NO2Na,M+Na]+的分子量336.1939相符，该结果进一步证实了产物分子结构为(E)-N-2-(4-叔丁基苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例10
一种生物正交前体试剂与五氟苯乙烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入五氟苯乙烯(58.2mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率87％。
N-(环己基-2-烯-1-基氧基)乙酰胺与五氟苯乙烯的脱氢偶联反应方程式如下：
Figure PCTCN2015074897-appb-000021
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,DMSO)δ10.90(s,1H),7.35(d,J＝16.6Hz,1H),6.88(d,J＝16.6Hz,1H),6.28(s,1H),4.62(s,1H),2.40–2.06(m,3H),1.76(s,3H),1.71–1.61(m,1H),1.55(m,1H),1.37(m,1H)；13C NMR(75MHz,DMSO)δ167.01,146.12,142.86,139.96,139.29,136.01,133.91,113.58,112.07,74.22,26.44,25.13,20.00,16.54。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为370.0837，与计算[C16H14NO2F5Na,M+Na]+的分子量370.0842相符，该结果进一步证实了产物分子结构为(E)-N-2-(五氟苯乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例11
一种生物正交前体试剂与乙烯基萘的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入2-乙烯基萘(58.2mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率88％。
N-(环己基-2-烯-1-基氧基)乙酰胺与2-乙烯基萘的脱氢偶联反应方程式如下：
Figure PCTCN2015074897-appb-000022
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ7.85(s,1H),7.82–7.74(m,3H),7.70(d,J＝8.6Hz,1H),7.49(d,J＝16.4Hz,1H),7.46–7.34(m,2H),6.87(d,J＝16.3Hz,1H),6.17(m,1H),4.77(s,1H),2.29(m,1H),2.26(m,1H),2.23–2.11(m,1H),1.93(s,3H),1.90–1.80(m,1H),1.70–1.57(m,1H),1.56–1.43(m,1H)；13C NMR(101MHz,MeOD)δ168.87,135.69,135.21,133.92,133.62,132.91,129.87,127.64, 127.48,127.35,127.22,125.89,125.77,125.17,123.09,76.20,25.92,25.33,18.31,16.34。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为330.1467，与计算[C20H21NO2Na,M+Na]+的分子量330.1470相符，该结果进一步证实了产物分子结构为(E)-N-2-(2-萘乙烯基环己基-2-烯-1-基氧基)乙酰胺。
实施例12
一种生物正交前体试剂与乙烯基噻吩的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入2-乙烯基噻吩(33.1mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率89％。
N-(环己基-2-烯-1-基氧基)乙酰胺与2-乙烯基噻吩的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000023
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.50(d,J＝16.1Hz,1H),7.19(d,J＝5.1Hz,1H),7.04(d,J＝3.3Hz,1H),6.96(dd,J＝5.1,3.6Hz,1H),6.52(d,J＝16.1Hz,1H),6.23–6.01(m,1H),4.68(s,1H),2.50–2.11(m,3H),1.91(s,3H),1.88–1.74(m,1H),1.70–1.56(m,1H),1.56–1.40(m,1H).13C NMR(75MHz,MeOD)δ168.81,143.57,134.84,133.21,129.14,127.06,125.31,123.20,120.97,76.00,25.88,25.26,18.26,16.29。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为286.0874，与计算[C14H17NO2SNa,M+Na]+的分子量286.0878相符，该结果进一步证实了产物分子结构为(E)-N-2-(2-噻吩乙烯基环己-2-烯-1-基氧基)乙酰胺。
实施例13
一种生物正交前体试剂与甲基乙烯基噻唑的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入4-甲基-5-乙烯基噻唑(37.6mg，0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率81％。
N-(环己基-2-烯-1-基氧基)乙酰胺与4-甲基-5-乙烯基噻唑的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000024
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ8.68(s,1H),7.63(d,J＝15.9Hz,1H),6.46(d,J＝15.9Hz,1H),6.13(m,1H),4.76(s,1H),2.53(s,3H),2.20(m,3H),1.91(s,3H),1.81(m,1H),1.68–1.56(m,1H),1.56–1.41(m,1H)； 13C NMR(101MHz,MeOD)δ168.62,149.97,149.34,136.17,133.41,132.89,132.13,117.33,75.49,25.95,25.08,18.27,16.18,13.75。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为301.0962，与计算[C14H17NO2SNa,M+Na]+的分子量301.0987相符，该结果进一步证实了产物分子结构为(E)-N-(2-(2-(4-甲基噻唑-5-基)乙烯基)环己-2-烯-1-基氧基)乙酰胺。
实施例14
一种生物正交前体试剂与乙基乙烯基酮的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入乙基乙烯基酮(21.0mg，0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率65％。
N-(环己基-2-烯-1-基氧基)乙酰胺与乙基乙烯基酮的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000025
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ7.19(d,J＝16.0Hz,1H),7.05(d,J＝16.0Hz,1H),6.70–6.32(m,1H),4.62(s,1H),2.66(qd,J＝7.3,1.1Hz,2H),2.35(dt,J＝20.3,4.5Hz,1H),2.26(dd,J＝23.0,11.6Hz,2H),1.90(s,3H),1.89–1.78(m,1H),1.72–1.57(m,1H),1.50(tt,J＝14.2,3.2Hz,1H),1.10(t,J＝7.3Hz,3H)；13C NMR(101MHz,MeOD)δ203.12,168.85,144.54,143.45,132.74,124.63,75.46,48.25,48.04,47.83,47.61,47.40,47.19,46.97,34.02,26.43,24.99,18.22,15.93,7.25。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为260.1260，与计算[C13H19NO3Na,M+Na]+的分子量260.1263相符，该结果进一步证实了产物分子结构为(E)-N-((2-(3-羰基-1-烯-1-基戊基)环己基-2-烯-1-基)氧基)乙酰胺。
实施例15
一种生物正交前体试剂与烯丙醛的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙醛(16.8mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到，收率67％。
N-(环己基-2-烯-1-基氧基)乙酰胺与烯丙醛的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000026
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ9.52(d,J＝7.9Hz,1H),7.23(d,J＝15.8Hz,1H),6.87(dd,J＝15.7,7.9Hz,1H),6.74–6.56 (m,1H),4.63(s,1H),2.57–2.33(m,1H),2.34–2.20(m,2H),1.97–1.87(m,3H),1.86(s,1H),1.69–1.59(m,1H),1.53(ddd,J＝14.0,8.8,3.3Hz,1H)；13C NMR(101MHz,MeOD)δ195.51,168.86,154.91,146.03,132.99,127.81,75.31,26.53,24.95,18.14,15.84。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为232.0945，与计算[C11H15NO3Na,M+Na]+的分子量232.0950相符，该结果进一步证实了产物分子结构为(E)-N-((2-(3-羰基-1-烯-1-基丙基)环己基-2-烯-1-基)氧基)乙酰胺。
实施例16
一种生物正交前体试剂与辛烯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入1-辛烯(33.7mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再50℃下搅拌约48小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率30％。
N-(环己基-2-烯-1-基氧基)乙酰胺与1-辛烯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000027
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ6.18–6.04(m,1H),5.41(s,1H),4.90(s,1H),4.58(s,1H),2.36–2.10(m,5H),1.89(s,1H),1.90–1.81(m,3H),1.57(s,1H),1.43(dd,J＝16.1,5.3Hz,3H),1.32(d,J＝7.8Hz,6H),0.90(dd,J＝8.8,4.7Hz,3H)；13C NMR(101MHz,MeOD)δ168.60,147.10,134.32,130.46,110.29,76.90,33.75,31.51,28.85,28.36,25.57,25.54,22.31,18.16,16.02,13.01。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为288.1944，与计算[C16H27NO2Na,M+Na]+的分子量288.1939相符，该结果进一步证实了产物分子结构为(E)-N-(2-(1-烯-1-基辛基)环己基-2-烯-1-基氧基)乙酰胺。
实施例17
一种生物正交前体试剂与烯丙基甲酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基甲酯(25.8mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率89％。
N-(环己基-2-烯-1-基氧基)乙酰胺与烯丙基甲酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000028
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.24(d,J＝15.9Hz,1H),6.68(d,J＝15.9Hz,1H),6.49–6.44(m,1H),4.61(s,1H),3.73(s, 3H),2.39–2.29(m,1H),2.26–2.24(m,1H),2.21–2.20(m,1H),1.89(s,3H),1.85–1.74(m,1H),1.67–1.56(m,1H),1.54–1.44(m,1H)；13C NMR(75MHz,MeOD)δ168.79,168.62,145.85,143.85,132.44,116.59,75.31,50.55,26.29,24.97,18.24,15.93。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为262.1051，与计算[C12H17NO4Na,M+Na]+的分子量262.1055相符，该结果进一步证实了产物分子结构为(E)-甲基3-(6-(乙酰胺-基氧基)环己基-1-烯-1-基)丙烯酸酯。
实施例18
另一例生物正交前体试剂与烯丙基甲酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例2中所示的生物正交前体苄基-5-(乙酰氨基)-5,6-二氢吡啶-1(2H)-羧酸(29.0mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基甲酯(25.8mg，0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率80％。
苄基-5-(乙酰氨基)-5,6-二氢吡啶-1(2H)-羧酸与烯丙基甲酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000029
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.35(d,J＝8.1Hz,5H),7.28(d,J＝15.9Hz,1H),6.90(d,J＝15.9Hz,1H),6.41(d,J＝21.0Hz,1H),5.18(s,2H),4.73(s,1H),4.53(dd,J＝14.5,10.0Hz,2H),3.98–3.76(m,1H),3.73(s,3H),2.99(d,J＝14.6Hz,1H),1.83(d,J＝41.2Hz,3H)；13C NMR(75MHz,MeOD)δ169.32,168.17,156.16,143.51,137.53,136.52,130.92,128.23,127.85,127.56,118.72,73.95,67.26,50.68,43.80,41.95,18.27。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为397.1368，与计算[C19H22N2O6Na,M+Na]+的分子量397.1376相符，该结果进一步证实了产物分子结构为(E)-苄基5-(乙酰胺-基氧基)-4-(3-甲氧基-3-氧-1-烯-1-基)-5,6-二氢吡啶-1(2H)-羧酸酯。
实施例19
另一例生物正交前体试剂与烯丙基甲酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例4中所示的生物正交前体N-(环戊基-2-烯-1-基氧基)乙酰胺(16.9mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基甲酯(25.8mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率85％。
N-(环戊基-2-烯-1-基氧基)乙酰胺与烯丙基甲酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000030
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.43(d,J＝15.9Hz,1H),6.65–6.26(m,2H),5.35–5.05(m,1H),3.74(s,3H),2.75–2.56(m,1H),2.44(m,1H),2.21–2.13(m,2H),1.87(s,3H)；13C NMR(75MHz,MeOD)δ169.08,168.20,146.08,138.67,138.57,119.35,88.16,50.62,30.86,28.86,18.17。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为248.0895，与计算[C11H15NO4Na,M+Na]+的分子量248.0899相符，该结果进一步证实了产物分子结构为(E)-甲基3-(5-(乙酰胺-基氧基)环庚基-1-烯-1-基)丙烯酸酯。
实施例20
一种生物正交前体试剂与烯丙基乙酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基乙酯(55.9mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率84％。
N-(环己基-2-烯-1-基氧基)乙酰胺与烯丙基乙酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000031
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.24(d,J＝15.9Hz,1H),6.64(d,J＝15.9Hz,1H),6.57–6.28(m,1H),4.61(s,1H),4.19(q,J＝7.1Hz,2H),2.44–2.28(m,1H),2.28–2.23(m,1H),2.22–2.16(m,1H),1.89(s,3H),1.85–1.76(m,1H),1.70–1.56(m,1H),1.55–1.43(m,1H),1.29(t,J＝7.1Hz,3H)；13C NMR(75MHz,MeOD)δ168.81,168.19,145.66,143.70,132.45,116.93,75.31,59.93,26.27,24.98,18.19,15.94,13.25。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为276.1207，与计算[C13H19NO4Na,M+Na]+的分子量276.1212相符，该结果进一步证实了产物分子结构为(E)-乙基3-(6-(乙酰胺-基氧基)环己基-1-烯-1-基)丙烯酸酯。
实施例21
一种生物正交前体试剂与烯丙基叔丁酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基叔丁酯(38.5mg，0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率85％。
N-(环己基-2-烯-1-基氧基)乙酰胺与烯丙基叔丁酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000032
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(400MHz,MeOD)δ7.14(d,J＝15.9Hz,1H),6.48(d,J＝15.9Hz,1H),6.43(m,1H),4.59(s,1H),2.34(m,1H),2.24(m,1H),2.21(m,1H),1.89(s,3H),1.83(m,1H),1.61(m,1H),1.50(s,9H),1.45(m,1H)；13C NMR(101MHz,MeOD)δ168.83,167.64,144.75,143.11,132.40,118.55,79.90,75.42,27.09,26.23,25.05,18.18,15.98。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为304.1521，与计算[C15H23NO4Na,M+Na]+的分子量304.1525相符，该结果进一步证实了产物分子结构为(E)-叔丁基3-(6-(乙酰胺-基氧基)环己基-1-烯-1-基)丙烯酸酯。
实施例22
另一例生物正交前体试剂与烯丙基叔丁酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例3中所示的生物正交前体N-(环庚基-2-烯-1-基氧基)乙酰胺(16.9mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基叔丁酯(38.5mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率62％。
N-(环庚基-2-烯-1-基氧基)乙酰胺与烯丙基叔丁酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000033
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(500MHz,MeOD)δ7.19(d,J＝15.9Hz,1H),6.66–6.44(m,1H),5.95(d,J＝15.9Hz,1H),4.90(m,1H),2.70–2.51(m,1H),2.37–2.21(m,2H),2.11–2.02(m,1H),1.89–1.79(m,4H),1.78–1.68(m,1H),1.63–1.55(m,1H),1.48(m,10H)；13C NMR(126MHz,MeOD)δ168.66,167.27,147.63,147.02,138.18,116.74,79.98,78.99,28.37,27.97,26.99,26.08,23.93,18.04。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为318.1681，与计算[C16H25NO4Na,M+Na]+的分子量318.1676相符，该结果进一步证实了产物分子结构为(E)-叔丁基3-(7-(乙酰胺-基氧基)环庚基-1-烯-1-基)丙烯酸酯。
实施例23
一种生物正交前体试剂与烯丙基苄酯的脱氢偶联反应制备生物正交试剂片段：
在15ml的封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(15.5mg,0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,0.005mmol)。向封管中加入烯丙基苄酯(48.7mg,0.3mmol)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约6小时。反应结束后过滤，浓缩得到粗品。将粗品经柱层析纯化得到产物，收率93％。
N-(环己基-2-烯-1-基氧基)乙酰胺与烯丙基苄酯的脱氢偶联反应方程式如下:
Figure PCTCN2015074897-appb-000034
将制备的产物进行核磁共振谱图(NMR)分析，其结果为：1H NMR(300MHz,MeOD)δ7.46–7.28(m,5H),7.27(d,J＝12.7Hz,1H),6.70(d,J＝15.9Hz,1H),6.45(m,1H),5.18(s,2H),4.61(s,1H),2.29(m,1H),2.24(m,1H),2.19(m,1H),1.86(s,3H),1.84–1.74(m,1H),1.66–1.53(m,1H),1.54–1.39(m,1H)；13C NMR(75MHz,MeOD)δ168.84,167.88,146.17,144.08,136.43,132.43,128.13,127.82,127.73,116.61,75.29,65.70,26.29,24.97,18.19,15.91。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为338.1362，与计算[C18H21NO4Na,M+Na]+的分子量338.136相符，该结果进一步证实了产物分子结构为(E)-苄基3-(6-(乙酰胺-基氧基)环己基-1-烯-1-基)丙烯酸酯。
实施例24
一种生物正交活化剂与生物分子的脱氢偶联反应用以对生物分子安装化学报告基团：
向封管中加入实施例1中所示的生物正交活化剂N-(环己基-2-烯-1-基氧基)乙酰胺(3.7mg,0.024mmol)，氯化血红素的二甲基酯化合物(20.4mg,0.03mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(0.9mg,5mol％)和二甲基亚砜/水(0.5ml，20％DMSO)，滴加完后再室温下搅拌约8小时。反应结束后，用二氯甲烷进行稀释，转移到圆底烧瓶中，然后向其中加入0.3g硅胶，浓缩得到粗品。将粗品经柱层析纯化得到产物2(9.2mg)和3(2.2mg)，总收率55％，产物2和产物3的摩尔比为5：1。
生物正交活化剂N-(环己基-2-烯-1-基氧基)乙酰胺对铁卟啉IX的脱氢偶联选择性修饰反应方程式如下:
Figure PCTCN2015074897-appb-000035
进行高分辨质谱HRMS(ESI)分析，得到2的分子量其结果为797.2905，与计算[C44H47FeN5O6,M-Cl-]+的分子量797.2876相符，得到3的分子量其结果为950.3702，与计算[C44H47FeN5O6,M-Cl-]+的分子量950.3666相符，该结果进一步证实了活化剂对生物分子修饰后的产物分子结构，并且证实了反应可以选择性地进行生物分子的单靶点活化。
实施例25
一种生物正交活化剂与生物分子的脱氢偶联反应用以对生物分子安装化学报告基团：
向封管中加入实施例1中所示的生物正交活化剂N-(环己基-2-烯-1-基氧基)乙酰胺(11.6 mg,0.075mmol)，氯化血红素的二甲基酯化合物1(20.4mg,0.03mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(0.9mg,5mol％)和二甲基亚砜/水(0.5ml，20％DMSO)，滴加完后再室温下搅拌约14小时。其余步骤与实施例24相同。得到产物2(3mg)和3(10.6mg)，总收率48％，产物2和产3的摩尔比为1：3，该该结果进一步证实了活化剂对生物分子修饰后的产物分子结构，并且证实了反应可以选择性地进行生物分子的多靶点活化。
实施例26
一种生物正交前体试剂与氟硼荧类荧光分子的脱氢偶联反应用以对荧光分子安装触发基团制备生物正交试剂:
向封管中加入实施例1中所示的生物正交前体N-(环己基-2-烯-1-基氧基)乙酰胺(18.6mg,0.12mmol)和烯基化的氟化硼络合二吡咯甲川类底物(35mg，0.1mmol)，双(2-胺基-4,6-二羟基嘧啶)合醋酸钯钠盐(2.8mg,5mol％)和二甲基亚砜/水(1.0ml，20％DMSO)，滴加完后再室温下搅拌约14小时。反应结束后，用二氯甲烷进行稀释，转移到圆底烧瓶中，然后向其中加入0.3g硅胶，浓缩得到粗品。将粗品经柱层析纯化得到修饰后的氟化硼络合二吡咯甲川类荧光分子(35mg)，收率70％。
生物正交试剂前体N-(环己基-2-烯-1-基氧基)乙酰胺对氟化硼络合二吡咯甲川类荧光分子的脱氢偶联选择性修饰反应方程式如下:
Figure PCTCN2015074897-appb-000036
将制备的产物化合物进行核磁共振谱图(NMR)分析，其结果为:1H NMR(500MHz,CD2Cl2)δ8.04(s,1H),7.64(d,J＝7.3Hz,2H),7.57(d,J＝15.9Hz,1H),7.26(d,J＝8.2Hz,2H),6.85(d,J＝16.3Hz,1H),6.24(s,1H),6.04(s,2H),4.85(s,1H),2.53(s,6H),2.40–2.16(m,3H),1.95(s,3H),1.88–1.67(m,3H),1.48(s,6H)。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为526.2449，与计算[C29H32BF2N3O2Na,M+Na]+的分子量526.2453相符，该结果进一步证实了氟化硼络合二吡咯甲川类荧光分子已被修饰，修饰位点为氟化硼络合二吡咯甲川类荧光分子的稀基上，产生双烯，并且生物正交试剂前体中的乙酰胺被保存且被保护，其产生的双烯基团可作为第一生物正交的化学报告基团。
实施例27
生物正交试剂前体N-(环己基-2-烯-1-基氧基)乙酰胺修饰的氟化硼络合二吡咯甲川类荧光分子脱保护制备含氟化硼络合二吡咯甲川类荧光分子的生物正交试剂：
将实施例26中得到的生物正交试剂修饰的氟化硼络合二吡咯甲川类荧光分子(25mg，0.05mmol)溶于THF/H2O(0.2mL/0.2mL)，然后向其中加入CuCl2(6.6mg,0.05mmol)和HCl(5M,20μL)。室温下搅拌约36小时。加入饱和的碳酸氢钠进行中和，用二氯甲烷萃取三 次，收集有机层，然后用MgSO4进行干燥。最后加入0.3g硅胶，浓缩得到粗品。将粗品经柱层析纯化得到含脱保护羟胺结构的氟化硼络合二吡咯甲川类荧光分子(9.2mg)，收率40％。
修饰后氟化硼络合二吡咯甲川类荧光分子中生物正交片段中羟胺基团的脱保护反应方程式如下:
Figure PCTCN2015074897-appb-000037
将制备的产物化合物进行核磁共振谱图(NMR)分析，其结果为:1H NMR(500MHz,CD2Cl2)δ7.60(d,J＝8.2Hz,2H),7.26(d,J＝8.2Hz,2H),6.85–6.75(m,2H),6.16–6.14(m,1H),6.04(s,2H),4.48(s,1H),2.53(s,6H),2.40–2.11(m,3H),1.68(m,3H),1.48(s,6H)；13C NMR(126MHz,CD2Cl2)δ155.23,143.31,142.01,138.76,135.65,134.51,133.27,131.59,131.39,128.25,126.80,125.10,120.99,26.40,25.98,16.97,14.21。进行高分辨质谱HRMS(ESI)分析，得到分子量其结果为462.2492，与计算[C29H33BF2N3O2,M+H]+的分子量462.2528相符，该结果进一步证实了修饰氟化硼络合二吡咯甲川类荧光分子的羟胺基团已被脱保护，其产生的非保护羟胺基团可作为第一生物正交的化学报告基团。
实施例28
含实施例27所述修饰氟化硼络合二吡咯甲川类荧光分子的生物正交试剂和活化细胞的活化剂组成的生物正交试剂盒及其使用方法。
试剂盒组成：生物正交试剂，此处为实施例27所述修饰氟化硼络合二吡咯甲川类荧光分子或其衍生物；活化剂，此处为用于生物分子标签化的高碘酸盐或其类似物。
试剂盒使用方法：活化剂活化生物分子表面形成化学报告基团，活化后的生物分子再与生物正交试剂进行生物正交的缩合反应。
以Hela细胞为例说明生物正交试剂盒的使用方法：
细胞表面唾液酸的高碘酸盐氧化：Hela贴壁细胞先在培养皿中培养48小时，再用磷酸缓冲盐溶液(pH 7.4)洗涤三次，然后让其在冰浴条件下保存在含有1mM高碘酸钠的磷酸缓冲盐溶液(pH 7.4)中半小时，以室温下作为其对照试验。该氧化反应被1mM的甘油淬灭，然后再用磷酸缓冲盐溶液(pH 7.4)洗涤三次。
苯胺催化的缩合反应生成肟：Hela细胞浸没在含有5％胎牛血清、100μM实施例36所述修饰氟化硼络合二吡咯甲川类荧光分子、和10mM苯胺的磷酸缓冲盐溶液(pH 6.7)中，将其置于黑暗和冰浴条件下，通过摇床轻轻地搅动，然后用细胞胰蛋白酶化，离心，再用磷酸缓冲盐溶液(pH 7.4)洗涤三次。将细胞置于含有HaltTM蛋白酶抑制剂(Thermo)的RIPA裂解液中，保证浓度为1x107cells/mL。然后将该裂解物通过10％聚丙烯酰胺凝胶 电泳以检测相应的蛋白。通过Typhoon FLA 9500直接检测原位荧光。
如图2所示，经过10mM高碘酸钠处理之后，Hela细胞在100μM实施例29所述修饰氟化硼络合二吡咯甲川类荧光分子和10mM苯胺存在下孵育30到90分钟。30分钟后，荧光强度有明显的加强，60分钟后与不经过NaIO4处理相比，荧光强度有非常明显的改变。这些数据表明实施例27所述修饰氟化硼络合二吡咯甲川类荧光分子可以作为生物正交试剂通过苯胺催化下缩合反应用于标记含有醛的细胞表面。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种生物正交前体，其含有至少可作为偶联反应的定位基团和生物正交基团的多功能官能基团，其中，所述多功能官能基团为羟胺基团。
  2. 根据权利要求1所述的生物正交前体，其特征在于：其分子结构通式为下述(A)：
    Figure PCTCN2015074897-appb-100001
    所述A式中，R1、R2相同或不相同的为H、含有直链或支链的烷基、含有直链或支链的酰基、烷氧羰基中的任一种，R3表示H、烷基、烯基、芳基、杂芳基中的任一种，弧形所表示的结构为链状结构或环状结构。
  3. 根据权利要求2所述的生物正交前体，其特征在于：所述R1、R2表示含有直链或支链的酰基时，所述酰基选自乙酰基、苯甲酰基、苄酰基、特戊酰基、十二酰基、三氟乙酰基中的任一种或两种；或所述R1、R2表示烷氧羰基时，所述烷氧羰基选自叔丁氧羰基、苄氧羰基、烯丙氧羰基、乙氧羰基、三氯乙氧羰基、芴甲氧羰基中的任一种或两种；和/或
    所述R3表示烯基时，所述烯基选自乙烯基、丙烯基、异戊烯基、辛烯基、十二烯基、环戊烯基中的任一种；或
    所述R3表示芳基时，所述芳基选自苯环或其衍生物；或
    所述R3表示杂芳基时，所述杂芳基选自吡咯基、呋喃基、咪唑基、吡啶基、噻唑基、嘧啶基、噻吩基、吡唑基中的任一种。
  4. 一种生物正交试剂，由权利要求2或3所述的分子结构通式(A)所示
    的生物正交前体经脱氢偶联反应而成，其分子结构通式为下述(B)：
    Figure PCTCN2015074897-appb-100002
    所述B式中，P1为光学探针分子片段或结构基团。
  5. 根据权利要求4所述的生物正交试剂，其特征在于：所述P1是通过不饱和键与权利要求2或3所述的分子结构通式(A)所示的生物正交前体发生脱氢偶联反应连接至所述生物正交前体上，其中，所述不饱和键为双键、三键、醛羰基或酮羰基，且所述不饱和键为所述光学探针分子片段或结构基团的一部分。
  6. 根据权利要求5所述的生物正交试剂，其特征在于：所述不饱和键为碳-碳双键，所述生物正交试剂分子结构通式为下述(B1)：
    Figure PCTCN2015074897-appb-100003
    所述B1式中，P1’为光学探针分子片段或结构基团。
  7. 根据权利要求4-6任一所述的生物正交试剂，其特征在于：所述分子结构通式中的所述R1、R2均为H。
  8. 根据权利要求4-6任一所述的生物正交试剂，其特征在于：所述光学探针分子片段为荧光探针片段或比色探针片段。
  9. 一种生物正交活化剂，为权利要求2或3所述的分子结构通式为(A)所示的生物正交前体，所述生物正交活化剂与生物分子经脱氢偶联反应生成活化生物分子，其分子结构通式为下述(C)：
    Figure PCTCN2015074897-appb-100004
    所述C式中，P2表示为目标生物分子片段或构件基团。
  10. 根据权利要求9所述的生物正交活化剂，其特征在于：所述P2是通过不饱和键与权利要求2或3所述的分子结构通式为(A)所示的生物正交前体发生脱氢偶联反应活化生物分子，其中，所述不饱和键由C、O、S、N或P构成的不饱和键，其中，所述不饱和键为所述目标生物分子片段或构件基团的一部分。
  11. 根据权利要求10所述的生物正交活化剂，其特征在于：所连接的不饱和键由碳-碳构成的双键，所述活化的生物分子结构通式为下述(C1)：
    Figure PCTCN2015074897-appb-100005
    所述C1式中，P2’表示为生物分子片段或构件基团。
  12. 根据权利要求9-11任一所述的生物正交试剂，其特征在于：所述分子结构通式中的所述R1、R2均为H。
  13. 根据权利要求9-11任一所述的生物正交活化剂，其特征在于：所述生物分子片段或构件基团为蛋白质、核酸、多糖片段中的任一种。
  14. 一种用于生物标记的生物正交试剂盒，其包括含有第二生物正交基团的生物正交试剂和能与所述生物正交试剂发生生物正交反应且能直接或间接产 生第一生物正交基团的活化剂，其特征在于：所述生物正交试剂为如权利要求4-8任一所述的生物正交试剂；或所述活化剂为如权利要求1-3任一所述的生物正交前体或如权利要求9-13任一所述的生物正交活化剂。
  15. 根据权利要求14所述的用于生物标记的生物正交试剂盒，其特征在于：所述生物正交试剂为如权利要求5-6任一所述的含有双烯基团的生物正交试剂，所述活化剂为亲双烯体。
  16. 根据权利要求14所述的用于生物标记的生物正交试剂盒，其特征在于：所述生物正交试剂为如权利要求7所述的生物正交试剂，所述活化剂为能直接或间接产生所述第一生物正交基团为醛羰基或酮羰基的活化剂。
  17. 根据权利要求14所述的用于生物标记的生物正交试剂盒，其特征在于：所述活化剂为如权利要求1-3所述的生物正交前体或如权利要求9-13任一所述的生物正交活化剂，其产生的第一生物正交基团为羟胺基团；所述生物正交试剂为非生物探针分子，所述非生物探针分子含有的所述第二生物正交基团为醛羰基或酮羰基。
  18. 根据权利要求14所述的用于生物标记的生物正交试剂盒，其特征在于：所述生物正交活化剂为如权利要求1-3所述的生物正交前体或如权利要求9-13任一所述的生物正交活化剂，所述生物正交前体或所述生物正交活化剂与偶联修饰目标生物分子产生的所述第一生物正交基团为双烯基团；所述生物正交试剂为亲双烯体。
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