Tetrazine-based bio-orthogonal coupling reagents and methods
AssigneeUniversity of Delaware
InventorsJoseph Michael Fox, Melissa Blackman, Maksim Royzen, Ni Yan
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Abstract
Coupling reactions, suitable for use in organic or aqueous media, are performed by contacting a 1,2,4,5-tetrazine with a dienophile. The dienophile may be covalently bonded to a protein, and the coupling reaction may be performed in biological media such as those containing cells or cell lysates. The reactions may be performed in the presence of primary amines, thiols, acetylenes, azides, phosphines, and products of Staudinger and/or Sharpless-Huisgen reactions Novel 3-substituted cyclopropene compounds and trans-cyclooctenes are exemplary dienophiles for these reactions.
Claims30
A method of performing a coupling reaction, comprising contacting a 1,2,4,5-tetrazine with a dienophile in an organic or aqueous medium comprising at least one species selected from the group consisting of primary amines, thiols, Staudinger reactants, Staudinger adducts, Sharpless-Huisgen reactants, and Sharpless-Huisgen adducts;
- said contacting performed under conditions sufficient to convert at least 50% of the 1,2,4,5-tetrazine or the dienophile to a Diels-Alder adduct of the 1,2,4,5-tetrazine with the dienophile.
A method of performing a coupling reaction, comprising contacting a 1,2,4,5-tetrazine with a dienophile in an organic or aqueous medium;
- said contacting performed under conditions sufficient to convert at least 50% of the 1,2,4,5-tetrazine or the dienophile to a Diels-Alder adduct of the 1,2,4,5-tetrazine with the dienophile, wherein the dienophile is covalently bonded to a protein.
A method of performing a coupling reaction, comprising contacting a 1,2,4,5-tetrazine selected from the group consisting of compounds according to formulas (4), (22), (23), (30), (31), (32), (33) and (34)
- with a substituted cyclopropene selected from the group consisting of compounds according to formulas (7), (8) and (27)
- in an organic or aqueous medium under conditions sufficient to convert at least 50% of the 1,2,4,5-tetrazine or the substituted cyclopropene to a Diels-Alder adduct of the 1,2,4,5-tetrazine with the substituted cyclopropene.
A method of performing a coupling reaction, comprising contacting a 1,2,4,5-tetrazine selected from the group consisting of compounds according to formulas (4), (22), (23), (30), (31), (32), (33) and (34)
- with trans-cyclooctene or a substituted trans-cyclooctene in an organic or aqueous medium under conditions sufficient to convert at least 50% of the 1,2,4,5-tetrazine, the trans-cyclooctene, or the substituted trans-cyclooctene to a Diels-Alder adduct of the 1,2,4,5-tetrazine with the trans-cyclooctene or the substituted trans-cyclooctene.
A method of preparing a substituted or unsubstituted trans-cyclooctene, comprising the steps of
- (a) exposing a solution of the cis-isomer of the substituted or unsubstituted trans-cyclooctene to ultraviolet radiation, thereby forming a solution comprising the substituted or unsubstituted trans-cyclooctene;
- (b) contacting the solution formed in step (a) with AgNO3 supported on a stationary phase, thereby binding at least a portion of the substituted or unsubstituted trans-cyclooctene to the stationary phase and producing a solution comprising the cis-isomer and a reduced amount of the substituted or unsubstituted trans-cyclooctene;
- and
- (c) freeing the substituted or unsubstituted trans-cyclooctene from the stationary phase.
A method of preparing cycloprop-2-enecarboxylic acid, comprising the steps of
- (a) contacting a mixture comprising ethyl diazoacetate with acetylene in the presence of a rhodium catalyst to form an intermediate mixture;
- (b) contacting the intermediate mixture with an alkali metal hydroxide to form an alkali metal salt of cycloprop-2-enecarboxylic acid;
- and
- (c) contacting the alkali metal salt with a strong acid to form cycloprop-2-enecarboxylic acid.
A method of preparing a compound according to formula (7)
- comprising contacting cycloprop-2-enecarboxylic acid with triethylamine and either pivaloyl chloride or 1-adamantyl chloride, and subsequently contacting the resulting mixture with lithium chloride, phenyloxazolidinone and 4-dimethylamino pyridine.
A method of preparing a compound according to formula (8)
- comprising contacting cycloprop-2-enecarboxylic acid with triethylamine and either pivaloyl chloride or 1-adamantyl chloride, and subsequently contacting the resulting mixture with lithium chloride, oxazolidinone and 4-dimethylamino pyridine.
A method of preparing a compound according to formula (27)
- comprising contacting cycloprop-2-enecarboxylic acid with triethylamine and either pivaloyl chloride or 1-adamantyl chloride, and subsequently contacting the resulting mixture with lithium chloride, 2-benzoxazolinone and 4-dimethylamino pyridine.
A 1,2,4,5-tetrazine selected from the group consisting of compounds according to formulas (4), (22), (23), (30), (31), (32), (33) and (34)
A 3-substituted cyclopropene selected from the following compounds
A trans-cyclooctene selected from the following compounds
A method of quantitative analysis comprising the steps of exposing an adduct of a 1,2,4,5-tetrazine with a dienophile to UV radiation, and measuring either the fluorescent emission or UV absorption of the sample.
Dependencies are inferred from the claim text.