Methods for inducing bioorthogonal reactivity
Inventors
Fox, Joseph M. • Thorpe, Colin • TALLON, Amanda M. • Xie, Yixin • Trout, William • CANTREL, Ashlyn S. • ROSENBERGER, Julia E.
Assignees
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Publication Number
US-11485987-B2
Publication Date
2022-11-01
Expiration Date
Abstract
A method for catalytically converting a dihydrotetrazine 1 into a tetrazine 2, wherein the dihydrotetrazine 1 comprises a first R group and a second R group, wherein the first R group is a substituted or unsubstituted aryl, heteroaryl, alkyl, alkenyl, alkynyl, carbonyl, or heteroatom-containing group, and the second R group is selected from the group consisting of H and substituted or unsubstituted aryl, heteroaryl, alkyl, alkenyl, alkynyl, carbonyl, and heteroatom-containing groups; wherein the method comprises (a) providing the dihydrotetrazine 1 in a reaction medium, and (b) adding an enzyme as a catalyst and an oxidant to the reaction medium, whereby the dihydrotetrazine 1 is converted to the tetrazine 2.
Core Innovation
A method provides a dihydrotetrazine 1 in a reaction medium, wherein the dihydrotetrazine 1 comprises a first R group and a second R group. The first R group is selected from substituted or unsubstituted aryl, heteroaryl, alkyl, alkenyl, alkynyl, carbonyl, or heteroatom-containing groups, and the second R group is selected from H, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl-containing groups, and substituted or unsubstituted heteroatom-containing groups. The dihydrotetrazine 1 is catalytically converted to a tetrazine 2.
An enzyme is added as a catalyst to the reaction medium, and the reaction medium comprises an oxidant. The enzyme is horseradish peroxidase (HRP) or an ascorbate peroxidase, and the ascorbate peroxidase consists of an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1. The enzymatic oxidation framework converts the dihydrotetrazine to the corresponding tetrazine.
The disclosure further includes conjugatable aryl ether/aryl oxyacrylic-acid derivatives and β-aryloxyacrylamide derivatives, including conjugates involving dihydrotetrazines and tetrazines linked to prodrugs. It also describes APEX-catalyzed oxidation of dihydrotetrazine/tetrazine-caged fluorogenic and prodrug molecules, with phenol release including 4-nitrophenol and umbelliferone, and refers to APEX2 variants identified as APEX2 F41A.
Claims Coverage
The consolidated claim coverage includes one independent claim directed to catalytic enzyme/oxidant conversion of a structurally defined dihydrotetrazine to a tetrazine, with enzyme selection between HRP and ascorbate peroxidase constrained by sequence identity to SEQ ID NO: 1. The inventive features center on the substrate scaffold, the enzyme catalyst, the oxidant, and the ascorbate peroxidase identity limitation.
Dihydrotetrazine to tetrazine conversion with enzyme and oxidant
Providing a dihydrotetrazine 1 in a reaction medium, adding an enzyme as a catalyst, wherein the reaction medium comprises an oxidant, whereby the dihydrotetrazine 1 is catalytically converted to a tetrazine 2, wherein the enzyme is horseradish peroxidase (HRP) or an ascorbate peroxidase that consists of an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1.
Substituent-defined dihydrotetrazine scaffold
The dihydrotetrazine 1 comprises a first R group and a second R group, wherein the first R group is a substituted or unsubstituted aryl, heteroaryl, alkyl, alkenyl, alkynyl, carbonyl, or heteroatom-containing group, and the second R group is selected from H, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl containing groups, and substituted or unsubstituted heteroatom-containing groups.
Overall, the claim coverage centers on enzyme-catalyzed oxidation of a substituent-defined dihydrotetrazine 1 into a tetrazine 2 using an oxidant, with catalyst selection between HRP and ascorbate peroxidase constrained by identity to SEQ ID NO: 1.
Stated Advantages
Not explicitly described in patent.
Documented Applications
APEX-catalyzed oxidation of dihydrotetrazine/tetrazine-caged fluorogenic and prodrug molecules to release phenols, including 4-nitrophenol and umbelliferone [procedural detail omitted for safety].
Oxidation and characterization studies for tetrazine/dihydrotetrazine-related compounds, including enzymatic HRP oxidation and catalytic photooxidation monitored by absorbance.
Photooxidation studies using LEDs and catalytic photosensitizers including methylene blue, fluorescein, and rose bengal.
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