{"id":{"repo_id":"cornell","oai_identifier":"oai:ecommons.cornell.edu:1813/120795"},"canonical_url":"https://search.dev.ndltd.org/etd/cornell/oai:ecommons.cornell.edu:1813/120795","repository":{"repo_id":"cornell","name":"Cornell University","base_url":"https://ecommons.cornell.edu/server/oai/request"},"display":{"title":"Development and Evolution of ‘Ene’-Reductases for Selective Radical Chemistry","abstract":"Chemical synthesis over recent decades has made increasing use of enzymatic transformations, taking advantage of biological machinery to produce and evolve highly-specialized catalysts. Though natural enzymes can perform a broad array of chemical reactions with extraordinary selectivity and efficiency, those that have proven the most amenable to interfacing with traditional organic synthesis catalyze only a relatively small range of transformations. However, chemists have found that these robust scaffolds can be repurposed in the laboratory to engage in chemical mechanisms outside of their known roles. ‘Ene’-reductases are one such class of repurposable biocatalyst. In Nature, these enzymes use a flavin-based cofactor to afford hydrogenated analogs of activated olefins via a hydride-transfer mechanism. Using different substrates, the very same flavin cofactor can be engaged in single-electron transfer events for radical initiation, offering the capability to generate high-energy open-shell intermediates in the controlled environment of the enzyme active site. This feature has been used to effect various radical-mediated transformations, offering exceptional selectivity for these otherwise challenging to control species. Furthermore, this approach can be expanded to reactions that initiate from electronically-excited states accessed via photoexcitation of an enzyme-flavin-substrate complex. Described herein are efforts made to apply these reaction design principles to new selective radical reactions catalyzed by ‘ene’-reductases. In each of these cases, the ‘ene’-reductase is responsible for initiating, controlling, and terminating the radical intermediate, generating new carbon-carbon bonds and building molecular complexity. First, we apply directed evolution to develop an ‘ene’-reductase capable of enantiocontrol over the carbon-carbon bond forming event in the hydroalkylation of olefins with dichloroacetamides. This mode of stereocontrol is otherwise inaccessible to these enzymes, but through modification of residues in the enzyme active site, we have developed a catalyst for the highly asymmetric synthesis of valuable α-chloroamide structures. Second, we demonstrate that the classes of radical precursors tolerated by these radical hydroalkylations can be broadened to include chloromethylpyridines, offering access to enantioenriched structures bearing azine motifs which are desirable for the synthesis of pharmaceutically-relevant molecules. Furthermore, we describe efforts to evolve an ‘ene’-reductase that can accept pyridinemethanols, tuning the enzyme environment to enable cleavage of strong carbon-oxygen bonds. Third, we demonstrate that non-natural reactivity in ‘ene’-reductases can be employed alongside native reactivity of imine reductases to produce 2,5-disubstituted piperidines from α-haloketones and allyl amines in a one-pot two-enzyme cascade reaction, enabling a convergent and highly-stereoselective preparation of these unsaturated heterocycles. Lastly, a collection of methods by which ‘ene’-reductases can be used to initiate radical intermediates oxidatively, and preliminary studies demonstrating that enzyme control is essential for these activation modes. In one of these cases, a modification of the approach is applied to a different family of enzymes—pyridoxal-5’-phosphate-dependent threonine aldolases and transaminases. In broadening the versatility of biocatalytic radical chemistry, these studies demonstrate that enzymatic methods can offer new approaches to synthetic chemistry and offer extraordinary degrees of control over reactions once thought to be exclusive to small-molecule catalysis. Through a mechanism-focused approach, these reactions can be developed to offer access to different types of compounds, previously-inaccessible modes of stereocontrol, and unprecedented activation pathways.","abstract_html":"Chemical synthesis over recent decades has made increasing use of enzymatic transformations, taking advantage of biological machinery to produce and evolve highly-specialized catalysts. Though natural enzymes can perform a broad array of chemical reactions with extraordinary selectivity and efficiency, those that have proven the most amenable to interfacing with traditional organic synthesis catalyze only a relatively small range of transformations. However, chemists have found that these robust scaffolds can be repurposed in the laboratory to engage in chemical mechanisms outside of their known roles. ‘Ene’-reductases are one such class of repurposable biocatalyst. In Nature, these enzymes use a flavin-based cofactor to afford hydrogenated analogs of activated olefins via a hydride-transfer mechanism. Using different substrates, the very same flavin cofactor can be engaged in single-electron transfer events for radical initiation, offering the capability to generate high-energy open-shell intermediates in the controlled environment of the enzyme active site. This feature has been used to effect various radical-mediated transformations, offering exceptional selectivity for these otherwise challenging to control species. Furthermore, this approach can be expanded to reactions that initiate from electronically-excited states accessed via photoexcitation of an enzyme-flavin-substrate complex. Described herein are efforts made to apply these reaction design principles to new selective radical reactions catalyzed by ‘ene’-reductases. In each of these cases, the ‘ene’-reductase is responsible for initiating, controlling, and terminating the radical intermediate, generating new carbon-carbon bonds and building molecular complexity. First, we apply directed evolution to develop an ‘ene’-reductase capable of enantiocontrol over the carbon-carbon bond forming event in the hydroalkylation of olefins with dichloroacetamides. This mode of stereocontrol is otherwise inaccessible to these enzymes, but through modification of residues in the enzyme active site, we have developed a catalyst for the highly asymmetric synthesis of valuable α-chloroamide structures. Second, we demonstrate that the classes of radical precursors tolerated by these radical hydroalkylations can be broadened to include chloromethylpyridines, offering access to enantioenriched structures bearing azine motifs which are desirable for the synthesis of pharmaceutically-relevant molecules. Furthermore, we describe efforts to evolve an ‘ene’-reductase that can accept pyridinemethanols, tuning the enzyme environment to enable cleavage of strong carbon-oxygen bonds. Third, we demonstrate that non-natural reactivity in ‘ene’-reductases can be employed alongside native reactivity of imine reductases to produce 2,5-disubstituted piperidines from α-haloketones and allyl amines in a one-pot two-enzyme cascade reaction, enabling a convergent and highly-stereoselective preparation of these unsaturated heterocycles. Lastly, a collection of methods by which ‘ene’-reductases can be used to initiate radical intermediates oxidatively, and preliminary studies demonstrating that enzyme control is essential for these activation modes. In one of these cases, a modification of the approach is applied to a different family of enzymes—pyridoxal-5’-phosphate-dependent threonine aldolases and transaminases. In broadening the versatility of biocatalytic radical chemistry, these studies demonstrate that enzymatic methods can offer new approaches to synthetic chemistry and offer extraordinary degrees of control over reactions once thought to be exclusive to small-molecule catalysis. Through a mechanism-focused approach, these reactions can be developed to offer access to different types of compounds, previously-inaccessible modes of stereocontrol, and unprecedented activation pathways.","abstract_has_math":false,"creators":["Bender, Sophie"],"institution":"Cornell University","degree_name":"Ph. D., Chemistry and Chemical Biology","degree_level":"Doctor of Philosophy","degree_discipline":"Chemistry and Chemical Biology","degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":["Hyster, Todd","Milner, Phillip"],"year":2025,"date_issued":"2025-08","date_published":"2025-08","updated_at":"2026-07-24T01:49:00Z","subjects":["asymmetric catalysis","biocatalysis","photoenzymes","radical chemistry"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7298/bz5n-4s89"],"render_values":[{"text":"https://doi.org/10.7298/bz5n-4s89","href":"https://doi.org/10.7298/bz5n-4s89","code":true}]},{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["ProQuest Submission ID: 15016","ProQuest Publication ID: 32043955"],"render_values":[{"text":"ProQuest Submission ID: 15016","href":null,"code":true},{"text":"ProQuest Publication ID: 32043955","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/1813/120795","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Hyster, Todd","Milner, Phillip"]},{"key":"dc:creator","label":"Author","values":["Bender, Sophie"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-04-02T18:49:55Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-08"]},{"key":"dc:type","label":"Dc Type","values":["dissertation or thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry and Chemical Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Doctor of Philosophy"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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Though natural enzymes can perform a broad array of chemical reactions with extraordinary selectivity and efficiency, those that have proven the most amenable to interfacing with traditional organic synthesis catalyze only a relatively small range of transformations. However, chemists have found that these robust scaffolds can be repurposed in the laboratory to engage in chemical mechanisms outside of their known roles. ‘Ene’-reductases are one such class of repurposable biocatalyst. In Nature, these enzymes use a flavin-based cofactor to afford hydrogenated analogs of activated olefins via a hydride-transfer mechanism. Using different substrates, the very same flavin cofactor can be engaged in single-electron transfer events for radical initiation, offering the capability to generate high-energy open-shell intermediates in the controlled environment of the enzyme active site. This feature has been used to effect various radical-mediated transformations, offering exceptional selectivity for these otherwise challenging to control species. Furthermore, this approach can be expanded to reactions that initiate from electronically-excited states accessed via photoexcitation of an enzyme-flavin-substrate complex. Described herein are efforts made to apply these reaction design principles to new selective radical reactions catalyzed by ‘ene’-reductases. In each of these cases, the ‘ene’-reductase is responsible for initiating, controlling, and terminating the radical intermediate, generating new carbon-carbon bonds and building molecular complexity. First, we apply directed evolution to develop an ‘ene’-reductase capable of enantiocontrol over the carbon-carbon bond forming event in the hydroalkylation of olefins with dichloroacetamides. This mode of stereocontrol is otherwise inaccessible to these enzymes, but through modification of residues in the enzyme active site, we have developed a catalyst for the highly asymmetric synthesis of valuable α-chloroamide structures. Second, we demonstrate that the classes of radical precursors tolerated by these radical hydroalkylations can be broadened to include chloromethylpyridines, offering access to enantioenriched structures bearing azine motifs which are desirable for the synthesis of pharmaceutically-relevant molecules. Furthermore, we describe efforts to evolve an ‘ene’-reductase that can accept pyridinemethanols, tuning the enzyme environment to enable cleavage of strong carbon-oxygen bonds. Third, we demonstrate that non-natural reactivity in ‘ene’-reductases can be employed alongside native reactivity of imine reductases to produce 2,5-disubstituted piperidines from α-haloketones and allyl amines in a one-pot two-enzyme cascade reaction, enabling a convergent and highly-stereoselective preparation of these unsaturated heterocycles. Lastly, a collection of methods by which ‘ene’-reductases can be used to initiate radical intermediates oxidatively, and preliminary studies demonstrating that enzyme control is essential for these activation modes. In one of these cases, a modification of the approach is applied to a different family of enzymes—pyridoxal-5’-phosphate-dependent threonine aldolases and transaminases. In broadening the versatility of biocatalytic radical chemistry, these studies demonstrate that enzymatic methods can offer new approaches to synthetic chemistry and offer extraordinary degrees of control over reactions once thought to be exclusive to small-molecule catalysis. Through a mechanism-focused approach, these reactions can be developed to offer access to different types of compounds, previously-inaccessible modes of stereocontrol, and unprecedented activation pathways."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Development and Evolution of ‘Ene’-Reductases for Selective Radical Chemistry"]}]}],"canonical_facts":{"dc:contributor.committeemember":["Hyster, Todd","Milner, Phillip"],"dc:creator":["Bender, Sophie"],"dc:date.accessioned":["2026-04-02T18:49:55Z"],"dc:date.issued":["2025-08"],"dc:description":["488 pages"],"dc:description.abstract":["Chemical synthesis over recent decades has made increasing use of enzymatic transformations, taking advantage of biological machinery to produce and evolve highly-specialized catalysts. Though natural enzymes can perform a broad array of chemical reactions with extraordinary selectivity and efficiency, those that have proven the most amenable to interfacing with traditional organic synthesis catalyze only a relatively small range of transformations. However, chemists have found that these robust scaffolds can be repurposed in the laboratory to engage in chemical mechanisms outside of their known roles. ‘Ene’-reductases are one such class of repurposable biocatalyst. In Nature, these enzymes use a flavin-based cofactor to afford hydrogenated analogs of activated olefins via a hydride-transfer mechanism. Using different substrates, the very same flavin cofactor can be engaged in single-electron transfer events for radical initiation, offering the capability to generate high-energy open-shell intermediates in the controlled environment of the enzyme active site. This feature has been used to effect various radical-mediated transformations, offering exceptional selectivity for these otherwise challenging to control species. Furthermore, this approach can be expanded to reactions that initiate from electronically-excited states accessed via photoexcitation of an enzyme-flavin-substrate complex. Described herein are efforts made to apply these reaction design principles to new selective radical reactions catalyzed by ‘ene’-reductases. In each of these cases, the ‘ene’-reductase is responsible for initiating, controlling, and terminating the radical intermediate, generating new carbon-carbon bonds and building molecular complexity. First, we apply directed evolution to develop an ‘ene’-reductase capable of enantiocontrol over the carbon-carbon bond forming event in the hydroalkylation of olefins with dichloroacetamides. This mode of stereocontrol is otherwise inaccessible to these enzymes, but through modification of residues in the enzyme active site, we have developed a catalyst for the highly asymmetric synthesis of valuable α-chloroamide structures. Second, we demonstrate that the classes of radical precursors tolerated by these radical hydroalkylations can be broadened to include chloromethylpyridines, offering access to enantioenriched structures bearing azine motifs which are desirable for the synthesis of pharmaceutically-relevant molecules. Furthermore, we describe efforts to evolve an ‘ene’-reductase that can accept pyridinemethanols, tuning the enzyme environment to enable cleavage of strong carbon-oxygen bonds. Third, we demonstrate that non-natural reactivity in ‘ene’-reductases can be employed alongside native reactivity of imine reductases to produce 2,5-disubstituted piperidines from α-haloketones and allyl amines in a one-pot two-enzyme cascade reaction, enabling a convergent and highly-stereoselective preparation of these unsaturated heterocycles. Lastly, a collection of methods by which ‘ene’-reductases can be used to initiate radical intermediates oxidatively, and preliminary studies demonstrating that enzyme control is essential for these activation modes. In one of these cases, a modification of the approach is applied to a different family of enzymes—pyridoxal-5’-phosphate-dependent threonine aldolases and transaminases. In broadening the versatility of biocatalytic radical chemistry, these studies demonstrate that enzymatic methods can offer new approaches to synthetic chemistry and offer extraordinary degrees of control over reactions once thought to be exclusive to small-molecule catalysis. Through a mechanism-focused approach, these reactions can be developed to offer access to different types of compounds, previously-inaccessible modes of stereocontrol, and unprecedented activation pathways."],"dc:format.mimetype":["application/pdf"],"dc:identifier.doi":["https://doi.org/10.7298/bz5n-4s89"],"dc:identifier.other":["ProQuest Submission ID: 15016","ProQuest Publication ID: 32043955"],"dc:identifier.uri":["https://hdl.handle.net/1813/120795"],"dc:language.iso":["en"],"dc:subject":["asymmetric catalysis","biocatalysis","photoenzymes","radical chemistry"],"dc:title":["Development and Evolution of ‘Ene’-Reductases for Selective Radical Chemistry"],"dc:type":["dissertation or thesis"],"thesis:degree_discipline":["Chemistry and Chemical Biology"],"thesis:degree_level":["Doctor of Philosophy"],"thesis:degree_name":["Ph. D., Chemistry and Chemical Biology"],"thesis:institution_name":["Cornell University"]},"updated_at":"2026-07-24T01:49:00Z"}