Periodic Reporting for period 1 - BIO-LIGHT (Photoexcitation for New-to-Nature Enzymatic Reactions)
Reporting period: 2021-08-01 to 2023-07-31
• What is the problem/issue being addressed?
Despite the changing face of chemistry, enantioselective synthesis (the ability to generate chiral molecules in a controlled fashion) continues to play a centrally important role. New reactivity concepts and strategies are needed to address the increasingly complex synthetic problems being posed by Nature, medicine, and materials. Biocatalysis has a central role in the transition towards a more sustainable stereocontrolled chemistry. In spite of its potential, natural enzymes use only a relatively small section of ‘reaction space’. This implies that only a limited number of stereocontrolled reaction classes, which are characterized by a similar reaction mechanism, can be effectively promoted. For example, a natural enzyme co-opted for new chemistry has the limitation that the new reactivity must be quite closely related to the naturally established catalytic function. The main aim of the BIO-LIGHT project is to identify new strategies that allow enzymes to catalyse new-to-nature asymmetric reactions, thus expanding the reactivity boundaries of biocatalysis. To achieve this goal, BIO-LIGHT combined biocatalysis with visible light photocatalysis, a modern strategy of chemical reactivity which offers a potent way to sustainably build complex organic frameworks. The effective combination of these two strategies was exemplified for the development of asymmetric synthetic strategies based on chiral enantiopure radical precursors a long lasting challenge in organic chemistry due to the intrinsic configurational instability and fast racemization of prochiral radicals. This finding underscores the active site's ability to transfer stereochemical information from the chiral radical precursor into the product, effectively preventing racemization of prochiral radicals. The resulting ‘memory of chirality’ scenario is a rarity in enantioselective radical chemistry
• Why is it important for society?
The project contributes to the excellence of basic science in the EU in the field of methodology or the preparation of biorelevant compounds. On a more extended timescale, to scientific innovation, positively impacting society and the economy through the development of a new photobiocatalytic platform versatile enough to enable the advancement of other unconventional stereocontrolled radical functionalization processes
• What are the overall objectives?
The main goal is therefore to access non-natural enzyme activities via photoexcitation. Specifically, the potential of specific native intermediates in the active sites of existing enzymes to reach an excited state upon light absorption and enable new-to nature asymmetric radical-mediated reactions has been demonstrated. Visible light serves to elicit mechanistic divergence within enzymes with an established ground-state reactivity, thus allowing them to promote completely different processes than those for which they evolved. This approach provides unexplored possibilities for developing stereoselective processes driven by light that cannot be realized using thermal activation. This provides advances in the development of more responsible and sustainable stereoselective synthetic methods while strengthening the chemistry toolbox to better face the challenges of modern organic chemistry.
Despite the changing face of chemistry, enantioselective synthesis (the ability to generate chiral molecules in a controlled fashion) continues to play a centrally important role. New reactivity concepts and strategies are needed to address the increasingly complex synthetic problems being posed by Nature, medicine, and materials. Biocatalysis has a central role in the transition towards a more sustainable stereocontrolled chemistry. In spite of its potential, natural enzymes use only a relatively small section of ‘reaction space’. This implies that only a limited number of stereocontrolled reaction classes, which are characterized by a similar reaction mechanism, can be effectively promoted. For example, a natural enzyme co-opted for new chemistry has the limitation that the new reactivity must be quite closely related to the naturally established catalytic function. The main aim of the BIO-LIGHT project is to identify new strategies that allow enzymes to catalyse new-to-nature asymmetric reactions, thus expanding the reactivity boundaries of biocatalysis. To achieve this goal, BIO-LIGHT combined biocatalysis with visible light photocatalysis, a modern strategy of chemical reactivity which offers a potent way to sustainably build complex organic frameworks. The effective combination of these two strategies was exemplified for the development of asymmetric synthetic strategies based on chiral enantiopure radical precursors a long lasting challenge in organic chemistry due to the intrinsic configurational instability and fast racemization of prochiral radicals. This finding underscores the active site's ability to transfer stereochemical information from the chiral radical precursor into the product, effectively preventing racemization of prochiral radicals. The resulting ‘memory of chirality’ scenario is a rarity in enantioselective radical chemistry
• Why is it important for society?
The project contributes to the excellence of basic science in the EU in the field of methodology or the preparation of biorelevant compounds. On a more extended timescale, to scientific innovation, positively impacting society and the economy through the development of a new photobiocatalytic platform versatile enough to enable the advancement of other unconventional stereocontrolled radical functionalization processes
• What are the overall objectives?
The main goal is therefore to access non-natural enzyme activities via photoexcitation. Specifically, the potential of specific native intermediates in the active sites of existing enzymes to reach an excited state upon light absorption and enable new-to nature asymmetric radical-mediated reactions has been demonstrated. Visible light serves to elicit mechanistic divergence within enzymes with an established ground-state reactivity, thus allowing them to promote completely different processes than those for which they evolved. This approach provides unexplored possibilities for developing stereoselective processes driven by light that cannot be realized using thermal activation. This provides advances in the development of more responsible and sustainable stereoselective synthetic methods while strengthening the chemistry toolbox to better face the challenges of modern organic chemistry.
During the fellowship new enzymatic cascade reactions were developed based on a genetically modified 4-oxalocrotonate tautomerase (4-OT) enzyme. The 4-OT enzyme could promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. This was achieved by utilizing its ability to form both enamines and iminium ions and combine their mechanisms of catalysis in complex sequences. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences. Having established the formation of such photoreactive intermediates (i.e. enamine and iminium ion) by the catalytic machinery of some enzymes we then developed artificial photoenzymes that use a fundamentally novel mechanism of excitation. Specifically, we harnessed the ability of iminium ions, transiently generated from enals within the active site of an engineered class I aldolase, to absorb violet light and function as single-electron oxidants.
We have shown that activation of chiral carboxylic acids, followed by decarboxylation, generates two radicals that undergo stereospecific cross-coupling, yielding products with two stereocenters Using the appropriate enantiopure chiral substrate, the desired diastereisomeric product was selectively obtained with complete enantiocontrol. This finding underscores the active site's ability to transfer stereochemical information from the chiral radical precursor into the product, effectively preventing racemization of prochiral radicals, which is a very challenging target for small molecule catalysis.
We have shown that activation of chiral carboxylic acids, followed by decarboxylation, generates two radicals that undergo stereospecific cross-coupling, yielding products with two stereocenters Using the appropriate enantiopure chiral substrate, the desired diastereisomeric product was selectively obtained with complete enantiocontrol. This finding underscores the active site's ability to transfer stereochemical information from the chiral radical precursor into the product, effectively preventing racemization of prochiral radicals, which is a very challenging target for small molecule catalysis.
The results of the project have significant implications positively impacting the fields of biocatalysis and photochemistry, while stimulating further research development. For example, the developed enzymatic cascade processes demonstrate that biocatalysis can match and even surpass in efficiency the potential of organocascade catalysis while offering a rare example of the use of a single enzyme to advance the rapidly developing field of enzymatic cascade catalysis. On this side, the development of artificial photoenzymes based on the photoexcitation of enzyme-bound intermediates shows how biocatalysis can expand the catalytic boundaries of conventional small-molecule catalysts. For example, a longstanding limitation in asymmetric radical chemistry arises from the inability to use chiral enantiopure radical precursors for asymmetric synthesis, as their stereochemical information is erased immediately after radical formation. The project successfully addressed this challenge by introducing a photoenzyme that preserves the stereochemical information encoded within a chiral sp3-hybridized carbon radical precursor. The resulting ‘memory of chirality’ scenario is a rarity in enantioselective radical chemistry.
Moreover, we have developed an artificial photodecarboxylase using a strategy that fundamentally diverges from the cofactor-based approach, which is currently the state of the art. This novel photoenzymatic strategy is versatile enough to enable the advancement of other unconventional stereocontrolled radical functionalization processes. On this basis, our findings have the potential to immediately impact the scientific community.
Moreover, we have developed an artificial photodecarboxylase using a strategy that fundamentally diverges from the cofactor-based approach, which is currently the state of the art. This novel photoenzymatic strategy is versatile enough to enable the advancement of other unconventional stereocontrolled radical functionalization processes. On this basis, our findings have the potential to immediately impact the scientific community.