Periodic Reporting for period 1 - Co-ARMs (Development of new-to-nature reactions under the concept of synergistic catalysis through the design of novel cobalt-based artificial metalloenzymes.)
Reporting period: 2022-09-01 to 2025-02-28
Enzyme catalysis has experienced a remarkable renaissance in research over the past decade. Its growing popularity is related to easy reaction set-up, environmentally friendly reaction conditions, such as aqueous media as solvent and non-cryogenic temperatures, and its renewable, biodegradable nature. However, its broader application in synthetic chemistry, especially at industrial level, has been constrained due to a series of limitations related to a relatively narrow reaction scope, substrate-dependence issues, and restricted product variability. Only recently, with the advancement of directed evolution and modern genetic engineering techniques, the discovery and development of biocatalysts for new-to-nature reactions started to flourish.
With the aim to develop and expand new methods under the framework of sustainable chemistry, expanding the scope of homogeneous catalytic transformations into the realm of enzymes is imperative. The expansion of homogeneous catalytic reactions into the realm of enzymes is an absolute necessity to develop sustainable chemistry. A promising approach to this objective is the creation of artificial metalloenzymes (ARMs), engineered metalloproteins which cannot be found in nature and are capable to catalyze abiological chemical reactions. A transformation of high relevance to medicinal, agrochemical, and pharmaceutical chemistry is allylic and benzylic functionalization. These transformations can generate structural frameworks common to many biologically active molecules but currently lack natural enzymatic counterparts. The Co-ARMs (Cobalt-containing ARtificial Metalloenzymes) project aims to fill this gap by designing novel ARMs relying on the unique reactivity of cobalt in mediating these challenging transformations.
To push the boundaries of ArM-catalyzed reactivity, we propose a synergistic catalysis strategy that combines two abiological catalytic elements within a single protein scaffold: (1) an unnatural amino acid (uAA) capable of performing nucleophilic activation (i.e. enamine formation); and (2) a synthetic cobalt complex designed to activate electrophilic allylic or benzylic substrates. This dual-site approach would allow the design of a powerful, versatile catalyst capable of unlocking new chemical transformations in a sustainable and selective manner.
With the aim to develop and expand new methods under the framework of sustainable chemistry, expanding the scope of homogeneous catalytic transformations into the realm of enzymes is imperative. The expansion of homogeneous catalytic reactions into the realm of enzymes is an absolute necessity to develop sustainable chemistry. A promising approach to this objective is the creation of artificial metalloenzymes (ARMs), engineered metalloproteins which cannot be found in nature and are capable to catalyze abiological chemical reactions. A transformation of high relevance to medicinal, agrochemical, and pharmaceutical chemistry is allylic and benzylic functionalization. These transformations can generate structural frameworks common to many biologically active molecules but currently lack natural enzymatic counterparts. The Co-ARMs (Cobalt-containing ARtificial Metalloenzymes) project aims to fill this gap by designing novel ARMs relying on the unique reactivity of cobalt in mediating these challenging transformations.
To push the boundaries of ArM-catalyzed reactivity, we propose a synergistic catalysis strategy that combines two abiological catalytic elements within a single protein scaffold: (1) an unnatural amino acid (uAA) capable of performing nucleophilic activation (i.e. enamine formation); and (2) a synthetic cobalt complex designed to activate electrophilic allylic or benzylic substrates. This dual-site approach would allow the design of a powerful, versatile catalyst capable of unlocking new chemical transformations in a sustainable and selective manner.
Within our enzyme design work package, we successfully expressed a protein host harboring the uAA para-aminophenylalanine (pAF), following established protocols. However, in our hands, this designer protein did not demonstrate the expected nucleophilic activation via enamine formation. To overcome this obstacle, we embarked in the design and genetic incorporation development of novel uAA’s bearing cyclic secondary amines, which are known to promote enamine chemistry. However, this task proved technically challenging, and no successful candidates were obtained
In parallel, within our cofactor development work package, we synthesize a small library of transition metal complexes with phenanthroline derivatives, including various cobalt-containing species. These were evaluated with a panel of allylic electrophilic substrates under different conditions. Yet, the desired allylic functionalization was not observed.
At this stage, as our initial objectives faced substantial challenges to be achieved, likely due to the incompatibility of the targeted transformation with aqueous media, we reassessed our proposed plan and decided to pivot towards late-transition metal catalysis, whose reaction conditions were more compatible with our biocatalytic platform proposal. With the aim of achieving allylic or benzylic functionalization, we adjusted our objective: developing a gold-mediated benzylic alcohol formation., a structural motif present in drug candidates with, i.e. immunosuppressant activity. Moreover, we set our attention to developing an asymmetric version of this transformation by taking advantage of the chiral environment given by our protein host. Inspired by in-house preliminary work, we explored the coordination of gold via canonical and non-canonical amino acid residues. Upon reaction optimization, these systems successfully gave chiral secondary alcohols in a wide range of yield and enantioselectivities. One of these systems was further refined under a directed evolution campaign to improve its catalytic performance in terms of both activity and selectivity.
The outcomes of this project were presented in six international congresses. The work on uAA contributed to the publication of two open-access articles in internationally recognized high impact journals.
In parallel, within our cofactor development work package, we synthesize a small library of transition metal complexes with phenanthroline derivatives, including various cobalt-containing species. These were evaluated with a panel of allylic electrophilic substrates under different conditions. Yet, the desired allylic functionalization was not observed.
At this stage, as our initial objectives faced substantial challenges to be achieved, likely due to the incompatibility of the targeted transformation with aqueous media, we reassessed our proposed plan and decided to pivot towards late-transition metal catalysis, whose reaction conditions were more compatible with our biocatalytic platform proposal. With the aim of achieving allylic or benzylic functionalization, we adjusted our objective: developing a gold-mediated benzylic alcohol formation., a structural motif present in drug candidates with, i.e. immunosuppressant activity. Moreover, we set our attention to developing an asymmetric version of this transformation by taking advantage of the chiral environment given by our protein host. Inspired by in-house preliminary work, we explored the coordination of gold via canonical and non-canonical amino acid residues. Upon reaction optimization, these systems successfully gave chiral secondary alcohols in a wide range of yield and enantioselectivities. One of these systems was further refined under a directed evolution campaign to improve its catalytic performance in terms of both activity and selectivity.
The outcomes of this project were presented in six international congresses. The work on uAA contributed to the publication of two open-access articles in internationally recognized high impact journals.
The redirection of our objectives toward the exploration of late-transition metal catalysis lead us to the development of a novel class of artificial metalloenzymes for benzylic alcohol formation, a challenging and unexplored area in biocatalysis with high potential for the medicinal chemistry industry. The results from this project pave the way for further development and expansion of this concept to other metal cofactors and reaction types.