RESEARCH HIGHLIGHTS + From Caltech's Resnick Fellows

Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry

Christopher Prier, PhD

Resnick Prize Postdoctoral Fellow

Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

Global Significance Biocatalysis – the use of enzymes in chemical synthesis – has emerged as a powerful technology for the clean, green, and highly efficient production of the complex chemicals desired by society. The worldwide demand for chemical products (including material goods, fuels, and pharmaceuticals) is continually increasing, compelling us to produce these materials with the absolute minimum consumption of resources and energy, while generating as little waste as possible. This challenge requires the implementation of creative new strategies in chemical synthesis. Enzymatic processes, in contrast to many conventional chemical processes, typically proceed in benign solvents under non-energy intensive conditions (ambient temperature and pressure). Biocatalysis typically avoids the use of toxic metals, and often shortens synthetic routes, leading to less costly and less resource-intensive overall production schemes. Biocatalysis thus holds much promise as an environmentally benign and green technology.

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Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

Project Summary Enzymes (nature’s catalysts) have evolved over millennia into extremely efficient catalysts for their given natural functions. Although we might wish to use enzymes for our own purposes in the production of valuable chemicals, our needs often differ greatly from those of nature – in particular, for many of the reactions we desire, no natural enzymes exist.

Synthetic chemists have developed catalysts for a broad range of reactions that are completely absent in biology. Taking these reactions as inspiration, and using chemical intuition based on mechanism or structure as a guide, we have introduced some of these non-biological reactions into enzymatic catalysis (an approach we term “chemomimetic biocatalysis”).

Expanding the utility of biocatalysis thus requires developing new enzymes having functions that are absent from nature’s catalytic repertoire. We have engineered cytochrome P450s (iron heme-dependent enzymes found in all kingdoms of life) to perform nitrene transfer, a class of reactions that introduce nitrogen into organic substrates.

In doing so, we create enzymes with new catalytic capabilities while also in many cases improving on the efficiencies and selectivities of the chemical catalysts that inspired them.

In particular, we have developed an approach for the P450-promoted synthesis of valuable allylic amines, via nitrene transfer and rearrangement steps that are both absent from nature. As nitrogen-containing compounds are ubiquitous in pharmaceutical and agrochemical products, the efficient enzymatic synthesis of these molecules has significant potential to contribute to the greening of chemical manufacturing.

This project draws from the fields of organic chemistry, biochemistry, protein engineering, and structural biology, and is enabled by the methods of directed evolution.

Figure 1: Cytochrome P450s are iron heme-dependent enzymes present in all kingdoms of life. The protein is shown in green, iron in orange, heme in red, and certain active-site residues in yellow.

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Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

Potential Impact The development of enzymatic catalysts for nitrene transfer has the potential to impact the manufacture of amine-containing compounds, particularly in the fine chemical and pharmaceutical fields. These processes may remove toxic metals from manufacturing routes, reduce costs, and reduce the amount of waste generated. For instance, when a biocatalytic process was implemented for the synthesis of Januvia, an anti-diabetic compound, waste was reduced by 19% compared to an alternative, highly-optimized precious metal-catalyzed process. Demonstrating how to exploit existing enzymes for non-natural reactions may also inspire and guide further efforts to introduce new functions into biology.

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Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

The Science We developed an enzymatic route to chiral allylic amines, a class of nitrogen-containing compounds that are biologically active, valuable synthetic intermediates, and also difficult to produce using existing biocatalytic methods. We adopted a strategy for their synthesis involving an enzyme-catalyzed nitrene transfer followed by a rearrangement reaction – neither step is part of any known natural biological pathway. Certain cytochrome P450s having altered ligation to the iron center, and which we term “cytochrome P411s”, are especially adept at nitrene transfer. In our reaction design, a P411 variant first reacts with an azide reagent to give a key metal nitrenoid intermediate (Figure 2). In the nitrene transfer step, a sulfide intercepts this intermediate, accepting the nitrogen species from the enzyme. The rearrangement step then forges a new carbon–nitrogen bond and delivers the desired allylic amine. In initial studies, certain P411 variants were found capable of promoting this reaction, but only at very low levels of activity. Directed evolution via iterative cycles of gene mutagenesis and screening was then employed, first to obtain enzyme variants capable of accepting the large allylic sulfide substrates, and then to enhance the new activity to useful levels (Figure 3). We identified five beneficial mutations, yielding a P411 variant having greater than 200-fold improved activity for the desired reaction compared to the initial variant.

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Figure 2: Strategy for P411-promoted synthesis of chiral allylic amines.

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Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

The Science

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P-I263F P-I263F P-I263F P-I263F P-I263F P-I263F P-I263F V87A V87A V87A V87A A328V V87A A328V A328V A328V A328V A268G A268G A268G (P-3) % yield TTN A82I A82L (P-4) (P-5)

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Figure 3: Directed evolution of a P411 enzyme for the synthesis of allylic amines.

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Cytochrome P450-Catalyzed Nitrene Transfer: A Platform for Green Amination Chemistry Christopher Prier, PhD

Key Results We have successfully executed our reaction design, and have evolved an enzyme capable of delivering allylic amines in high yield and efficiency (up to 79% yield with greater than 6,000 turnovers) – far exceeding the turnover numbers achievable with chemical catalysts for this transformation. The catalysts also perform highly selective sulfimidation reactions (up to 98% enantioselectivity), which may also be synthetically valuable.

Future Steps The development of commercially applicable enzymes for nitrene transfer will require further evolution to improve their activities. Certain promising strategies to do so require the screening of very large enzyme libraries (>1,000 clones in a single round of evolution). To enable such an endeavor, we developed a highthroughput screen for activity based on production of a colored byproduct, and we are using this method to evolve highly active enzymes for nitrene transfer. Finally, we will explore these catalysts for other non-natural functions, including valuable transformations such as the direct amination of C–H bonds.

Publications •

“Chemomimetic Biocatalysis: Exploiting the Synthetic Potential of Cofactor-Dependent Enzymes to Create New Catalysts,” C. K. Prier, F. H. Arnold, Journal of the American Chemical Society, 2015, 137, 13992.

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