Development of a versatile and efficient C–N lyase platform for asymmetric hydroamination via computational enzyme redesign

Abstract

Although C-N bonds are ubiquitous in natural products, pharmaceuticals, and agrochemicals, biocatalysts that forge these bonds with high atom e ciency and enantioselectivity have primarily been limited to a few select enzymes. In particular, the use of ammonia lyases has emerged as a powerful strategy to access C-N bond formation through hydroamination reactions, which has no counterpart in traditional synthetic chemistry. However, the broad utility of ammonia lyases is rather restricted due to their narrow synthetic scope, and the conjugate addition of a matrix of nucleophilic donors to electrophilic acceptors remains a longstanding challenge. Herein, we report the computational redesign of aspartase, a highly speci c ammonia lyase, to yield C-N lyases with unprecedented cross-compatibility of nonnative nucleophiles and electrophiles. A wide range of noncanonical amino acids (ncAAs) are afforded with excellent conversion (up to 99%), regioselectivity > 99%, and product enantiomeric excess > 99%, and the process is scalable under industrially relevant protocols (demonstrated in kilogram-scale synthesis). Furthermore, the redesigned enzymes can be facilely integrated in cascade reactions, as we demonstrated the synthesis of β-lactams with different substitution patterns at the N-1 and C-4 positions in a one-pot reaction. This versatile and e cient C-N lyase platform supports the preparation of diverse libraries of ncAAs and their derivatives and will present opportunities in medicinal chemistry and synthetic biology.

Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics

“…A structure‐guided redesign of aspartase from Bacillus sp. YM55‐1 significantly enhanced the substrate scope towards non‐native nucleophiles and electrophiles [20,21] …”

“…[20] Recently, the computer-aided redesign of AspB resulted in novel CÀ N lyases with cross-compatibility of nonnative nucleophiles and electrophiles enabling the production of a wide range of unnatural amino acids with excellent conversion, regioselectivity and enantiotopic selectivity at industrially relevant scale. [21] Because enzyme immobilization may increase the range of conditions where the enzyme can be applied, and also may enhance enzyme activity, selectivity or specificity, [22] it is understandable that immobilization of the industrially relevant AALs has been carried out with several methods already. The first approaches captured AAL expressing whole cells enabling the synthesis of Asp due to the high half-life of the immobilized whole-cell biocatalysts.…”

“…Furthermore, engineering of AspB enabled the redesigned mutants to catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic β‐amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds [20] . Recently, the computer‐aided redesign of AspB resulted in novel C−N lyases with cross‐compatibility of non‐native nucleophiles and electrophiles enabling the production of a wide range of unnatural amino acids with excellent conversion, regioselectivity and enantiotopic selectivity at industrially relevant scale [21] …”

Selective Biocatalytic Defunctionalization of Raw Materials

“…Among the various enzymes involved in formally removing nitrogen from amino acids, the amino acid lyases are straightforward to use in N ‐defunctionalizations, as they are the only ones that do not require oxygen, an external nicotinamide adenine dinucleotide (NAD + ) cofactor, or an amine acceptor [148] . The reversible elimination of nitrogen from l ‐amino acids, in the form of ammonia, is catalyzed by l ‐amino acid ammonia lyases, which have attracted much interest for applications in the reverse direction [149,150] . On the other hand, making available selected l ‐amino acids, α,ω‐dicarboxylic acids, and α,β‐unsaturated carboxylic acids from l ‐amino acids may not only provide opportunities for valorizing biobased raw materials, [148,151] but also for N ‐defunctionalization applications (see Scheme 11).…”

“…[148] The reversible elimination of nitrogen from l-amino acids, in the form of ammonia, is catalyzed by l-amino acid ammonia lyases, which have attracted much interest for applications in the reverse direction. [149,150] On the other hand, making available selected l-amino acids, α,ω-dicarboxylic acids, and α,β-unsaturated carboxylic acids from l-amino acids may not only provide opportunities for valorizing biobased raw materials, [148,151] but also for N-defunctionalization applications (see Scheme 11). Aromatic amino acid ammonia lyases are valuable for the sustainable preparation of phenylpropanoids, for example, in the synthesis of trans-cinnamic acid and p-coumaric acid, [152] also as starting reaction in a biocatalytic reaction cascade in one-pot to valuable chiral chemicals.…”