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 efficiency 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 specific 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 efficient 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.

Rotamer-free protein sequence design based on deep learning and self-consistency

“…Computational protein design brings the capability of inventing de novo proteins [1,2] to fulfill various structural and functional needs from therapeutics [3,4] to bio-catalysis [5,6]. One of the general problems to be solved in computational protein design is inverse protein folding, which is to select amino acid sequences that autonomously fold into a given backbone target.…”

Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates

“…S2-S3) with only limited catalytic solutions [24][25][26][27][28][29] . There are natural enzymes that catalyze direct hydroaminations of internal alkenes, yet, due to their underlying mechanism they depend on activated internal alkenes such as a,b unsaturated carboxylic acids 41,42 . As internal unactivated alkenes are easily accessible and ketones are substrates in many chemical transformations using enzymes 21,23,[43][44][45][46] or small molecule catalysts 47,48 , many new synthetic routes can be envisioned using evolved ketone synthases.…”

Enhancing lactose recognition of a key enzyme in 2′‐fucosyllactose synthesis: α‐1,2‐fucosyltransferase

“…These sites could have some complex potential negative effects on the execution of the catalytic function, in large part by affecting the spatial arrangement of the surrounding amino acid side chains to induce changes in the microenvironment near the substrate pocket. 45 Hydrogen bonds in proteins are typically found in networks and play integral roles in protein folding, molecular recognition, catalysis, and allostery. [46][47][48] Hydrogen bonds can provide greater positional restraints than individual hydrophobic interactions.…”