R.B. Leveson-Gower

Short peptide-based amyloid nanotubes were able to demonstrate enantioselective covalent catalysis by exploiting chiral binding pockets with multiple solvent exposed residues. To achieve this, lysine was used for reversible imine formation, leucine for hydrophobic binding surface, and imidazole for the hydrolytic cleavage of the substrates.

Abstract

Extant enzymes with precisely arranged multiple residues in their three-dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide-based nanotubes that could exploit multiple solvent-exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double-site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide-based folds and argues their possible role in the emergence of homochiral chemical inventory.

Nature, Published online: 01 November 2023; doi:10.1038/s41586-023-06613-4

A new type of transformation converting a heteroaromatic carbon atom into a nitrogen atom, turning quinolines into quinazolines to enable manipulation of molecular properties, is reported.

Atroposelective metathesis catalyzed by artificial enzymes in aqueous solution would provide an attractive and sustainable route to drug molecules and other compounds of interest. We demonstrate that this is possible using artificial metalloenzymes harboring a ruthenium cofactor.

Abstract

Atropisomers – separable conformers that arise from restricted single-bond rotation – are frequently encountered in medicinal chemistry. However, preparing such compounds with the desired configuration can be challenging. Herein, we present a biocatalytic strategy for achieving atroposelective synthesis relying on artificial metalloenzymes (ArMs). Based on the biotin-streptavidin technology, we constructed ruthenium-bearing ArMs capable of producing atropisomeric binaphthalene compounds through ring-closing metathesis in aqueous media. Further, we show that atroposelectivity can be fine-tuned by engineering two close-lying amino acid residues within the streptavidin host protein. The resulting ArMs promote product formation with enantiomeric ratios of up to 81 : 19, while small-molecule catalysts for atroposelective metathesis under aqueous reaction conditions are yet unknown. This study represents the first demonstration that stereoselective metathesis can be achieved by an artificial metalloenzyme.

Aldolases are prodigious C-C bond forming enzymes, but their reactivity has only been extended past activated carbonyl electrophiles in special cases. We have used a pair of pyridoxal-phosphate-dependent aldolases to probe the mechanistic origins of this limitation. Our results reveal how aldolases are limited by thermodynamically favorable proton transfer with solvent, which undermines aldol addition into ketones. However, we show how a transaldolase can circumvent this limitation by protecting the enzyme-bound enolate from solvent protons and thereby enabling efficient addition into unactivated ketones. The resulting products are non-canonical amino acids with side chains that contain chiral tertiary alcohols. This study reveals the principles for extending aldolase catalysis beyond its previous limits and enables convergent, enantioselective C-C bond formation from simple starting materials.

Dinuclear monooxygenases mediate challenging C-H bond oxidation reactions throughout Nature. Many of these enzymes are presumed to exclusively utilize diiron cofactors. Herein, we report the bioinformatic discovery of an orphan dinuclear monooxygenase that preferentially utilizes a heterobimetallic manganese-iron (Mn/Fe) cofactor to mediate an O2 dependent C-H bond hydroxylation reaction. Unlike the structurally similar Mn/Fe-dependent monooxygenase AibH2, the diiron form of this enzyme (SfbO) exhibits nascent enzymatic activity. This behavior raises the possibility that many other dinuclear monooxygenases may be endowed with the capacity to harness cofactors with variable metal content.

Nucleosides functionalized at the 2′-position play a crucial role in therapeutics, serving as both small molecule drugs and modifications in therapeutic oligonucleotides. However, the synthesis of these molecules often presents significant synthetic challenges. In this study, we present an approach to the synthesis of 2′-functionalized nucleosides based on enzymes from the purine nucleoside salvage pathway. Initially active-site variants of DERA aldolase were generated for the highly stereoselective synthesis of D-ribose-5-phosphate analogs with a broad range of functional groups at the 2-position. Thereafter these 2-modified pentose phosphates were converted into 2′-modified purine analogs by construction of one-pot multi-enzyme cascade reactions, leading to the synthesis of guanosine (2′-OH) and adenosine (2′-OH, 2′-Me, 2′-F) analogues. Our findings demonstrate the capability of these biocatalytic cascades to efficiently generate 2′ functionalized nucleosides, starting from simple starting materials.

Biocatalytic oxidations have the potential to address many synthetic chemistry challenges, enabling the selective synthesis of chiral intermediates such as carbonyl compounds, alcohols, or amines. The use of oxygen-dependent enzymes can dramatically reduce the environmental footprint of redox transformations at manufacturing scale. Here, as part of the biocatalytic cascade to an anti-HIV investigational drug islatravir 1, we describe the development of an aerobic oxidation process delivering (R)-ethynylglyceraldehyde 3-phosphate 3 using an evolved galactose oxidase enzyme. Integrated enzyme and reaction engineering were critical for achieving a robust, high-yielding oxidation performed at pilot plant scale (>20 kg, 90% yield).

Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of (R)- or (S)-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2’-Bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2’-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel-Crafts reaction with SCP_Q111BpyAla achieving the best selectivity with an enantioselectivity of 72% ee (S). Interestingly, despite using the same protein scaffold, different attachment strategies for Bpy at the same residue (Q111) led to a switch in the enantiopreference of the ArM.

Proceedings of the National Academy of Sciences, Volume 120, Issue 43, October 2023.

Nature, Published online: 25 October 2023; doi:10.1038/d41586-023-03330-w

Debate rages in scientific community over Michael Eisen’s removal from prominent open-access journal.

N-hydroxy-γ-lactams are produced through an enzymatic sequence combining a lipase-catalyzed hydroxylamidation with an oxidase/peroxidase-induced ene-type cyclization. This methodology provides a mild and scalable access to N-heterocyclic building blocks from basic γ,δ-unsaturated esters and aqueous hydroxylamine, and its utility is illustrated by the formal total synthesis of the tetracyclic alkaloid cephalotaxine.

Abstract

The assembly of enzymatic cascades and multi-step reaction sequences represents an attractive alternative to traditional synthetic-organic approaches. The biocatalytic reaction mediators offer not only mild conditions and permit the use of environmentally benign reagents, but the high compatibility of different enzymes promises more streamlined reaction setups. In this study, a triple-enzymatic strategy was developed that enables the direct conversion of γ,δ-unsaturated esters to N-hydroxy-γ-lactam building blocks. Hereby, a lipase-catalyzed hydroxylaminolysis generates hydroxamic acid intermediates that are subsequently aerobically activated by horseradish peroxidase and glucose oxidase to cyclize in an intramolecular nitroso ene reaction. Utilizing the hydroxylaminolysis/ene-cyclization sequence for the preparation of an aza-spirocyclic lactam, the multi-enzymatic methodology was successfully employed in the synthesis of key intermediates en route to alkaloids of the Cephalotaxus family.

Modern, highly evolved, nucleoside-processing enzymes are known to exhibit perfect regioselectivity over the glycosylation of purine nucleobases at N9. We herein report an exception to this paradigm. Wild-type nucleoside phosphorylases also furnish N7- xanthosine, the “non-native” ribosylation regioisomer of xanthosine. This unusual nucleoside possesses several atypical physicochemical properties such as redshifted absorption spectra, a high equilibrium constant of phosphorolysis and low acidity. Ultimately, the biosynthesis of this previously unknown natural product illustrates how even highly evolved, essential enzymes from the primary metabolism are imperfect catalysts.

Despite the unique reactivity of vitamin B12 and its derivatives, B12-dependent enzymes remain underutilized in biocatalysis. In this study, we repurpose the B12-dependent transcription factor CarH to enable non-native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ- and δ-lactams via either redox-neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B12 cofactor. CarH* also catalyzes an unusual spirocyclization via dearomatization of pendant arenes to produce bicyclic 1,3-diene products instead of 1,4-dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B12 and expanding the synthetic utility of B12-dependent enzymes.

Science, Volume 382, Issue 6668, Page 325-329, October 2023.

Reciprocal stapling of two helices, an aromatic foldamer helix and a peptide α-helix, was observed in the conformation of a hybrid foldamer–peptide macrocycle when bound to the protein target against which it was selected. This intriguing shape and the possible contribution of the foldamer to protein binding highlight potential benefits of inserting a foldamer segment in display selection of macrocyclic peptides.

Abstract

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer–peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.