R.B. Leveson-Gower

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.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing a covalent bond between a flavin cofactor and its hosting protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that ‘dissociable’ flavoproteins can be turned into covalent flavoproteins. Specifically, three different FMN-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage (LOV) domain protein, a mini singlet-oxygen-generator (miniSOG), and a nitroreductase (BtNR). Optimizing the flavinylation motif and expression conditions led to covalent flavinylation of all three flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance such as higher thermostability or catalytic performance. Crystal structures of all three covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins which cannot lose their cofactor, boosting their performance.

Artificial metalloenzymes based on a cobalt cofactor show higher activity than the free cobalt complex for the oxidation of benzylic C(sp³)−H bonds in aqueous medium. The cobalt cofactor is presumably stabilized by the hydrophobic cleft provided by the protein NB4.

Abstract

Artificial metalloenzymes have emerged as biohybrid catalysts that allow to combine the reactivity of a metal catalyst with the flexibility of protein scaffolds. This work reports the artificial metalloenzymes based on the β-barrel protein nitrobindin NB4, in which a cofactor [Co^IIX(Me₃TACD-Mal)]⁺X⁻ (X=Cl, Br; Me₃TACD=N,N^',N''-trimethyl-1,4,7,10-tetraazacyclododecane, Mal=CH₂CH₂CH₂NC₄H₂O₂) was covalently anchored via a Michael addition reaction. These biohybrid catalysts showed higher efficiency than the free cobalt complexes for the oxidation of benzylic C(sp³)−H bonds in aqueous media. Using commercially available oxone (2KHSO₅ ⋅ KHSO₄ ⋅ K₂SO₄) as oxidant, a total turnover number of up to 220 and 97 % ketone selectivity were achieved for tetralin. As catalytically active intermediate, a mononuclear terminal cobalt(IV)-oxo species [Co(IV)=O]²⁺ was generated by reacting the cobalt(II) cofactor with oxone in aqueous solution and characterized by ESI-TOF MS.

A novel insect-derived flavoenzyme from the honeybee Apis mellifera (beeHMFO) can selectively oxidize 5-hydroxymethylfurfural (HMF) to the corresponding dialdehyde 2,5-diformylfuran (DFF), which is an interesting bio-based polymer precursor. Moreover, activity toward a number of other aromatic alcohols is observed. The predicted structure of beeHMFO shows high similarity to other flavoprotein oxidases capable of oxidizing HMF.

Abstract

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts.

NRPSs are an important source of pharmaceutically valuable natural products. The large sequence space of NRPS variant libraries requires a robust, high-throughput sorting and screening platform. Here we present a novel high-throughput microfluidic screening platform for the investigation of mutants of NRP producing bacteria in a highly parallel manner. Our results demonstrate the power of this platform for studying large libraries of NRPS variants.

Abstract

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

A nucleotidoprotein engineered via facile “green synthesis” exhibits strong electrostatic attraction and hydrogen bonding with complementary base-modified polyethyleneimine to form salt-resistant nanocomplexes with robust cytosolic delivery efficiency. The acidic endolysosomal environment enables traceless restoration of the nucleotidoprotein and consequently promotes the intracellular release of native protein.

Abstract

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

Science, Volume 382, Issue 6666, Page 19-20, October 2023.