Titia

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce a reactive enethiol. This enzyme activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugation of the enethiol with Cβ of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization.

Abstract

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S-[2-aminovinyl](3-methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C-terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two-stage bioinformatics mining of posttranslational modifications (PTMs) related to C-terminal Cys processing, we report herein that LanD activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.

Given the role of human intuition in current drug design efforts, crowd-sourced 'citizen scientist' games have the potential to greatly expand the pool of potential drug designers. Here, we introduce ‘Drugit', the small molecule design mode of the online ‘citizen science’ game Foldit. We demonstrate its utility for design with a use case to identify novel binders to the von Hippel Lindau E3 ligase. Several thousand molecule suggestions were obtained from players in a series of 10 puzzle rounds. The proposed molecules were then evaluated by in silico methods and by an expert panel and selected candidates were synthesized and tested. One of these molecules, designed by a player, showed dose-dependent shift perturbations in protein-observed NMR experiments. The co-crystal structure in complex with the E3 ligase revealed that the observed binding mode matched in major parts the player’s original idea. The completion of one full design cycle is a proof of concept for the Drugit approach and highlights the potential of involving citizen scientists in early drug discovery.

Here, we combined unsupervised machine learning (ML), non-natural amino acids, and affinity-selection mass-spectrometry (AS-MS) for the discovery of ultra-high affinity peptidomimetics that bind to a protein target. Peptides and peptidomimetics were discovered using AS-MS, encoded using diverse representations, and decomposed into two-dimensional “maps” of the chemical space by dimensionality reduction. These maps showed well-defined clusters of target-specific binders distinct from the remaining chemical space that included nonspecific and nonbinding peptides. Experimental testing of abiotic peptidomimetics confirmed the discovery of low nanomolar to picomolar binders and the accurate mapping of high-affinity binders across the co-learned sequence space. With ML and AS-MS, we anticipate this cartographic approach will accelerate the definition of chemical design spaces for the prediction and generation of functional peptidomimetics.

Precisely designed peptide sequences decorated with phenylboronic acid derivative and sugar residues (fructonic or galacturonic acid) in appropriate positions show reversibly forming ester residue and switching between cyclic (closed, stapled) and linear (open, non-stapled) forms. The boronate ester stapling is stabilized in mild basic conditions and may be switched off by acidification leading to unfolded organization of the peptide chain.

Abstract

Stabilization of a peptide conformation via stapling strategy may be realized by the reversible or more often irreversible connection of side chains being in mutually appropriate geometry. An incorporation of phenylboronic acid and sugar residues (fructonic or galacturonic acid), attached to two lysine side chains via amide bonds and separated by 2, 3, or 6 other residues in the C-terminal fragment of RNase A introduces the intramolecular interaction stabilizing the α-helical organization. The boronate ester stapling is stabilized in mild basic conditions and may be switched off by acidification leading to unfolded organization of the peptide chain. We investigated the possibility of using switchable stapling by mass spectrometry, NMR and UV-CD spectroscopies, and DFT calculations.

Biaryl and heterobiaryl-containing cyclic peptides represent promising scaffolds in the development of bioactive molecules. The incorporation of heterobiaryl motifs continues to pose synthetic challenges, which is partially due to the difficulties in effecting late-stage metal-catalyzed cross-couplings. We report a new strategy to form heterobiaryls that is based on trapping nitrilium ions. The sequence is exemplified using oxadiazole- and oxazole-containing biaryl linkages. NMR analysis and molecular dynamics simulations reveal structural control elements common to each member of the heterobiaryl containing peptide family in this study. Strategic substitutions on the C-terminal aminobenzoic acid moiety paired with installation of oxadiazole or oxazole heterobiaryl backbone linkages allow for the modulation of peptide backbone conformation, which should assist efforts to optimize the biophysical properties of peptide macrocycles.

The Front Cover shows the single modification at the N-terminus of norvancomycin, especially lipo-sulfonium furnishment. This provides norvancomycin additional interaction with the bacterial membrane via the electrostatic interaction and hydrophobic moieties’ insertion into the membrane inner leaflet. Thus, it restores the antibacterial activity against glycopeptide-resistant bacteria. Cover design by Dongliang Guan. More information can be found in the Research Article by Dongliang Guan, Lefu Lan, Wei Huang et al.

The bioactivity of the enzyme HRP immobilized with the covalent-organic framework COF-LZU1 is shown to be promoted by a weak interaction between the enzyme and COF-LZU1, the easy access of the substrate to COF-LZU1, as well as an optimal conformation of enzyme inside the COF-LZU1. This comprehensive understanding of interfacial interactions, substrate diffusion, and enzyme conformation alteration aids the design of biocatalysts.

Abstract

Selecting a suitable support material for enzyme immobilization with excellent biocatalytic activity and stability is a critical aspect in the development of functional biosystems. The highly stable and metal-free properties of covalent-organic frameworks (COFs) make them ideal supports for enzyme immobilization. Herein, we constructed three kinds of COFs via a biofriendly and one-pot synthetic strategy at room temperature in aqueous solution. Among the three developed COFs (COF-LZU1, RT-COF-1 and ACOF-1), the horseradish peroxidase (HRP)-incorporated COF-LZU1 is found to retain the highest activity. Structural analysis reveals that a weakest interaction between the hydrated enzyme and COF-LZU1, an easiest accessibility by the COF-LZU1 to the substrate, as well as an optimal conformation of enzyme together promote the bioactivity of HRP-COF-LZU1. Furthermore, the COF-LZU1 is revealed to be a versatile nanoplatform for encapsulating multiple enzymes. The COF-LZU1 also offers superior protection for the immobilized enzymes under harsh conditions and during recycling. The comprehensive understanding of interfacial interactions of COF host and enzyme guest, the substrate diffusion, as well as the enzyme conformation alteration within COF matrices represents an opportunity to design the ideal biocatalysts and opens a broad range of applications of these nanosystems.

Biocatalytic processes are highly selective and specific. However, their utility is limited by the comparatively narrow scope of enzyme-catalysed transformations. To expand product scope, we are developing biocompatible processes that combine biocatalytic reactions with chemo-catalysis in single-flask processes. Here, we show that a chemocatalysed Pictet-Spengler annulation can be interfaced with biocatalysed alcohol oxidation. This two-step, one-pot cascade reaction converts tyramine and aliphatic alcohols to tetrahydroquinoline alkaloids in aqueous buffer at mild pH. Tryptamine derivatives are also efficiently converted to tryptolines. Optimization of stoichiometry, pH, reaction time, and whole-cell catalyst deliver the tetrahydroisoquinolines and tryptolines in >90% and >40% isolated yield, respectively, with excellent regioselectivity.

A primordial anti-messenger RNA composed only of adenosine and uracil nucleotides is proposed 1) to contain anticodons for a maximum of eight different amino acids (left) and, consequently, be able to form short active polypeptides and 2) to fold as a hairpin RNA adaptor exposing one of the anticodons for the formation of a specific amino acyl species (right).

Abstract

Examination of the genetic code (GeCo) reveals that amino acids coded by (A/U) codons display a large functional spectrum and bind RNA whereas, except for Arg, those coded by (G/C) codons do not. From a stereochemical viewpoint, the clear preference for (A/U)-rich codons to be located at the GeCo half blocks suggests they were specifically determined. Conversely, the overall lower affinity of cognate amino acids for their (G/C)-rich anticodons points to their late arrival to the GeCo. It is proposed that i) initially the code was composed of the eight (A/U) codons; ii) these codons were duplicated when G/C nucleotides were added to their wobble positions, and three new codons with G/C in their first position were incorporated; and iii) a combination of A/U and G/C nucleotides progressively generated the remaining codons.

Separation not required: Mass spectrometry without prior chromatographic separation, carried out on a single-quadrupole HPLC-MS, can be used for the qualitative and quantitative analysis of diverse biotransformations. This flow-injection analysis mass spectrometry (FIA-MS) approach represents an attractive alternative to more traditional photometric, fluorometric, and chromatographic methods for screening enzymatic reactions.

Abstract

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1–50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Herein, we report a novel strategy for the modification of peptides based on the introduction of highly reactive hypervalent iodine reagents - ethynylbenziodoxolones (EBXs)- onto peptides. These peptide-EBXs can be easily accessed, both via solution and solid phase peptide synthesis (SPPS). They can be used to couple the peptide to other peptides or a protein via reaction with cysteine, leading to thioalkynes in organic solvents and hypervalent iodine adducts in water buffer. In addition, a photocatalytic decarboxylative coupling to the C-terminus of peptides was developed using an organic dye. This later reaction was also successful in an intramolecular way, leading to macrocyclic peptides of an unprecedented shape. The rigid linear aryl alkyne linker was essential to achieve high affinity to Keap1 at the Nrf2 binding site with potential protein-protein interaction inhibition.

Nature, Published online: 09 March 2023; doi:10.1038/d41586-023-00717-7

Proof-of-concept mouse experiment will have a long road before use in humans is possible.

Deep learning networks offer considerable opportunities for accurate structure prediction and design of biomolecules. While cyclic peptides have gained significant traction as a therapeutic modality, developing deep learning methods for designing such peptides has been slow, mostly due to the small number of available structures for molecules in this size range. Here, we report approaches to modify the AlphaFold network for accurate structure prediction and design of cyclic peptides. Our results show this approach can accurately predict the structures of native cyclic peptides from a single sequence, with 36 out of 49 cases predicted with high confidence (pLDDT > 0.85) matching the native structure with root mean squared deviation (RMSD) less than 1.5 [A]. Further extending our approach, we describe computational methods for designing sequences of peptide backbones generated by other backbone sampling methods and for de novo design of new macrocyclic peptides. We extensively sampled the structural diversity of cyclic peptides between 7-13 amino acids, and identified around 10,000 unique design candidates predicted to fold into the designed structures with high confidence. X-ray crystal structures for seven sequences with diverse sizes and structures designed by our approach match very closely with the design models (root mean squared deviation < 1.0 [A]), highlighting the atomic level accuracy in our approach. The computational methods and scaffolds developed here provide the basis for custom-designing peptides for targeted therapeutic applications.

A strategy, named “aromatic residue scanning”, was developed for enzyme engineering, and an alcohol dehydrogenase was engineered to exhibit higher activity and substrate tolerance based on the implementation of the strategy to randomly mutate five identified potential plasticity sites. By using the best variant, an efficient bioprocess for the synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol was established.

Abstract

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named “aromatic residue scanning” was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as “exploratory residues” to introduce steric hindrance or potential π-π interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

A cysteine-selective, visible-light-mediated bioconjugation reaction that enables direct installation of a range of desired moieties into peptide and protein scaffolds via C(sp³)−C(sp³) bond formation is described. The method is rapid, operationally simple, tolerant to ambient atmosphere and can be utilized to incorporate general tools for chemical biology, including effective PTM mimics, via a stable, biogenic hydrocarbon linkage.

Abstract

The site-selective modification of peptides and proteins facilitates the preparation of targeted therapeutic agents and tools to interrogate biochemical pathways. Among the numerous bioconjugation techniques developed to install groups of interest, those that generate C(sp³)−C(sp³) bonds are significantly underrepresented despite affording proteolytically stable, biogenic linkages. Herein, a visible-light-mediated reaction is described that enables the site-selective modification of peptides and proteins via desulfurative C(sp³)−C(sp³) bond formation. The reaction is rapid and high yielding in peptide systems, with comparable translation to proteins. Using this chemistry, a range of moieties is installed into model systems and an effective PTM-mimic is successfully integrated into a recombinantly expressed histone.

Since 1858, aryldiazonium salts have been inspiring chemists to use them as dye precursors and as substrates for ionic or radicalar transformations. The latter is experiencing a renaissance among the reliable methodologies in chemical synthesis due to the discovery of catalysts and reaction protocols. Herein, these modern examples were compiled to highlight the general methods to produce aryl radicals from aryldiazonium salts in route to complexes arylated molecules.

Abstract

This review provides an overview of the use of aryldiazonium salts as the source of aryl radicals and their application in the development of selective transformations over the last 13 years. Examples were organized under thermal and photocatalytic transformations. This review provides an accessible follow-up in this field for both radical and catalytic chemists working in industry as well as academia.

The catalytic promiscuity of a copper oxidase was expanded to accommodate new-to-nature enantioselective oxidative cross-coupling of 3-hydroxyindole esters with various nucleophiles. The resulting functionalized 2,2-disubstituted indolin-3-ones showed excellent optical purity as well as anticancer activity.

Abstract

Herein, we disclose the highly enantioselective oxidative cross-coupling of 3-hydroxyindole esters with various nucleophilic partners as catalyzed by copper efflux oxidase. The biocatalytic transformation delivers functionalized 2,2-disubstituted indolin-3-ones with excellent optical purity (90–99 % ee), which exhibited anticancer activity against MCF-7 cell lines, as shown by preliminary biological evaluation. Mechanistic studies and molecular docking results suggest the formation of a phenoxyl radical and enantiocontrol facilitated by a suited enzyme chiral pocket. This study is significant with regard to expanding the catalytic repertoire of natural multicopper oxidases as well as enlarging the synthetic toolbox for sustainable asymmetric oxidative coupling.

Organocatalysis : Asymmetric desymmetrization of prochiral enal-tethered cyclohexadienones has been developed by using organocatalytic iminium-enamine activation. The reaction provides an access to bicyclic enones with excellent enantioselectivity and diastereoselectivity, which further undergo acid-mediated intramolecular annulation to afford highly strained and complex cyclohepta[b]indoles with five contiguous stereocentres.

Abstract

The expeditious construction of complex molecules having multiple stereocentres is highly desirable in organic chemistry. In the present communication, we report the development of an organocatalytic asymmetric desymmetrization of prochiral enal-tethered cyclohexadienones via the C3-selective Friedel−Crafts alkylation of indoles triggered by LUMO-lowering iminium activation/HOMO-raising enamine activation. The reaction provides access to bicyclic enones, which further undergo acid-mediated intramolecular annulation from C2-position to afford highly strained cyclohepta[b]indoles with five contiguous stereocentres and three new C−C bonds in excellent enantioselectivity and diastereoselectivity.