A novel tyrosine hyperoxidation enables selective peptide cleavage

A novel tyrosine hyperoxidation enabling selective peptide cleavage is reported. The scission of the N-terminal amide bond of tyrosine was achieved with Dess–Martin periodinane under mild conditions, generating a C-terminal peptide fragment bearing the unprecedented hyperoxidized tyrosine motif, 4,5,6,7-tetraoxo-1H-indole-2-carboxamide, along with an intact N-terminal peptide fragment. This reaction proceeds with high site-selectivity for tyrosine and exhibits broad substrate scope for various peptides, including those containing post-translational modifications. More importantly, this oxidative cleavage was successfully applied to enable sequencing of three naturally occurring cyclic peptides, including one depsipeptide and one lipopeptide. The linearized peptides generated from the cleavage reaction significantly simplify cyclic peptide sequencing by MS/MS, thus providing a robust tool to facilitate rapid sequence determination of diverse cyclic peptides containing tyrosine. Furthermore, the highly electrophilic nature of the hyperoxidized tyrosine unit disclosed in this work renders it an important electrophilic target for the selective bioconjugation or synthetic manipulation of peptides containing this unit.

A Tyr-selective peptide cleavage was reported using Dess–Martin periodinane. The cleavage generates an unprecedented hyperoxidized tyrosine motif in the C-terminal fragment and showed excellent site-specificity and broad scope for various peptides.     

Late-stage modification of peptides and proteins at cysteine with diaryliodonium salts

The modification of peptides and proteins has emerged as a powerful means to efficiently prepare high value bioconjugates for a range of applications in chemical biology and for the development of next-generation therapeutics. Herein, we report a novel method for the chemoselective late-stage modification of peptides and proteins at cysteine in aqueous buffer with suitably functionalised diaryliodonium salts, furnishing stable thioether-linked synthetic conjugates. The power of this new platform is showcased through the late-stage modification of the affibody zEGFR and the histone protein H2A.

New operationally simple platform for the chemoselective arylation of cysteine in peptides and proteins to access a variety of high value bioconjugates.     

Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins

New methods for chemo-selective modifications of peptides and native proteins are important in chemical biology and for the development of therapeutic conjugates. Less abundant and uncharged amino-acid residues are interesting targets to form less heterogeneous conjugates and preserve biological functions. Phenylurazole (PhUr), N-methylphenylurazole (NMePhUr) and N-methylluminol (NMeLum) derivatives were described as tyrosine (Y) anchors after chemical or enzymatic oxidations. Recently, we developed the first electrochemical Y-bioconjugation method coined eY-click to activate PhUr in biocompatible media. In this work, we assessed the limitations, benefits and relative efficiencies of eY-click conjugations performed with a set of PhUr, NMePhUr and NMeLum derivatives. Results evidenced a high efficiency of NMeLum that showed a complete Y-chemoselectivity on polypeptides and biologically relevant proteins after soft electrochemical activation. Side reactions on nucleophilic or heteroaromatic amino-acids such as lysine or tryptophan were never observed during mass spectrometry analysis. Myoglobine, bovine serum albumin, a plant mannosidase, glucose oxidase and the therapeutically relevant antibody trastuzumab were efficiently labelled with a fluorescent probe in a two-step approach combining eY-click and strain-promoted azide–alkyne cyclization (SPAAC). The proteins conserved their structural integrity as observed by circular dichroism and the trastuzumab conjugate showed a similar binding affinity for the natural HER2 ligand as shown by bio-layer interferometry. Compared to our previously described protocol with PhUr, eY-click with NMeLum species showed faster reaction kinetics, higher (complete) Y-chemoselectivity and reactivity, and offers the interesting possibility of the double tagging of solvent-exposed Y.

We assessed the relative efficiencies of tyrosine anchors in the electrochemical conjugation of peptides and proteins. Luminol derivatives showed faster reaction kinetics, complete tyrosine-chemoselectivity, and possible double modification.     

Rapid and robust cysteine bioconjugation with vinylheteroarenes

Methods for residue-selective and stable modification of canonical amino acids enable the installation of distinct functionality which can aid in the interrogation of biological processes or the generation of new therapeutic modalities. Herein, we report an extensive investigation of reactivity and stability profiles for a series of vinylheteroarene motifs. Studies on small molecule and protein substrates identified an optimum vinylheteroarene scaffold for selective cysteine modification. Utilisation of this lead linker to modify a number of protein substrates with various functionalities, including the synthesis of a homogeneous, stable and biologically active antibody–drug conjugate (ADC) was then achieved. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology.

Vinylheteroarene linkers can chemoselectively modify cysteine residues in proteins and antibodies. These linkers give stable bioconjugates, and were used to synthesise efficacious antibody-drug conjugates.     

Tunable heteroaromatic azoline thioethers (HATs) for cysteine profiling

Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions. Although the resulting cysteine–azoline conjugate is stable, we highlight traceless decoupling of the conjugate to afford unmodified starting components in response to reducing conditions. We demonstrated that HAT probes reverse the reactivity of nucleophilic cysteine to electrophilic dehydroalanine (Dha) under mild basic conditions. We demonstrated the umpolung capability of HAT probes for the modification of cysteine on peptides and proteins with various nucleophiles. We demonstrated that HAT probes increase the mass sensitivity of the modified peptides and proteins by 100 fold as compared to the classical methods. Finally, we extended the application of HAT probes for specific modification of cysteines in a complex cell lysate mixture.

Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions.     

Functional and protective hole hopping in metalloenzymes

Electrons can tunnel through proteins in microseconds with a modest release of free energy over distances in the 15 to 20 Å range. To span greater distances, or to move faster, multiple charge transfers (hops) are required. When one of the reactants is a strong oxidant, it is convenient to consider the movement of a positively charged “hole” in a direction opposite to that of the electron. Hole hopping along chains of tryptophan (Trp) and tyrosine (Tyr) residues is a critical function in several metalloenzymes that generate high-potential intermediates by reactions with O₂ or H₂O₂, or by activation with visible light. Examination of the protein structural database revealed that Tyr/Trp chains are common protein structural elements, particularly among enzymes that react with O₂ and H₂O₂. In many cases these chains may serve a protective role in metalloenzymes by deactivating high-potential reactive intermediates formed in uncoupled catalytic turnover.

Hole hopping through tryptophan and tyrosine residues in metalloenzymes facilitates catalysis and prolongs survival.     

A bioorthogonal chemical reporter for the detection and identification of protein lactylation

l-Lactylation is a recently discovered post-translational modification occurring on histone lysine residues to regulate gene expression. However, the substrate scope of lactylation, especially that in non-histone proteins, remains unknown, largely due to the limitations of current methods for analyzing lactylated proteins. Herein, we report an alkynyl-functionalized bioorthogonal chemical reporter, YnLac, for the detection and identification of protein lactylation in mammalian cells. Our in-gel fluorescence and chemical proteomic analyses show that YnLac is metabolically incorporated into lactylated proteins and directly labels known lactylated lysines of histones. We further apply YnLac to the proteome-wide profiling of lactylation, revealing many novel modification sites in non-histone proteins for the first time. Moreover, we demonstrate that lactylation of a newly identified substrate protein PARP1 regulates its ADP-ribosylation activity. Our study thus provides a powerful chemical tool for characterizing protein lactylation and greatly expands our understanding of substrate proteins and functions of this new modification.

YnLac is an alkynyl-functionalized l-lactate analogue that is metabolically incorporated into l-lactylated proteins in live cells, enabling the fluorescence detection and proteomic identification of novel l-lactylated proteins.     

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

A modular strategy to convert commercially available antibodies into fluorogenic probes has been developed, enabling selective recognition and wash-free imaging of endogenous membrane proteins.      

Borinostats: solid-phase synthesis of carborane-capped histone deacetylase inhibitors with a tailor-made selectivity profile

The elevated expression of histone deacetylases (HDACs) in various tumor types renders their inhibition an attractive strategy for epigenetic therapeutics. One key issue in the development of improved HDAC inhibitors (HDACis) is the selectivity for single HDAC isoforms over unspecific pan inhibition to minimize off-target toxicity. Utilizing the carborane moiety as a fine-tuning pharmacophore, we herein present a robust solid phase synthetic approach towards tailor-made HDACis meeting both ends of the selectivity spectrum, namely pan inhibition and highly selective HDAC6 inhibition.

This work describes a versatile solid phase synthesis of carborane-capped histone deacetylase inhibitors with a tunable selectivity profile and synergistic anticancer activity with bortezomib.     

Exploring Tyrosine‐Triazolinedione (TAD) Reactions for the Selective Conjugation and Cross‐Linking of N‐Carboxyanhydride (NCA) Derived Synthetic Copolypeptides

Highly efficient functionalization and cross‐linking of polypeptides is achieved via tyrosine‐triazolinedione (TAD) conjugation chemistry. The feasibility of the reaction is demonstrated by the reaction of 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) with tyrosine containing block copolymer poly(ethylene glycol)‐Tyr₄ as well as a statistical copolymer of tyrosine and lysine (poly(Lys₄₀‐st‐Tyr₁₀)) prepared form N‐carboxyanhydride polymerization. Selective reaction of PTAD with the tyrosine units is obtained and verified by size exclusion chromatography and NMR spectroscopy. Moreover, two monofunctional and two difunctional TAD molecules are synthesized. It is found that their stability in the aqueous reaction media significantly varied. Under optimized reaction conditions selective functionalization and cross‐linking, yielding polypeptide hydrogels, can be achieved. TAD‐mediated conjugation can offer an interesting addition in the toolbox of selective (click‐like) polypeptide conjugation methodologies as it does not require functional non‐natural amino acids.

     

Umpolung strategies for the functionalization of peptides and proteins

Umpolung strategies, defined as synthetic approaches which reverse commonly accepted reactivity patterns, are broadly recognized as enabling tools for small molecule synthesis and catalysis. However, methods which exploit this logic for peptide and protein functionalizations are comparatively rare, with the overwhelming majority of existing bioconjugation approaches relying on the well-established reactivity profiles of a handful of amino acids. This perspective serves to highlight a small but growing body of recent work that masterfully capitalizes on the concept of polarity reversal for the selective modification of proteinogenic functionalities. Current applications of umpolung chemistry in organic synthesis and chemical biology as well as the vast potential for further innovations in peptide and protein modification will be discussed.

This perspective highlights the growing body of literature that leverages polarity reversal (umpolung reactivity) for the selective modification of proteinogenic functionalities and identifies opportunities for further innovation.     

Diverse protein manipulations with genetically encoded glutamic acid benzyl ester

Site-specific modification of proteins has significantly advanced the use of proteins in biological research and therapeutics development. Among various strategies aimed at this end, genetic code expansion (GCE) allows structurally and functionally distinct non-canonical amino acids (ncAAs) to be incorporated into specific sites of a protein. Herein, we genetically encode an esterified glutamic acid analogue (BnE) into proteins, and demonstrate that BnE can be applied in different types of site-specific protein modifications, including N-terminal pyroglutamation, caging Glu in the active site of a toxic protein, and endowing proteins with metal chelator hydroxamic acid and versatile reactive handle acyl hydrazide. Importantly, novel epigenetic mark Gln methylation is generated on histones via the derived acyl hydrazide handle. This work provides useful and unique tools to modify proteins at specific Glu or Gln residues, and complements the toolbox of GCE.

Herein, we genetically encode an esterified glutamic acid analogue (BnE) into proteins, and demonstrate that BnE can be applied in different types of site-specific protein modifications.     

Cleavable and tunable cysteine-specific arylation modification with aryl thioethers

Cysteine represents an attractive target for peptide/protein modification due to the intrinsic high nucleophilicity of the thiol group and low natural abundance. Herein, a cleavable and tunable covalent modification approach for cysteine containing peptides/proteins with our newly designed aryl thioethers via a S_NAr approach was developed. Highly efficient and selective bioconjugation reactions can be carried out under mild and biocompatible conditions. A series of aryl groups bearing different bioconjugation handles, affinity or fluorescent tags are well tolerated. By adjusting the skeleton and steric hindrance of aryl thioethers slightly, the modified products showed a tunable profile for the regeneration of the native peptides.

A cleavable and tunable covalent modification approach for cysteine by aryl thioethers via a S_NAr approach was developed. The highly efficient and selective bioconjugation reactions can proceed under the mild and biocompatible conditions.     

Determinants for intrinsically disordered protein recruitment into phase-separated protein condensates

Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often biased residues of IDPs are also likely involved in selective recruitment of many other IDPs into condensates. However, determining factors for this IDP recruitment into protein condensates are not understood yet. Here, we quantitatively examined recruitment tendencies of various IDPs with different sequence compositions into IDP-clustered condensates both in vitro as well as in cells. Condensate-forming IDP scaffolds, recruited IDP clients, and phase separation conditions were carefully varied to find key factors for selective IDP partitioning in protein condensates. Regardless of scaffold sequences, charged residues in client IDPs assured potent IDP recruitment, likely via strong electrostatic interactions, where positive residues could further enhance recruitment, possibly with cation–pi interactions. Notably, poly-ethylene glycol, a widely used crowding reagent for in vitro phase separation, abnormally increased IDP recruitment, indicating the need for careful use of crowding conditions. Tyrosines of IDP clients also strongly participated in recruitment both in vitro and in cells. Lastly, we measured recruitment degrees by more conventional interactions between folded proteins instead of disordered proteins. Surprisingly, recruitment forces by an even moderate protein interaction (K_d ∼ 5 μM) were substantially stronger than those by natural IDP–IDP interactions. The present data offer valuable information on how cells might organize protein partitioning on various protein condensates.

Diverse interactions between folded and disordered proteins collectively dictate selective protein recruitment into bimolecular condensates.     

Scalable synthesis and coupling of quaternary α-arylated amino acids: α-aryl substituents are tolerated in α-helical peptides

Quaternary amino acids are important tools for the modification and stabilisation of peptide secondary structures. Here we describe a practical and scalable synthesis applicable to quaternary alpha-arylated amino acids (Q4As), and the development of solid-phase synthesis conditions for their incorporation into peptides. Monomeric and dimeric α-helical peptides are synthesised with varying degrees of Q4A substitution and their structures examined using biophysical methods. Both enantiomers of the Q4As are tolerated in folded monomeric and oligomeric α-helical peptides, with the (R)-enantiomer slightly more so than the (S).

Both R and S enantiomers of Fmoc-protected amino acids bearing α-aryl substituents may be made on gram scale. Solid-phase synthesis leads to helical peptides unperturbed by the presence of these additional α-aryl groups.     

Small peptide diversification through photoredox-catalyzed oxidative C-terminal modification

A photoredox-catalyzed oxidative decarboxylative coupling of small peptides is reported, giving access to a variety of N,O-acetals. They were used as intermediates for the addition of phenols and indoles, leading to novel peptide scaffolds and bioconjugates. Amino acids with nucleophilic side chains, such as serine, threonine, tyrosine and tryptophan, could also be used as partners to access tri- and tetrapeptide derivatives with non-natural cross-linking.

A photoredox approach for the generation of N-acyliminiums derived from peptides enabling diversification via Friedel–Crafts reactions.     

An evolution-inspired strategy to design disulfide-rich peptides tolerant to extensive sequence manipulation

Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.

A general method was developed to design multicyclic peptides with diverse disulfide frameworks amenable to random peptide library design, enabling the development of new disulfide-rich peptide ligands and therapeutics with structures not derived from natural peptides.     

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Genome mining for pathways containing YcaO proteins revealed a widespread novel family of RiPP gene clusters. A model gene cluster was characterised through genetic and chemical analyses, which yielded streptamidine, a novel amidine-containing RiPP.     

Selective covalent capture of collagen triple helices with a minimal protecting group strategy

Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge–pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine–glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally.

A minimal protecting group strategy is developed to allow selective covalent capture of collagen-like triple helices. This allows stabilization of this critical fold while preserving charge–pair interactions critical for biological applications.     

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization

Recent dramatic expansion in potential uses of protein conjugates has fueled the development of a wide range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge. Herein, we present the application of 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) as advantageous alternatives to widely used maleimides for the chemo- and site-selective labeling of cysteine residues within proteins. A variety of 5HP2O building blocks have been synthesized using a one-pot photooxidation reaction starting from simple and readily accessible furans and using visible light and oxygen. These novel reagents display excellent cysteine selectivity and also yield thiol conjugates with superior stability. 5HP2O building blocks offer a unique opportunity to introduce multiple new functionalities into a protein at a single site and in a single step, thus, significantly enhancing the resultant conjugate''s properties.

Recent expansion in potential uses of protein conjugates has fueled the development of a range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge.