Nickel-catalyzed cross-electrophile allylation of vinyl bromides and the modification of anti-tumour natural medicine β-elemene

Herein, we present a facile and efficient allylation method via Ni-catalyzed cross-electrophile coupling of readily available allylic acetates with a variety of substituted alkenyl bromides using zinc as the terminal reductant. This Ni-catalyzed modular approach displays excellent functional group tolerance and a broad substrate scope, which the creation of a series of 1,4-dienes including several structurally complex natural products and pharmaceutical motifs. Moreover, the coupling strategy has the potential to realize enantiomeric control. The practicality of this transformation is demonstrated through the potent modification of the naturally antitumor active molecule β-elemene.

Herein, we present a facile and efficient allylation method via Ni-catalyzed cross-electrophile coupling of readily available allylic acetates with a variety of substituted alkenyl bromides using zinc as the terminal reductant.     

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.     

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.     

Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy

Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosine-click’ reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.

A new strategy for selective tryptophan modification using triazolinedione (TAD) chemistry at pH 4 is shown on peptides and proteins. Additionally, off-target modification of tryptophan residues during the classical TAD-Y click reaction is uncovered.     

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and β-arrestin-1 (βarr1). Using this strategy, we monitored the subtle but functionally important conformational change of βarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

A facile bioconjugation reaction for site-specific protein modification was developed for smFRET measurement, which detected the subtle but important conformational change of the β-arrestin/GPCR complex for the first time.     

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.     

Engineering proteinaceous colloidosomes as enzyme carriers for efficient and recyclable Pickering interfacial biocatalysis

Despite Pickering interfacial biocatalysis being a popular topic in biphasic biocatalysis, the development of water-in-oil (w/o) emulsion systems stabilized by single particles remains a challenge. For the first time, hydrophobized proteinaceous colloidosomes with magnetic-responsiveness are developed to function as both an enzyme carrier and emulsifier, achieving a breakthrough in protein-based w/o Pickering bioconversion. Enzyme-loaded protein colloidosomes are synthesized by a facile and mild method via emulsion templating. This system exhibits superior catalytic activity to other systems at the oil–water interface. Besides, feasible enzyme recovery and reusability ensure that this novel system can be employed as an efficient and eco-friendly recyclable platform.

Engineering proteinaceous colloidosomes with magnetic-responsiveness are designed as both enzyme carrier and emulsifier, achieving a breakthrough in protein-based w/o Pickering interfacial biocatalysis.     

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.     

Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Affinity-based benzotriazole (BTA) probes selectively and covalently label native proteins or endogenous proteins in cells with a fast reaction rate. It is enormously useful for characterizing protein function in biological systems and for drug development.     

Short peptide-based cross-β amyloids exploit dual residues for phosphoesterase like activity

Herein, we report that short peptides are capable of exploiting their anti-parallel registry to access cross-β stacks to expose more than one catalytic residue, exhibiting the traits of advanced binding pockets of enzymes. Binding pockets decorated with more than one catalytic residue facilitate substrate binding and process kinetically unfavourable chemical transformations. The solvent-exposed guanidinium and imidazole moieties on the cross-β microphases synergistically bind to polarise and hydrolyse diverse kinetically stable model substrates of nucleases and phosphatase. Mutation of either histidine or arginine results in a drastic decline in the rate of hydrolysis. These results not only support the argument of short amyloid peptides as the earliest protein folds but also suggest their interactions with nucleic acid congeners, foreshadowing the mutualistic biopolymer relationships that fueled the chemical emergence of life.

Amyloid based short peptide assemblies use antiparallel registry to expose multiple catalytic residues to bind and cleave kinetically stable phosphoester bonds of nucleic acid congeners, foreshadowing interactions of protein folds with nucleic acids.     

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.     

Rapid one-pot iterative diselenide–selenoester ligation using a novel coumarin-based photolabile protecting group

The development of an iterative one-pot peptide ligation strategy is described that capitalises on the rapid and efficient nature of the diselenide–selenoester ligation reaction, together with photodeselenisation chemistry. This ligation strategy hinged on the development of a novel photolabile protecting group for the side chain of selenocysteine, namely the 7-diethylamino-3-methyl coumarin (DEAMC) moiety. Deprotection of this DEAMC group can be effected in a mild, reagent-free manner using visible light (λ = 450 nm) without deleterious deselenisation of selenocysteine residues, thus enabling a subsequent ligation reaction without purification. The use of this DEAMC-protected selenocysteine in iterative DSL chemistry is highlighted through the efficient one-pot syntheses of 60- and 80-residue fragments of mucin-1 as well as apolipoprotein CIII in just 2–4 hours.

A method for the rapid one-pot iterative assembly of proteins via diselenide–selenoester ligation (DSL) chemistry is described that capitalises on a novel coumarin-based photolabile protecting group for selenocysteine.     

Electrochemical oxidative N–H/P–H cross-coupling with H2 evolution towards the synthesis of tertiary phosphines

Tertiary phosphines(iii) find widespread use in many aspects of synthetic organic chemistry. Herein, we developed a facile and novel electrochemical oxidative N–H/P–H cross-coupling method, leading to a series of expected tertiary phosphines(iii) under mild conditions with excellent yields. It is worth noting that this electrochemical protocol features very good reaction selectivity, where only a 1 : 1 ratio of amine and phosphine was required in the reaction. Moreover, this electrochemical protocol proved to be practical and scalable. Mechanistic insights suggested that the P radical was involved in this reaction.

A facile and novel electrochemical oxidative N–H/P–H cross-coupling method for obtaining tertiary phosphines(iii) was developed.     

Photodriven water oxidation initiated by a surface bound chromophore-donor-catalyst assembly

In photosynthesis, solar energy is used to produce solar fuels in the form of new chemical bonds. A critical step to mimic photosystem II (PS II), a key protein in nature''s photosynthesis, for artificial photosynthesis is designing devices for efficient light-driven water oxidation. Here, we describe a single molecular assembly electrode that duplicates the key components of PSII. It consists of a polypyridyl light absorber, chemically linked to an intermediate electron donor, with a molecular-based water oxidation catalyst on a SnO₂/TiO₂ core/shell electrode. The synthetic device mimics PSII in achieving sustained, light-driven water oxidation catalysis. It highlights the value of the tyrosine–histidine pair in PSII in achieving efficient water oxidation catalysis in artificial photosynthetic devices.

We describe a single molecular assembly electrode that mimics PSII. Flash photolysis revealed the electron transfer steps between chromophore light absorption and the creation and storage of redox equivalents in the catalyst for water oxidation.     

Primary trifluoroborate-iminiums enable facile access to chiral α-aminoboronic acids via Ru-catalyzed asymmetric hydrogenation and simple hydrolysis of the trifluoroborate moiety

This work describes the first preparation and application of primary trifluoroborate-iminiums (pTIMs) as a new, easily accessible and valuable class of organoboron derivatives. An array of structurally diverse pTIMs was prepared from potassium acyltrifluoroborates in excellent yields. Highly efficient and enantioselective [(R,R)-TethTsDpen-RuCl] complex-catalyzed hydrogenation of pTIMs provided direct access to chiral primary trifluoroborate-ammoniums (pTAMs). Moreover, facile synthesis of a series of structurally diverse chiral α-aminoboronic acids from chiral pTAMs was accomplished through novel, operationally simple and efficient conversion using hexamethyldisiloxane/aqueous HCl. Using no chromatography at any point, this work allowed easy access to chiral α-aminoboronic acids, as exemplified by the synthesis of optically pure anti-cancer drugs bortezomib and ixazomib.

Starting with potassium acyltrifluoroborates (KATs), N-unprotected chiral α-aminoboronic acids are prepared in three simple steps without chromatography. This facile methodology will tap the broad potential of these valuable compounds.     

A site-differentiated [4Fe–4S] cluster controls electron transfer reactivity of Clostridium acetobutylicum [FeFe]-hydrogenase I

One of the many functions of reduction–oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme. Diversity in relay design is exemplified among different members of hydrogenases, enzymes which catalyze reversible H₂ activation, which also couple to diverse types of donor and acceptor molecules. The [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is a member of a large family of structurally related enzymes where interfacial electron transfer is mediated by a terminal, non-canonical, His-coordinated, [4Fe–4S] cluster. The function of His coordination was examined by comparing the biophysical properties and reactivity to a Cys substituted variant of CaI. This demonstrated that His coordination strongly affected the distal [4Fe–4S] cluster spin state, spin pairing, and spatial orientations of molecular orbitals, with a minor effect on reduction potential. The deviations in these properties by substituting His for Cys in CaI, correlated with pronounced changes in electron transfer and reactivity with the native electron donor–acceptor ferredoxin. The results demonstrate that differential coordination of the surface localized [4Fe–4S]His cluster in CaI is utilized to control intermolecular and intramolecular electron transfer where His coordination creates a physical and electronic environment that enables facile electron exchange between electron carrier molecules and the iron–sulfur cluster relay for coupling to reversible H₂ activation at the catalytic site.

Histidine coordination of the distal [4Fe–4S] cluster in [FeFe]-hydrogenase was demonstrated to tune the cluster spin-states, spin-pairing and surrounding molecular orbitals to enable more facile electron transfer compared to cysteine coordination.     

Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application

The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone''s size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

A new technique for single molecular peptide sequencing is demonstrated by translocation of ssDNA-conjugated-peptide through MspA nanopore which is regulated by a DNA helicase motor.     

Enantioselective palladaelectro-catalyzed C–H olefinations and allylations for N–C axial chirality

Enantioselective palladaelectro-catalyzed C–H alkenylations and allylations were achieved with easily-accessible amino acids as transient directing groups. This strategy provided access to highly enantiomerically-enriched N–C axially chiral scaffolds under exceedingly mild conditions. The synthetic utility of our strategy was demonstrated by a variety of alkenes, while the versatility of our approach was reflected by atroposelective C–H allylations. Computational studies provided insights into a facile C–H activation by a seven-membered palladacycle.

Enantioselective palladaelectro-catalyzed C–H alkenylations and allylations were achieved by the means of an easily-accessible amino acid for the synthesis of N–C axially chiral indole biaryls.