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.     

Sulfur(iv)-mediated umpolung α-heterofunctionalization of 2-oxazolines

The α-umpolung of carbonyl compounds significantly expands the boundaries of traditional carbonyl chemistry. Despite various umpolung methods available today, reversing the inherent reactivity of carbonyls still remains a substantial challenge. In this article, we report the first use of sulfonium salts, in lieu of well-established hypervalent iodines, for the carbonyl umpolung event. The protocol enables the incorporation of a wide variety of heteroatom nucleophiles into the α-carbon of 2-oxazolines. The success of this investigation hinges on the following factors: (1) the use of sulfoxides, which are abundant, structurally diverse and tunable, and easily accessible, ensures the identification of a superior oxidant namely phenoxathiin sulfoxide for the umpolung reaction; (2) the “assembly/deprotonation” protocol previously developed for rearrangement reactions in our laboratory was successfully applied here for the construction of α-umpoled 2-oxazolines.

Aryl sulfonium salts serving as a new type of carbonyl umpolung reagent enable direct α-heterofunctionalization of 2-oxazolines.     

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.     

Ruthenium(ii)-catalyzed regioselective 1,6-conjugate addition of umpolung aldehydes as carbanion equivalents

One of the most efficient and reliable approaches to construct C–C bonds involves the conjugate addition of carbon nucleophiles to electron-deficient ketones. Yet, 1,6-conjugate additions of extended conjugated systems largely remain underexplored due to difficulties in controlling the regioselectivity. Herein, we report umpolung aldehydes as carbanion equivalents for highly regioselective 1,6-conjugate addition reactions to unsaturated ketones, with preliminary studies of the enantioselective variant. The synergy of ruthenium(ii) catalyst and electron-rich, bidentate phosphine ligand is essential for the reactivity and selectivity under mild reaction conditions.

Highly regioselective 1,6-conjugate addition was developed using hydrazone as carbanion equivalent catalyzed by ruthenium under mild conditions.     

Stimuli-responsive temporary adhesives: enabling debonding on demand through strategic molecular design

Stimuli-responsive temporary adhesives constitute a rapidly developing class of materials defined by the modulation of adhesion upon exposure to an external stimulus or stimuli. Engineering these materials to shift between two characteristic properties, strong adhesion and facile debonding, can be achieved through design strategies that target molecular functionalities. This perspective reviews the recent design and development of these materials, with a focus on the different stimuli that may initiate debonding. These stimuli include UV light, thermal energy, chemical triggers, and other potential triggers, such as mechanical force, sublimation, electromagnetism. The conclusion discusses the fundamental value of systematic investigations of the structure–property relationships within these materials and opportunities for unlocking novel functionalities in future versions of adhesives.

Stimuli-responsive temporary adhesives emerge as next-generation multifunctional materials with advantages that include strong temporary adhesion, debonding on demand, and tunable reactivity.     

Combining flavin photocatalysis with parallel synthesis: a general platform to optimize peptides with non-proteinogenic amino acids

Most peptide drugs contain non-proteinogenic amino acids (NPAAs), born out through extensive structure–activity relationship (SAR) studies using solid-phase peptide synthesis (SPPS). Synthetically laborious and expensive to manufacture, NPAAs also can have poor coupling efficiencies allowing only a small fraction to be sampled by conventional SPPS. To gain general access to NPAA-containing peptides, we developed a first-generation platform that merges contemporary flavin photocatalysis with parallel synthesis to simultaneously make, purify, quantify, and even test up to 96 single-NPAA peptide variants via the unique combination of boronic acids and a dehydroalanine residue in a peptide. We showcase the power of our newly minted platform to introduce NPAAs of diverse chemotypes-aliphatic, aromatic, heteroaromatic-directly into peptides, including 15 entirely new residues, and to evolve a simple proteinogenic peptide into an unnatural inhibitor of thrombin by non-classical peptide SAR.

We report a non-classical approach to interrogate peptides with non-proteinogenic amino acids via flavin photocatalysis. We establish a new platform to make, purify, quantify, and biochemically test up to 96 peptide variants in batch.     

Sulfur stereogenic centers in transition-metal-catalyzed asymmetric C–H functionalization: generation and utilization

Transition-metal-catalyzed enantioselective C–H functionalization has emerged as a powerful tool for the synthesis of enantioenriched compounds in chemical and pharmaceutical industries. Sulfur-based functionalities are ubiquitous in many of the biologically active compounds, medicinal agents, functional materials, chiral auxiliaries and ligands. This perspective highlights recent advances in sulfur functional group enabled transition-metal-catalyzed enantioselective C–H functionalization for the construction of sulfur stereogenic centers, as well as the utilization of chiral sulfoxides to realize stereoselective C–H functionalization.

This perspective highlights sulfur functional groups enabled enantioselective C–H functionalization for the construction of sulfur stereogenic centers, and the utilization of chiral sulfoxide to realize stereoselective C–H functionalization.     

Ruthenium catalyzed β-selective alkylation of vinylpyridines with aldehydes/ketones via N2H4 mediated deoxygenative couplings

Umpolung (polarity reversal) tactics of aldehydes/ketones have greatly broadened carbonyl chemistry by enabling transformations with electrophilic reagents and deoxygenative functionalizations. Herein, we report the first ruthenium-catalyzed β-selective alkylation of vinylpyridines with both naturally abundant aromatic and aliphatic aldehyde/ketones via N₂H₄ mediated deoxygenative couplings. Compared with one-electron umpolung of carbonyls to alcohols, this two-electron umpolung strategy realized reductive deoxygenation targets, which were not only applicable to the regioselective alkylation of a broad range of 2/4-alkene substituted pyridines, but also amenable to challenging 3-vinyl and steric-embedded internal pyridines as well as their analogous heterocyclic structures.

Ruthenium-catalyzed β-selective alkylation of vinylpyridines with carbonyls (both aromatic and aliphatic ketones/aldehydes) via N₂H₄ mediated deoxygenative couplings was achieved.     

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3—a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3–histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic ‘groove’ of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

We describe the development of covalent cyclic peptide ligands which target a chromatin methylation reader domain using a proximity-reactive sulfonyl fluoride moiety.     

An umpolung strategy for intermolecular [2 + 2 + 1] cycloaddition of aryl aldehydes and nitriles: a facile access to 2,4,5-trisubstituted oxazoles

We have described the first example of an umpolung strategy for intermolecular [2 + 2 + 1] cycloaddition between two aryl aldehydes and a nitrile under the influence of TMSOTf that proceeds through the formation of N–C, O–C and C–C bonds providing a simple synthetic protocol for obtaining 2,4,5-trisubstituted oxazoles.

An unprecedented intermolecular [2 + 2 + 1] cycloaddition strategy between two aryl aldehydes and a nitrile, wherein one of the aryl aldehydes serves as a carbanion (or equivalent) in the presence of TMSOTf for obtaining oxazole framework is presented.     

Proton to hydride umpolung at a phosphonium center via electron relay: a new strategy for main-group based water reduction

Generation of dihydrogen from water splitting, also known as water reduction, is a key process to access a sustainable hydrogen economy for energy production and usage. The key step is the selective reduction of a protic hydrogen to an accessible and reactive hydride, which has proven difficult at a p-block element. Although frustrated Lewis pair (FLP) chemistry is well known for water activation by heterolytic H–OH bond cleavage, to the best of our knowledge, there has been only one case showing water reduction by metal-free FLP systems to date, in which silylene (Si^II) was used as the Lewis base. This work reports the molecular design and synthesis of an ortho-phenylene linked bisborane-functionalized phosphine, which reacts with water stoichiometrically to generate H₂ and phosphine oxide quantitatively under ambient conditions. Computational investigations revealed an unprecedented multi-centered electron relay mechanism offered by the molecular framework, shuttling a pair of electrons from hydroxide (OH⁻) in water to the separated proton through a borane-phosphonium-borane path. This simple molecular design and its water reduction mechanism opens new avenues for this main-group chemistry in their growing roles in chemical transformations.

A (bisborane)triarylphosphine was developed to spontaneously generate H₂ from water under ambient conditions, revealing an unprecedented multi-centered electron relay mechanism for a metal-free umpolung of proton to hydride.     

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids

Development of multiple chemical tools for deoxyribonucleic acid (DNA) labeling has facilitated wide use of their functionalized conjugates, but significant practical and methodological challenges remain to achievement of site-specific chemical modification of the biomacromolecule. As covalent labeling processes are more challenging in aqueous solution, use of nonaqueous, biomolecule-compatible solvents such as an ionic liquid consisting of a salt with organic molecule architecture, could be remarkably helpful in this connection. Herein, we demonstrate site-specific chemical modification of unprotected DNAs through a tetrazene-forming amine–azide coupling reaction using an ionic liquid. This ionic liquid-enhanced reaction process has good functional group tolerance and precise chemoselectivity, and enables incorporation of various useful functionalities such as biotin, cholesterol, and fluorophores. A site-specifically labeled oligonucleotide, or aptamer interacting with a growth factor receptor (Her2) was successfully used in the fluorescence imaging of breast cancer cell lines. The non-traditional medium-promoted labeling strategy described here provides an alternative design paradigm for future development of chemical tools for applications involving DNA functionalization.

Site-specific chemical modification of unprotected DNAs through a phosphine-mediated amine–azide coupling reaction in ionic liquid.     

Catalytic asymmetric synthesis of quaternary trifluoromethyl α- to ε-amino acid derivatives via umpolung allylation/2-aza-Cope rearrangement

In this study, we developed an efficient Ir-catalyzed cascade umpolung allylation/2-aza-Cope rearrangement of tertiary α-trifluoromethyl α-amino acid derivatives for the preparation of a variety of quaternary α-trifluoromethyl α-amino acids in high yields with excellent enantioselectivities. The umpolung reactivity empowered by the activation of the key isatin-ketoimine moiety obviates the intractable enantioselectivity control in Pd-catalyzed asymmetric linear α-allylation. In combination with quasi parallel kinetic resolution or kinetic resolution, the generality of this method is further demonstrated by the first preparation of enantioenriched quaternary trifluoromethyl β-, γ-, δ- and ε-amino acid derivatives.

In this study, we developed an efficient Ir-catalyzed cascade umpolung allylation/2-aza-Cope rearrangement for the preparation of a variety of quaternary trifluoromethyl α-ε-amino acids in high yields with excellent enantioselectivities.     

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.     

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.      

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.     

Torsional strain inversed chemoselectivity in a Pd-catalyzed atroposelective carbonylation reaction of dibenzothiophenium

A palladium-catalyzed enantioselective ring-opening/carbonylation of cyclic diarylsulfonium salts is reported. In comparison to thioethers, the sulfonium salts displayed high reactivity and enabled the reaction to be performed under mild conditions (room temperature). The steric repulsion of the two non-hydrogen substituents adjacent to the axis led cyclic diarylsulfonium salts to be distorted, which enabled the ring-opening reaction to proceed with significant preference for breaking the exocyclic C–S bond.

Torsional strain inversed chemoselectivity: from bond a to bond b.     

A naphthalimide-based peptide conjugate for concurrent imaging and apoptosis induction in cancer cells by utilizing endogenous hydrogen sulfide

The excessive production of endogenous hydrogen sulfide (H₂S) in cancer cells leads to enhanced tumor growth and metastasis. On the other hand, decreased endogenous H₂S suppresses tumor growth. The reported approaches for inhibiting tumor growth are selective silencing of the tumor-promoting genes and pharmacological inhibition of these proteins. To enhance the antitumor efficacy of frontline chemotherapeutic agents, herein, we synthesized a highly sensitive endogenous H₂S responsive fluorescent probe, i.e., a hydrogen sulfide-sensing naphthalimide-based peptide conjugate (HSNPc), which showed selective inhibition of proliferation of cancer cells due to apoptosis induction. Furthermore, HSNPc suppressed the glycolytic reserve, a critical energy source for the proliferation of cancer cells. HSNPc also decreased the Young''s modulus of HeLa cells compared to the control cells, which demonstrated a direct relation between cell apoptosis and cell stiffness. Taken together, we demonstrated the dual function of detection and killing of cancer cells by HSNPc that can be likened to a theranostic role.

A hydrogen sulfide sensing naphthalimide based peptide conjugate (HSNPc) worked as a novel cancer cell imaging agent and showed selective cell apoptosis.