Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

Divinylpyrimidine reagents generate antibody-drug conjugates with excellent in vivo efficacy and tolerability.

The development of divinylpyrimidine (DVP) reagents for the synthesis of antibody-drug conjugates (ADCs) with in vivo efficacy and tolerability is reported. Detailed structural characterisation of the synthesised ADCs was first conducted followed by in vitro and in vivo evaluation of the ADCs' ability to safely and selectively eradicate target-positive tumours.

Multifunctional Probes with High Utilization Rates: Self-Assembled Merocyanine Nanoparticles in Water as Acid-Base Indicators and Mitochondrion-Targeting Chemotherapeutic Agents.

Multifunctional probes with high utilization rates have great value in practical applications in various fields such as cancer diagnosis and therapy. Here we have synthesized two organic molecules based on merocyanine. They can self-assemble in water to form ∼1.5 nm nanoparticles. Both of them have good application potential in fluorescent anticounterfeit printing ink and pH detection. More importantly, they have excellent mitochondrial targeting ability, intracellular red light and near-infrared dual-channel imaging ability, strong antiphotobleaching ability, and in vivo and in vitro near-infrared imaging capabilities, showing superior chemotherapy capabilities and biocompatibility in the 4T1 tumor-bearing mouse model.

A novel design of multiple ligands for ultrasensitive colorimetric chemosensor of glutathione in plasma sample.

In this study, we developed a novel colorimetric chemosensor for selective and sensitive recognition of Glutathione (GSH) using a simple binary mixture of commercially accessible and inexpensive metal receptors with names, Bromo Pyrogallol Red (BPR) and Xylenol Orange (XO). This procedure is based on the synergistic coordination of BPR and XO with cerium ion (Ce3+) for the recognition of GSH over other available competitive amino acids (AAs) especially thiol species in aqueous media. Generally, cysteine (Cys) and homocysteine (hCys) can seriously interfere with the detection of GSH among common biological species because they possess similar chemical behavior. Using all the information from 1HNMR and FT-IR studies, the proposed interaction is presented in which GSH acts as a tri-dentate ligand with three N donor atoms in conjunction with BPR and XO as mono and bi-dentate ligands respectively. This approach opens a path for selective detection of other AAs by argumentatively selecting the ensemble of mixed organic ligands from commercially available reagents, thereby eliminating the need for developing synthetic receptors, sample preparation, organic solvent mixtures, and expensive equipment. Evaluating the feasibility of the existing method was led to the determination of GSH in human plasma samples.

Synthesis of a TNF inhibitor, flurbiprofen and an i-Pr analogue in enantioenriched forms by copper-catalyzed propargylic substitution with Grignard reagents.

The copper-catalyzed substitution reaction of diethyl phosphate derived from TMSCCCH(OH)CH2CH2OTBDPS with 3-c-C5H9-4-MeOC6H3MgBr, followed by several transformations, afforded a tumor necrosis factor inhibitor possessing a Ph-acetylene moiety. The inhibitor was also synthesized from phenylacetylene phosphate PhCCCH(OP(O)(OEt)2)CH2CH2OTBDPS. Furthermore, the substitution of phosphates derived from TMSCCCH(OH)CH3 and TMSCCCH(OH)-i-Pr with 3-F-4-PhC6H3MgBr gave the corresponding substitution products, which were transformed to flurbiprofen and its i-Pr analogue, respectively. The copper-catalyzed substitutions in these syntheses proceeded in a regio- and stereoselective manner.

Fluorinated S-Adenosylmethionine as a Reagent for Enzyme-Catalyzed Fluoromethylation.

Strategic replacement of protons with fluorine atoms or functional groups with fluorine-containing fragments has proven a powerful strategy to optimize the activity of therapeutic compounds. For this reason, the synthetic chemistry of organofluorides has been the subject of intense development and innovation for many years. By comparison, the literature on fluorine biocatalysis still makes for a slim chapter. Herein we introduce S-adenosylmethionine (SAM) dependent methyltransferases as a new tool for the production of fluorinated compounds. We demonstrate the ability of halide methyltransferases to form fluorinated SAM (S-adenosyl-S-(fluoromethyl)-L-homocysteine) from S-adenosylhomocysteine and fluoromethyliodide. Fluorinated SAM (F-SAM) is too unstable for isolation, but is accepted as a substrate by C-, N- and O-specific methyltransferases for enzyme-catalyzed fluoromethylation of small molecules.

Multiplexed single-cell proteomics using SCoPE2.

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting.

Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic α-carbon and electrophilic γ-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells. We highlight the broad utility of this conjugation method to enable site-specific proteome-wide stoichiometry analysis of S-sulfenylation and to visualize redox-dependent changes in mitochondrial cysteine oxidation and redox-triggered triphenylphosphonium generation for the controlled delivery of small molecules to mitochondria.

An Atom-Economic Inverse Solid-Phase Peptide Synthesis Using Bn or BcM Esters of Amino Acids.

An atom-economic N-to-C-directed solid-phase peptide synthesis is reported that uses benzyl (Bn) or (benzhydryl-carbamoyl)-methyl (BcM) esters of amino acids as the building blocks, which facilitate efficient hydrazinolysis, convenient conversion to acyl azide, and robust amidation with the next amino acid ester. This method is free of coupling reagents and free of protection on the side-chain OH, CO2H, CONH2, etc., therefore exhibiting a significantly improved atom economy compared to those of BOC- or Fmoc-based C-to-N-directed approaches.

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents.

Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 µM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

Supramolecular Bioimaging through Signal Amplification by Combining Indicator Displacement Assay with Förster Resonance Energy Transfer.

Fluorescent chemosensors are powerful imaging tools in the fields of life sciences and engineering. Based on the principle of supramolecular chemistry, indicator displacement assay (IDA) provides an alternative approach for constructing and optimizing chemosensors, which has the advantages of simplicity, tunability, and modularity. However, the application of IDA in bioimaging continues to face a series of challenges, including interfering signals, background noise, and inconsistent spatial location. Accordingly, we herein report a supramolecular bioimaging strategy of Förster resonance energy transfer (FRET)-assisted IDA by employing macrocyclic amphiphiles as the operating platform. By merging FRET with IDA, the limitations of IDA in bioimaging were addressed. As a proof of concept, the study achieved mitochondria-targeted imaging of adenosine triphosphate in live cells with signal amplification. This study opens a non-covalent avenue for bioimaging with advancements in tunability, generality, and simplicity, apart from the covalent approach.

A Review of Chemical Tools for Studying Small Molecule Persulfides: Detection and Delivery.

Hydrogen sulfide (H2S) has gained significant attention as a potent bioregulator in the redox metabolome, but it is just one of many reactive sulfur species (RSS). Recently, small molecule persulfides (structure RSSH) have emerged as RSS of particular interest due to their enhanced antioxidant abilities compared to H2S and their ability to directly convert protein thiols into protein persulfides, suggesting that persulfides may have distinct physiological functions from H2S. However, persulfides exhibit instability and cross-reactivity that hampers the elucidation of their precise biological roles. As such, chemists have designed chemical tools and techniques to facilitate the study of persulfides under various conditions. These molecules and methods include persulfide trapping reagents and sensors, as well as compounds that degrade in response to various triggers to release persulfides, termed persulfide donors. There now exist a variety of persulfide donor classes, some of which possess tissue-targeting capabilities designed to mimic localized endogenous production of RSS. This Review briefly covers the physicochemical properties of persulfides, the endogenous production of small molecule persulfides, and their reactions with protein thiols and other reactive species. These introductory sections are followed by a discussion of chemical tools used in persulfide chemical biology, with critical analysis of recent advancements in the field and commentary on potential directions for future research.

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites.

Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.

Multifunctional nanoparticles as theranostic agents for therapy and imaging of breast cancer.

Over the last decade, there has been significant developments in nanotechnology, in particular for combined imaging and therapeutic applications (theranostics). The core or shell of nanoemulsions (NEs) can be loaded with various therapeutic agents, including drugs with low solubility for effective treatment, or various imaging agents for specific imaging modalities (e.g., MRI, fluorescence). In this work, perfluorohexane (PFH) NEs were synthesized for theranostic applications and were coupled to silica coated gold nanoparticles (scAuNPs) to increase the generation of PFH bubbles upon laser induced vaporization (i.e., optical droplet vaporization). The localized heat generated from the absorption properties of these nanoparticles (used to provide photoacoustic signals) can also be used to treat cancer without significantly damaging nearby healthy tissues. The theranostic potential of these PFH-NEs for contrast imaging of tumors and as a drug-delivery vehicle for therapeutic purposes were demonstrated for both in vitro and in vivo systems using a combination of photoacoustic, ultrasound and fluorescence imaging modalities. The ability of PFH-NEs to couple with scAuNPs, attach to the membranes of cancer cells and internalize within cancer cells, are encouraging for targeted chemotherapeutic applications for directly inducing cancer cell death via vaporization in clinical settings.

Peptide Bond Formation of Amino Acids by Transient Masking with Silylating Reagents.

A one-pot peptide bond-forming reaction has been developed using unprotected amino acids and peptides. Two different silylating reagents, HSi[OCH(CF3)2]3 and MTBSTFA, are instrumental for the successful implementation of this approach, being used for the activation and transient masking of unprotected amino acids and peptides at C-termini and N-termini, respectively. Furthermore, CsF and imidazole are used as catalysts, activating HSi[OCH(CF3)2]3 and also accelerating chemoselective silylation. This method is versatile as it tolerates side chains that bear a range of functional groups, while providing up to >99% yields of corresponding peptides without any racemization or polymerization.

Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures.

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt intramolecular secondary structure, and a 3' blocker to prevent its elongation by DNA polymerase. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates, did not demonstrate any improving effect.

Substituted Cysteine Modification and Protection with n-Alkyl- Methanethiosulfonate Reagents Yields a Precise Estimate of the Distance between Etomidate and a Residue in Activated GABA Type A Receptors.

The anesthetic etomidate modulates synaptic α1ß2/3γ2 GABAA receptors via binding sites located in transmembrane ß+/α- interfaces. Various approaches indicate that etomidate binds near ß2/3M286 side chains, including recent cryogenic electron microscopy images in α1ß2γ2L receptors under nonphysiologic conditions with ∼3.5-Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized n-alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and ß3M286 side chains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1ß3M286Cγ2L GABAA receptors, we measured functional changes after exposing GABA-activated receptors to n-alkyl-MTS reagents, from methyl-MTS to n-decyl-MTS. Based on previous studies using a large sulfhydryl reagent, we anticipated that cysteine modifications large enough to overlap etomidate sites would cause persistently increased GABA sensitivity and decreased etomidate modulation and that etomidate would hinder these modifications, reducing effects. Based on altered GABA or etomidate sensitivity, ethyl-MTS and larger n-alkyl-MTS reagents modified GABA-activated α1ß3M286Cγ2L GABAA receptors. Receptor modification by n-propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked ß3M286C modification by n-propyl-MTS, n-butyl-MTS, and n-hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, and ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and n-propyl-MTS, from which we infer that -S-(n-propyl) is the smallest ß3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and -S-ethyl and -S-(n-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the ß3M286 side chain. SIGNIFICANCE STATEMENT: Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. This study combined electrophysiology, a cysteine substitution, and n-alkyl-methanethiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1ß3γ2L GABA type A receptor residue and etomidate bound in the transmembrane ß+/α- interface.

Gold(III) Aryl Complexes as Reagents for Constructing Hybrid Peptide-Based Assemblies via Cysteine S-Arylation.

Organometallic complexes have recently gained attention as competent bioconjugation reagents capable of introducing a diverse array of substrates to biomolecule substrates. Here, we detail the synthesis and characterization of an aminophosphine-supported Au(III) platform that provides rapid and convenient access to a wide array of peptide-based assemblies via cysteine S-arylation. This strategy results in the formation of robust C-S covalent linkages and is an attractive method for the modification of complex biomolecules due to the high functional group tolerance, chemoselectivity, and rapid reaction kinetics associated with these arylation reactions. This work expands upon existing metal-mediated cysteine arylation by introducing a class of air-stable organometallic complexes that serve as robust bioconjugation reagents enabling the synthesis of conjugates of higher structural complexity including macrocyclic stapled and bicyclic peptides as well as a peptide-functionalized multivalent hybrid nanocluster. This organometallic-based approach provides a convenient, one-step method of peptide functionalization and macrocyclization, and has the potential to contribute to efforts directed toward developing efficient synthetic strategies of building new and diverse hybrid peptide-based assemblies.

Highly Sensitive Determination of Amino Acids by LC-MS under Neutral Conditions.

Peptide drug leads possess unusual structural features that allow them to exert their unique biological activities and ideal physicochemical properties. In particular, these peptides often have D-amino acids, and therefore the absolute configurations of the component amino acids have to be elucidated during the structural determination of newly isolated peptide drug leads. Recently, we developed the highly sensitive labeling reagents D/L-FDVDA and D/L-FDLDA for the structural determination of the component amino acids in peptides. In an LC-MS-based structural study of peptides, these reagents enabled us to detect infinitesimal amounts of amino acids derived from mild degradative analysis of the samples. Herein, we firstly report the improved LC-MS protocols for the highly sensitive analyses of amino acids. Second, two new labeling reagents were synthesized and their detection sensitivities evaluated. These studies increase our understanding of the structural basis of these highly sensitive labeling reagents, and should provide opportunities for future on-demand structural modifications of the reagents to enhance their hydrophobicity, stability, and affinity for applications to specialized HPLC columns.