Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells

Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine‐tuning of the EBX reagents allows optimization of their reactivity and physical properties.     

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.     

Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins

A series of fluoroalkylated cyclic λ³-iodanes and their hydrochloride salts was prepared and used in a combination with sodium ascorbate in buffer or aqueous methanol mixtures for radical fluoroalkylation of a range of substituted indoles, pyrroles, tryptophan or its derivatives, and Trp residues in peptides. As demonstrated on several peptides, the aromatic amino acid residues of Trp, Tyr, Phe, and His are targeted with high selectivity to Trp. The functionalization method is biocompatible, mild, rapid, and transition-metal-free. The proteins myoglobin, ubiquitin, and human carbonic anhydrase I were also successfully functionalized.     

Expanding the Scope of Hypervalent Iodine Reagents for Perfluoroalkylation: From Trifluoromethyl to Functionalized Perfluoroethyl

A series of new hypervalent iodine reagents based on the 1,3‐dihydro‐3,3‐dimethyl‐1,2‐benziodoxole and 1,2‐benziodoxol‐3‐(1H)‐one scaffolds, which contain a functionalized tetrafluoroethyl group, have been prepared, characterized, and used in synthetic applications. Their corresponding electrophilic fluoroalkylation reactions with various sulfur, oxygen, phosphorus, and carbon‐centered nucleophiles afford products that feature a tetrafluoroethylene unit, which connects two functional moieties. A related λ³‐iodane that contains a fluorophore was shown to react with a cysteine derivative under mild conditions to give a thiol‐tagged product that is stable in the presence of excess thiol. Therefore, these new reagents show a significant potential for applications in chemical biology as tools for fast, irreversible, and selective thiol bioconjugation.     

Metal Binding Ability of Small Peptides Containing Cysteine Residues

The Cd(II)-, Pb(II)-, Ni(II)- and Zn(II)-complexes of small terminally protected peptides containing CXXX, XXXC, XCCX, CX_nC (n=1–3) sequences have been studied with potentiometric, UV/Vis and CD spectroscopic techniques. The cysteine thiolate group is the primary binding site for all studied metal ions, but the presence of a histidyl or aspartyl side chain in the molecule contributes to the stability of the complexes. For two-cysteine containing peptides the (S⁻,S⁻) coordinated species are formed in the physiological pH range and the stability increases in the Ni(II)<Zn(II)<Pb(II)<Cd(II) order. As a conclusion, the inserting of −CXXC− sequence into the peptide makes the synthesis of peptides with high selectivity to toxic Cd(II) or Pb(II) ion possible. In addition, the spectroscopic characterization of these complexes can contribute to the discovery of the exact binding site and binding mode of longer peptides mimicking the biologically important proteins.     

Cyclic Haloiodanes: Syntheses,Applications and Fundamental Studies

Hypervalent iodine reagents are powerful tools in contemporary organic synthesis. They have found numerous applications in modern oxidative transformations. The unique reactivity of hypervalent iodine allows access to unconventional electrophilic synthons. For example, electrophilic halogenation chemistry has been greatly expanded by the study of various haloiodanes. Cyclic λ³-haloiodanes are versatile reagents which can promote reactions such as halogenations, halocyclizations and oxidations. Their peculiar reactivity sets them apart from traditional sources of electrophilic halogens. Furthermore, they offer a broad range of reactivities which have been exploited in more diversified transformations. This review summarizes the different syntheses and derivatives of these cyclic haloiodanes, their applications and mechanistic insights as well as the relevant computational, structural and kinetic studies.     

Synthesis and Structure of Hypervalent Iodine(III) Reagents Containing Phthalimidate and Application to Oxidative Amination Reactions

A new class of hypervalent iodine reagents containing phthalimidate was synthesized, and structurally characterized by X‐ray analysis. The benziodoxole‐based reagent displays satisfactory solubility in common organic solvents and is reasonably stable in solution as well as in the solid state. The reagent was used for the oxidative amination of the C(sp³)? H bond of N,N‐dimethylanilines. In addition, the reagent was also applicable to oxidative amination with rearrangement of trialkylamines as well as enamines that were prepared in situ from secondary amines and aldehydes.     

Oxidation of BINOLs by Hypervalent Iodine Reagents: Facile Synthesis of Xanthenes and Lactones

Xanthene derivatives have broad applications in medicines, fluorescent probes, dyes, food additives, etc. Therefore, much attention was focused on developing the synthetic methods to prepare these compounds. Binaphthyl-based xanthene derivatives were prepared through the oxidation of BINOLs promoted by the hypervalent iodine reagent iodosylbenzene (PhIO). Nine-membered lactones were obtained through a similar oxidative reaction when iodoxybenzene (PhIO₂) was used. Additionally, one-pot reactions of BINOLs, PhIO and nucleophiles such as alcohols and amines were also investigated to provide alkoxylated products and amides in good to excellent yields.     

Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology

Cys‐tematic modification : Cysteine is a versatile amino acid for selective chemical modification of proteins. Both chemical and biological innovations made possible by cysteine modification are highlighted in this Focus Review.

     

Access to Vinyl Ethers and Ketones with Hypervalent Iodine Reagents as Oxy-Allyl Cation Synthetic Equivalents

We report an Umpolung strategy of enol ethers to generate oxy-allyl cation equivalents based on the use of hypervalent iodine reagents. Under mild basic conditions, the addition of nucleophiles to aryloxy-substituted vinylbenziodoxolone (VBX) reagents, easily available in two steps from silyl alkynes, resulted in the stereoselective formation of substituted aryl enol ethers. The reaction was most efficient with phenols as nucleophiles, but preliminary results were also achieved for C- and N- nucleophiles. In absence of external nucleophiles, the 2-iodobenzoate group of the reagent was transferred. The obtained aryl enol ethers could then be transformed into α-difunctionalized ketones by oxidation. The described “allyl cation”-like reactivity contrast with the well-established “vinyl-cation” behavior of alkenyl iodonium salts.     

Intramolecular Metal‐Free Oxidative Aryl–Aryl Coupling: An Unusual Hypervalent‐Iodine‐Mediated Rearrangement of 2‐Substituted N‐Phenylbenzamides

Hypervalent‐iodine‐mediated oxidative coupling of the two aryl groups in either 2‐acylamino‐N‐phenyl‐benzamides or 2‐hydroxy‐N‐phenylbenzamides, with concomitant insertion of the ortho‐substituted N or O atom into the tether, has been described for the first time. This unusual metal‐free rearrangement reaction involves an oxidative C(sp²)? C(sp²) aryl–aryl bond formation, cleavage of a C(sp²)? C(O) bond, and a lactamization/lactonization. Furthermore, unsymmetrical diaryl compounds can be easily obtained by removing the tether within the cyclized product.     

Access to Vinyl Ethers and Ketones with Hypervalent Iodine Reagents as Oxy‐Allyl Cation Synthetic Equivalents

We report an Umpolung strategy of enol ethers to generate oxy‐allyl cation equivalents based on the use of hypervalent iodine reagents. Under mild basic conditions, the addition of nucleophiles to aryloxy‐substituted vinylbenziodoxolone (VBX) reagents, easily available in two steps from silyl alkynes, resulted in the stereoselective formation of substituted aryl enol ethers. The reaction was most efficient with phenols as nucleophiles, but preliminary results were also achieved for C‐ and N‐ nucleophiles. In absence of external nucleophiles, the 2‐iodobenzoate group of the reagent was transferred. The obtained aryl enol ethers could then be transformed into α‐difunctionalized ketones by oxidation. The described “allyl cation”‐like reactivity contrast with the well‐established “vinyl‐cation” behavior of alkenyl iodonium salts.     

Continuous‐Flow Electrochemical Generator of Hypervalent Iodine Reagents: Synthetic Applications

An efficient and reliable electrochemical generator of hypervalent iodine reagents has been developed. In the anodic oxidation of iodoarenes to hypervalent iodine reagents under flow conditions, the use of electricity replaces hazardous and costly chemical oxidants. Unstable hypervalent iodine reagents can be prepared easily and coupled with different substrates to achieve oxidative transformations in high yields. The unstable, electrochemically generated reagents can also easily be transformed into classic bench‐stable hypervalent iodine reagents through ligand exchange. The combination of electrochemical and flow‐chemistry advantages largely improves the ecological footprint of the overall process compared to conventional approaches.     

Photocatalytic Modification of Amino Acids,Peptides, and Proteins

In the last decade, visible-light photoredox catalysis has emerged as a powerful strategy to enable novel transformations in organic synthesis. Owing to mild reaction conditions (i.e., room temperature, use of visible light) and high functional-group tolerance, photoredox catalysis could represent an ideal strategy for chemoselective biomolecule modification. Indeed, a recent trend in photoredox catalysis is its application to the development of novel methodologies for amino acid modification. Herein, an up-to-date overview of photocatalytic methodologies for the modification of single amino acids, peptides, and proteins is provided. The advantages offered by photoredox catalysis and its suitability in the development of novel biocompatible methodologies are described. In addition, a brief consideration of the current limitations of photocatalytic approaches, as well as future challenges to be addressed, are discussed.     

Site-Selective Modification of Peptides and Proteins via Interception of Free-Radical-Mediated Dechalcogenation

The development of site-selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free-radical-mediated dechalcogenation. By exploiting phosphine-induced homolysis of the C−Se and C−S bonds of selenocysteine and cysteine, respectively, we demonstrate the site-selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low-oxidation-state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.     

Chemo‐ and Regioselective Ethynylation of Tryptophan‐Containing Peptides and Proteins

Ethynylation of various tryptophan‐containing peptides and a single model protein was achieved using Waser's reagent, 1‐[(triisopropylsilyl)ethynyl]‐1,2‐benziodoxol‐3(1 H)‐one (TIPS‐EBX), under gold(I) catalysis. It was demonstrated by NMR that the ethynylation occured selectively at the C2‐position of the indole ring of tryptophan. Further, MS/MS showed that the tryptophan residues could be modified selectively with ethynyl functionalities even when the tryptophan was present as a part of the protein. Finally, the terminal alkyne was used to label a model peptide with a fluorophore by means of copper‐catalyzed click chemistry.     

Electron Transfer in Peptides with Cysteine and Methionine as Relay Amino Acids

Caught on the hop : In multistep electron transfer (ET) reactions through peptides, aliphatic amino acids can also act as relay stations. With cysteine, the reaction occurs as a proton‐coupled electron transfer (PCET) with water used as a mediator for the proton transfer (see picture).

     

A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full‐length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε‐amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site‐specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site‐specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.     

Metal‐free Synthesis of Aryl Amines: Beyond Nucleophilic Aromatic Substitution

A mild and metal‐free approach to C?N coupling is described that employs diaryliodonium salt electrophiles and secondary aliphatic amine nucleophiles. This reaction results in direct ipso‐substitution of the iodonium moiety and unsymmetrical aryl(TMP)iodonium salts are primarily employed. Moreover, arene substituents and substitution patterns that currently pose a challenge to classical metal‐free methods are accommodated and the alicyclic amine nucleophiles used here are unprecedented in other contemporary metal‐free C?N coupling reactions.