Palladium in the Chemical Synthesis and Modification of Proteins

The field of site‐specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium‐mediated C−C bond‐forming and C−O bond‐cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.     

Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins

In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent‐labeling studies demonstrated that the installation of a C‐terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site‐specific labeling of cysteine‐containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.     

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.

     

A Rhodium Catalyst Superior to Iridium Congeners for Enantioselective Radical Amination Activated by Visible Light

A bis‐cyclometalated rhodium(III) complex catalyzes a visible‐light‐activated enantioselective α‐amination of 2‐acyl imidazoles with up to 99 % yield and 98 % ee. The rhodium catalyst is ascribed a dual function as a chiral Lewis acid and, simultaneously, as a light‐activated smart initiator of a radical‐chain process through intermediate aminyl radicals. Notably, related iridium‐based photoredox catalysts reported before were unsuccessful in this enantioselective radical C?N bond formation. The surprising preference for rhodium over iridium is attributed to much faster ligand‐exchange kinetics of the rhodium complexes involved in the catalytic cycle, which is crucial to keep pace with the highly reactive and thus short‐lived nitrogen‐centered radical intermediate.     

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid‐phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.     

Pushing the Boundaries of Chemical Protein Synthesis: The Case of Ubiquitin Chains and Polyubiquitinated Peptides and Proteins

Chemical synthesis offers unique opportunities to prepare proteins with precise control of the atomic composition. Thanks to recent breakthroughs in synthetic methods, the preparation of large and complex proteins composed of 200–300 residues has now become possible. With these advances, a unique toolbox has been created to enable chemical biologists to investigate proteins that are difficult or even impossible to achieve otherwise, such as posttranslationally modified proteins and proteins composed of d ‐amino acids. In this review we describe the latest achievements in constructing protein conjugates of record sizes, such as those that are involved in the ubiquitin system.

     

Direct Utilization of Near-Infrared Light for Photooxidation with a Metal-Free Photocatalyst

Near-infrared (NIR) light-triggered photoredox catalysis is highly desirable because NIR light occupies almost 50% of solar energy and possesses excellent penetrating power in various media. Herein we utilize a metal-free boron dipyrromethene (BODIPY) derivative as the photocatalyst to achieve NIR light (720 nm LED)–driven oxidation of benzylamine derivatives, sulfides, and aryl boronic acids. Compared to blue light–driven photooxidation using Ru(bpy)₃Cl₂ as a photocatalyst, NIR light–driven photooxidation exhibited solvent independence and superior performance in large-volume (20 mL) reaction, presumably thanks to the neutral structure of a BODIPY photocatalyst and the deeper penetration depth of NIR light. We further demonstrate the application of this metal-free NIR photooxidation to prodrug activation and combination with Cu-catalysis for cross coupling reaction, exhibiting the potential of metal-free NIR photooxidation as a toolbox for organic synthesis and drug development.     

Merger of Visible Light Induced Oxidation and Enantioselective Alkylation with a Chiral Iridium Catalyst

A single chiral octahedral iridium(III) complex is used for visible light activated asymmetric photoredox catalysis. In the presence of a conventional household lamp and under an atmosphere of air, the oxidative coupling of 2‐acyl‐1‐phenylimidazoles with N,N‐diaryl‐N‐(trimethylsilyl)methylamines provides aminoalkylated products in 61–93 % yields with high enantiomeric excess (90–98 % ee). Notably, the iridium center simultaneously serves three distinct functions: as the exclusive source of chirality, as the catalytically active Lewis acid, and as a central part of the photoredox sensitizer. This conceptionally simple reaction Scheme may provide new avenues for the green synthesis of non‐racemic chiral molecules.     

Single-Step N-Terminal Modification of Proteins via a Bio-Inspired Copper(II)-Mediated Aldol Reaction

The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The N-terminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the N-terminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.     

A PEGylated Photocleavable Auxiliary Mediates the Sequential Enzymatic Glycosylation and Native Chemical Ligation of Peptides

Research aimed at understanding the specific role of glycosylation patterns in protein function would greatly benefit from additional approaches allowing direct access to homogeneous glycoproteins. Herein the development and application of an efficient approach for the synthesis of complex homogenously glycosylated peptides based on a multifunctional photocleavable auxiliary is described. The presence of a PEG polymer within the auxiliary enables sequential enzymatic glycosylation and straightforward isolation in excellent yields. The auxiliary‐modified peptides can be directly used in native chemical ligations with peptide thioesters easily obtained by direct hydrazinolysis of the respective glycosylated peptidyl resins and subsequent oxidation. The ligated glycopeptides can be smoothly deprotected by UV irradiation. We apply this approach to the preparation of variants of the epithelial tumor marker MUC1 carrying one or more Tn, T, or sialyl‐T antigens.     

Fast and Cysteine-Specific Modification of Peptides,Proteins and Bacteriophage Using Chlorooximes

This work reports a novel chlorooxime mediated modification of native peptides and proteins under physiologic conditions. This method features fast reaction kinetics (apparent k₂=306±4 M⁻¹s⁻¹ for GSH) and exquisite selectivity for cysteine residues. This cysteine conjugation reaction can be carried out with just single-digit micromolar concentrations of the labeling reagent. The conjugates show high stability towards acid, base, and external thiol nucleophiles. A nitrile oxide species generated in situ is likely involved as the key intermediate. Furthermore, a bis-chlorooxime reagent is synthesized to enable facile Cys-Cys stapling in native peptides and proteins. This highly efficient cysteine conjugation and stapling was further implemented on bacteriophage to construct chemically modified phage libraries.     

Development of a DNA‐Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins

Histone post‐translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA‐templated technology and a photo‐crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications.     

CO2‐Triggered Switchable Hydrophilicity of a Heterogeneous Conjugated Polymer Photocatalyst for Enhanced Catalytic Activity in Water

Water compatibility for heterogeneous photocatalysts has been pursued for energy and environmental applications. However, there exists a trade‐off between hydrophilicity and recyclability of the photocatalyst. Herein, we report a conjugated polymer photocatalyst with tertiary amine terminals that reversibly binds CO₂ in water, thereby generating switchable hydrophilicity. The CO₂‐assisted hydrophilicity boosted the photocatalytic efficiency in aqueous medium with minimum dosage. When CO₂ was desorbed, the photocatalyst could be simply regenerated from reaction media, facilitating the repeated use of photocatalyst. Hydrophilicity/hydrophobicity control of the polymer photocatalyst was successfully showcased through a variety of organic photoredox reactions under visible‐light irradiation in water.     

Peptide‐Templated Acyl Transfer: A Chemical Method for the Labeling of Membrane Proteins on Live Cells

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N‐terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N‐terminally tagged with a 22 aa long Cys‐E3 peptide (acceptor), which is capable of forming a coiled‐coil with a reporter‐armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y₂ receptor on living cells.     

Site‐Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site‐directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site‐selective modifications since most proteins contain disulfide bonds. In this Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Synthetic Strategies for Artificial Lipidation of Functional Proteins

Biosynthesis of natural lipidated proteins is linked to important signal pathways, and therefore analyzing protein lipidation is crucial for understanding cellular functions. Artificial lipidation of proteins has attracted attention in recent decades as it allows modulation of the amphiphilic nature of the protein of interest, and is used in the design of drug-delivery systems containing antibodies anchored on a lipid bilayer carrier. However, the intrinsic hydrophobicity of lipids makes the synthesis of lipid–protein conjugates challenging with respect to the yield and selectivity of the lipidation. In this Minireview, the development of chemical and enzymatic synthetic strategies for the preparation of a range of lipid–protein conjugates that do not compromise the functions of the proteins are discussed as well as applications of the conjugates.