基于非天然氨基酸的蛋白质生物正交标记

生物正交化学反应正日益成为在活体内对生物大分子进行特异标记的一种有效方法. 最近涌现出的一个突出的例子是将金属钯催化的碳碳偶联反应这一在有机合成领域具有里程碑意义的反应拓展到生物大分子的标记上. 在活细胞上进行生物正交反应的一个先决条件是需要将参与这类反应的正交官能团特异地引入到目标生物大分子当中. 遗传密码子拓展技术是将多种生物正交活性基团引入到蛋白质当中的一种先进的手段; 最近发展出的基于吡咯赖氨酸氨酰合成酶和tRNA的体系能够将携带有生物正交官能团的非天然氨基酸有效地引入到原核生物、真核生物, 甚至是动物体内的蛋白质上. 在这一展望中, 我们首先介绍在生物正交反应和遗传密码子拓展这两个领域内的研究前沿与进展. 接着我们将讨论将这些新发展的研究工具, 尤其是基于钯催化的生物正交反应和基于吡咯赖氨酸氨酰合成酶的遗传密码子拓展技术, 应用于标记和修饰哺乳动物细胞蛋白质上的优势和诱人前景. 生物相兼容性更好的正交反应和更为灵活的非天然氨基酸引入技术必将有力地增强和拓宽人们在活细胞环境下特异操纵蛋白质的能力.     

Bioorthogonal chemistry:a covalent strategy for the study of biological systems

The development of genetically encoded,wavelength-tunable fluorescent proteins has provided a powerful imaging tool to the study of protein dynamics and functions in cellular and organismal biology.However,many biological functions are not directly encoded in the protein primary sequence,e.g.,dynamic regulation afforded by protein posttranslational modifications such as phosphorylation.To meet this challenge,an emerging field of bioorthogonal chemistry has promised to offer a versatile strategy to selective...     

Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules

The chemical labeling of biomolecules continues to be an important tool for the study of their function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. In addition, with the advances in biocompatible synthetic organic chemistry, a whole new field of opportunity has opened up, affording high diversity in the nature of the label as well as a choice of ligation reactions. In recent years, several different two-step labeling strategies have emerged. These rely on the introduction of a bioorthogonal attachment site into a biomolecule, then ligation of a reporter molecule to this site using bioorthogonal organic chemistry. This Perspective focuses on these techniques, their implications and future directions.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.     

Nanostructured Heterogeneous Catalysts for Bioorthogonal Reactions

Bioorthogonal chemistry has inspired a new subarea of chemistry providing a powerful tool to perform novel biocompatible chemospecific reactions in living systems. Following the premise that they do not interfere with biological functions, bioorthogonal reactions are increasingly applied in biomedical research, particularly with respect to genetic encoding systems, fluorogenic reactions for bioimaging, and cancer therapy. This Minireview compiles recent advances in the use of heterogeneous catalysts for bioorthogonal reactions. The synthetic strategies of Pd-, Au-, and Cu-based materials, their applicability in the activation of caged fluorophores and prodrugs, and the possibilities of using external stimuli to release therapeutic substances at a specific location in a diseased tissue are discussed. Finally, we highlight frontiers in the field, identifying challenges, and propose directions for future development in this emerging field.     

Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification

In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.     

Photo-controllable bioorthogonal chemistry for spatiotemporal control of bio-targets in living systems

The establishment of bioorthogonal chemistry is one of the most significant advances in chemical biology using exogenous chemistry to perturb and study biological processes. Photo-modulation of biological systems has realized temporal and spatial control on biomacromolecules in living systems. The combination of photo-modulation and bioorthogonal chemistry is therefore emerging as a new direction to develop new chemical biological tools with spatiotemporal resolution. This minireview will focus on recent development of bioorthogonal chemistry subject to spatiotemporal control through photo-irradiation. Different strategies to realize photo-control on bioorthogonal bond-forming reactions and biological applications of photo-controllable bioorthogonal reactions will be summarized to give a perspective on how the innovations on photo-chemistry can contribute to the development of optochemical biology. Future trends to develop more optochemical tools based on novel photochemistry will also be discussed to envision the development of chemistry-oriented optochemical biology.

The establishment of photo-controllable bioorthogonal chemistry is one of the most significant advances in chemical biology to perturb and study biological processes.     

Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.     

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.     

Bioorthogonal chemistry: Optimization and application updates during 2013–2017

Bioorthogonal chemical groups to tag naturally occurring biomolecules in their native setting is a powerful tool for studying and manipulating biological processes, whereby unique functional groups incorporated into target biomolecules can be detected in a second step by using selective partners. On the other hand, bioorthogonal cleavage reactions enables chemically controlled spatiotemporal activation of intracellular proteins and prodrugs. Considerable attention has been focused on the bioorthogonal reactions, not only optimizing the known bioorthogonal reagents to gain fast reaction kinetics, high stability of the reagents and the products, but also extending new applications as well as developing new types of bioorthogonal reactions.     

Effect of Azide Position on the Rate of Azido Glucose–Cyclooctyne Cycloaddition

The strain-promoted azide–alkyne cycloaddition (SPAAC) is the most widely used bioorthogonal reaction for imaging azide-labeled glycans in living systems. Rapid SPAAC reactions are essential for visualizing biological processes that occur on a short timescale, and efforts to increase SPAAC reaction rates by modulating the cyclooctyne structure have been highly successful. However, optimizing azido sugar structure for improved SPAAC rates has not been explored. In this study, we show that altering azide position on the sugar ring can have a modest but significant impact on SPAAC reaction rate, which has implications for designing and interpreting experiments involving azide-specific bioorthogonal reactions.     

Building and Breaking Bonds via a Compact S‐Propargyl‐Cysteine to Chemically Control Enzymes and Modify Proteins

Analogous to reversible post‐translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S‐propargyl‐cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol–yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd‐cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.     

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.     

Luciferase‐Induced Photouncaging: Bioluminolysis

Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein–protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc‐Halotag chimera protein (H‐Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t_1/2<2 min). We applied this BRET uncaging system to release a potent kinase inhibitor, ibrutinib, in living cells, highlighting its broad utility in controlling the supply of bioactive small molecules in vivo.     

A biosynthetic route to photoclick chemistry on proteins

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.     

A bioorthogonal quadricyclane ligation

New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.     

Monitoring Endocytic Trafficking of Anthrax Lethal Factor by Precise and Quantitative Protein Labeling

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site‐specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF’s membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild‐type LF. This allowed time‐lapse imaging of LF’s natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.     

Breakable Hybrid Organosilica Nanocapsules for Protein Delivery

The direct delivery of specific proteins to live cells promises a tremendous impact for biological and medical applications, from therapeutics to genetic engineering. However, the process mostly involves tedious techniques and often requires extensive alteration of the protein itself. Herein we report a straightforward approach to encapsulate native proteins by using breakable organosilica matrices that disintegrate upon exposure to a chemical stimulus. The biomolecule‐containing capsules were tested for the intracellular delivery of highly cytotoxic proteins into C6 glioma cells. We demonstrate that the shell is broken, the release of the active proteins occurs, and therefore our hybrid architecture is a promising strategy to deliver fragile biomacromolecules into living organisms.     

Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5’ cap of Eukaryotic mRNAs

Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5’ cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5’ cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N²-modified 5’ cap is a suitable dipolarophile for photoclick reactions, we used Kohn–Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5’ cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.     

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.