生物正交化学在活体标记及药物传递中的研究进展

生物正交化学反应是一类可以在生理条件下发生的化学反应,具有简单、高效、高特异性的特点,在生物医学的研究中被广泛应用.基于生物体天然生命过程的代谢工程,可对生物分子进行无损、高效的生物代谢修饰,是一种理想的生物修饰技术.通过生物代谢途径可有效地将各种化学报告基团引入靶标物的生物分子中,有利于携带配对基团的标记物与其发生生物正交反应,从而在活体系统中实现生物分子的标记示踪和药物递送.这种基于代谢工程与生物正交化学的标记策略因为具有两者之间的优势,在生物医学工程中的标记、成像示踪、诊断等领域展现出巨大的研究价值与应用潜力.本文介绍了生物正交和代谢工程的原理与生物医学研究进展,阐述了生物正交化学在分子成像和药物传递等方面的研究与应用.     

99mTc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a ^99mTc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with ^99mTc at an efficiency of >95% and was radiochemically stable. ^99mTc–HYNIC tetrazine reacted with the TCO–CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of ^99mTc–HYNIC–tetrazine for tumor imaging with pretargeted mAbs.     

Immobilization strategies for small molecule,peptide and protein microarrays

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site‐selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol‐ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct

The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be incorporated into a protein site-specifically using the pyrrolysine-based genetic code expansion platform, and subsequently used for ligation chemistry. Additionally, we exploited the photolability of the QC ligation product to render the adduct cleavable with a handheld UV lamp. We further developed a protein purification method that involves QC ligation of biotin to a protein of interest, capture on streptavidin resin, and finally release using only UV light. The QC ligation thus brings novel chemical manipulations to the realm of bioorthogonal chemistry.     

Tandem photoaffinity labeling–bioorthogonal conjugation in medicinal chemistry

Photoaffinity labeling has a longstanding history as a powerful biochemical technique. However, photoaffinity labeling has significantly evolved over the past decade principally due to its coupling with bioorthogonal/click chemistry reactions. This review aims to highlight tandem photoaffinity labeling–bioorthogonal conjugation as a chemical approach in medicinal chemistry and chemical biology. In particular, recent examples of using this strategy for affinity-based protein profiling (AfBPP), drug target identification, binding ensemble profiling, studying endogenous biological molecules, and imaging applications will be presented. Additionally, recent advances in the development of ‘all-in-one’ compact moieties possessing a photoreactive group and clickable handle will be discussed.     

The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.     

Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics

Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.     

Chemical strategies for functional proteomics

With complete genome sequences now available for several prokaryotic and eukaryotic organisms, biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and protein function. Here we review a promising new class of proteomic strategies that utilizes synthetic chemistry to create tools and assays for the characterization of protein samples of high complexity. These approaches include the development of chemical affinity tags to measure the relative expression level and post-translational modification state of proteins in cell and tissue proteomes. Additionally, we discuss the emerging field of activity-based protein profiling, which aims to synthesize and apply small molecule probes that monitor dynamics in protein function in complex proteomes.     

Rapid oligonucleotide-templated fluorogenic tetrazine ligations

Template driven chemical ligation of fluorogenic probes represents a powerful method for DNA and RNA detection and imaging. Unfortunately, previous techniques have been hampered by requiring chemistry with sluggish kinetics and background side reactions. We have developed fluorescent DNA probes containing quenched fluorophore-tetrazine and methyl-cyclopropene groups that rapidly react by bioorthogonal cycloaddition in the presence of complementary DNA or RNA templates. Ligation increases fluorescence with negligible background signal in the absence of hybridization template. Reaction kinetics depend heavily on template length and linker structure. Using this technique, we demonstrate rapid discrimination between single template mismatches both in buffer and cell media. Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences.     

蛋白质组研究中的化学"探针"

随着蛋白质组学概念的提出以及诸如血浆蛋白质组等有影响力的计划开展, 蛋白质组研究迅速发展起来, 这门基于分析化学和物理化学的领域也逐渐为广大生物学家所关注, 同时也相应地在细胞生物学、生物化学等领域的研究中崭露头角。蛋白质表达量的变化以及各种各样的修饰无不反映出机体对环境变化的应激和自身功能的需要。因此, 定量蛋白质组和修饰化的蛋白质组成为了目前蛋白质组研究的重要领域之一。文章着重从采用化学标记实现定量和修饰化研究这个角度来介绍近些年来在这方面取得的进展, 希望对生物学领域的研究有所借鉴。     

Proteomic tools for biomedicine

Proteomic tools measure gene expression, protein activity and interactions of biological events at the protein level. Proteins are the major catalysts of biological functions and contain several dimensions of information that collectively indicate the actual rather than the potential functional state as indicated by mRNA analysis. Measurements can be made in terms of protein quantity, location, and time-point. For the future we see a further integration of existing and new technologies for proteomics from a wide range of areas of biochemistry, chemistry, physics, computing science and molecular biology. This will further advance our knowledge of how biological systems are built up and what mechanisms control these systems. However, the potential of proteomics to comprehensively answer all biological questions is limited as only protein activity is measured. A unification of genomics, proteomics, and other technologies is needed if we are to start to understand the complexity of biological function in the context of disease and health.     

Recent advances in quantitative and chemical proteomics for autophagy studies

Macroautophagy/autophagy is an evolutionarily well-conserved cellular degradative process with important biological functions that is closely implicated in health and disease. In recent years, quantitative mass spectrometry-based proteomics and chemical proteomics have emerged as important tools for the study of autophagy, through large-scale unbiased analysis of the proteome or through highly specific and accurate analysis of individual proteins of interest. At present, a variety of approaches have been successfully applied, including (i) expression and interaction proteomics for the study of protein post-translational modifications, (ii) investigating spatio-temporal dynamics of protein synthesis and degradation, and (iii) direct determination of protein activity and profiling molecular targets in the autophagic process. In this review, we attempted to provide an overview of principles and techniques relevant to the application of quantitative and chemical proteomics methods to autophagy, and outline the current landscape as well as future outlook of these methods in autophagy research.     

Proteome informatics for cancer research: from molecules to clinic

Proteomics offers the most direct approach to understand disease and its molecular biomarkers. Biomarkers denote the biological states of tissues, cells, or body fluids that are useful for disease detection and classification. Clinical proteomics is used for early disease detection, molecular diagnosis of disease, identification and formulation of therapies, and disease monitoring and prognostics. Bioinformatics tools are essential for converting raw proteomics data into knowledge and subsequently into useful applications. These tools are used for the collection, processing, analysis, and interpretation of the vast amounts of proteomics data. Management, analysis, and interpretation of large quantities of raw and processed data require a combination of various informatics technologies such as databases, sequence comparison, predictive models, and statistical tools. We have demonstrated the utility of bioinformatics in clinical proteomics through the analysis of the cancer antigen survivin and its suitability as a target for cancer immunotherapy.     

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry - one of the most commonly used analytical tools in proteomics - for high-throughput analyses.     

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry – one of the most commonly used analytical tools in proteomics – for high-throughput analyses.     

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.     

Metal-free click and bioorthogonal reactions of aggregation-induced emission probes for lighting up living systems

In 2002, two transformative research paradigms emerged: ‘click chemistry’ and ‘aggregation-induced emission (AIE),’ both leaving significant impacts on early 21st-century academia. Click chemistry, which describes the straightforward and reliable reactions for linking two building blocks, has simplified complex molecular syntheses and functionalization, propelling advancements in polymer, material, and life science. In particular, nontoxic, metal-free click reactions involving abiotic functional groups have matured into bioorthogonal reactions. These are organic ligations capable of selective and efficient operations even in congested living systems, therefore enabling in vitro to in vivo biomolecular labelling. Concurrently, AIE, a fluorogenic phenomenon of twisted π-conjugated compounds upon aggregation, has offered profound insight into solid-state photophysics and promoted the creation of aggregate materials. The inherent fluorogenicity and aggregate-emission properties of AIE luminogens have found extensive application in biological imaging, characterized by their high-contrast and photostable fluorescent signals. As such, the convergence of these two domains to yield efficient labelling with excellent fluorescence images is an anticipated progression in recent life science research. In this review, we intend to showcase the synergetic applications of AIE probes and metal-free click or bioorthogonal reactions, highlighting both the achievements and the unexplored avenues in this promising field.     

Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation

1,2,4,5-Tetrazines have been established as effective dienes for inverse electron demand [4 + 2] Diels-Alder cycloaddition reactions with strained alkenes for over 50 years. Recently, this reaction pair combination has been applied to bioorthogonal labeling and cell detection applications; however, to date, there has been no detailed examination and optimization of tetrazines for use in biological experiments. Here, we report the synthesis and characterization of 12 conjugatable tetrazines. The tetrazines were all synthesized in a similar fashion and were screened in parallel to identify candidates most ideally suited for biological studies. In depth follow-up studies revealed compounds with varying degrees of stability and reactivity that could each be useful in different bioorthogonal applications. One promising, highly stable, and water-soluble derivative was used in pretargeted cancer cell labeling studies, confirming its utility as a bioorthogonal moiety.