Activity-based proteomic profiling: The application of photoaffinity probes in the target identification of bioactive molecules

Accepted Manuscript Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules Jin Wang, Qinhua Chen, Yuanyuan Shan, Xiaoyan Pan, Jie Zhang PII:

S0165-9936(19)30032-9

DOI:

https://doi.org/10.1016/j.trac.2019.03.028

Reference:

TRAC 15464

To appear in:

Trends in Analytical Chemistry

Received Date: 22 January 2019 Revised Date:

28 March 2019

Accepted Date: 28 March 2019

Please cite this article as: J. Wang, Q. Chen, Y. Shan, X. Pan, J. Zhang, Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2019.03.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Article title: Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules Reference: TRAC15464 Journal title: Trends in Analytical Chemistry Corresponding author: Professor Jie Zhang First author: Dr Jin Wang

Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules

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Jin Wanga,1, Qinhua Chenb,1, Yuanyuan Shanc, Xiaoyan Pan a, Jie Zhanga,∗ a

School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, China c Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China

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• Title: Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules • Author names and affiliations.

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Jin Wanga,1, Qinhua Chenb,1, Yuanyuan Shanc, Xiaoyan Pan a, Jie Zhanga,* School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, China c Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China E-mail address: Jin Wang ([email protected]); Qinhua Chen ([email protected]); Yuanyuan Shan ([email protected]); Xiaoyan Pan ([email protected]); Jie Zhang ([email protected])

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• Corresponding author. Correspondence should be addressed to Prof. Jie Zhang at the following address, phone and fax number, and email address: School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province, 710061, China; Tel/Fax: +86-29-826555451; E-mail: [email protected] • Present/permanent address. No

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Corresponding author. E-mail: [email protected] (Jie Zhang). Both authors contributed equally to this work and should be regarded as joint first authors.

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Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in the Target Identification of Bioactive molecules Abstract Identifying cellular targets for bioactive molecules is a major challenge and a key issue in drug discovery. Small molecule probes play an essential role in identifying targets of bioactive compounds. Photoaffinity labelling (PAL) has been developed as an emerging strategy owing to its capability of investigating the interaction between ligands and proteins. However, non-convalent interaction are too weak to precisely detect the target protein. PAL can covalently capture the target protein and offer an bioorthogonal reporter group to identify the target protein. Moreover, with its function in structural insight and instant binding site validation, PAL has the most potential to accelerate drug discovery. Herein, we will present an overview of the photoactive groups, reporter groups, and bio-orthogonal reactions involved in PAL, with a focus on their application in target identification of bioactive molecules. A number of comparative studies are described in which the efficiency of various photoaffinity probes are compared. Key words: Photoaffinity labelling; Intracellular targets identification; Photoactive groups; Reporter groups; Bio-orthogonal reactions. 1. Introduction Target protein identification is a key step in early-stage drug discovery [1]. It has been verified that biotechnology at the genetic level cannot thoroughly identify molecular targeting for certain diseases. Meanwhile, photoaffinity labeling (PAL) has been developed as a quintessential strategy to identify the targets of active molecules, owing to its capability of investigating the interctions between ligands and proteins. PAL is a quintessential technique in the elucidation of protein structure, function and conformational changes, as well as the identification of novel or alternative binding sites in proteins. Recently, two key functional groups, the photoaffinity group (PAG) and bio-orthogonal handle, have been incorporated into probes for target identification of bioactive molecules. A PAG can generate a photoactive intermediate following irradiation with light of a specific wavelength, and the intermediate can produce a stable and irreversible covalent bond to capture the target protein. After capturing the target protein, a biorthogonal handle is used to trace or purify the target protein. At last, the captured proteins are analyzed and identified using bioanalytical techniques (Fig. 1).

Fig. 1. Photoaffinity labelling technique schematic.

As is known, proteins are an essential part of cell life activity. Proteins also participate in regulating cells and their signals. Thus, the exploration of protein-protein interaction is quite important. Traditional methods of exploring protein-protein interactions focused on known protein-protein interactions, which may ignore unknown protein-protein interactions. However, the unknown protein-protein interactions play important roles in exploring cell signals, which help us to understand the complex environment of the cell. Based on PAL principles to explore the

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interaction of an active molecule probe and target protein, researchers found that this technology can also be used to explore protein-protein interactions. PAL can therefore be used to explore unknown protein-protein interactions. For this method, we just need to know the prey-protein interaction to explore the prey-bait protein interaction. At first, we can design and synthesize the photoaffinity probe of the bait-protein. Then, using the specific wavelength of light to irradiate the probe, the probe can rapidly covalently bind to the prey-protein. Next, we can utilize the corresponding analysis methods to analyze and identify the prey-protein. In the near future, we can use the technology of PAL to explore unknown protein-protein interactions. Traditional fluorescent labeling directly introduces a fluorescent group onto a ligand forming a probe. However, fluorescent labeling exhibits the following disadvantages: (1) ligands and receptors form specific and noncovalent reversible interactions, which show poor stability and are prone to false positive results, and (2) the existing fluorescent molecules are bulky and might affect the ligand’s physical and chemical properties, as well as biological activity. Compared to traditional fluorescent labeling, PAL could improve upon these deficiencies. First, through the introduction of two functional groups onto the nonpharmacological unit of a bioactive molecule, there is little effect on the physical and chemical properties and biological activity of original molecule. Second, the PAG could form a very stable covalent bond between the ligand and receptor [2]. Consequently, the incidence of false positive results is greatly reduced. In this review, we will discuss the general principles of PAL, concerning the two key functional groups of the photoaffinity probe (PAP) and the biorthogonal unit. We will focus on successful examples of PAL application on the identification of intracellular targets of bioactive molecules. Most examples presented here were published within the past five years, and a greater emphasis has been given to recent examples illustrative of the biorthogonal PAL reaction. 2. Photoaffinity groups PAPs exhibit several necessary characteristics. First, they form a reactive intermediate with the target protein to generate an irreversible covalent bond. Second, they are stable under a variety of conditions. Third, they display similar potency to the parent compound. Finally, the luminescence can be detected at a minimum wavelength without damage to the protein. Currently, diaziridine/phenyldiaziridine, benzophenone, tetrazole and phenyl azide are common PAGs with highly reactive intermediates, such as carbene, diradical, nitrile imine and nitrene, formed after irradiation with specific wavelengths of light (Fig. 2).

Fig. 2. The major photoaffinity functional groups and their reactive intermediates.

As several wavelengths of light may damage a protein, the half-life of these highly reactive intermediates must be shorter than the dissociation of the ligand-protein complex. Meanwhile, the

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activation energy should be greater than the absorption wavelength of the protein to avoid damaging the biomolecule. Absorbance at 280 nm is attributed to the aromatic amino acids tryptophan, tyrosine and phenylalanine, and the peptide bond structure produces absorption at 200 nm. The protein absorbs a specific wavelength of ultraviolet light and then converts it into an electronically excited molecule, which is chemically converted after degradation. These characteristics meet the requirement that PAPs do not cause photodamage to proteins. The diaziridine group can transform into a carbene under irradiation at 350-380 nm; benzophenone generates a diradical intermediate under irradiation at 350-360 nm; tetrazole transforms into a nitrile imine at 302 nm; and phenylazide generates a nitrene at 250-350 nm [3]. Among these four PAGs, the excitation wavelengths of diaziridine and benzophenone are longer than the others, thus, diaziridine and benzophenone need less energy to be activated, which causes less damage to the organism. Furthermore, carbenes are more reactive than nitrenes and nitrile imines. Therefore, diaziridine and benzophenone are generally used as the photoactive groups to capture target proteins. Previously, researchers used commercially available phenylazides as photoreactive groups [4]. Unfortunately, phenylazides are activated by shorter wavelengths than other photoreactive groups, which can cause protein damage. Particularly, the nitrene intermediate formed is less reactive to photocrosslinking compared with carbenes [5]. Therefore, we expected to find another high-activity group. Benzophenones form a reactive triplet diradical when irradiated with light and are activated by long wavelengths, which decreases the risk of biomolecule damage. Benzophenone is not only highly active, but it is also commercially available. A disadvantage is that a long period of time is often needed to increase the nonspecific labeling [6]. Recently, researchers have focused on photoreactive groups with high reactivity and specificity. They found that aryl diazirines, particularly the trifluoromethyl derivative, are the most popular PAGs and are favored due to their excellent chemical stability. Aryl diazirines require long wavelengths to become active, which is beneficial, and they rapidly form a covalent crosslink to the protein. However, due to their high reactivity, carbenes are often quickly quenched by water. This may affect the yield of the photoaffinity label, but it can decrease the nonspecific binding. 3. Reporting groups After capturing the target protein, it is necessary to identify the protein beginning with the separation of the photolabeled protein. To facilitate the successful separation of photolabeled biomacromolecules, a reporter group needs to be incorporated either directly or indirectly into the probe. There are two methods for separating target proteins. One is the utilization of a click reaction to detect the target protein, which is often then separated by fluorescence techniques. Usually, the fluorophore is attached to the end of a click-reactive moiety to trace, separate and identify the target protein. Fluorescein, BODIPY (boron difluoride dipyrromethene), rhodamine, cyanine dyes (Cy-5 and Cy-3) and NBD (nitrobenzene-2-oxa-1,3-dioxazole) are used as fluorophores, with the main advantages being that these labels are hydrophobic and can usually penetrate the cell membrane. Each fluorophore has its own advantages and disadvantages as well. Fluorescein and rhodamine are cheaper than other alternatives; however, they are susceptible to photobleaching. BODIPY and the cyanine dyes have high absorption coefficients, narrow absorption peaks and high quantum yields, which provide clearer detection and identification. Another method is the application of an affinity tag. The most commonly used affinity tag is the biotin-avidin system, which takes advantage of the high affinity of avidin for biotin. This type

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of probe is called a biotin probe. Affinity tags allow avidin-containing proteins to easily enrich and separate labeled proteins and can detect tag-specific related antibodies. However, affinity tags are usually too large to exhibit favorable cell permeability. In addition to the use of fluorescent labels and biotin-avidin systems for the identification and screening of target proteins, recent studies have shown that isotopic labels, which incorporate major isotopic labels, can also be used to identify target proteins. After capturing the target protein by the photoreaction, the reporter label can be directly reflected, thereby realizing the structural identification of the target. 4. Applications of Photoaffinity Labeling PAL is mainly used for the confirmation targets of bioactive molecules. Herein, we will summarize the PAL studies applied in drug target identification. First, the photocrosslinking ability of several photoactive groups was evaluated. After crosslinking to known target proteins, the photocrosslinking efficiencies were compared. The efficiency and selectivity of single-stranded oligonucleotides (ODN-B, ODN-D and ODN-S) containing three different photoreactive moieties for the SSB protein were compared [7].The classical photoreactive groups benzophenone (B) and trifluoromethyl diaziridine (D) were selected, while 4-thiothymidine (S) was used as a PAG for re-evaluation. These reagents can affect the PAL efficiency and selectivity. A set of photoaffinity nanoprobes with small molecule ligands and photoreactive groups were also developed [8]. Due to the typical hydrophobicity of these functional groups, a hydrophilic structure was introduced to functionalize the nanoprobes to maintain their dispersibility in aqueous buffer solution. Simultaneous PAL studies using nanoprobes consisting of these three functional groups in varying proportions indicated that the inclusion of high-density spacer groups reduces crosslinking efficiency. At last, a comparative analysis of the reactivity between the three major photoreactive groups indicates that the phenylazide group showed the highest activity compared to conventional PAPs. A trifunctional PAP bearing a benzophenone or diaziridine group was prepared and evaluated as glycolipid PAP [9]. A comparative analysis involving competitive ligands showed that the diazirine PAPs were more efficient than the benzophenone and diazirine probes and can be used to distinguish specific binding proteins. Recently, many groups used PAL to confirm the biological targets of known active small molecules. First, suitable photoaffinity and reporter groups were selected to prepare a PAP based on the biological activity of the small molecule. The activity was evaluated ranging from the protein level to the cell level. Next, under specific-wavelength light irradiation, the probe covalently captured the target protein. At last, the captured protein was traced or purified using a reporter group. The LW6 target protein was identified adopting this principle [10]. PAPs of LW6 were prepared based on prior SAR studies. Placement of the probes in HCT116 cells irradiated click-conjugates with a fluorescent reporter tag. The proteins were separated by SDS-PAGE and visualized by in-gel fluorescence scanning, giving a prominent band at approximately 37 kDa. Following trypsin digestion and sequencing of the peptides by MS, the protein was identified as MDH2, a mitochondrial enzyme involved in the tricarboxylic acid cycle. Further studies showed that LW6 interacted with and inhibited MDH2, and that a known inhibitor of MDH2 potently inhibited HIF transcriptional activity. The intracellular target of CCG-1423 was also determined using this strategy [11]. First, benzophenone and the reporter Cy-5.5 were selected to design a synthetic PAP to identify the 24 kDa protein. Then, they found that the labeling of the protein was substantially and competitively reduced using excess unlabeled CCG1423, thus indicating that the protein is a true target. A

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similar approach was implemented to identify the target protein for the anticancer agent LBL1 [12]. Trifluoromethyl diaziridine PAG and an alkynyl-tag were used to construct probe LBL1-P. Then, it was found that LBL1 targets laminin, which is a V-type intermediate fiber (IF) protein. Further studies have indicated that LBL1 binds to the coiled-coil domain of lamin A. OSW-1 is a highly potent anticancer saponin with an unknown mechanism of action. To investigate its binding protein, a diaziridine photoactive group was incorporated onto OSW-1 to form a probe [13]. The PAP was clicked, and its photochemical reactivity was characterized. PAL studies indicate that probes are capable of crosslinking model sterol-binding proteins in affinity-dependent manner. In the same way, a photoswitching method was developed using fluorescent labeling to identify photosensitized peptides in target proteins [14]. The PAP was designed by introducing a trifluoromethyl-substituted diaziridine PAG and an avidin-biotin tag. The authors found that the PAL could rapidly identify labeled peptides, but the necessary amount of protein for analysis was in the range of 10 micrograms. A multifunctional probe was obtained from an HIF-1α inhibitor using trifluoromethyl diaziridine as the PAG [15]. Photoaffinity reaction markers and click reactions were combined to identify the target proteins, and HSP60 was ultimately identified as the primary target. A diaziridine analog of palmitic acid was developed to detect potential binding sites for purified rat liver peroxisomes [16], and the substance was found to be reproducible for the 80 kDa protein. Under competitive conditions, the PAL efficiency was reduced, confirming that the protein MFE2 is the target of the rat liver peroxisomes. The target proteins of Sortin1 were investigated using benzophenone as the photoreactive group and biotin as the tag [17]. It was found that Sortin1 could exhibit photocrosslinking stability against model binding proteins, which could then be used to capture and detect binding proteins. This study identified not only the target of the small molecule but also the target of a short peptide. A benzophenone PAG and a biotin tag were incorporated to modify a short peptide targeting HIV-1 integrase. Through photocrosslinking investigation, the researchers found that the probe bound directly to IN [18]. Sakurai developed a multivalent carbohydrate PAP based on gold nanoparticles (AuNP) to recognize carbohydrate binding proteins [19]. AuNP were used as the carbohydrate ligand, while benzophenone and phenylazide were used as photoreactive groups to design the PAP to directly enrich the crosslinked protein by illumination and centrifugation. These probes can remove nonspecific proteins and separate low-affinity carbohydrate-binding proteins from the selective markers and cell lysates. PAL is used to not only identify unknown targets of bioactive molecules, but also to explore protein-protein interactions. A releasable PAP was constructed with the genetically encoded multifunctional photocrosslinker DiZASeC [20]. DiZASeC contains three functional parts: a diaziridine photocrosslinking group, an alkyne bio-orthogonal handle, and a Se releasable linker. The PAP enables capture and identification of low-profile proteins. Combined with stable isotope labeling, the iso-CAPP strategy can be quantified. This strategy identified the fourteen proteolytic substrates for the identification of E. coli PQC factor DegP under heat shock conditions, including six newly identified proteins. The commonly used noncovalent binding tracer technology has low specificity, and therefore, it is particularly important to realize the protein trace by photocrosslinking. Trifluoromethyl diazirine was used as photoactive group to design a PAP, LEI121, to investigate the type 2 cannabinoid receptor (CB2R) [21]. Under irradiation, LEI121 covalently captured CB2R. It was

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possible to observe the endogenously expressed CB2R receptor in HL60 cells by covalently capturing the receptor protein. Three selective serotonin reuptake inhibitors (SSRIs) were derivated from citalopram. Benzophenone and phenylazide were used as PAGs, and an alkyne served as bio-orthogonal handle to capture target protein [22]. The clickable benzophenone (S)-citalopram probe had 11 times higher affinity compared to (S)-citalopram. In addition to verifying photocrosslinking efficiency, PAL can identify small molecules, macromolecular targets, and even study protein-protein interactions. A set of activity-based fluorescent probes were prepared as useful tools for screening HDAC inhibitors [23]. Based on these results, diaziridine was introduced to afford a dual-test fluorescent probe. The resulting probes were capable of not only reporting enzymatic activity, but also capable of directly identifying the target protein from complex cellular environments. In summary, a PAL is a chemical probe that covalently binds its target in response to activation by light. It has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location of binding sites. 5. Bio-orthogonal Reactions Bio-orthogonal reactions can be carried out in living cells or tissues without interfering with the biochemical reactions of the organism. This concept was first proposed by Carolyn R. Bertozzi in 2003. The bio-orthogonal reaction connotation refers to the study of biological macromolecules, such as proteins and lipids, without toxicity to cells. It has been widely used from glycan engineering to in vivo imaging. It has already made a tremendous scientific impact, helping us to understand glycosylation in cells and animals, providing tools for conjugating functional groups to therapeutically relevant proteins such as antibodies, and enabling the assembly of molecular imaging agents in vivo to detect disease. Bio-orthogonal chemistry has inspired chemical biologists to think about how to relate classical organic reactions to life systems and apply these reactions to explore biological systems. Bio-orthogonal chemistry is a powerful approach to the real-time study of biomolecules in living systems. It relies on rapid chemical ligation reactions between two bio-orthogonal functional groups. These two bio-orthogonal partners react with each other in a chemo-selective manner, and are inserted into other chemical entities. The reactions used in bio-orthogonal chemistry should occur quickly, with quantitative yield and be compatible with living systems. Thus, bio-orthogonal reaction–based hyperpolarization tagging appears to be an attractive strategy that can selectively highlight and localize the target-containing biorthogonal partner. The organisms used in bio-orthogonal chemistry must tolerate the reaction conditions. Recently, it has been found that bio-orthogonal reactions mainly include the following types of reactions: click, aldol condensation, and the biocrosslinking of a cyclopropene cation and phosphine. The click reaction can be divided into two types, namely, lossy and nondestructive. Among them, the lossy click reaction causes cell damage and the link is generated using a copper catalyst, while the nondestructive click reaction does not need copper as a catalyst. 5.1 Click reaction The concept of click chemistry was introduced by Sharpless in 2001. The main purpose is to quickly and reliably complete the chemical synthesis of various colored molecules through the splicing of small units. It emphasizes the development of new combinatorial chemistry methods based on the synthesis of carbon-heteroatom bonds (C-X-C). With these click reactions, molecular diversity is obtained simply and efficiently. Due to the availability of raw materials and reagents,

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Fig. 3. Click reaction under copper catalysis.

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simple reaction conditions, high yield, good stereoselectivity, easy separation and purification, and good product stability, the click reaction has been rapidly developed since it appeared. The representative click reaction is the copper-catalyzed azide-alkyne Huisgen cycloaddition reaction (copper-catalyzed azide–alkyne cycloaddition). In addition, click chemistry also includes cycloaddition, nucleophilic ring opening, the carbonyl chemistry of nonalkanes, and additional reactions to carbon-carbon multiple bonds. 5.1.1 The copper-catalyzed bio-orthogonal reaction The copper-catalyzed click reaction mainly refers to the reaction between a terminal alkynyl group and an azide as shown in Fig. 3. This method is generally used to label a target protein, which means that the probe molecule must bear not only a PAG but also a clickable handle. The bio-orthogonal handle undergoes a click reaction with the azide group to display or purify the captured protein. This method can be used to trace or purify target proteins.

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A strategy for labeling teichoic acid in the human pathogen S. pneumonia was developed using a click reaction between an azide and an alkynyl [24]. A dual-response metabolic precursor can produce azide-containing non-natural glycans with high selectivity on a targeted cancer cell membrane for the detection of various biological processes [25]. A water-soluble fluorescent light-emitting probe with aggregation-induced emission (AIE) that fluoresces upon reaction with the azide group on the surface of cancer cells could image specific cancer cells with low background signals. Moreover, these probes can produce O2 under light irradiation, achieving a dual function as imaging and therapeutic agents for cancer cells. Multifunctional peptide ligands for dense and biocompatible QD were also designed. The target-ligand formulation utilizes an effective non-activator Ugi reaction consisting of four functional components to incorporate a lipoic acid, a pyridine, a zwitterionic motif and a reactive functional group in a one-pot process [26]. Cap exchange of functional polypeptide ligands produces a hydrophilic QD dispersion that is colloidally stable over time. Simultaneous zwitterionic ligation provides a compact and small QD, and the presence of reactive functional groups enables the QD to prepare coupled biological agents, such as azide-modified QDs to DNA, by bio-orthogonal coupling (Fig. 4).

Fig. 4. Preparation of a novel multifunctional peptide ligand for QDs.

A copper-catalyzed alkynyl azide was used to develop a biological surface which can produce a uniformly covered surface through covalent bonding [27]. The authors chose solid silicon bound

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to an azide carrier, where the S. aureus protein A (SpA) and maltose binding protein (MBP) were alkynylated to construct an active and reproducible biolayer. A stable surface that is therefore capable of uniform coverage is produced in a repeatable manner. A clickable hydrogel was afforded through preparation of triblock copolymers based on dendrimer conjugates containing orthogonal functionalized dendrimers [28]. A cycloaddition reaction between an azide and an alkyne was used to adjust amount of protein immobilized and alter the amount of the ligand in the hydrogel. The localization of acylation to specific sites in complex proteomes is critical to understanding the molecular mechanisms that control redox signaling. Cu(I) catalyzed azide-alkyne cycloaddition was used to modify biotin-labeled cysteine [29]. Most of the click reactions are carried out with copper catalysts. However, copper may induce the degradation of viruses and nucleic acids, and may also produce cytotoxicity, which may cause damage to organisms [30]. Therefore, more and more copper-free click reactions have been studied, gradually replacing copper catalysts. 5.1.2 The copper-free bio-orthogonal reaction The presence of metals has been shown to affect the normal physiological activities of living organisms leading to an investigation of click chemistry without copper or even metal catalysts. Copper-free click chemistry not only maintains the characteristics of original click chemistry but also avoids its toxins. Copper-free click reactions mainly refer to following types (Fig. 5): addition of a thiol to an ene compound [a]; a Diels-Alder reaction [b]; a reverse-demand Diels-Alder reaction [c]; and a [3+2] cycloaddition reaction [d]. We will summarize the progress of the copper-free click reaction.

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Fig. 5. Copper-free bio-orthogonal reactions. a) the addition of a sulfhydryl group to an olefin; b) a D-A reaction; c) a D-A reaction for reverse electron demand; d) a [3+2] cycloaddition reaction.

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Copper-free click reactions, such as the thiol-ene coupling, Diels-Alder or azide-alkyl cycloaddition, were used to study lysozyme PEGylation in aqueous media [31]. A phospholipid-based bio-orthogonal labeling strategy was disclosed to confer exosome optical probes without affecting the function of their natural bioalcohols (Fig. 6) [32].

Fig. 6. Bio-orthogonal phospholipid labelling strategy.

Yevgeny utilized a Diels-Alder reaction of reverse electron demand using a bio-orthogonal click chemistry refillable hydrogel reservoir system [33]. The modified hydrogel reservoir was clicked to capture a prodrug supplement from the blood, followed by local release of the active drug in a sustained manner. Capturing systemically managed refills is an effective and nontoxic

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method that can be refilled repeatedly. The refillable depot together with the prodrug supplement achieves sustained release from the precancerous tumor site to improve cancer treatment while eliminating systemic side effects. Chemical modification of the engineered microenvironment surrounding living cells is a means of directing cell behavior through cell-matrix interactions. A time-controlled method for the regulation of biomimetic synthetic cells was developed using a fast bio-orthogonal tetrazine linkage with trans-cyclooctene (TCO) dienophiles during live cell culture [34].This method is compatible to a diffusion-controlled cell and is independent of light. The principles of bio-orthogonal click chemistry and metabolic sugar engineering were applied to produce targeted anticancer drug delivery via gelatine-oleic acid nanoparticles based on a fattening platform [35]. A sialic acid precursor (Ac4ManNAz) was first introduced to the cell surface. Conjugation with gelatine and oleic acid using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxy succinimide formed covalently attached dibenzocyclooctyne (DBCO) through a click reaction. The newly designed GON-DBCO-DOX provides a safe and effective drug delivery system that actively targets cancer cells. A switchable, pre-targeted nano-immunotherapy strategy was disclosed for the treatment of NHL [36]. The pre-targeting system consisted of a DBCO-functionalized anti-CD20 antibody (α-CD20), a tumor-targeting component, an azide and a Y90-bifunctional dendrimer composition. The physicochemical properties of these pre-targeting components have been extensively investigated. The optimized bifunctional dendrimers could undergo rapid strain-promoted azide-alkyne cycloaddition under physiological conditions with DBCO functionalized α-CD20. The therapeutic effect of this pre-targeting system not only selectively delivers radionucleotides to targeted tumor cells, but also increases the complement dependent cytotoxicity of α-CD20, thereby enhancing anticancer potency. A universal vaccine for the influenza A virus (IAV) that does not require seasonal changes has been developed globally. These vaccines specifically induce targeted T cell responses, which are more likely to be shared between different strains and subtypes of viral proteins, to provide effective cross-reactive IAV immunity. Various vaccine platforms were reported based on solid-phase synthesis and bio-orthogonal conjugation methods [37]. This method involves the covalent coupling of a long synthetic peptide to the potent adjuvant molecule α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition was an efficient conjugation method, and the pseudoproline method was used to increase the efficiency of peptide synthesis. A simple and well-controlled method of stem cell imaging was developed by combining metabolic sugar engineering with bio-orthogonal copper-free click chemistry [38]. The metabolic precursor tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) produces exogenous chemical receptors containing azide groups on the surface of stem cells. Bicyclo[6.1.0]decene-modified diol chitosan nanoparticles (BCN-CNP) were used as imageable nanoparticles to deliver different imaging agents including Cy-5.5. These nanoparticles achieve animal stem cell imaging by bio-orthogonal copper-free click chemistry, specifically binding to chemical receptors on the surface of stem cells treated with Ac4ManNAz. A schematic diagram of a copper-free catalytic stem cell imaging method is shown in Fig. 7.

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Fig 7. Schematic diagram of a copper-free catalytic stem cell imaging method

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A catalyst-free reverse-demand Diels-Alder reaction was performed to demonstrate the bio-orthogonal nature of crosslinking chemistry, which is chemically inert to proteins [39]. Various analytical characterizations indicated that Fab1 is capable of retaining antigen binding ability without any physical or chemical modifications. It was found that bio-orthogonal and catalyst-free aqueous chemistry could achieve efficient in situ protein encapsulation in a single step and provide sustained protein release. A small molecule anticoagulant bearing an azide as a safety needle has been developed [40]. It allows physiological coagulation by an in vivo click reaction with a suitable cyclooctyne-based neutralizer. This bio-orthogonal reaction provides an opportunity for drug neutralization in vivo, thus reducing side effects. The principle of bio-orthogonal labeling is based on inverse-electron demand Diels-Alder (IEDDA) cycloaddition. Eszter inserted the cyclooctyne structure into other basic structures, such as amino acids and nucleotides. Then, the modified amino acid or nucleotide becomes the raw material to construct a protein containing cyclooctyne by interfering with the synthesis of a protein or nucleic acid molecule [41]. The two proteins are labeled by a bio-orthogonal IEDDA reaction in combination with a fluorescent molecule containing azide. Thus, it is beneficial to understand the biochemical process at molecular level. The PTAD of tyrosine residues was functionalized as a hydrogel by selectively introducing azide into the ELP to crosslink to SPAAC [42], which could be used for stem cell encapsulation in regenerative medicine. The IEDDA between tetrazine and thiocarbamate-functionalized transcyclooctene was used to release carbonyl sulfide (COS), which is rapidly converted to H2S by CA, thereby providing active H2S gas molecules for living organisms [43]. A click reaction was also used to directly synthesize PROTAC in vitro, which can avoid the disadvantages of excessive molecular weight, poor solubility, and poor cell permeability [44]. Large PROTAC molecules are broken down into smaller molecules, and then resynthesized by click chemistry in cells to degrade target proteins. An azide-labeled arsenic, which was click-reacted with dibenzyl cyclooctene, was

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used to label in situ binding cellular proteins with trivalent arsenic groups to capture arsenic-containing proteins [45]. An azide group was incorporated into the metabolism of a monosaccharide analog and a copper-catalyzed azide-alkyne click reaction was able to directly image the glycans of Arabidopsis thaliana [46]. Ac4GalNAz was used to metabolize mucin-type O-glycan azide (Fig. 8). The alkynyl-PEG-b-CD was then crosslinked by CuAAC, achieving a host-guest interaction [47]. The results opened new ways to control contact-dependent cell-cell reversible interactions and facilitated the study on cellular communication.

Fig. 8. Illustration of engineering photoresponsive host-guest recognition on cell surfaces.

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Strain-promoted azide-alkyne cycloaddition (SPAAC), or Staudinger crosslinking, was then developed [48]. SPAAC crosslinking material forms a gel within a few seconds and gels within a few minutes to improve gelation kinetics, matrix mechanical properties and biochemical properties. RT-PCR analysis of flow cytometry was carried out, and it was found that the exposure time of the CuAAC reaction mixture caused a substantial significant impact on biocompatibility [49]. Copper-free SPAAC was more efficient than CuAAC for the sugar-engineered cell modification. A nontoxic azido-functionalized ceramide could allow bio-orthogonal click reactions to fluorescently label the incorporated ceramides to investigate the formation of ceramide-rich domains [50]. Azido-functionalized C6-ceramide was incorporated and localized in plasma membrane microdomains and proximal vesicles in T cells. The results indicated that there was a subcellular redistribution of ceramides during T cell activation. Wang identified relative synthetic and absolute quantification (iTRAQTM) by combining bio-orthogonal noncanonical amino acid markers (BONCAT) and isobaric tags to identify newly prepared proteins [51]. They He incorporated L-azido-homoalanine (AHA) into a newly synthesized protein and then enriched the protein for biotin after a click reaction between an alkyne-carrying biotin and bio-orthogonal azide portion of AHA. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LCMS/MS). Subsequently, they identified and quantified 1176 proteins. Irfan utilized a bio-orthogonal anti-electron demand Diels-Alder chemistry concept. Nanoparticles were embedded into tetrazine and the near-infrared fluorescent (NIRF) Cy5.5 dye was used to convert doxorubicin into a prodrug [52]. By clicking on the release process,

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nanoparticles taken up by breast cancer cells were effectively converted the prodrug into the biologically active doxorubicin. A novel enzyme-based cap system with effective and controlled drug delivery was developed for its on-demand release and targeting capabilities, selecting silica nanoparticles through tetrazine. The bio-orthogonal chemistry of the inverse-electron DA reaction with a cyclic olefin structure directly binds to the targeting ligand [53]. The capping system is based on the pH-responsive binding of arylsulfonamide-functionalized MSN to carbonic anhydrase (CA). A nonnatural amino acid bearing a norbornene moiety was genetically incorporated into CA. Thus, the cellular and stem cell uptake was mediated by specific receptors. A Z-lysine derivative (AmAzZLys) with both an amino and an azide on the benzyl moiety was designed [54]. The aniline and azide were operatively linked to the 5 kDa polyethylene glycol and fluorescent probe, respectively. The multifunctional probes could accommodate a plurality of reactive groups for protein conjugation. The Diels-Alder reaction principle of trans-cyclooctene and tetrazine was used to obtain a 34 nt-long aptamer synthesis and purification model of a hairpin RNA strand [55]. Functionalized random and block copolymers were used as microcontact printing inks to prepare tabular granules, which provided excellent size and quality control of the patch [56]. The authors selectively imaged the particles according to click type (amine/active ester, alkyne/azide, biotin/avidin) on the patch or on the particles. Bio-orthogonal reactions including SPAAC and IEDDA have become increasingly popular for live cell imaging. However, in the context of living cells, the stability and reactivity of reagents have never been systematically studied. A universal organelle-targetable system based on HaloTag protein technology was developed in various subcellular compartments [57]. The clickable HaloTag ligand was used to directly compare reactivity, specificity and stability of the bio-orthogonal reagents. This system provided detailed comparisons of bio-orthogonal reactions in living cells and provided information on the selection of optimal reagents and conditions for live cell imaging. It was found that reaction of sTCO with monosubstituted tetrazine was the fastest. However, both reagents have stability problems. To solve this problem, they introduced a new sTCO variant, Ag-sTCO, which was stable enough to be directly used in cells to rapidly undergo a bio-orthogonal reaction with tetrazine. Ag complexes using conformationally constrained trans-cyclooctene should greatly expand their application, especially paired with less reactive and more stable tetrazines. A viable platform for IFN-[α]2b PEGylation was developed by genetic code extension [58]. This method specifically incorporated an azide-containing amino acid into IFN-[α]2b, and then performed orthogonal and stoichiometric conjugation of various PEGs using copper-free click chemistry. In addition, only selected sites within IFN-[α]2b are PEGylated under mild conditions, resulting in a single, homogeneous conjugate. In addition, the side effects of PEGylation on biological and pharmacological properties were optimized. A two-step labeling scheme for the site-specific labeling of proteins was disclosed [59]. First, lipoic acid ligase A (LplAW37V) was used to attach a highly stable norbornene derivative to a specific peptide sequence mutant. Second, a tetrazine-modified fluorophore was covalently coupled with the norbornene moiety. Finally, this two-step labeling strategy was used to label proteins in cell lysates in a site-specific manner and perform cell surface labeling on live cells. The conjugation of polyethylene glycol (PEG) to bovine beta-lactoglobulin (BLG) was demonstrated by the photoinduced cycloaddition of tetrazole plus PEG and allyl-modified BLG [60]. It was found that the bio-orthogonal reaction was applied not only to biological

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macromolecular labeling and tracing, but also to protein-targeted chimera. The large chimera structure is directly synthesized in the body, thereby improving the protein-targeting chimera with a large molecule, poor solubility, low cell permeability, and poor biological activity. It was also found that such reflections were mainly applied to modify the surface molecules of various materials to improve the bioactive properties. 5.2 Other types of bio-orthogonal reactions 5.2.1 Aldol reactions Certain aldol conditions are mild and can be tolerated by organisms. Therefore, aldol it can be used for novel bio-orthogonal reactions. An organic catalyst based on amino acids was developed to catalyze an aldol coupling reaction (Fig. 9) [61]. This aldol reaction occurs quickly and under mild, biocompatible conditions (i.e., aqueous solution, moderate and neutral pH), and the bioconjugate was stable to abundant aldolase under both acidic and basic conditions. It has been demonstrated by cell imaging that various small molar dyes can be coupled to proteins.

Fig 9. Direct aldol coupling of organic catalysts derived from amino acids.

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Aldol condensation can be used to modify proteins [62]. Proteins were modified under mild conditions using 2,4-thiazolidinedione in an aldol reaction of a nucleophilic donor (Fig. 10). Various functional groups can be introduced without changing the conformation of a protein. For example, diaziridine, alkynyl, or azide units can be introduced to visualize the structure of a protein.

Fig. 10. Aldol condensation bio-orthogonal reaction structure to modify protein molecules.

5.2.2 Catalyst-free specific hydrazone binding modifications in proteins Bio-orthogonal reactions have become valuable tools for site-specific protein labeling and

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modification both in vitro and in vivo. The widespread use of ruthenium and osmium linkages in conjugation with biomolecules has attracted considerable attention. An electron-deficient benzaldehyde was developed. This benzaldehyde can be easily equipped with various biomolecules for catalyst-free sputum ligation. It can be equipped not only with small molecules, including fluorescent dyes or drugs, but also with macromolecules such as PEG. These moieties can be precisely linked to the C-terminus of a protein by an effective hydrazine reaction at neutral pH and room temperature (Fig. 11) [63].

Fig. 11.Catalyst-free specific hydrazone binding modifica-tions in proteins

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Dowine catalyzed the modification of Dha by a palladium-mediated cross-coupling reaction [64]. Various aryl boronic acids were coupled with dehydrated residues in proteins and peptides (Nisin) using Pd(EDTA)(OAc)2 as the catalyst. This cross-coupling reaction was used to obtain a Heck product in which sp2-hybridization of the α-carbon was retained. It is a bio-orthogonal reaction which can be carried out under mild aqueous conditions (Fig. 12).

Fig 12. The modification of Dha by a palladium mediated cross-coupling reaction

The bio-orthogonal reaction is precise, leading to its extensive application. Due to its clear concept, the bio-orthogonal reaction has a large space for development, as long as the organisms can tolerate the organic reaction conditions. Furthermore, the bio-orthogonal reaction provides a theoretical basis for materials chemistry, macromolecular identification and other aspects of chemistry.

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Acknowledgment

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6. Conclusions and Outlooks Increasingly more investigations on PAL have been disclosed in the past several years. These new findings emphasize the increasing importance of PAL in drug discovery. The progress highlighted in this review indicates that various PAGs can be incorporated to afford functional PAPs, followed by labeling and bio-orthogonal reactions. Among them, the bio-orthogonal reaction is quite important, leading to its fast development. The current bio-orthogonal reaction is not limited to the original click reaction, as well as other types, such as the aldol condensation reaction. With the in-depth study of these bio-orthogonal reactions, we have a new definition of the structural modification of probe molecules in PAL, which can be applied not only to click reactions but also to probes. The labeling of the target proteins of bioactive molecules is diverse and nondestructive, which will bring a historic advancement in the future diagnosis of diseases. PAL has the potential to accelerate drug discovery through providing structural insight and instant binding site validation. PAL has been thought of as an enormous approach to explore drug targets, and with the advanced strategy applications, its application has expanded from drug target identification to drug discovery. However, some limitations still exist, such as probe-labeling of nonspecific proteins, which is the main issue in this field. Luckily, the competitive ABPP strategy is commonly used to solve this problem by comparison with control [65]. The other issue is the probe itself and probe-specific hits, which are difficult to deal with. Enrichment in the presence and absence of a competitor is one approach widely used to test whether a protein is a probe-specific hit. Further work in this area may be helpful in providing resources to aid researchers in assessing whether putative targets are genuine or related to the probe moiety itself. To address this issue, follow-up relevant analytical verification methods of putative targets becomes very important.

This work was supported by the National Natural Science Foundation of China (NSFC, Grant No.

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81573285), the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2018JM7071 and 2014JM4101), and the Fundamental Research Funds for the Central Universities

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Abbreviations: PAL: Photoaffinity labelling PAG: Photoaffinity group PAP: Photoaffinity Probe BODIPY: Boron difluoride dipyrromethene NBD: Nitrobenzene-2-oxa-1,3-dioxazole IF: Intermediate fibre AuNP: Gold nanoparticles CB2R: Type 2 cannabinoid receptor

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SSRIs: Selective serotonin reuptake inhibitors AIE: Aggregation-induced emission TCO: Trans-cyclooctene DBCO: Dibenzocyclooctyne IEDDA: Inverse-electron required Diels-Alder SPAAC: Strain-promoted azide-alkyne cycloaddition LC-MS/MS: Liquid chromatography-tandem mass spectrometry NIRF: Near-infrared fluorescent

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ACCEPTED MANUSCRIPT Activity-Based Proteomic Profiling: The Application of Photoaffinity Probes in Target Identification of Bioactive molecules

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Highlights:
It presents an overview of the photoactive groups, reporter groups, and bio-orthogonal reactions involved in PAL strategies, with a focus on their application in target identification of bioactive molecules.
Photoaffinity labelling is an emerging technique in studying and analyzing the interaction between ligand and receptor.
It presents an interdisciplinary proposal to identify target protein of bioactive molecules using multifunctional photoaffinity probes.