Development of Î²- Caryophyllene as A Chemical ... - ACS Publications

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Nature-Inspired Bioorthogonal Reaction: Development of β‑Caryophyllene as a Chemical Reporter in Tetrazine Ligation Yunfei Wu,†,‡ Jiulong Hu,†,# Chen Sun,# Yu Cao,# Yuanhe Li,† Fayang Xie,† Tianyin Zeng,† Bing Zhou,*,# Juanjuan Du,*,† and Yefeng Tang*,†,‡

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School of Pharmaceutical Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China ‡ Collaborative Innovation Center for Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Medical School, Sichuan University, Chengdu 610041, China # State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: A nature-inspired bioorthogonal reaction has been developed, hinging on an inverse-electron-demand Diels−Alder reaction of tetrazine with β-caryophyllene. Readily accessible from the cheap starting material through a scalable synthesis, the newly developed β-caryophyllene chemical reporter displays appealing reaction kinetics and excellent biocompatibility, which renders it applicable to both in vitro protein labeling and live cell imaging. Moreover, it can be used orthogonally to the strain-promoted alkyne− azide cycloaddition for dual protein labeling. This work not only provides an alternative to the existing bioorthogonal reaction toolbox, but also opens a new avenue to utilize naturally occurring scaffolds as bioorthogonal chemical reporters.

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M−1 s−1), and thus are particularly applicable for the in vivo labeling of biomolecules with extremely low abundance. However, their metabolic stability and potential cross-reactivity with biological nucleophiles have remained arguable.40 In comparison, the less strained cyclopropenes (Cyps), a new class of chemical reporters independently developed by the Devaraj and Prescher groups,27−30 are notable for their improved bioorthogonality and appealing small sizes. Nevertheless,27−30 the reaction kinetics of Cyps-mediated tetrazine ligations (k2 = 10−2−101 M−1 s−1) are generally slower than those of TCOs. Besides the conflicting stability and reactivity issues, the accessibility of the currently known strained chemical reporters is not trivial. Indeed, only a few of them (e.g., the transcyclooctene and norbornene derivatives) are commercially available, whereas the others have to be prepared through multiple synthetic steps. In this context, despite the significant advances, striving for the right balance of reactivity, biocompatibility, and synthetic accessibility has remained a grand challenge in bioorthogonal reaction development. Owing to the remarkable structural novelty and diversity, natural products have been recognized as invaluable sources for the discovery of new chemistry, biology, and medicine.41−45 By comparison, the potential of natural products as bioorthogonal

INTRODUCTION Bioorthogonal chemistry, since its conceptualization by Bertozzi in the early 21st century,1−3 has evoked tremendous interest in the scientific community. So far, an array of bioorthogonal reactions have been developed,4−15 among which the inverse-electron-demand Diels−Alder (IEDDA) cycloadditions of tetrazines with strained alkenes are particularly attractive for their fast reaction kinetics, high specificity, and catalyst-free nature (Figure 1A).16−20 The first tetrazine ligation was introduced by the Fox group in 2008,21 with the trans-cyclooctene (TCO) derivative I (Figure 1A) employed as the dienophile component. Almost simultaneously, Hilderbrand and co-workers also reported a strain-promoted tetrazine ligation, using the norbornene derivative II as the reactant partner.22 Notably, both tetrazine ligations necessitate the usage of strained alkenes as the reactant partners for securing fast reaction speed. Inspired by these seminal studies, various strained alkene- and alkyne-based chemical reporters have been developed for tetrazine ligations over the past decade, which could be roughly classified into four groups: trans-cyclooctenes and cyclooctynes,21,23−25 norbornenes,22,26 cyclopropenes (Cyps),27−31 and cyclobutenes32,33 (Figure 1A). While the strain-promoted tetrazine ligations have found broad applications in biomedical studies,34−39 they are not without limitation, particularly in regard to their bioorthogonality and synthetic accessibility. For example, the highly strained TCOs show extraordinarily fast reaction kinetics (k2 = 103−106 © XXXX American Chemical Society

Received: April 24, 2018 Revised: May 26, 2018 Published: May 31, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00283 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. (A) IEDDA reaction of tetrazines with strained alkenes/alkynes (previous work). (B) Naturally occurring IEDDA of β-caryophyllene with o-quinone methide species (previous work). (C) Nature-inspired β-caryophyllene-tetrazine ligation (this study). POI = protein of interest.

Figure 2. Activation Gibbs free energies (ΔG‡, in kJ/mol) calculated at PWPB95-D3/def2-QZVPP//M06-2X-D3/def-TZVP level with SMD solvation in water for the IEDDA reactions between 3,6-di(2-pyridyl)-s-tetrazine (2a) and different strained alkene substrates.

caryophyllene, as a potential chemical reporter, would have two appealing advantages. First, it is an abundant natural product produced by living organisms in a biologically relevant environment. In this context, it can be viewed as a chemical reporter evolved through natural selection, which should have excellent biocompatibility. Second, it is commercially available and inexpensive (ca. $60/mol), which warrants the ready accessibility of β-caryophyllene-based chemical reporters. Herein we report the successful implementation of the abovementioned design in practice, which has culminated in the development of a conceptually novel and practically useful bioorthogonal reaction (Figure 1C).

chemical reporters has been underestimated. Our group has shown a keen interest in natural product synthesis over the past several years.46−51 During this process, we came to realize that some naturally occurring scaffolds hold great promise to function as chemical reporters in bioorthogonal reactions. βcaryophyllene (1a, Figure 1B), a naturally occurring sesquiterpene featuring a strained trans-cyclononene core,52−55 represents such a paradigm. Structurally, β-caryophyllene bears considerable structural resemblance to some wellknown TCOs such as the cyclopropane- or dioxolane-fused trans-cyclooctenes,23,25 which renders it a potential chemical reporter for tetrazine ligation. More encouragingly, it has been reported that β-caryophyllene could engage in the IEDDA reactions of with some electron-deficient dienes such as oquinone methide species (Figure 1B), as witnessed in a number of biomimetic total syntheses of natural products.56−61 Taken together, we envisaged that it was feasible to develop a new bioorthogonal reaction based on the IEDDA reaction of tetrazine and β-caryophyllene. We anticipated that β-

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RESULTS AND DISCUSSION Predicted Reactivity of β-Caryophyllene and Relevant Derivatives through Calculation Study. We initiated our work by conducting a computational study to predict the reactivity of β-caryophyllene in the IEDDA reaction with tetrazine. A seminal study was reported by Sauer and coB


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the resulting cycloadducts [for details, see Scheme S1, Supporting Information (SI)] gradually advanced to the 1,4dihydropyridazine 3 in a quantitative yield with the extension of reaction time. It should be noted that β-caryophyllene occurs in solution as two interconvertible conformers (βα:ββ = 3:1).64,65 It is likely that β-caryophyllene adopts the βα-conformer in the IEDDA reaction as it has lower activation energy (Figure S15, SI), thus leading to the observed product as a single diastereosiomer. Synthesis of the Functionalized β-Caryophyllene as Chemical Reporter. Promoted by the preliminary experimental result, we moved to elaborate β-caryophyllene into an operational chemical reporter such as 1b. Thus, 1a was treated with m-CPBA to give the epoxide 4,66 which then underwent hydroboration−oxidation to yield the primary alcohol 5.67 Treatment of 5 with TBSCl/Imidazole gave the silyl ether 6 which, after reductive de-epoxidation66 followed by deprotection, resulted in 1b as a crystalline solid. The structure of 1b was confirmed by X-ray crystallographic study (Scheme 2).68 The current synthetic route allows accessing 1b from the cheap starting material with excellent efficiency (40% overall yield). Moreover, no special reagent or instrument is required, which makes it amenable to scalable and practical synthesis. Determination of the Reaction Rate of IEDDA between Functionalized β-Caryophyllene and Tetrazine. With 1b in hand, we then evaluated the second-order rate constant (k2) of the IEDDA reaction of 1b with 2a (Figure S3, SI). The reaction displayed appealing reaction kinetics [k2 = 9 × 10−2 M−1 s−1 in methanol/PBS (9/1)], which is superior to some known bioorthogonal reactions such as the Staudinger ligation [k2 = 0.25 × 10−2 M−1 s−1 in H2O/CH3CN (1:19)],69 the IEDDA reaction of o-qinolinone methide with vinyl thioether [k2 = 1.5 × 10−3 M−1 s−1 in H2O/CH3CN (5:1)],70 and the strain-promoted azide−alkyne cycloaddition (k2 = 7.6 × 10−2 M−1 s−1 in CH3CN).71 Meanwhile, it can also compete with the tetrazine ligations with the N-acylazetine (IV, Figure 1A) [k2 = 4.5 × 10−3 in DMSO/H2O (1:8)]33 and the carboxylate-substituted cyclopropane [k2 = 5.1 × 10−2 in CH3CN/PBS (1:1)].27 Furthermore, in order to minimize the underlying solvent effect, we conducted a competitive experiment with a 1:1 ratio of 1b and 5-norbornene-2-methanol used

workers in the 1990s, which provided a systematic survey of the reactivity of various strained alkenes/alkynes in the IEDDA cycloadditions with tetrazines.62 Actually, most of the previously reported strained alkenes/alkynes chemical reporters in tetrazine ligation, such as the trans-cyclooctene, norbornene, cyclopropane, cyclobutene, and cyclooctyne, could be traced back to this pioneer work. However, we noticed that no information was provided for the trans-cyclononene, which may partially account for the fact that such a type of scaffold has never been explored as a chemical reporter in tetrazine ligations. Our computational result revealed that the IEDDA reaction of trans-cyclononene and 3,6-di(2-pyridyl)-s-tetrazine 2a had a higher ΔG# than the trans-cyclooctene, cyclopropane, and norbornene (Figure 2), indicating that it would display inferior reactivity as a dienophile. However, it was predicted that β-caryophyllene would be more reactive than the simple trans-cyclononene, presumably due to the presence of a cyclobutane appendage that can increase the ring-strain of the trans-cyclononene. More encouragingly, it was anticipated that 1b and 1c, two β-caryophyllene derivatives designed by us as potential chemical reporters, would display further enhanced reactivity close to that of norbornenes. It appeared that increasing the hydrophilicity of β-caryophyllene might exert a beneficial effect on the IEDDA reactions with tetrazine, which is in alignment with some previously reported results.30,63 Model Study on the IEDDA Reaction of β-Caryophyllene and Tetrazine. Encouraged by the computational results, a model study was first performed by us using 1a and 2a as the reactant pairs. To our delight, the IEDDA reaction proceeded smoothly in MeOH at 37 °C, with the tetrazine component consumed completely within 2 h (Scheme 1). Not surprisingly, Scheme 1. IEDDA Reaction of β-Caryophyllene with Tetrazine

Scheme 2. Synthesis of β-Caryophyllene Chemical Reporter 1b

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Figure 3. Labeling of β-caryophyllene-modified BSA (BSA-β-Cp) with 9 or 10. (A) Modification of BSA (10 mg/mL) with 8 (150 μM) and subsequent labeling with 9 or 10. (B) Western blot analysis of the BSA-β-Cp/Tz-Biotin conjugate (BSA-Biotin) in H460 cell lysate with anti-biotin antibody. (C) Gel analysis of BSA-β-Cp incubated with 9 (0−150 μM) for 90 min. (D) Gel analysis of BSA-β-Cp incubated with 9 (150 μM) for 0− 90 min. In B−D, protein loading was assessed by Coomassie staining (lower panels).

Figure 4. Dual labeling of BSA modified with β-caryophyllene (BSA-β-Cp) and azide (BSA-Az). (A) BSA was functionalized with 8, 11, or both chemical reporters and then reacted with Tz-BODIPY FL (12) (40 μM), DBCO-Cy5 (13) (40 μM), both reagents simultaneously or no reagent. (B) Gel analysis of BSA (lanes 1−4), BSA-Az (lanes 5−8), BSA-β-Cp (lanes 9−12), and BSA functionalized with both chemical reporters (lanes 13− 16), with each group of proteins treated with either Tz-BODIPY FL (12) or DBCO-Cy5 (13), both reagents simultaneously, or no reagent. In B, protein loading was assessed by Coomassie staining (lower panel).

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Figure 5. (A) Pretargeting of SK-BR-3 cells with trastuzumab labeled by β-caryophyllene, followed by tagging the living cells with tetrazine-Cy5 (14) via an inverse-electron-demand Diels−Alder reaction. (B) Confocal microscope images of SK-BR-3 human breast cancer cells including (a) control exposed to 14, (b) control antibody exposed to 14, and (c) antibody modified with β-caryophyllene and exposed to 14. Scale bar: 50 μm.

(lane 4, Figure 3B). In-gel fluorescence analysis of the BSA modified with β-caryophyllene and treated with 9 revealed that the tetrazine ligation proceeded effectively in a time- and dosedependent manner (Figure 3C,D). Not surprisingly, in both cases no labeling of the unmodified BSA was observed. Application of β-Caryophyllene-Tetrazine Ligation in Dual Protein Labeling. The complicated nature of biological processes often demands concurrent modification and tracking of biomolecules with dual or multiple tags. To secure the specificity of such a process, the engaged bioorthogonal reaction pairs must be mutually orthogonal. Inspired by some precedents,72−77 we envisioned that the present β-caryophyllene-tetrazine ligation could be used orthogonally with the wellknown azide-strained alkyne cycloaddition for dual labeling. To test this hypothesis, the azide and β-caryophyllene chemical reporters were individually or concurrently appended to BSA using standard coupling conditions (Figure 4A). The modified protein was then exposed to either green fluorescent βcaryophyllene-reacting Tz-BODIPY FL (12) or red fluorescent azide-reacting DBCO-Cy5 (13), or both. The results were then analyzed by in-gel fluorescence imaging. As shown in Figure 4B, the BSA modified with β-caryophyllene (lanes 9−12) were fluorescently labeled only when they were treated with TzBODIPY FL (12) (lanes 11−12). By comparison, there was no

as the dienophiles (Figure S4, SI). It turned out that 1b displayed the same magnitude of reaction kinetics to that of the norbornene scaffold, which was in agreement with our computational study (Figure 2). Besides the appealing reaction kinetics, 1b also exhibited super stability toward various biologically relevant conditions. For examples, it was inert toward some strong nucleophiles such as thiol and amine. It was also stable in the acidic and basic solution, without obvious double bond isomerization observed for 2 days (Figure S1, SI). Notably, it remained intact in 20% fetal bovine serum (FBS) over 24 h (Figure S2, SI). Application of β-Caryophyllene-Tetrazine Ligation to in Vitro Protein Labeling. To evaluate the applicability of the newly developed IEDDA reaction in a biologically relevant setting, we first appended 1b to bovine serum albumin (BSA), a model protein for in vitro labeling, by means of a conventional coupling method (Figure 3A).70 The β-caryophyllene-modified BSA was subsequently incubated with either fluorescent tetrazine-FITC (Tz-FITC, 9) in PBS buffer or tetrazine-biotin (Tz-Biotin, 10) in H460 cell lysate and the resulting products were assayed by in-gel fluorescence measurements or Western blots using anti-biotin antibody. Gratifyingly, robust biotinylation was observed for the BSA modified with the βcaryophyllene chemical reporter and coincubated with 10 E


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Figure 6. Flow cytometry analysis of the fluorescent labeling of the labeled and unlabeled SK-BR-3 cells (red, control cell; blue, control antibody; orange, antibody modified with β-caryophyllene; pink, control cell exposed to 14; dark green, control antibody exposed to 14; green, antibody modified with β-caryophyllene and exposed to 14 (left); mean fluorescence intensities (in arbitrary units, au) for the histograms (right). Antibody concentrations: (A) 60 nM; (B) 200 nM; (C) 600 nM. Error bars denote the standard deviation from three replicate experiments. Ab: antibody, AbM: antibody modified with β-caryophyllene. Probe refers to tetrazine-Cy5 (14).

reaction between DBCO-Cy5 and β-caryophyllene (lane 10). Similarly, BSA modified with azide (lanes 5−8) showed selective labeling in the presence of DBCO-Cy5 (13) (lanes 6 and 8, Figure 4B), without any detection of BODIPY fluorescence labeling (lane 7). For the BSA functionalized simultaneously with both β-caryophyllene and azide (lanes 13− 16), specific fluorescence labeling was shown in the presence of DBCO-Cy5 (13) (lane 14) or Tz-BODIPY FL (12) (lane 15). Comparably, the successful dual labeling was observed for the BSA exposed to both fluorophore reagents in one pot (lane 16). As a control, unmodified BSA showed no fluorescence labeling after exposure to Tz-BODIPY FL (12) and/or DBCOCy5 (13) (lanes 1−4). The results above suggest that the βcaryophyllene-tetrazine ligation shows excellent orthogonality to the azide-strained alkyne cycloaddition and could be used in dual protein labeling.

Application of β-Caryophyllene−Tetrazine Ligation in Live Cell Imaging. Finally, the utility of β-caryophyllene− tetrazine ligation for live cell imaging was investigated. To evaluate whether the β-caryophyllene derivatives could be applicable for cell culture studies, we first examined their cytotoxicity. Gratifyingly, no detectable cytotoxicity was observed for 1a or 1b in Hela cells at concentrations up to 500 μM (Figure S11, SI). Subsequently, we sought to apply the β-caryophyllene−tetrazine ligation to live cell imaging through the pretargeting strategy.22,34,35 For this end, we first labeled the monoclonal antibody trastuzumab (Herceptin) with 1b. The modified antibody was then incubated with human breast cancer cells SK-BR-3 that overexpress Her2/neu. The labeled SK-BR-3 cells were then treated with the tetrazine-Cy5 (14) (50 μM) in HBSS buffer for 30 min. After washing, the cells were counterstained with Hoechst 33342 before being imaged F


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Beijing Natural Science Foundation (2172026) and National Key R&D Program of China (2017YFA0207900).

on a confocal microscope. It was shown that the covalently bound tetrazine−Cy5 could be visualized clearly in the nearinfrared (NIR) channel (Figure 5B, group c). Comparably, the control experiments (groups a and b) only resulted in weak NIR fluorescence, possibly due to the nonspecific binding. Furthermore, we used flow cytometry to perform a more quantitative analysis of the living cell fluorescent labeling with the tetrazine-Cy5 (14). Modified antibodies of different concentrations were incubated with SK-BR-3 cells, which were then treated with tetrazine-Cy5 (14) (10 μM) in HBSS buffer for 30 min. Figure 6A,B,C shows the relative cellular fluorescence intensity observed for the labeled and unlabeled SK-BR-3 cells. As shown in Figure 6, the β-caryophyllenetetrazine−Cy5 labeling yields significantly higher cellular fluorescence intensity than the background signals observed in those control groups. Meanwhile, the fluorescence intensities enhanced with the increasing dose of modified antibody. These results prove that the β-caryophyllene−tetrazine ligation is highly efficient and selective on the living cells.

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CONCLUSION In summary, we have developed a novel bioorthogonal reaction that hinges on a nature-inspired inverse-electron-demand Diels−Alder of β-caryophyllene and tetrazine. The newly developed bioorthogonal reaction displays appealing reaction kinetics and remarkable bioorthogonality and has been successfully applied to both in vitro protein labeling and live cell imaging. As a new class of chemical reporter, the functionalized β-caryophyllene can be readily accessed from the cheap starting material in a scalable and practical manner, which renders it a valuable addition to the arsenal of bioorthogonal chemical reporters. Besides its potential applicability, a more important aspect is that the current work provides a proof-of-concept case of the utilization of naturally occurring scaffolds as chemical reporters in bioorthogonal reactions. We believe that more new bioorthogonal reactions could be developed under the guidance of this concept. Related studies are underway in our laboratory and will be reported in due course.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00283. Detailed experimental procedure, and 1H and 13C NMR spectra, as well as X-ray data information (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yefeng Tang: 0000-0002-6223-0608 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial supports from National Natural Science Foundation of China (21572112, 21772109), G


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