Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex

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

Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform Ryo Nishihara,† Emi Hoshino,† Yoshiki Kakudate,† Satoshi Kishigami,† Naoko Iwasawa,† Shin-ichi Sasaki,‡ Takahiro Nakajima,§ Moritoshi Sato,§ Shigeru Nishiyama,† Daniel Citterio,† Koji Suzuki,*,† and Sung Bae Kim*,∥ †

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan ‡ Nagahama Institute of Bio-Science and Technology, Nagahama, Kusatsu, Shiga 525-8577, Japan § Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan ∥ Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan S Supporting Information *

ABSTRACT: Native coelenterazine (nCTZ) is a common substrate to most marine luciferases and photoproteins. In this study, nine novel dye- and azideconjugated CTZ analogues were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of the nCTZ backbone to obtain bulkiness-driven substrate specificity and potential chemiluminescence/bioluminescence resonance energy transfer (C/BRET). The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly biased bioluminescence to ALucs and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living animal cells or lysates. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than nCTZ. Nile redconjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). No or less spectral overlap occurs among [Furimazine−NanoLuc], [6-N3-CTZ−ALuc26], [6-pi-OH-CTZ− RLuc8.6], and [6-N3-CTZ−RLuc8.6] pairs, because of the substrate-driven luciferase specificity and color shifts, providing a crosstalk-free multiplex bioassay platform. The unique bioluminescence system appends a new toolbox to bioassays and multiplex molecular imaging platforms. This study is the first example that systematically synthesized fluorescent dye-conjugated CTZs and applied them for a bioluminescence assay system.

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shrimp Oplophorus was introduced with the name of NanoLuc.5 It was further engineered with a fluorescent protein for creating an efficient BRET system, named “Antares”.12 In parallel, great efforts have been made to synthesize a specific substrate that potentially provides better optical performance13,14 and color variety to luciferases,15,16 since native coelenterazine (nCTZ) was first isolated from coelenterate Aequorea victoria.17 Many nCTZ analogues have been synthesized and conjugated with other heterogeneous components, like cages18 or alkyne/triazole groups.19 We previously demonstrated that optical performance of ALucs is dominated by the chemical structures of the substrates;11,14 for example, a conventional nCTZ analogue, CTZi, enabled specific bioluminescence to an ALuc, compared with RLuc8.6-535. Independently, we investigated the advantages of C-6-extended modification of nCTZ, in terms of

INTRODUCTION Fluorescence and bioluminescence are widely used optical readouts for monitoring various biological processes in living subjects. These optical readouts are utilized independently or cooperatively for illuminating many intracellular molecular events, for example, bioluminescence resonance energy transfer (BRET) for determining protein−protein interactions (PPIs) in mammalian cells.1−4 Compared with fluorescence, bioluminescence is a better optical readout in practical bioassays due to its sensitivity, versatility, and instrumental simplicity. However, it commonly has the drawbacks of relatively poor optical intensity and limited color variety. These drawbacks have been partly addressed by engineered variants of luciferases.5,6 Renilla reniformis luciferase (RLuc), derived from sea pansy, has been genetically modified to improve its optical stability and redshifted property.7,8 A series of artificial luciferases (ALucs) was also established by extracting frequently occurring amino acid sequences from the public database of copepod luciferases.9−11 Furthermore, an engineered version of a luciferase from the © XXXX American Chemical Society

Received: March 14, 2018 Revised: April 25, 2018

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DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. (A) Schematic diagram to exemplify modification of synthetic luciferins with a bulky side chain at C-6 position. The yellow shadows highlight the functional groups at C-6 position. (B) Relative substrate-specific optical intensities of marine luciferases (n = 3). Because of the copepod-derived luciferases (GLuc and ALucs) secretion, they were retained in the cells with an ER retention signal (KDEL), as explained in the Experimental Section. The X- and Y-axes specify newly synthesized nCTZ analogues and conventional marine luciferases, respectively. The Z-axis shows the corresponding optical intensities, normalized by time (s) and area (mm2) measurements. The percentage values on top of the bars indicate the remaining optical intensities after 20 min. Abbreviations: GLuc, Gaussia princeps luciferase; RLuc8.6-535, Renilla renifomis luciferase 8.6535; ALuc16, artificial luciferase 16. The asterisks denote the maximal optical intensity of each luciferase with newly synthesized nCTZ analogues. The relative optical intensity was summarized in Table S1. (C) Bioluminescence spectra of the newly synthesized azide-conjugated CTZ analogues with ALuc34. All the spectra were normalized as percentages (%) of maximal intensity. FWHM means the full width at half maximal intensity in wavelength. The red background color and the percentage value denotes the portion of red light emission above 600 nm relative to the total light emission.

tunable by simply incubating the engineered NanoLucs with fluorescent probes.21 The short distance between the small luciferase and the FP might have partly contributed to the improvement of their systems. The above studies encouraged us to create an efficient luciferin−luciferase assay system with azide- or fluorescent dyeconjugated CTZ analogues, allowing enhanced optical properties and/or potential resonance energy transfer (RET) in the presence of marine luciferases. We previously reviewed an earlier crystallographic study on an RLuc8−coelenteramide binding model, emphasizing the role of the C-6 hydroxyphenyl group of coelenteramide in the binding with RLuc8. In contrast, due to the low-sequence identity between RLuc8 and ALucs, ca. 17%, ALucs might be able to accommodate a bulkier moiety if they contain variant residues in the vicinity of their active site pockets. Based on this knowledge, we synthesized a series of nCTZ analogues, carrying an azide group (N3) or various fluorescent dyes at the C-2 or C-6 position. The optical performance was examined with existing marine luciferases and newly fabricated ALucs. The azide group-linked nCTZ was originally synthesized as an intermediate for further coupling reactions with organic dyes. However, the azide group-conjugated CTZ analogues unexpectedly exhibited biased optical intensities to artificial luciferases (ALucs) over the other marine luciferases, which may be driven by their unique host guest−chemistry. A study on kinetic factors revealed that the enhanced optical

bioluminescence, allowing a large spectral blue shift of the bioluminescence spectra, but causing little influence on the enzymatic recognition of RLucs.13 Furthermore, the C-6 and C2 positions of nCTZ were found to be the specificity core for marine luciferases.14 The above studies strongly suggest (i) that the luciferase−luciferin reactions adhere basically to the traditional host−guest docking models, which may be controllable by just modifying the functional groups in the luciferin. The above studies also revealed (ii) that bulky modification at the C-6 position of nCTZ with fluorescent dyes may be well tolerated for luciferases including artificial luciferases (ALucs) (Figure 1A; Figure S2). The advantages of fluorescent dye-conjugated substrates over the conventional fluorescent proteins (FPs) in BRET may be summarized as (i) fluorescent dye-conjugated substrates shorten the distance between the luciferase donor and fluorescent dye acceptor, contributing to better BRET efficiency, (ii) fluorescent dyes are much smaller than FPs and thus should less affect the physiological function in living systems, different from FPs.20 The requirement of labor intensive synthesis is a disadvantage of fluorescent dyeconjugated substrates. Recently, small luciferase-linked FPs have been reported for efficient BRET systems: e.g., Chu et al. developed an efficient and bright BRET system with one of the smallest luciferases, NanoLuc, and named it “Antares”.12 Further, Hiblot et al. created an innovative BRET system, whose colors are freely B

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. (A) Unrooted phylogenetic tree of newly fabricated artificial luciferases and conventional marine luciferases. The new ALucs (black) were fabricated by modifying the brightest luciferases, ALuc16, ALuc23, ALuc30, in Figure 1 (red). Conventional marine luciferases (blue) are phylogenetically distinctive from the ALucs. Inset a shows chemical structures of the selected nCTZ analogues. The yellow shadows highlight the characteristic functional groups. (B) Optical matrix showing relative optical intensities of the most potent nCTZ analogues in Figure 1 with newly fabricated ALucs. The optical image represents the integrated photon flux during the initial 5 min. Because of the copepod-derived luciferases (GLuc and ALucs) secretion, they were retained in the cells with an ER retention signal (KDEL), as explained in the Experimental Section. The newly synthesized nCTZ analogues have their own unique optical specificities with marine luciferases (n = 4). The specific values were listed in Table S2.

intensities of azide-conjugated CTZs with ALucs are explained by higher catalytic efficiency. Additionally, some of the dyeconjugated CTZ analogues exerted red-shifted CRET and BRET spectra, which are highly tissue-permeable. Although many attempts to modify nCTZ have been tried, this study is the first example where nCTZ was systematically engineered with fluorescent dyes. The present imaging system based on bioluminescence provides a useful toolbox to current bioassay and molecular imaging studies.

we introduced Nile Red and Chlorin. Nile Red is known to reflect the intracellular hydrophobic circumstance and Chlorin has the potential to generate singlet oxygen for cancer treatments. 4,6-Dimethoxy-1,3,5-triazin-2-yl (DMT) was introduced for increasing the chemical stability of the CTZ backbone and extending the π conjugation. Azide-Conjugated CTZ Analogues Showed Improved Optical Properties with ALucs. We previously investigated the advantages of C-6-extended modification of nCTZ in the bioluminescence capacity, allowing a large spectral shift of the bioluminescence spectra and little influence on the enzymatic recognition of RLucs.13 In this study, we examined if the C-2 or C-6 position of the imidazopyrazinone backbone can be extended by conjugating an azide group or fluorescent dyes (Figure 1 and Figure S2). The resulting relative optical intensities show that azideconjugated CTZ analogues with ALucs exerted approximately 2- or 4-fold brighter bioluminescence than nCTZ in combination with the same ALucs. Among the azide-conjugated CTZs at C-6 and C-2 positions, 6-N 3 -CTZ showed approximately 2-fold brighter bioluminescence than 2-N3CTZ with ALucs. The relative optical intensities of luciferases were compared in cell lysates and not with purified proteins (Figures 1 and 2). The reason for this is that the purpose of this study is to identify the best candidates for practical use under experimental conditions relevant for cell assays. Optical intensities obtained

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RESULTS AND DISCUSSION Native coelenterazine (nCTZ), with an imidazopyrazinone backbone, is the common substrate of most marine luciferases, including Renilla reniformis luciferase (RLuc) and copepoda luciferases.1 In this study, the C-2 and C-6 positions of the structure were conjugated with an azide group or organic fluorescent dyes to achieve (i) potential improvement in optical properties, (ii) potential specificity of the nCTZ analogues to marine luciferases, and (iii) blue- or red-shifted optical signals for multiplex assays via bioluminescence or chemiluminescence resonance energy transfer (B/CRET). The synthesis of fluorescein isothiocyanate (FITC)-linked CTZ was first undertaken because FITC is a common fluorescent dye for molecular imaging. Fluorescein succinimidyl ester (SFX) was introduced to the CTZ backbone for evaluating the dependency of the optical properties of FITC on the type of linker. To achieve red-shifted BRET emission, C

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 3. Collective bioluminescence image of living cells and time course. (A) Comparison of substrate-driven collective bioluminescence image of live cells on 6-channel microslides. The optical intensities of 6-N3-CTZ were compared with those of Furimazine and nCTZ. (B) Time course of the bioluminescence intensities activated by Furimazine, nCTZ, and 6-N3-CTZ. The optical intensities were determined every 5 min. Inset a shows the optical profile of 6-N3-CTZ according to marine luciferases. The optical image represents the integrated photon flux for the initial 5 min.

compact, compared with the other dye-conjugated CTZ analogues, the size of the dyes works as a burden to the bioluminescence reaction. The suppressed optical intensities were not improved even with 10 times higher concentrations of 2-SFX-CTZ and 6-Nile-R-CTZ (final concentration: 1 mM) (Figure 1B). Because the azide-conjugated CTZ analogues emitted excellent bioluminescence intensities (see Figure 1B), the corresponding bioluminescence spectra were determined with ALuc16 (Figure 1C). The results show that the optical maxima (λmax) of 2-N3CTZ and 6-N3-CTZ are ca. 514 and 490 nm with ALuc16, respectively. These λmax values represent 14 and 10 nm red- or blue-shifted spectra of the azide-conjugated CTZ analogues, compared with that of nCTZ. The intensity ratios of the wavelengths greater than 600 nm by 2-N3-CTZ and 6-N3-CTZ were found to be 6% and 5%, respectively. The wavelength region longer than 600 nm was marked with reddish background colors, considering the excellent tissue permeability. Host−Guest Chemistry Reasoning the Biased Luciferase Specificity of Azide-Conjugated CTZ Analogues to ALucs. The molecular mechanism behind the strong luminescence of azide-conjugated CTZ analogues (6-N3-CTZ and 2-N3-CTZ) with ALucs but not with other luciferases is unclear (Figure 1B). Furthermore, 6-N3-CTZ luminesces stronger than 2-N3-CTZ with ALucs. Similarly, 6-FITC-CTZ and 6-Nile-R-CTZ are brighter than 2-FITC-CTZ and 2-Nile-R-CTZ, respectively, upon reaction with ALucs. The common feature of 6-N3-CTZ, 6-FITC-CTZ, and 6-Nile-R-CTZ is the hydroxyl group (OH) at the C-2 position and the bulkiness at the C-6 position. The overall results may be explained by the host−guest chemistry between azide-conjugated CTZ analogues and ALucs: i.e., (i) the azide group just fits the space available inside the active site cavity of ALucs, (ii) ALucs tolerate a bulky side chain at the C-6 position of the substrate, and (iii) the OH group of the substrate at the C-2 position is an important moiety for binding to ALucs, as explained previously.14

under these conditions are not necessarily identical to those of the column-isolated luciferase variants. Regarding the evaluation of the expression levels of the luciferases, it is inadequate to conduct a Western blot analysis, because multiple primary antibodies have to be applied at once. Furthermore, due to the large cysteine content, copepod luciferases generally suffer from poor folding efficiency in bacterial cell lines. Alternatively, we referred to our Western blot analysis for investigating the total protein amounts with the same cell line, plasmids, luciferases, and protocol as those of the present study.14 The results show that no variance occurs in the applied protein amounts. Kinetic Factors of Azide-Conjugated CTZ Analogues with ALucs. We further investigated the kinetic factors because an azide-conjugated CTZ exceeds nCTZ in the optical intensities, which is exceptional in the history of nCTZ analogues.19,22 The results with column-purified ALuc16 show that Km values of nCTZ were similar to those of 6-N3-CTZ (less than 2% discrepancy), for example, 76.3 μM (nCTZ) and 77.5 μM (6-N3-CTZ) (in the case of a 4 s integration). In contrast, the corresponding Vmax values had an 18% discrepancy, that is, 8.4 × 107 and 9.9 × 107, respectively. These overall results show that (i) the binding affinity of ALuc16 to both substrates is almost equivalent; and (ii) the higher optical intensities of the azide-conjugated CTZ could arise from either kinetic factors like enzymatic turnover or an inherently higher efficiency of light production like quantum yield (QY), or both as explained previously.23 The 1.2-fold higher catalytic efficiency of 6-N3CTZ−ALuc16 pair than that of nCTZ−ALuc16 pair explains the better optical intensities. Similarly, between the FITC-conjugated CTZ analogues at C-6 and C-2 positions, 6-FITC-CTZ exerted approximately 5fold brighter bioluminescence than 2-FITC-CTZ with ALucs. These two cases above suggest that ALucs tolerate well the bulky C-6 position-modified CTZ analogues. The results correspond with our previous study.13 Different from the azide-conjugated cases, the dye-conjugated CTZ analogues generally emitted weaker bioluminescence intensities than nCTZ. As the azide group is relatively D

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. (A) Bioluminescence resonance energy transfer (BRET) spectra of the representative dye-conjugated coelenterazine analogues with ALuc16. The spectra of nCTZ, 2-FITC-CTZ, 6-FITC-CTZ, 2-Nile-R-CTZ, and 6-Nile-R-CTZ were determined with ALuc16. The spectrum of 2FITC-CTZ with ALuc16 is asymmetrical, compared to the others. The percentage in red indicates the portion of red light emission above 600 nm relative to the total light emission. Among the tested, spectra with poor absolute optical intensities were omitted in the figure. (B) BRET spectra of representative CTZ analogues with RLuc8. All the spectra were normalized as percentages (%) of maximal intensity. (C) BRET spectra of representative CTZ analogues with RLuc8.6-535. All the spectra were normalized as percentages (%) of maximal intensity.

Selected Coelenterazine Analogues Showed Unique Luciferase Preference. As azide-conjugated CTZs (2-N3CTZ and 6-N3-CTZ) emit relatively strong optical intensities with ALuc16, ALuc23, and ALuc34, we expanded the experimental boundary to the other ALuc variants, newly fabricated from ALuc 25 (GenBank MF958967) and ALuc 34 in the hope of finding the best host−guest fit between the active site cavity of ALucs and the azide-conjugated CTZs (Figure 2; Figure S1). The ALuc variants were chosen for their superior optical intensity and stability compared to the others. The sibling ALucs were examined with 2-N3-CTZ and 6-N3CTZ. The newly synthesized ALucs showed unique phylogenetic positions, which are clearly different from those of conventional marine luciferases, like MpLuc1, and MLuc (Figure 2A). The optical matrix in Figure 2B reveals that nCTZ commonly luminesces with a broad range of marine luciferases, including GLuc, RLuc8.6-535, ALucs17-19, and ALuc26 (GenBank MF958968), where RLuc8.6-535 was chosen because it is the most improved RLuc variant in terms of stability and optical intensity, and shares the same substrate CTZ as the ALucs. Particularly, nCTZ strongly luminesces with both ALuc18 and ALuc26, whereas azideconjugated CTZ analogues optically, biased to ALuc26 rather than ALuc18, did not luminesce with RLuc8.6-535 and GLuc. In contrast, 6-pi-OH-CTZ, which is known to be selective for both RLuc and ALuc variants,14 interestingly emitted the strongest bioluminescence with RLuc8.6-535. The above results show that every substrate has its preferred luciferin−luciferase pairs; for example, the [6-N3-CTZ−ALuc26], [nCTZ−ALuc18], and [6-pi-OH-CTZ−Rluc8.6-535] pairs suggest the best matches for optical crosstalk-free bioassays. These optimal pairs may be utilized in a multiplex assay system, which simultaneously determines ligands with multiple optical readouts. Discrepancy in the selectivity patterns was observed in the comparison of the lysate study (Figure 2B) and the live cell study (Figure 3); for example, the nCTZ−ALuc26 pair shows almost identical intensity to the one of 6-N3-CTZ−ALuc26 in lysates (Figure 2B), whereas the nCTZ−ALuc26 pair is significantly lower in the case of live cells (Figure 3). This discrepancy may be caused by two different factors: one is the cell membrane permeability of the substrates, and the other is

the ingredients of the lysis buffer influencing the enzymatic reaction, such as the acidic pH and detergents. The KDELlinked ALuc26 is sequestered into the lumen of the endoplasmic reticulum (ER) after expression. The substrates have to penetrate two membrane barriers (plasma membrane and endomembrane) and finally reach ALuc26 in the ER of live cells. The higher optical intensity of 6-N3-CTZ may be interpreted in 6-N3-CTZ carrying a single OH group, which has higher membrane permeability than nCTZ carrying two OH groups. Similarly, CTZ-h carrying a single OH group also has higher membrane permeability than nCTZ.24 This membranedriven effects do not work in the cell lysates. The best optical stability was found with ALuc26 (Table S2), which sustained 64% and 52% of its optical intensity, until 20 min after injection of nCTZ and 6-pi-OH-CTZ, respectively. Optical stability of 6-N3-CTZ was found to be generally poor with the tested marine luciferases; for example, ALuc19 sustained 54% of optical intensity with 2-N3-CTZ for 20 min, but 6-N3-CTZ in the same condition sustained only 8%. Considering this significant optical intensity decay over time in Figure 3B, the immediate measurement is advantageous for obtaining brighter images. The mechanism behind the rapid decay of bioluminescence is unclear, but the less durability of luciferases and oxyluciferins after the reaction may hamper the luciferase activities. 6-N3-CTZ Selectively Luminesces with ALuc26 in Living Mammalian Cells in Microslides. As the [6-N3CTZ−ALuc26] pair showed excellent optical properties in mammalian cell lysates (Figure 2), we further investigated the optical properties in living animal cells (Figure 3). Besides 6-N3-CTZ, we simultaneously examined nCTZ and Furimazine for references, because nCTZ is the most commonly used substrate in many laboratories, and Furimazine is chosen because it is specific to NanoLuc; thus, it potentially provides a multiplex system with the present ALuc-based imaging modality.5 Furimazine emitted strong bioluminescence only with NanoLuc in COS-7 cells. Similarly, 6-N3-CTZ also exerted biased optical intensities only with ALuc26. There were no cross-reactions between [Furimazine−NanoLuc] and [6-N3CTZ-ALuc26], which proved that these two luminescent systems can be combined in a single-assay model because E

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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quantum yield (QY) for Nile Red emission is less than 0.1 in water, causing weak RET efficiency,30 whereas the one of fluorescein is more than 0.9.31 The CRET spectra revealed that 6-Nile-R-CTZ has a clear RET peak because the Nile Red exerts fluorescence in polar organic solvents such as DMSO and DMF, but not in aqueous solution32 (Figure 4A,B). In addition, the orientation of the dyes to the conjugated CTZ could also be suboptimal for the RET in the vicinity of the pocket of ALuc16. Further comparison of the overall FWHMs of the spectra revealed that the largest widths were the Nile Red-conjugated CTZs, and the narrowest widths were 6-FITC-CTZ. The overall order of the widths was as follows: 6-Nile-R-CTZ = 2Nile-R-CTZ > 2-FITC-CTZ > 6-FITC-CTZ. This tendency corresponds with the expected FWHM values of net Nile Red and Fluorescein: i.e., Nile Red for 62 nm and Fluorescein for 39 nm.33 The BRET efficiency of the synthetic nCTZ analogues was further investigated with RLuc8.6-535 (Figure 4C). The spectrum of 6-N3-CTZ with RLuc8.6-535 has the unique peak only at 400 nm. However, the dye-conjugated form, 6FITC-CTZ, shows a unique spectrum peak at 522 nm in addition to that at 400 nm. We noted that the peak of RLuc8.6-535 with nCTZ is coincidently close to that with 6-FITC-CTZ. Thus, we further conducted a similar study with RLuc8, because the emission peak of RLuc8 with nCTZ is clearly separated from that with 6FITC-CTZ (Figure 4B). Figure 4B shows that nCTZ has a peak at ca. 477 nm, but modification of the C-6 position OH with an azide group causes a blue shift of the emission peak at ca. 400 nm. Upon replacing the OH group with FITC, the emission peak is shifted to ca. 522 nm in the presence of RLuc8. The overall results are summarized as follows: (i) the almost identical emission spectra of 6-FITC-CTZ in Figure 4B,C demonstrate that the BRET indeed occurs via the conjugated FITC, although the energy sources (luciferases) differ; (ii) considering that the emission spectrum at 400 nm is attributed to the neutral intermediate of nCTZ,13,34 the unique spectra at 400 and 522 nm may be explained that the BRET of 6-FITCCTZ occurs between the neutral form of CTZ intermediates and the adjacent FITC by their spectral overlap, although the overlapping area is small; (iii) the FWHM of the nCTZ spectra at 522 nm is significantly wider than that of 6-FITC-CTZ at 522 nm (41 nm, Figure 4C), which reasonably points to a BRET-type radiation from the fluorescent dye and much more unlikely to the other radiation forms of CTZ; and (iv) the [6N3-CTZ-RLuc8.6-535] pair generates a spectral mixing-free signature in multiplex assays, considering the ca. 130 nm blueshifted spectrum. The large blue shifts of the spectra depending on CTZ analogues were previously explained by the authors13 and others.34 In brief, CTZ shows a broad emission peak from blue to green (400−535 nm), because of four different energy levels of intermediates, i.e., neutral species, amide anion, phenolate anion, and pyrazine anion. The modification of the OH group at C-6 position of native CTZ is known to prevent the formation of the pyrazine anion upon the enzymatic oxidation, which results in blue-shifted emission around 400 nm. In summary, (i) we synthesized a new series of dye- or azideconjugated CTZ analogues, by adding a series of organic fluorescent dyes or an azide group to the C-2 or C-6 position of nCTZ backbone. (ii) Moreover, we newly fabricated ALuc

they both carry fully independent and noninterfering reporters. In contrast, nCTZ allowed bioluminescence with all the tested luciferases. The corresponding optical profile highlights that 6N3-CTZ specifically illuminates ALuc26 in living COS-7 cells, where the COS-7 cell line was chosen because it is derived from mammalian tissues and expresses a large amount of fusion proteins in the cell context. The overall absolute optical intensities were in the order of [Furimazine−NanoLuc] > [6N3-CTZ−ALuc26] > [nCTZ−NanoLuc] = [nCTZ−ALuc26] > [nCTZ−Rluc8.6-535]. The time course of collective bioluminescence intensities in live cells shows that the best optical stability is found in the order of [Furimazine−NanoLuc] > [6-N3-CTZ−ALuc26] > [nCTZ−Rluc8.6-535]. The bioluminescence emission half-lives of [6-N3-CTZ−ALuc26] and [nCTZ−Rluc8.6-535] were ca. 10 and 3 min, respectively, whereas that of [Furimazine− NanoLuc] pair was longer than 25 min. CRET and BRET Spectra of Various Pairs of CTZ Analogues and Luciferases. The CRET and BRET spectra were obtained with various dye-conjugated CTZ analogues (Figure S3; Figure 4). The chemiluminescence spectrum of nCTZ was determined as a reference, and its maximal peak (λmax) was found only at 468 nm. In contrast, the spectra of 6-FITC-CTZ, 6-Nile-RCTZ, and 6-Chlorin-CTZ exerted the maximal peaks at ca. 538, 650, and 680 nm, respectively, although they commonly kept small chemiluminescence peaks near 468 nm. These peak positions are almost the same as the predicted emission peak positions of each conjugated dye after RET.25−29 These two characteristic peaks at 468 nm (nCTZ) and the other wavelengths (dyes) in the spectra indicate that the resonance energy of chemiluminescence, generated by chemical degradation of coelenterazine, was indeed transferred to the adjacent, conjugated dyes. The authors further examined the BRET spectra of dyeconjugated CTZ analogues with affinity column-purified ALuc16 (Figure 4B). Among the synthesized CTZ analogues, 6-Nile-R-CTZ showed a characteristic RET peak at 650 nm, where ca. 11% of the total photons were emitted in the red region longer than 600 nm, which is a highly tissue-permeable region, called “optical window”. The λmax at 650 nm is generally accepted as the optical emission peak of Nile Red.25 The comparison of the optical intensities reveals that the Nile Redconjugated CTZ at the C-6 position provides better BRET efficiency with ALuc16 than that at the C-2 position. This result corresponds with the previous conclusion, that is, ALucs can tolerate bulky and extended chemical structures of the side chain at the C-6 position.14 In the spectra of FITC-conjugated CTZ analogues, no distinctly separate optical peaks of the donor and the acceptor were found around 500 to 520 nm; instead, strong red-shifted optical peaks were found at 524 and 536 nm, which look reasonable as the RET emission, considering that reference studies reporting that (i) the emission peak of FITC is between 516 and 525 nm,30 and (ii) a FITC-conjugated Cypridina luciferin analogue (FCLA) shows chemiluminescence at 532 nm.27 It should be noted that the spectrum of 2-FITC-CTZ with ALuc16 shows an asymmetrical shape. The results suggest that the donor’s peak at 500 nm is almost superimposed by emission peaks at 524 and 536 nm. The above results in the BRET efficiency among Nile Redand FITC-conjugated CTZs may be explained with the dye’s optical characteristics in aqueous media; for example, the F

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

microtube was set in a dark chamber of a high-precision spectrophotometer (AB-1850, ATTO). The corresponding chemiluminescence spectra were measured by integrating the signal over the entire wavelength range for 5 s, immediately after injecting 80 μL of DMSO. Determination of Relative Bioluminescence Intensities of nCTZ Analogues with Conventional Marine Luciferases. The pcDNA3.1(+) vectors encoding each copepod luciferase were obtained from our previous studies,9,37 where each copepod luciferase was tagged with a “KEDL” sequence for the intracellular retention. The pcDNA3.1(+) vector encoding Renilla reniformis luciferase 8.6-535 (RLuc8.6535) was also obtained from our previous study.9 COS-7 cells, derived from the kidney of an African green monkey, were grown in Dulbecco minimal essential medium (high glucose, DMEM) (Gibco), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) and 1% penicillin/ straptomycin (P/S) (Gibco). The COS-7 cells were subcultured in a 96-well optical-bottom microtiter plate (Nunc) and transiently transfected with the pcDNA3.1(+) vectors encoding each marine luciferase using TransIT-LT1 (Mirus), as shown along the Y-axis of Figure 1B. The marine luciferases include Gaussia princeps luciferase (GLuc; GenBank AAG54095.1)37 or RLuc8.6-535,7 and Aluc16-34.9 The GenBank accession numbers were as follows: ALuc16, MF817967; ALuc23, MF817968; ALuc24, MF817969; ALuc30, MF817970; ALuc34, MF958969. ALuc16, 25, and 34 are the ALuc family members that have been previously developed by the authors through extracting frequently occurring amino acids and lining up the amino acids to make a full length of artificial luciferases. Sixteen hours after the transfection, the cells in each well were lysed with 50 μL of a lysis buffer (E291A; Promega), and an aliquot of the lysates (10 μL) was transferred into a fresh 96well optical bottom microplate (Thermo Scientific), using an 8channel micropipette. 40 μL of RLuc assay buffer, carrying nCTZ (E2820; Promega assay kit), was injected into the aliquot, using a 12-channel micropipette, and immediately after that, the relative optical intensities of the lysates in the microplate were determined every 5 min, using an image analyzer (LAS-4000, FujiFilm), equipped with a cooled CCD camera. The bioluminescence intensities were normalized by integration time (second) and light-emitting area (mm2). The unit of the normalized bioluminescence intensity was subsequently expressed in terms of relative luminescence unit per second area (RLU/sec/mm2). The relative optical intensities after substrate injection were listed in Table S1, and the sustained optical intensities of azideconjugated CTZ 20 min after the substrate injection was briefly noted on the bars of Figure 1B. Determination of Kinetic Properties of nCTZ and 6N3-CTZ with Purified ALuc16. The bioluminescence kinetic properties (Km and Vmax) of affinity column-purified ALuc16 according to nCTZ and 6-N3-CTZ were determined. The column-purified ALuc16 was prepared with the same method as that of Kim et al.38 Briefly, the cDNA encoding ALuc16 was subcloned into pOPTHM vector and expressed in the bacterial strain Shuffle T7 Express lysS (New England Biolabs). The lysate was purified with HisTrap HP column (GE Healthcare) in an Ä KTA Purifier system (GE Healthcare). nCTZ and 6-N3-CTZ were dissolved in methanol and diluted to appropriate concentration in PBS buffer (pH 7.4) for each measurement. The ALuc16 was also dissolved in PBS buffer (1.0 μg mL−1). An aliquot of the luciferase solution (50

variants for screening their best optical matches with the synthetic CTZ analogues. We found that azide-conjugated CTZ analogues preferably activate ALuc variants, whose optical intensities were superior to those of nCTZ. Some of the unique pairs, like [6-N3-CTZ−ALuc26], have less optical cross-talk with other substrate−luciferase pairs, such as [FurimazineNanoLuc] and [6-pi-OH-CTZ−RLuc8.6-535] pairs. Further, the [6-N3-CTZ−RLuc8.6-535] pair makes a largely blue-shifted optical peak, which is clearly isolated from those of the other pairs. By combining the substrate−luciferase specificity and the color variance, one may create many different kinds of ideal multiplex bioassay platforms. Although many attempts to modify nCTZ have been applied to date, this study is the first systematic approach to conjugate many organic dyes to nCTZ. This unique bioluminescence system appends a new toolbox to bioassays and molecular imaging. This study provides new insights into how azide- or dye-conjugated CTZ analogues luminesce with marine luciferases with their unique optical selectivity and functionality.

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EXPERIMENTAL SECTION General Aspects of Organic Synthesis. All reagents and solvents for organic synthesis were purchased from commercial suppliers (Tokyo Kasei, Aldrich Chemical, and Wako Pure Chemical) and used without further purification. All moisturesensitive reactions were carried out under the atmosphere of argon. The composition of mixed solvents is given in volume ratio (v/v). 1H NMR and 13C NMR spectra were recorded on an ECA-500 (JEOL Ltd.), ECS-400 (JEOL Ltd.), or LA-300 (JEOL Ltd.) spectrometer at room temperature at 500, 400, or 300 MHz (1H NMR), and at 125 MHz (13C NMR), respectively. All chemical shifts are relative to an internal standard of tetramethylsilane (δ = 0.0 ppm) or solvent residual peaks (CDCl3: δ = 7.26 ppm, CD3OD: δ = 3.31 ppm, DMSOd6: δ = 2.50 ppm for 1H; CDCl3: δ = 77.16 ppm, CD3OD: δ = 49.00 ppm for 13C), and coupling constants are given in Hz. HPLC purification was performed on a reversed-phase column, Intersil ODS-3 (30 × 50 mm) (GL Sciences Inc.), fitted on an LC-918 recycling preparative HPLC system (Japan Analytical Industry Co. Ltd.). High-resolution MS spectra (HR-MS) were recorded on a Waters LCT premier XE, with MeOH as the eluent. The detailed information on the organic synthesis process of the nCTZ analogues were described in Supporting Information with corresponding NMR data. Design and Organic Synthesis of CTZ Analogues. The analogues having an azide group at C-2 or C-6 position of CTZ were newly synthesized according to the synthesis routes reported in the literature.35,36 In order to synthesize the fluorescent dye-conjugated CTZ derivatives, we conducted the reduction of the azide group to the amine group in the azidesubstituted CTZ analogue, and the condensation reaction of the amine group and the corresponding fluorescent dye, generating the dye-conjugated CTZ derivatives. The detailed information on the organic synthesis process of the nCTZ analogues is described in the Supporting Information with corresponding NMR data. Determination of Chemiluminescence Resonance Energy Transfer (CRET) Spectra of Newly Synthesized CTZs. The CRET spectra were determined as follows (Figure S3): a methanolic solution of the respective dye-conjugated CTZ analogue was mixed with PBS buffer (final concentration, 0.5 mM, 8 μL) and transferred to a 200 μL microtube. The G

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

(Invitrogen) for mammalian cell expression, where the “KDEL” tag was added for cell retention. COS-7 cells were first grown over 6-channel microslides (μSlide VI0.4, ibidi) and transiently transfected with NanoLuc, RLuc8.6-535, or ALuc26. Sixteen hours after incubation, the cells on each microslide were rinsed with HBSS buffer (Gibco) and immediately bathed with 60 μL of Furimazine, nCTZ, or 6N3-CTZ dissolved in an HBSS buffer (final concentration: 0.1 mM), using a multichannel pipet (Gilson). The microslides were immediately transferred to the dark chamber of the LAS4000 (FujiFilm), and their optical intensities integrated every 5 min. The optical images of the microslides were adjusted according to the optical intensity scales. The optical image profile of 6-N3-CTZ on the microslide was displayed, additionally, in terms of RLU per distance (mm). Measurement of the BRET Spectra of Dye-Conjugated CTZ Analogues. The corresponding BRET spectra were obtained with the dye-conjugated CTZ analogues (Figure 4). The affinity column-purified ALuc16 was prepared as the same method as that of our previous study.21 The BRET spectra were determined as follows (Figures 4B): the purified ALuc16 stock was diluted 500-fold to 2 μg/mL with a universal or an assay buffer before experiments. Twenty microliters of the diluted ALuc16 was transferred into a 200 μL PCR microtube and mixed with 80 μL of azide- or dyeconjugated CTZ analogues (final concentration: 0.1 mM). The microtube was immediately moved into the chamber of the precision spectrophotometer (AB-1850, ATTO), and the resulting spectra were taken in an integration of 30 s (Figure 4B). The spectra of the 6-N3-CTZ and 6-FITC-CTZ with RLuc8.6-535 were further determined as follows: COS-7 cells expressing RLuc8.6-535 were lysed with a lysis buffer (Promega) and an aliquot of the lysate (4 μL) was transferred to a 200 μL microtube. The lysate in the microtube was mixed with 200 μL of nCTZ analogue dissolved in PBS buffer (final concentration: 20 μM), and the resulting spectra were immediately determined using a precision spectrophotometer (AB-1850, ATTO) (Figure 4C).

μL) was tranfered into a 1.5 mL microtube. The bioluminescence intensities, immediately after quick manual injection of each nCTZ analogue (50 μL) into the microtube, were determined every second with a luminometer (GloMax 20/20n, Promega). There was time lapse of two seconds between the substrate injection and intensity measurement. The initial bioluminescence signal over seconds was taken for the measurement of the kinetic properties of ALuc16 (the final nCTZ concentrations were 0, 0.25, 0.5, 1, 2, 5, 10, and 20 μM, whreas the final concentration of ALuc16 was 0.5 μg mL−1). The corresponding Km and Vmax values were calculated using a specific software, Prism v 7 (GraphPad). The measurements were performed in triplicate, and the data shown represent the averages of three runs. Determination of the Optical Spectra of AzideConjugated CTZ Analogues. The cell lysates were made following the same method as that shown in Figure 1B. An aliquot of the lysates (20 μL) was transferred into a 200 μL microtube and mixed with 80 μL of the RLuc assay buffer (E2820; Promega), carrying 2-N3-CTZ or 6-N3-CTZ (final concentration: 0.1 mM). The microtube was immediately moved into the chamber of a precision spectrophotometer (AB-1850, ATTO), equipped with a cooled charge-coupled device (CCD) camera that can capture the entire light in one shot, and the resulting spectra were taken in an integration of 30 s. The optical spectra were normalized in percentages (%). Among all the spectra recorded, only those of ALuc34 were shown in Figure 1C for simplicity. Fabrication of New ALuc Variants and Screening Their Best Matches with Azide-Conjugated CTZ Analogues. The new series of ALuc variants derived from ALuc16, 25 (GenBank MF958967), and 34 were fabricated according to the basic concept of the precedent studies of the authors:9,10 The specific experimental procedure is described in the Experimental Procedure S1. Relative Bioluminescence Intensity Matrix of Selected nCTZ Analogues with New ALuc Variants. The relative bioluminescence intensities of selected nCTZ analogues, with newly fabricated ALuc variants, were addressed in a heat plot matrix in the hope of having better optical properties (Figure 2B; Table S2). For highlighting the best substrate−luciferase matches, the results were expressed in a heat plot in Figure 2B. The COS-7 cells were prepared following the same method as that shown in Figure 1B. The COS-7 cells, in each well of a 96-well optical bottom microplate, were transiently transfected with pcDNA3.1 vector (Invitrogen), encoding GLuc, RLuc8.6535, or one of the new variants of ALucs, using TransIT-LT1 (Mirus). Sixteen hours after the transfection, the cells were lysed, and an aliquot of the lysates transferred into a fresh 96well optical bottom microplate. The corresponding optical intensities, which were developed with the protocol shown in Figure 1B, were determined by LAS-4000 and finally analyzed with the software, Multi Gauge v 3.2 (FujiFilm). The specific optical intensity and stability were summarized in Table S2. Cell-Based Bioluminescence Imaging on the Basis of the New ALuc−Luciferin Systems. The collective bioluminescence image of live cells and optical profiles of 6-N3CTZ were compared with those of Furimazine and nCTZ (Figure 3). As a reference, the cDNA construct encoding fulllength NanoLuc with an N-terminal secretion peptide and a “KDEL” tag was custom-synthesized by the help of Eurofins Genomics (Tokyo), and subcloned into pcDNA3.1 vector

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00188. Sequence alignment and phylogenetic tree of newly fabricated ALucs; Chemical structures of novel coelenterazine analogues; Table of optical intensities of the newly synthesized azide- or dye-conjugated CTZ analogues with existing luciferases; The chemiluminescence resonance energy transfer (CRET) spectra of the dyeconjugated CTZ analogues; Table of the optical intensities of the azide- or dye-conjugated CTZ analogues with newly fabricated ALucs; Experimental procedures for the synthesis of the new ALucs and the C2 and C-6 modified CTZ derivatives, which include the NMR data. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry ORCID

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Daniel Citterio: 0000-0001-7420-045X Sung Bae Kim: 0000-0002-7033-9056 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Japan Society for the Promotion of Science (JSPS), through grant numbers 26288088, 15KK0029, 16K14051, 17H01215, and 24225001. This work is also supported by a Research Fellowship Grant of JSPS for Young Scientists.

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DOI: 10.1021/acs.bioconjchem.8b00188 Bioconjugate Chem. XXXX, XXX, XXX−XXX