Superior Fluorogen-Activating Protein Probes Based on 3-Indole

Letter pubs.acs.org/OrgLett

Superior Fluorogen-Activating Protein Probes Based on 3‑Indole− Malachite Green Qingyang Zhang,†,‡,∥ Qinghua Wang,†,‡,∥ Ying Sun,‡ Limin Zuo,§ Verena Fetz,⊥ and Hai-Yu Hu*,†,‡ †

State Key Laboratory of Bioactive Substances and Function of Natural Medicine and ‡Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China § Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China ⊥ Department of Chemical Biology, Helmholtz-Zentrum für Infektionsforschung (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany S Supporting Information *

ABSTRACT: A series of novel fluorogenic dyes based on 3-indole− Malachite Green, MGs 5−7, have been developed that are dark in solution but highly fluorescent when bound to the cognate reporter, fluorogen-activating protein (FAP). Significantly, the new MGs 5−7 probes are superior to the traditional MG 1 with high fluorescent efficiency and low toxicity to cells while maintaining the large “pseudo-Stokes” shifts (Δλ = λex − λem) and the malachite green (MG)-like fluorescence OFF−ON switching mechanism in both live mammalian cells and bacterial cells when bound to FAP.

F

luorescence imaging is a powerful approach for noninvasive and real-time visualization and tracking of biomolecules and biological processes in living cells and animals.1 The development of convenient and high-efficiency fluorophores is essential for the rapidly developing imaging technology.2 Toward this end, numbers of fluorophores have been developed, including naphthalimide,3 styryl,4 coumarin,5 BODIP,Y6 and cyanine.7 However, to achieve a high signal-to-noise ratio for visualization and quantification, extensive and time-consuming washing of cells to remove free fluorophore is required, rendering the continuous monitoring of biological processes virtually impossible.8 This has led to a push in the development of fluoromodule-based reporter/probe systems that feature robustly activatable probes that could be optimized for real-time fluorescence imaging.9 Fluorogen-activating proteins (FAPs) are a class of fluoromodules that bind a nonfluorescent small molecule in a noncovalent fashion and exhibit fluorescence.10 FAP technology was first introduced in 2008 and has been successfully used to monitor receptor trafficking in live cells.11 MG (Figure 1a) is one such fluorogen whose fluorescence is strongly quenched by conformational flexibility in solution. FAP/MGs display two excitation (absorbance) bands, a main band (X) contributed by rings A and B and a minor band (Y) contributed by ring C.12 For instance, the most commonly used cell-permeable FAP fluorogen, FAP/MG 1 (see Figure 3), exhibits two excitation bands, the minor Y band with maximum excitation at 470 nm and the main X band with maximum excitation at 630 nm (Figure 1b). Both of the excitation Y and X bands give an emission maximum at 650 nm. However, since FAP/MG 1 displayed very © 2017 American Chemical Society

Figure 1. (a) Framework of malachite green (MG). (b) Absorbance spectrum of MG 1 in PBS.

weak fluorescence when excited by the Y band, only the X band was used as an excitation band for fluorescence imaging studies (λex 630 nm/λem 650 nm) and exhibited small Stokes shifts (Δλ = 20 nm). A small Stokes shift can cause self-quenching and measurement error by excitation light and scattered light. To address this issue, great effects have been dedicated, and three series of Förster resonance energy transfer (FRET)-based MG fluorogens with large pseudo-Stokes shifts have been developed.13 However, some problems are encountered with them because they contain complicated structures, require multistep reactions, and give low yields, and FRET strategies do not entirely eliminate the self-quenching and inner-filter effects.14 Fluorescent labels that have a large Stokes shift, low toxicity, and are bright and cell permeable are preferred for biological imaging.15 Herein, we demonstrate that a series of novel MG Received: July 5, 2017 Published: August 18, 2017 4496

DOI: 10.1021/acs.orglett.7b02055 Org. Lett. 2017, 19, 4496−4499

Letter

Organic Letters analogues with rational modifications of the Y band contributor ring C show improved quantum yield, large “pseudo-Stokes” shift (Δλ = λex − λem = 115 nm), and lower cell toxicity, improving the application of FAP/MGs in live-cell fluorescence microscopy. To our knowledge, use of a single MG fluorogen to achieve a large wavelength difference between maximum excitation and emission peaks by internal charge transfer (ICT) mechanism has not been reported. A large Stokes shift is desirable in fluorescent labeling applications of dyes, as it reduces self-quenching effects and interference from an excitation source.16 Exploration of multifluorophores with energy donor−acceptor architectures to achieve large pseudo-Stokes shifts of MG fluorogens has been reported.17 We intend to explore an alternative approach to increase the “pseudo-Stokes” shift in MGs by stabilizing the excited state through structural relaxation. ICT has been utilized with the aim of achieving a large wavelength difference between the maximum excitation and emission peaks in common organic systems,18 while this strategy has not been adopted in MGs and their analogues. Quantum chemical calculations have been proven to be an important tool for investigation of the relationships between structures and spectral properties of the organic molecules and for the interpretation of experimental results. Lately, timedependent density functional theory (TD-DFT) appears to be one of the most successful methods in terms of the balance between accuracy and speed, and it has been widely used in electronic transition energy predictions.19 To gain insight into the relationship between molecular structure and photophysical properties, TD-DFT calculations were performed on MG 1 using B3LYP/TZP (basis set) in ADF 2016. Figure 2 shows the

Theoretical calculations suggested that increasing the charge transfer and enhancing the π-conjugation of the bottom half could improve the fluorescence efficiency of MGs. Therefore, we hypothesized modulation of the photophysical properties of MG via fusion of the aromatic rings to the ring C phenyl unit. Six new MG derivatives with naphthalene (MG 2), quinolone (MG 3), or an indole group (MGs 4−7) and, for comparison, control MG fluorogen MG 1 (Figure 3) were synthesized in high

Figure 3. Structures of malachite green (MG) synthesized.

yields (see the synthetic procedures in the SI for details).We first explored the effects of the aromatic substitution on the fluorogen’s absorption spectrum, FAP_dH6.2 binding ability, and fluorescent spectrum in PBS. The photophysical properties of fluorogen MGs 2−7 along with control fluorogen MG 1 are summarized in Table 1 and Figures S3 and 4. As expected, the presence of a conjugated polycyclic aromatic moiety on ring C could impact on the absorption of X or/and Y band. However, the fluorescence quantum yields of MGs 2−4 are much lower than that of MG 1 after binding with FAP_dH6.2 and showed no fluorescence or only weak fluorescence. The fluorescence quantum yields of MGs 5−7 are comparable to MG 1 and showed a good fluorescence turn-on response to FAP_dH6.2. Moreover, MGs 5−7 retained the remarkable selectivity of FAP over various tested human and mice proteins in vitro (Figure S5). Crystallography of the FAP/MG complex reveals that MG binds FAP as left-handed propeller conformers with somewhat variable rotation angles along the C2 axis (Figure S1).12,20 TD-DFT calculations showed that ring C of MG 1 and MGs 5−7 were left-handed propellers, while MGs 2−4 were right-handed propellers (Figure S2). We inferred that only lefthanded propeller MG fluorogen could become fluorescent when binding with FAP. Figure 4a shows the UV/vis spectra of MG 1 and 3-indole conjugated fluorogens MGs 5−7. Compared with MG 1, MGs 5−7 were found to exhibit efficient ICT emission (Y band), resulting in large “pseudo-Stokes” shifts along with significantly enhanced emission intensity. Concentration−effect relationships between MGs and FAP_dH6.2 were observed (Figure S6−9). When excited at a long wavelength band (X band), maximum fluorescence intensities showed an unambiguous enhancement from MGs 5−7 compared to MG 1 (2213, 2007, 2384 vs 1283). More interestingly, fluorescence intensities of MG 5, MG 6, and MG 7 were 3.8, 2.6, and 4.1 times higher compared with MG 1 at short Y band excitation, respectively (Table 1 and Figure S4). In general, the electron-donating substituent on the 3-indole group could increase the fluorescence intensity. The “pseudoStokes” shifts of MG 5 (115 nm), MG 6 (125 nm), and MG 7 (115 nm) were shorter than MG 1 (180 nm); nevertheless, they

Figure 2. Frontier orbital of MG 1 and its electron transition from HOMOs to LUMO estimated by TD-DFT calculations to produce X and Y bands.

electron transition of MG 1 from the highest occupied molecular orbitals (HOMOs) to the lowest unoccupied molecular orbital (LUMO). The HOMO and LUMO densities of MG 1 are mainly located on the top half of the fluorogen, whereas HOMO−1 and HOMO−2 densities are mainly localized on ring C on the down half of MG 1. Theoretical calculations showed the ICT transition in MG 1 appears at the excitation Y band attributed to HOMO− 2 to LUMO transition; meanwhile, both HOMO → LUMO and HOMO−1 → LUMO transitions contribute to the excitation of X band, which are in good agreement with the literature.12 4497

DOI: 10.1021/acs.orglett.7b02055 Org. Lett. 2017, 19, 4496−4499

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Organic Letters Table 1. Photophysical Properties of Fluorogen MGs 1−7g fluorogen

λaba (nm)

λexb (nm)

λemc (nm)

ε × 103 (M−1 cm−1)

FId

ΦFe

KD (pM)

MG 1

460 615 505 620 640 625 515 585 510 585 515 575

470 630 510 630 NDf 625 515 590 510 600 515 575

650 650 665 665 ND 670 630 630 635 635 630 630

13.0 30.7 9.8 31.6 38.4 35.6 25.2 31.6 24.8 30.8 25.2 32.0

278 1283 77 398 ND 192 1055 2213 716 2007 1147 2384

0.154 0.296 ND 0.105 ND 0.068 0.215 0.153 0.224 0.196 0.140 0.112

1.533

MG 2 MG 3 MG 4 MG 5 MG 6 MG 7

0.813 ND 2.061 1.487 2.346 1.721

λab= absorption maximum. bλex= excitation maximum. cλem= emission maximum. dFI = fluorescence intensity. eΦF = fluorescence quantum yield. ND= not detectable. gSolvent conditions: 0.01 M PBS containing 1.0 % DMSO and 2.0 μM FAP_dH6.2, pH 7.4.

a f

Figure 4. (a) Absorption spectra of MG 1 and MGs 5−7. (b) Fluorescence spectra of MG 5, MG 5/FAP, MG 5/FAP + 1.0 equiv of MG 1, and MG 5/FAP + 4.0 equiv of MG 1. Figure 5. Confocal microscopic images of HEK293A_GFP_ dH6.2 cells incubated with MG 1, MG 5, and MG 5 for 15 min and then 1.0 equiv of MG 1 for 15 min, respectively. Blue fluorescence signals from Hoechst 33258 stain; green from GFP (λex = 488 ± 20 nm, λem = 500 ± 25 nm); red from MG/FAP (λex = 514 ± 20 nm, λem = 630 ± 25 nm); merge from bright-field and blue, green, red fluorescence signals. Scale bar = 20 μm.

are sufficient for avoiding the overlap of excitation peak and emission peak. MG 5 was later used in the cellular studies. MG 5 is fluorescence-off in solution, but intensely fluorescent, with the excitation and emission maxima at 515 and 630 nm, respectively, upon binding to FAP_dH6.2. However, FAP_dH6.2/MG 1 shows no fluorescence when excited at 515 nm, while the binding abilities of MG 1 and MG 5 to FAP_dH6.2 are comparable. We hypothesized that the MG 5/FAP_dH6.2/ MG 1 system could exhibit an OFF−ON−OFF fluorescence intensity change in the fluorescence spectra. Figure 4b and Figure S10 show the fluorescence spectral changes of MG 5 before and after addition of FAP_dH6.2 and the changes during the titration of MG 1. Accordingly, the fluorescence brightness of MG 5 increased by 817-fold upon binding with FAP_dH6.2 and then exhibited 90% fluorescence decrease after addition of 4.0 equiv of MG 1. The fluorescence modulation of MG 5 with FAP_dH6.2 and MG 1 clearly demonstrates an OFF−ON−OFF switch as expected. Inspired by the large “pseudo-Stokes” shifts results and an OFF−ON−OFF switch for FAP_dH6.2 imaging in PBS solution, we investigated the applicability of MG 5 for live cell imaging. In this study, FAP_dH6.2 expressing mammalian cells HEK293A and bacterial cells E. coli were generated and used, respectively.21 Typically, the HEK293A_dH6.2 cells and E. coli_dH6.2 bacterial strains were separately incubated with 2 μM of MG 5 or MG 1 for 15 min at 37 °C and subsequently subjected to confocal microscopy for fluorescence imaging (Figure 5). As shown in Figures S12−S14, strong fluorescence emission was observed after incubation of MG 5 with FAP_dH6.2 expressing mammalian and bacterial cells, whereas similar cellular incubation with MG 1 led to no obvious fluorescence when a 514 nm laser excitation was applied. These results demonstrated

that MG 5 with large “pseudo-Stokes” shifts allowed for no-wash imaging of FAP inside mammalian cells and bacterial cells with reasonable incubation time. Similar to the PBS solution, the MG 5/FAP/MG 1 system could exhibit an OFF−ON−OFF fluorescence switch. The fluorescent OFF mode of FAP_dH6.2 expressed cells could be switched ON in the presence of MG 5 and then exhibited ∼50% fluorescence decrease after addition of 1.0 equiv of MG 1. To localize FAP and further verify the selectivity of MG 5 to FAP in cells, HEK293A cells transiently expressing FAP_GFP fusion protein were generated by transfection of the plasmid pcDNA3.1 encoding FAP_GFP and incubated with MG 5. Confocal images of the GFP channel present the location of the FAP_GFP protein in the cell. MG 5/FAP signal confocal microscopy analysis reveals a high level of colocalization of the GFP signal with the MG 5/FAP signal, which indicates the remarkable selectivity of MG 5 to FAP. Z-Stack confocal imaging studies showed a clear intracellular distribution of both of the GFP and MG 5/FAP signals, including the nucleus in living cells (Figure S14 and S15). Moreover, it is noteworthy that MG 5 was found to be much less toxic than MG 1 in three tested human cell lines (for details, see the SI, Table S1). In summary, we have rationally designed and synthesized a series of novel 3-indole−Malachite Green-based FAP fluorogens, MGs 5−7. The important features of this class of FAP fluorogens 4498

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Organic Letters

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are the efficient internal charge transfer resulting significant fluorescence enhancements, remarkable large “pseudo-Stokes” shifts, low toxicity to cells, as well as very fast onset in response to FAP in both live mammalian cells and bacterial cells. They have the potential to be an alternative to FRET-based MG fluorogens with large pseudo-Stokes shifts in multiplexing applications with FAP imaging.

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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.orglett.7b02055. Synthetic procedures and spectral data for all new compounds, UV−vis, FI spectral studies, cell cytotoxic studies, and confocal imaging studies (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hai-Yu Hu: 0000-0002-4760-6865 Author Contributions ∥

Q.Z. and Q.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Mark Brönstrup (Helmholtz Centre for Infection Research) for providing the FAP_dH6.2 plasmid and for valuable discussions. Financial support from the Sino-German research project GZ 1271, PUMC Youth Fund (3332016056), and Beijing Nova Program (Z16111000490000) is acknowledged.

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REFERENCES

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DOI: 10.1021/acs.orglett.7b02055 Org. Lett. 2017, 19, 4496−4499