S-Cis Diene Conformation: A New Bathochromic Shift Strategy for

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S-Cis Diene Conformation: A New Bathochromic Shift Strategy for NearInfrared Fluorescence Switchable Dye and the Imaging Applications Hsiang-Jung Chen, Chee Ying Chew, En-Hao Chang, Yu-Wei Tu, Li-Yu Wei, Bo-Han Wu, ChienHung Chen, Ya-Ting Yang, Su-Chin Huang, Jen-Kun Chen, I-Chia Chen, and Kui-Thong Tan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01159 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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Journal of the American Chemical Society

S-Cis Diene Conformation: A New Bathochromic Shift Strategy for Near-Infrared Fluorescence Switchable Dye and the Imaging Applications Hsiang-Jung Chen,† Chee Ying Chew,† En-Hao Chang,† Yu-Wei Tu,† Li-Yu Wei,† Bo-Han Wu,† ChienHung Chen,# Ya-Ting Yang,# Su-Chin Huang,# Jen-Kun Chen,# I-Chia Chen†,‡,* and Kui-Thong Tan†,‡,* †

Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd, Hsinchu 30013, Taiwan (ROC) Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Sec. 2, Kuang-Fu Rd, Hsinchu 30013, Taiwan (ROC) # Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan (ROC) ‡

ABSTRACT: In this paper, we present a novel charge-free fluorescence switchable near-infrared (IR) dye based on merocyanine for target specific imaging. In contrast to the typical bathochromic shift approach by extending -conjugation, the bathochromic shift of our merocyanine dye to the near-IR region is due to an unusual S-cis diene conformer. This is the first example where a fluorescent dye adopts the stable S-cis conformation. In addition to the novel bathochromic shift mechanism, the dye exhibits fluorescence-switchable properties in response to polarity and viscosity. By incorporating a protein specific ligand to the dye, the probes (for SNAP-tag and hCAII proteins) exhibited dramatic fluorescence increase (up to 300-fold) upon binding with its target protein. The large fluorescence enhancement, near-IR absorption/emission and charge-free scaffold enabled no-wash and sitespecific imaging of target proteins in living cells and in vivo with minimum background fluorescence. We believe that our unconventional approach for a near-IR dye with the S-cis diene conformation can lead to new strategies for the design of near-IR dyes.

INTRODUCTION Fluorescent dyes are a rapidly expanding area of research in chemical and biological sciences with multiple applications as biomolecule labels, enzyme substrates, environmental indicators and cellular stains.1-4 Fluorescent dyes that are excited and emit in the near-infrared (IR) region are especially biocompatible and advantageous as they cause minimum damage to biological samples, have deep tissue penetration, and come under minimal interference from background autofluorescence by biomolecules in the living systems.5-7 Currently, most nearIR dyes are based on the cyanine and rhodamine scaffolds. Conventional strategies to obtain cyanine and rhodamine derivatives with appreciable bathochromic shift covering near-IR spectra include extending the-conjugation and introducing heteroatoms and rigid bridges.8-10 Although these dyes show exceptional brightness and photostability, cyanine and rhodamine tend to bind preferentially and accumulate in the mitochondria due to their permanent cationic charge, rendering them unsuitable for organelle-specific imaging.10-14 Thus, cumbersome additional manipulations are needed to reduce the background signal from unspecific binding.15,16 Besides being bright, near-IR and exhibiting no unspecific binding in certain cellular compartment, an ideal fluorescent dye should also exhibit significant fluorescent enhancement (fluorogenic) upon target molecule interaction to reduce the background fluorescence and substantially increase the signalto-noise ratio. Of all the fluorogenic dyes, those that show large changes in fluorescence intensities, as well as shifts in absorption and emission wavelength in response to H-bonding, polarity and viscosity from the surrounding are particularly

valuable in chemistry and biology.17,18 These environmentsensitive dyes, including derivatives of NBD, AIEgens, oxazine, dansyl, prodan, naphthalimide and merocyanine, have been used to study protein structure, 19-21 to report protein binding interactions22,23 and as fluorescent probes for rapid sensing and labeling of proteins.24-29 Although most environment-sensitive dyes have excellent performance in clean buffer solutions, their applications in living cells often suffer from one or more limitations, including less than adequate fluorescence increase in response to the target protein, insufficient brightness, short excitation and emission wavelengths that are toxic to the cells and overlap with autofluorescence, low plasma membrane permeability and/or potential preferential binding of the fluorescent dyes to other intracellular compartments. In this paper, we introduce a novel near-IR fluorescence switchable merocyanine dye, P-Mero4, that can be coupled with different protein ligands to interact with non-enzymatic proteins for the rapid fluorescence turn-on labeling and imaging in living cells and in vivo. Typically, merocyanine dyes are characterized by an electron acceptor and a donor component linked by a linear polymethine chain.30 As compared to the cyanine dyes which carry a permanent cationic charge, meroyanines are neutral and are sensitive to the hydrogen bonding (H-bonding) and solvent polarity due to the resonance delocalization of the push-pull system across the polyene system. The absorption and emission wavelength of merocyanine dyes can be tuned by varying the length of polymethine. For example, Mero2, which has two methine carbons between the acceptor and donor components has absorption and emission maxima at about 495 nm and 516 nm, while Mero4 with four

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(a)

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Scheme 1. Synthesis of P-Mero4 and fluorogenic probe PMero4SA and P-Mero4BG for sensing hCAII and SNAPtag proteins.

Figure 1. Chemical structures of (a) Mero2 and Mero4, (b) PMero4.

methine linker exhibits maxima at around 589 nm and 617 nm (Figure 1a). Certain merocyanine dyes such as MC540 and MeroCaM have been used for measuring membrane potential and calcium levels.31,32 As compared with the typical merocyanine dye, our PMero4 consists of a -substituted phenyl group at the tetramethinine bridge which resulted in red-shifted absorption and emission (bathochromic) of about 45 nm to the near-IR region (Figure 1b). In contrast to the typical -conjugation extension approach, P-Mero4 achieves bathochromic shift by the formation of an unusual S-cis diene conformer. Although bathochromic shifts due to S-cis conformation was first reported by Woodward several decades ago and are well documented in many literatures, the application of this conformation in fluorescent dyes has not been demonstrated.33-35 This is the first time that a stable S-cis conformation has been successfully identified in a near-IR dye. In addition to the novel bathochromic shift mechanism, our new P-Mero4 dye also exhibits fluorescence-switchable properties in response to polarity and viscosity. When different protein specific ligands were conjugated to P-Mero4, the probes show a dramatic increase in fluorescence (up to 300-fold) in the presence of target proteins (SNAP-tag and hCAII proteins). Therefore, no-wash labeling and imaging of the SNAP-tag protein in different subcellular compartments was possible with minimum background staining. The protein probes were successfully applied in living cells to visualize the incomplete posttranslational processing of the nuclear envelope protein and in vivo for site-specific tumor cells imaging. RESULTS AND DISCUSSION Synthesis of P-Mero4 dye. The synthesis of P-Mero4 can be accomplished in two major steps, beginning with the condensation of the 2,3,3-trimethylindolenine derivative 1 with dialdehyde linker 2 to give intermediate 3 (Scheme 1). The final product P-Mero4 can be obtained by treatment of compound 3 with 1,3-indandione. The carboxylic acid group on PMero4 can be further functionalized with different protein ligands via the standard peptide coupling reaction. In this paper, sulfonamide and benzylguanine moieties were coupled to the dye to obtain fluorescent probes P-Mero4SA and PMero4BG, respectively. P-Mero4SA can be used to bind and sense human Carbonic Anhydrase II (hCAII) via its sulfonamide ligand,36 while P-Mero4BG can be employed to covalent label the SNAP-tag protein, which is one of the most

prominent labeling techniques in living cells.37 Spectroscopic Characterization of P-Mero4 dye. To characterize and compare the spectroscopic properties of Mero4 and P-Mero4, the solvents dichloromethane (DCM), toluene, DMSO, ACN, phosphate buffered saline (PBS), methanol, dioxane, THF, ethylene glycol and glycerol were used to evaluate the solvent dependency of dye absorption, emission, extinction coefficient and quantum yield. These solvents were selected to provide a wide range of polarities, H-bonding and viscosity and the results were summarized in Table 1. In DMSO, P-Mero4 displays an absorption maxima at around 640 nm (Figure 2a). As compared to Mero4, the absorption maxima of P-Mero4 in different solvents are generally about 45 nm red-shifted and with a slightly lower extinction coefficient than Mero4 (Table 1 and Figure S1). As for the emission spectra, the maxima of P-Mero4 occurs at around 660 nm which is about 40 nm red-shifted as compared to Mero4 (Figure 2b and Figure S2). By incorporating a phenyl substituent at the polymethine chain, P-Mero4 displays similar absorption and emission properties as the classical Cy5 dye. Therefore, our new P-Mero4 dye can be easily detected using the existing laser light and optical filters developed for the Cy5 dyes. Generally, solvents with different polarity always influence the quantum yields and brightness of merocyanine dyes. As a typical environment-sensitive merocyanine dye, Mero4 shows weak fluorescence and low quantum yields in protic solvents but exhibits higher quantum yields in polar and hydrophobic solvents (Φ = 0.0002 in PBS buffer, Φ = 0.018 in methanol and Φ = 0.35 in DMSO, Table 1). Greater emission yield in polar solvent was also reported by Kulinich et al. and MacNevin et al. for merocyanine dyes.38-40 For P-Mero4, the quantum yield is not only influenced by the solvent polarity and Hbonding, but is also sensitive to solution viscosity. The dye exhibits the highest quantum yield in a very viscous glycerol solution (Φ = 0.0002 in PBS buffer, Φ = 0.012 in methanol, Φ = 0.033 in DMSO and Φ = 0.15 in glycerol). P-Mero4 and Mero4 show similar solubility in aqueous solution up to 100 M (Figure S3). This indicates that the additional phenyl moiety on the tetramethine chain does not affect its aqueous solubility. As with many cyanine and merocyanine dyes that exhibit aggregation-caused fluorescence quenching (ACQ) at


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Table 1. Spectroscopic data of Mero4 and P-Mero4 in different solvents. Absorption (nm)

Emission (nm)

εb x104

Фa

λP-Mero4 (λMero4)

λP-Mero4 (λMero4)

P-Mero4 (Mero4)

P-Mero4 (Mero4)

Toluene

630 (577)

652 (610)

7.67 (12.6)

0.0043 (0.029)

DCM

633 (587)

656 (618)

7.61 (9.83)

0.0035 (0.10)

Dioxane

629 (578)

650 (606)

5.89 (6.88)

0.0097 (0.067)

THF

631 (578)

653 (612)

8.72 (13.1)

0.0057 (0.036)

ACN

632 (578)

653 (612)

7.11 (9.94)

0.0088 (0.26)

DMSO

637 (582)

660 (626)

7.95 (9.01)

0.033 (0.35)

MeOH

634 (593)

655 (612)

8.72 (12.8)

0.012 (0.018)

Ethylene glycol

638 (591)

657 (612)

8.11 (9.09)

0.050 (0.028)

Glycerol

642 (603)

654 (627)

6.76 (10.7)

0.15 (0.061)

PBS

629 (599)

655 (619)

3.26 (8.55)

0.0002 (0.0002)

Solvents

a

Cy5 (Ф = 0.27 in PBS) and rhodamine B (Ф = 0.31 in PBS) were used as quantum yield references for P-Mero4 and Mero4, respectively. The unit of extinction coefficient is M-1cm-1.

b

(a)

(b)

(c)

(d)

Figure 3. Chemical structure of the five P-Mero4 derivatives. Figure 2. Spectroscopic characterization of P-Mero4 and Mero4 dyes. (a) Normalized absorption spectra and (b) emission spectra of P-Mero4 and Mero4. Fluorescence response of (c) P-Mero4 and (d) Mero4 in methanol (5 M) with increasing concentrations of glycerol.

high concentration, ACQ occurs at around 25 M for PMero4 and Mero4 (Figure S4). We have also evaluated the photostability of P-Mero4 and Mero4 (Figure S5). The dyes (10 M in DMSO) in a cuvette were continuously irradiated for 1 hour. The results showed that both P-Mero4 and Mero4 displayed good photostability and their fluorescence intensities remained unchanged after 60 minutes of irradiation. In a titration experiment in which glycerol was added to the methanol solution of P-Mero4, the fluorescence was enhanced gradually as viscosity increases (Figure 2c). In contrast, the increase in fluorescence for Mero4 only begins

after 70% of glycerol was added to the methanol solution (Figure 2d). Thus, the incorporation of one phenyl moiety on the tetramethine chain can change the fluorescence response of the merocyanine dye in different solvents. P-Mero4 Derivatives to Understand the Mechanism of Bathochromic Shift. Although many derivatives of cyanine and merocyanine dyes have been synthesized and characterized spectroscopically, the incorporation of a phenyl group at the -position of the polymethine chain that results the substantial bathochromic shift is quite unusual and has not been reported previously. To better understand the mechanism of this interesting phenomenon, we decided to modify the phenyl moiety at the polymethine chain. Based on the same synthetic strategy, electron-donating para-methoxyphenyl (MP-Mero4),


Journal of the American Chemical Society Table 2. Spectroscopic data for different P-Mero4 derivatives. λabs

λem

εb x104

Фa

MeOH

634

664

6.43

0.004

MP-Mero4 DMSO

644

672

6.99

0.020

Glycerol

644

656

5.05

0.084

MeOH

628

644

5.75

0.001

DMSO

637

652

6.00

0.009

Glycerol

636

644

5.08

0.035

MeOH

634

648

7.40

0.005

CH-Mero4 DMSO

643

656

8.93

0.015

Glycerol

642

652

6.43

0.045

MeOH

634

658

5.08

0.01

Dyes

NP-Mero4

Solvents

(a)

(b)

(c)

(d)

1.0

NAPHMero4S

DMSO

646

668

5.31

0.04

Glycerol

644

656

5.77

0.23

MeOH

653

674

21.2

0.018

DMSO

663

684

17.3

0.013

0.8 0.8 0.6 0.6

Energy (eV)

P-Mero4S

Energy (eV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.4 0.2 0.0 -0.2

Mero4 trans/S-cis Mero4 trans/S-trans -200

-150

-100

-50

0

50

Scan Coordinate

Glycerol

663

677

16.1

0.29

electron-withdrawing para-nitrophenyl (NP-Mero4) and cyclohexyl (CH-Mero4) groups were incorporated at the position of the polymethine chain (Figure 3). Furthermore, two highly water soluble sulfonic acid derivatives P-Mero4S and NAPH-Mero4S were also synthesized to investigate the effect of electron-donation from the terminal group of merocyanine dye. The absorption and emission data of the five P-Mero4 derivatives in DMSO, MeOH, and glycerol were summarized in Table 2, Figure S6 and S7. We found that all the five new derivatives of P-Mero4 exhibit environment-sensitive properties and substantial bathochromic shift as the P-Mero4 dye. With increase of the electron-donating ability of phenylsubstituents at the -position from NP-Mero4 to MP-Mero4, we observed a gradual increase of absorption and emission wavelengths at the near-IR region among the P-Mero4 derivatives. NAPH-Mero4S, which has a stronger electron-donating naphthalene moiety at the terminal of the dye, shows the most bathochromic shift with maximum absorption and emission at 663 nm and 684 nm in DMSO, respectively. However, it is surprising to note that CH-Mero4, which has a cyclohexy group that is inefficient in contributing electron resonance to the polymethine linker, also shows a significant red-shifted absorption and emission similar to the P-Mero4 dye. This result suggests that bathochromic shift based on the typical conjugation extension may not be the major factor leading to the significant red-shifted absorption and emission of the PMero4 derivatives.

100

0.4

0.2

P-Mero4 trans/S-trans

0.0

P-Mero4 trans/S-cis 150

200

-0.2 -50

0

50

100

150

200

250

300

350

400

Scan Coordinate

Figure 4. Calculated energy and stability of Mero4 and P-Mero4 under different conformations. (a) Mero-4 adopts the more stable trans/S-trans conformer. (b) P-Mero4 adopts the trans/S-cis conformer due to the steric hindrance between the phenyl substituent and the indandione moiety. (c) Energy variation of Mero4 from trans/S-trans to trans/S-cis. (d) Energy variation of P-Mero4 from trans/S-cis to trans/S-trans.

Computational Calculation and 2D NMR Measurements to Reveal the Bathochromic Shift Mechanism of P-Mero4. In order to better understand the bathochromic shift mechanism of P-Mero4, we performed a computational study using density functional theory (DFT) and time-dependent DFT (TD-DFT) method with B3LYP basis set up to 6-31G level.41,42 The Gaussian 09 program package was used for all the calculations. In view of geometric isomerism, the cis/trans isomers and S-trans/S-cis conformers of Mero4, P-Mero4 and CH-Mero4 were considered. The results were summarized in Table S1 and Figure S8, respectively. For Mero4, the trans/Strans structure is the most stable conformer which has 10.9 kJ/mol lower in energy than the trans/S-cis conformer (Figure 4a). Surprisingly, the most stable conformer for P-Mero4 and CH-Mero4 is trans/S-cis conformer which is 13.3 kJ/mol and 22.7 kJ/mol lower than the trans/S-trans conformer, respectively (Figure 4b). The calculated vertical transition for the trans/S-cis conformer of P-Mero4 is red shifted by about 34 nm (16 kJ/mol, 0.16 eV) as compared to the trans/S-trans conformer of Mero-4 which correlates well with the experimental observations. The results using SCAN by varying the torsional angle show that the energy barriers for S-trans-S-cis conformational change are about 80 kJ/mol (Figure 4c and 4d). A higher energy barrier of about 100 kJ/mol is found for the cistrans isomerization (Figure S9). Hence, it would be difficult to


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Journal of the American Chemical Society (a)

(b)

Figure 5. 2D ROESY NMR spectra of (a) Mero4 and (b) P-Mero4 in d6-DMSO at 298 K.

convert to the other isomer/conformer at room temperature on the ground state. Based on the computational calculations and analogs of P-Mero4, we believe that steric hindrance between the phenyl substituent and the indandione moiety is the reason to cause P-Mero4 and its derivatives to adopt the unusual Scis conformer. The introduction of a bulky phenyl or cyclohexyl group at the -position of tetramethinine chain is critical to generate the steric hindrance as the similar substituent on a longer pentamethinine chain showed no apparent bathochromic shift.43,44 Furthermore, the geometries of indandione and indole groups for P-Mero4 were found to be near coplanar, with a 60⁰ dihedral angle between the phenyl substituent and the backbone which indicates that electrons from the phenyl substituents can partially delocalize onto the polymethine chain (Table S2). As a result, MP-Mero4 with a stronger electron-donating para-methoxyphenyl substituent displays longer red-shifted absorption and emission spectra than NP-Mero4. However, it is important to note that the electron-donation from the phenyl substituent to the tetramethinine chain only contributes a minor effect on the bathochromic shift as the differences in the maximum absorption and emission wavelength is less than 10 nm between MP-Mero4 and NP-Mero4. To further validate that the incorporation of a bulky substituent at the -position of tetramethinine chain results in the formation of the S-cis conformation, we used 2D COSY and ROESY NMR spectroscopy to determine the proton chemical shifts and conformation of Mero4 and P-Mero4. From the 2D ROESY spectra of Mero4, Hc proton was found to exhibit strong NOE effect with Ha and He (Figure 5a and Figure S10). For P-Mero4, we found that Ha’ has strong NOE with Hc’ and Hg’ (Figure 5b and Figure S11). Furthermore, we also observed that Hf’ has correlation with both Hb’ and He’. Another

proof of S-cis conformer comes from the 2D COSY spectra of P-Mero4 which shows that chemical shift of Hc’ is downfield shifted to 10.2 ppm (Figure S12). Such a large downfield shift occurs when the indandione moiety is in a S-cis conformation to exert anisotropy effect to the adjacent Hc’ proton. Compounds with similar deshielding effect have been reported previously.45,46 These results clearly indicate that P-Mero4 exists as S-cis conformation and Mero4 as S-trans conformer. For Mero4, the major conformer is the S-trans form which has an absorption maximum at around 585 nm. The small redshifted absorption band due to the minor S-cis conformer of Mero4 can only be observed when the absorption spectrum was recorded at 77 K, at which the hot band transition was suppressed (Figure S13). Bathochromic shifts associated with the S-cis conformation in a -chromophore are relatively well known. In fact, it is an integral part of the Woodward-Fieser rule for UV-absorption properties of unsaturated systems where an 39 nm is added to the base values of homoannular dienes.33-35 The S-cis conformer has also been proposed as an important intermediate that cause the absorption maximum of bathorhodopsin to redshift about 45 nm.47,48 As compared to the S-trans structures, S-cis conformers are normally less stable and exist only in smaller proportions (≈ 2 %).49 By introducing a bulky group at the -position, we have successfully created a more stable Scis conformer which is formed to be the major species in a polyene system. These derivatives of P-Mero4 are the first examples to showcase bathochromic shift based on the formation of a stable S-cis conformer. We believe that our novel approach to generate bathochromic shift based on the unusually stable S-cis conformation will open up new strategy to



(a)

(b)

Figure 6. Molecular orbitals (MO) involving in transitions S2-S0 and S1-S0 of (a) Mero4 and (b) P-Mero4 calculated with TD-DFT at B3LYP/6-31G level using polarizable continuum model (PCM) assuming solvent ACN.

design new fluorescent dyes for the applications in bioanalysis, chemical biology and material science. Computational Study to Understand the FluorescenceSwitchable Properties of Mero4 and P-Mero4. For both Mero4 and P-Mero4 dyes, the results of TD-DFT calculations show that excitation to S1 has the larger oscillator strength (1.85 for Mero4 and 0.98 for P-Mero4) and is a π to π* transition between the highest occupied molecular orbital (HOMO) and the lowest unoccupied MO (LUMO) which are largely delocalized on the tetramethine chain (Figure 6). The higher oscillator strength calculated for Mero4 is consistent with the larger extinction coefficient obtained for Mero4 as compared to P-Mero4 (Table 1). Considering the effect of solvent polarity, the polarizable continuum model (PCM) assuming ACN as the solvent, revealed that the second transition S 0-S2 is HOMOLUMO+1 which is a π-π* transition with a smaller oscillator strength (0.06 for Mero4 and 0.17 for P-Mero4). LUMO+1 is a π* orbital which is localized largely on the in-

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dandione moiety. Hence, S2 is a charge-transfer state. In more polar solvents the transition S0-S2 is expected to be lower in energy but remains above the delocalized π-π* transition (S1S0) in ACN. From the absorption and emission spectra measured in all solvents, there were no large bathochromic shift in polar solvents and the same electronic bands were observed. Hence, we can conclude that photochemistry involving the charge transfer transition state S0-S2 is insignificant in this system. Our experimental data reveal that the emission quantum yield of both Mero4 and P-Mero4 is strongly affected by Hbonding (φ = 0.012 for P-Mero4 and 0.018 for Mero4 in methanol). However, in ethylene glycol and glycerol solution, the effect of H-bonding is superseded by viscosity, with the phenyl substituent on the tetramethine chain as the main contributor for this phenomenon. This additional one-bondrotation relaxation channel in P-Mero4 would result in lower emission and quantum yield in polar and H-bonding solvents than for Mero4 (Table 1). However, in viscous solvents (e.g. glycerol), the one-bond-rotation relaxation is restricted causing the fluorescence intensity and quantum yield to increase. This feature provides a significant advantage when designing fluorogenic probes for protein labeling and sensing. 50-54 We envisaged that the binding of the ligand to the protein would bring P-Mero4 closer to the crowded protein surrounding. As a result, torsional motion and ring deformation should be sufficiently restricted to trigger the emission of a stronger fluorescence signal. Fluorescence Turn-On Labeling of P-Mero4BG to SNAP-tag protein. Currently, most of the fluorogenic probes are reaction-based and designed to monitor enzyme activities and reactive small molecule species.3 Conversely, only a limited number of fluorescent dyes have been developed for fluorescence turn-on protein labeling and non-enzymatic protein sensing in living cells.55,56 As most of the ligand binding sites in proteins are crowded and hydrophobic, we expect that protein probes based on the conjugation of P-Mero4 and a protein ligand could afford a large fluorescence increase upon the interaction of the ligand to the target protein. To test our probe design, we incorporate a benzylguanine (BG) ligand on P-Mero4 to obtain fluorogenic probe, PMero4BG, for the covalent labeling of the SNAP-tag protein (Figure 7a). Being one of the most prominent labeling tools in cell biology, SNAP-tag is well-known for its non-toxicity in cells and rapid reaction rate with BG derivatives.37 SNAP-tag labeled with an affinity ligand or fluorescent probe has been used for protein localization and interaction studies,57 drug discovery,58 super-resolution imaging applications59 and the construction of fluorescent sensors.60,61 P-Mero4BG showed extremely weak fluorescence in aqueous PBS buffer (ε = 2.31x104 M-1cm-1, Φ = 0.0006, Figure 7b). Upon the addition of the SNAP-tag protein, the fluorescence was enhanced dramatically with a high turn-on ratio of more than 300-fold (ε = 5.81x104 M-1cm-1, Φ = 0.096) and can be observed easily under handheld UV lamp (Figure 7b, inset). The fluorescence enhancement was suppressed when O6benzylguanine inhibitor (O6-BG, 100 M) was pre-incubated with SNAP-tag protein. The fluorescence increase was so effective that the P-Mero4BG-SNAP-tag complex showed similar emission intensity as the fluorescence of P-Mero4BG recorded in DMSO solution (Figure S14). In comparison, covalent labeling of polarity-sensitive probe Mero4BG to SNAP-


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Journal of the American Chemical Society (a)

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(b)

(c)

(b)

Figure 8. (a) Schematic illustration of the fluorescence switchable probe P-Mero4SA for the sensing of hCAII. (b) Fluorescence spectra of 5 M P-Mero4SA in the absence and presence of 5 M hCAII and after the addition of 100 M ethoxzolamide, a hCAII competitive inhibitor. The inset shows that the fluorescence response was linear in the range of 0.025−2.5 μM with the LOD of 27 nM hCAII. R2 = 0.99.

Figure 7. (a) Schematic illustration of the fluorescence turn-on labeling of SNAP-tag protein with P-Mero4BG. (b) Fluorescence spectra of 5 M P-Mero4BG in the absence and presence of 5 M SNAP-tag protein and 100 M O6-benzylguanine. The inset shows the images of the P-Mero4BG solution in a cuvette before (left) and after (right) addition of SNAP-tag under excitation with a UV lamp (254 nm). (c) Selectivity test of 1 M P-Mero4BG with SNAP-tag and nine non-targeted proteins (1 M). Error bars were calculated from three independent measurements.

tag protein gave about 50-fold fluorescence enhancement (Figure S15). This result indicates that although both polarity and viscosity exhibit synergistic effect in fluorescence turn-on, viscosity has a more significant contribution toward the higher fluorescence enhancement of P-Mero4 dye. From the titration experiment, the limit of detection (LOD) for P-Mero4BG to detect SNAP-tag was determined to be as low as 1.5 nM, reflecting the high sensitivity of this fluorogenic probe (Figure S16). To show that fluorescence increase of P-Mero4BG is highly specific and occurs only in the presence of SNAP-tag protein, we incubated P-Mero4BG with eleven other nontarget proteins (Figure 7c). In all cases, dramatic fluorescence increase was observed only when SNAP-tag was present. Thus, this design presents a very valuable approach for target protein detection. The specific covalent bond formation between P-Mero4BG and SNAP-tag has also been characterized by in-gel fluorescence analysis (Figure S17).

Furthermore, we also constructed a SNAP-tag probe based on the sulfonated NAPH-Mero4S (ε = 4.98x104 M-1cm-1, Φ = 0.0004) which has maximum absorption and emission at 663 nm and 684 nm, respectively. In the presence of SNAP-tag protein, NAPH-Mero4SBG exhibits fluorescence enhancement of about 270-fold (ε = 8.21x104 M-1cm-1, Φ = 0.14, Figure S18). A kinetic analysis of the SNAP-tag labeling reaction with NAPH-Mero4SBG revealed that full labeling can be achieved within three minutes with t1/2 = 41 s (Figure S19), which is comparable to other fast SNAP-tag fluorogenic labeling probes.25,59,62,63 Currently, most of the near-IR dyes are based on the cyanine and rhodamine scaffold which tend to bind preferentially and accumulate in the mitochondria due to their permanent cationic charge, rendering them unsuitable for organelle-specific imaging. Although P-Mero4BG and NAPH-Mero4SBG might not be as bright as the previously reported near-IR fluo-rogenic SNAP-tag probes that are based on the Si-Rhodamine dye,59,62,64 the advantages of our new probes are their high fluorescence turn-on ratio upon SNAPtag labeling, and their charge-free and simple synthetic protocols which can be applied in no-wash and site-specific imaging of target proteins in living cells and in vivo to achieve minimum background fluorescence. Fluorogenic Response of P-Mero4SA with hCAII Protein. To illustrate the modular nature of P-Mero4 dye as a fluorogenic protein probe, we replaced the BG group with a benzenesulfonamide (SA) ligand to generate P-Mero4SA for the fluorescence turn-on sensing of human carbonic anhydrase II (hCAII, Figure 8a). hCAII and many of its isoforms are important proteins in the regulation of numerous physiological process, including pH and CO2 homeostasis, bone resorption, calcification, and tumorigenicity.65 In the presence of hCAII, the benzenesulfonamide ligand can bind very tightly to the hydrophobic and crowded active site to turn on the fluorescence of P-Mero4SA.



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Figure 10. No-wash live cell imaging of CHO cells expressing SNAP-Lamin A proteins treated with different drugs followed by treatment of 2.5 M P-Mero4BG. (a) No drug, (b) 10 M Lopinavir and (c) 10 M Lovastatin for 20 hours, respectively. Scale bar: 20 m. Figure 9. No-wash live cell imaging of CHO cells expressing proteins fused with SNAP-tag at different subcellular locations by P-Mero4BG. (a) SNAP-tag protein only for both cytosol and nucleus, (b) SNAP-H2B for nucleus, (c) PDHA1-SNAP for mitochondria, (d) SNAP-Lamin A for nuclear envelope. SNAP-tagged proteins labeled with P-Mero4BG are shown in red, nuclei labeled with Hoechst 34580 are shown in blue. Scale bar: 20 m.

As expected, P-Mero4SA displayed very weak fluorescence in PBS buffer (ε = 1.99x104 M-1cm-1, Φ = 0.0007), while strong fluorescence enhancement of around 70-fold was achieved upon addition of hCAII protein (ε = 3.10x104 M-1cm1 , Φ = 0.041, Figure 8b). The fluorescence increase was reduced dramatically when a competitive inhibitor of hCAII, ehthoxzolamide was added. This demonstrates the dynamic fluorescence switching mechanism where fluorescence can be regulated by the recognition of the benzenesulfonamide ligand on P-Mero4SA by hCAII. From the titration experiment, the LOD for P-Mero4SA to detect was determined to be as low as 27 nM (Figure 8b, inset). Organelle-Specific and No-wash Live Cell Imaging of SNAP-tagged Proteins with P-Mero4BG. Although many fluorescent probes have been reported for labeling proteins in living cells, most of these absorb and emit light in UV-visible range.66 The development of near-IR fluorescent probes to label intracellular proteins at the specific subcellular compartments of living cells still remains a challenge. This is because most of the near-IR dyes possess permanent cationic charges which tend to bind preferentially to and accumulate in the mitochondria. One recent example of near-IR fluorescent dye for specific protein imaging within living cells is siliconrhodamine which binds to its target protein while keeping the fluorophore in the fluorescent-ON zwitterionic form.59 In this paper, we applied P-Mero4BG for the labeling of SNAP-tag that have been expressed in different subcellular compartments and imaged the proteins under no-wash conditions. We transiently transfected Chinese Hamster Ovary (CHO) cells with genes encoding SNAP-tag, PDHA1-SNAP, SNAP-H2B and SNAP-Lamin A fusion proteins. Without the localization tag, SNAP-tag protein will be expressed in the cytosol and nucleus. For SNAP-tag fused with PDHA1, H2B

and Lamin A, the proteins will be localized on the mitochondria, nucleus and nuclear membrane, respectively. These CHO cells were incubated with 2.5 M P-Mero4BG for 1 hour and fluorescence images were taken without wash out of the excess P-Mero4BG probe. In all the cases, we observed a specific fluorescence labeling of SNAP-tag fusion proteins in the corresponding organelles of living cells (Figure 9). In the nontransfected cells, low background fluorescence was observed in the presence of P-Mero4BG. In comparison, when the BG ligand was conjugated with Cy5 and Rhodamine dyes for the labeling of SNAP-tagged Lamin A fusion proteins, a substantial amount of unspecific fluorescence signals in the mitochondria besides the nucleus envelope was observed (Figure S20). Even with extensive washing, this unspecific fluorescence in the mitochondria cannot be removed. We also investigated the permeability and labeling rate of P-Mero4BG to SNAP-tagged Lamin A (Figure S21). Consecutive capture of cell images without the washing procedure showed timedependent fluorescence enhancement along the nuclear membrane and complete labeling was achieved within 50 minutes. Thus, by using our near-IR fluorogenic probe P-Mero4BG coupled with SNAP-tag labeling technology, we are able to obtain organelle-specific fluorescent images with minimal background fluorescence. Specific Labeling of Pre-Mature Lamin A. Lamin A is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane of the nucleus membrane.67 Maturation of Lamin A requires multistep posttranslational modification at its C-terminal CaaX motif. Initially, a farnesyl moiety is added to the cysteine by farnesyltransferase, followed by ZMPSTE24 protease cleavage. Incomplete Lamin A processing is associated with the rare premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) which is characterized by the rapid appearance of aging, beginning at childhood. The truncated Lamin A, termed progerin, lacks the second cleavage site for ZMPSTE24, resulting in an accumulation of farnesyl-prelamin A. An obvious effect of progerin expression in cells is a significant change in nuclear morphology, such as lobulations and folds in the nuclear envelope, loss of peripheral heterochromatin and accumulation of Lamin A aggregates in the nucleo-


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(a)

(b)

Figure 11. In vivo and ex vivo imaging of tumor cells with NAPH-Mero4SBG. (a) Biodistribution of NAPH-Mero4SBG within 9 days after tail vein injection. Red arrows indicate NAPHMero4SBG accumulation in HeLa-SNAP tumor. (b) The ex vivo images of organs, tissues and tumors collected at 24 and 240 hours post-injection of NAPH-Mero4SBG. Abbreviation: Ht (heart), Lg (lung), Lv (liver), Spl (spleen), Kd (kidney), Ms (muscle), Sk (skin), GI (gastrointestinal tract), Con (contralateral), TmS (tumor).

plasm. Currently, most of the fluorescent images of Lamin A were acquired using Lamin A antibodies or fluorescent protein tagged Lamin A. To demonstrate that our P-Mero4BG can be used to visualize incomplete Lamin A processing in living cells, we treated the Lamin A expressing CHO cells with Lopinavir and Lovastatin. Lopinavir is an inhibitor of ZMPSTE24,68 while Lovastatin is an inhibitor of HMG-CoA reductase, which catalyzes mevalonic acid synthesis, a key precursor of isoprenoid synthesis. Treatment of Lovastatin will eventually block Lamin A prenylation involving both farnesyl (C15) and geranylgeranyl (C20).69 6 hours after transfection, the drugs were added to the cells and incubated for 20 hours. The drug treated cells were incubated with 2.5 M P-Mero4BG for 1 hour, and fluorescence images were taken without washing out of the probe. For the cells expressing mature Lamin A, the nuclear envelope shows a circular shape (Figure 10). Treatment of Lopinavir led to the accumulation of pre-lamin A, and the cells acquired irregular nuclear envelope profiles with decreased circularity. In contrast, blocking the farnesylation of progerin by Lovastatin led to the redistribution from the nuclear membrane and aggregation in the nucleoplasm. Our results are consistent with

all the previously reported images that were taken using immunocytochemistry, fluorescent protein tagged Lamin A and fluorescent probes.63,70,71 In Vivo Imaging of Tumor Cells with NAPH-Mero4SBG. Near-IR probes have the advantages of deep tissue imaging and reduced interference from autofluorescence in more complex biological samples, such as tissues or living animals. The promising results in living cell imaging encouraged us to further explore the applicability of P-Mero4 scaffold as an in vivo near-IR imaging probe. To demonstrate the application, NAPH-PMero4SBG probe was applied in vivo for imaging HeLa-SNAP tumors in nude mice. We used NAPHPMero4SBG to image HeLa-SNAP tumors because the probe has the most red-shifted absorption/emission wavelength among our series of P-Mero4 dyes and contains a sulfonated moiety to increase the aqueous solubility. As shown in Figure 11a, the fluorescent images show the biodistribution of NAPH-Mero4SBG probe after tail vein injection. The probe remained in the blood circulatory system after 6 hours and a strong fluorescent signal was observed in the abdominal region. We observed that NAPH-Mero4SBG can specifically target subcutaneous tumors developed by HeLa-SNAP cells in xenograft, with significant accumulation in tumor at 24, 48, 144, 216 hours after injection. To examine the excretion pathway of NAPH-Mero4SBG, plasma samples were collected at 6 and 24 hours after injection (Figure S22). Fluorescence intensity of NAPH-Mero4SBG in plasma samples dramatically dropped back to the background level 24 hours after injection. This observation led us to predict that probe might have been washed out from the blood pool rapidly and subsequently redistributed into tumor, organs and tissues. Indeed, NAPHMero4SBG was found to be excreted effectively through feces and urine, and the fluorescence signals in feces were 40 ‒ 80 times more intensive than that in urine. We therefore suggest that NAPH-Mero4SBG is predominantly excreted through the hepato-intestinal metabolism pathway. To verify the observation of in vivo images, mice were sacrificed on day 1 (N = 3) and day 10 (N = 3) after the injection of NAPH-Mero4SBG. Organs and tissues were collected for ex vivo imaging and the ROIs were quantified to calculate the tissue distribution (Figure 11b and Table S3). High tumor to normal ratios (T/N) of 10.11±1.88 and 7.48±2.66 at 24 and 240 hours were obtained, suggesting that NAPH-Mero4SBG is a highly specific tumor-targeting probe to diagnose the growth of tumor. NAPH-Mero4SBG was also found to accumulate in the gastrointestinal tract, kidney and liver. As compared to the previous cyanine-based near-IR SNAP-tag probes for in vivo imaging, our fluorescent turn-on NAPHMero4SBG probe gave a much lower background fluorescence.72,73 Thus, the fluorescent enhancements of P-Mero4 dye and its derivatives in near-IR region upon target protein binding present a novel solution to reduce the background fluorescence for target-specific in vivo imaging. CONCLUSIONS In summary, we have developed a novel class of fluorescence switchable near-IR dye, P-Mero4, with a phenyl substituent at the -position of its tetramethine chain. P-Mero4 and its derivatives show about 45 nm red-shifted absorption and emission as compared to the linear merocyanine dye. Mechanistic studies using computational calculations, 2D ROESY NMR spectroscopy and analogs of P-Mero4 reveal



that steric hindrance between the phenyl substituent and the indandione moiety in P-Mero4 and its derivatives causes it to adopt the unusual S-cis conformer. This is the first time that a stable S-cis conformation is observed in fluorescent dyes. Besides being sensitive to polarity and H-bonding, the dye also exhibits strong fluorescence in viscous solvents and crowded surroundings due to the restricted one-bond rotation of the phenyl substituent on the tetramethine chain. This environment-sensitive feature of P-Mero4 was exploited to generate fluorogenic probes, NAPH-Mero4SBG, P-Mero4BG and PMero4SA, for sensing SNAP-tag and hCAII proteins. In the presence of the target proteins, the probes showed dramatic fluorescence increase of up to 300-fold for SNAP-tag and 70fold for hCAII. With P-Mero4BG, no-wash labeling of intracellular proteins fused with SNAP-tag was successfully achieved and the localization of the protein was specifically visualized with minimal background staining. Furthermore, PMero4BG and NAPH-Mero4SBG can also be harnessed to image the incomplete Lamin A processing in living cells and for in vivo imaging. We believe that these near-IR fluorescentswitchable P-Mero4 dye derivatives can spearhead the design and development of novel fluorogenic probes to become an important tool in target-specific imaging in living cells and animals.

ASSOCIATED CONTENT Supporting Information. For complete experimental methods and figures see Supplementary Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected]

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We are grateful to the Ministry of Science and Technology (Grant No.: 106-2113-M-007-027) Taiwan (ROC) for financial support. We thank Prof. Lily Hui-Ching Wang and Mr. Tian-Neng Li for preparing the stable SNAP-PDGFR HeLa cells. Authors also thank the Animal Molecular Imaging Core Facility and Miss ShuHsien Wu for technical support of animal care in NHRI (MOHW106-TDU-B-211-144-001 and NHRI-BN-106-PP-31). We also thank Miss Li-Ching Shen of the Center for Advanced Instrumentation of National Chiao Tung University for the 2D NMR measurements.

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