Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by

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Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group Wei Wang,†,‡ Liping Zhu,‡ Yoshinori Hirano,§ Marziyeh Kariminavargani,‡,¶ Seiichi Tada,⊥ Guanxin Zhang,∥ Takanori Uzawa,‡,⊥ Deqing Zhang,∥ Takuji Hirose,¶ Makoto Taiji,§ and Yoshihiro Ito*,‡,⊥ †

High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031, P. R. China Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan § Laboratory for Computational Molecular Design, Computational Biology Research Core, RIKEN Quantitative Biology Center, 2F, QBiC Building B, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan ¶ Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan ∥ Key Laboratory of Organic Solids, Beijing National Laboratory of Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ⊥ Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ‡

S Supporting Information *

ABSTRACT: To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.

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Solvatochromic amino acids whose side chains are environment-sensitive fluorophores have been replaced with conserved hydrophobic aromatic amino acids of natural ligands,11 or, alternatively, an environment-sensitive fluorophore is conjugated to a small molecule ligand that targets a specific protein.12 These strategies have been applied for the detection of 14-33,13 calmodulin,14,15 class II MHC proteins,16 trypsin,12 and PDZ domains.17

etection systems using antibodies are very important for various analytical fields, because the antibody can be prepared to bind any antigen target.1−3 However, antibodies only have binding affinity to the targets, and thus the antibody must be labeled and requires bound/free (B/F) isolation for analytical applications. To avoid B/F isolation, several elegant strategies have been reported to develop turn-on fluorescent probes that can both bind and signal to targets. Environmentsensitive fluorophores have been widely used for signaling the binding of a target molecule.4−6 Various environment-sensitive solvatochromic fluorophores have been prepared and conjugated at a particular position of the ligand for detecting biomolecular interactions. 7−10 © XXXX American Chemical Society

Received: March 15, 2016 Accepted: July 12, 2016

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DOI: 10.1021/acs.analchem.6b01032 Anal. Chem. XXXX, XXX, XXX−XXX

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10.0 mL), then 5 M aqueous pyridine-HCl pH 5.0 (10.0 mL) was added, and the solution was stirred at 37 °C overnight. The solvent was evaporated under reduced pressure, and the crude residue was purified by flash chromatography on silica gel (CH2Cl2:CH3OH, 100:1, v/v) to obtain Dansylaa (3 in Scheme S1) as a pale green solid and 4-DMNaa (6 in Scheme S2) as a yellow solid. The final compounds were confirmed using NMR and mass spectrum analysis. Dansylaa: 1H NMR (400 MHz, hexadeuterodimethyl sulfoxide (DMSO-d6)) δ (ppm): 12.72 (s, 1H), 9.74 (s, 1H), 8.44 (d, 1H, J = 8.0 Hz), 8.30 (d, 1H, J = 8.4 Hz), 8.09 (d, 1H, J = 7.2 Hz, 8.90−8.86 (m, 3H), 7.71 (d, 1H, J = 8.0 Hz), 7.66− 7.56 (m, 4H), 7.48 (d, 2H, J = 8.4 Hz), 7.39 (dd, 2 H, J1 = 7.6 Hz, J2 = 14.4 Hz), 7.33−7.23 (m, 3H), 7.17 (d, 2H, J = 8.4 Hz), 4.21−4.09 (m, 4H), 3.02 (dd, 1H, J1 = 4.4 Hz, J2 = 13.6 Hz), 2.81 (s, 6H), 2.83−2.75 (m, 2H), 2.13 (t, 2H, J = 7.6 Hz), 1.41−1.28 (m, 4H), and 1.19−1.12 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.27, 170.77, 155.80, 151.20, 143.64, 143.62, 140.55, 137.62, 136.04, 132.25, 129.20, 129.16, 128.97, 128.93, 128.09, 127.65, 127.50, 126.94, 125.18, 125.10, 123.47, 119.97, 119.04, 118.71, 114.97, 65.49, 55.50, 46.23, 44.94, 42.17, 36.08, 35.801, 28.86, 25.55, and 24.52. ESI-QTOF MS (QSTAR ELITE, AB Sciex) for the Fmocdeprotected Dansylaa: theoretical mass for [M + Na]+ 549.2142, observed 549.2139 (on average 10 times measurements). 4-DMNaa: 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.77 (s, 1H), 8.50 (d, 1H, J = 8.8 Hz), 8.45 (d, 1H, J = 7.2 Hz), 7.56 (d, 1H, J = 7.2 Hz), 7.75 (t, 1H, J = 7.6 Hz), 7.68−7.61 (m, 3H), 7.48 (d, 2H, J = 8.8 Hz), 7.40 (apparent t, 2H, J = 7.2 Hz), 7.33−7.26 (m, 2H), 7.20 (d, 1H, J = 8.0 Hz), 7.16 (d, 2H, J = 8.4 Hz), 4.22−4.11 (m, 4H), 4.03 (t, 2H, J = 7.2 Hz), 3.09 (s, 6H), 3.02 (dd, J1 = 4.4 Hz, J2 = 14.0 Hz), 2.82 (apparent dd, 1H, J1 = 10.8 Hz, J2 = 13.6 Hz), 2.91 (t, 2H, J = 7.2 Hz), 1.69− 1.60 (m, 4H) and 1.40−1.33 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.27, 170.87, 163.52, 162.87, 156.45, 155.80, 143.65, 143.62, 140.55, 137.62, 132.25, 132.18, 131.41, 130.47, 129.49, 129.16, 127.49, 126.95, 125.19, 125.10, 124.92, 124.12, 122.22, 119.97, 118.75, 113.22, 112.90, 65.50, 55.49, 46.34, 44.27, 36.12, 35.79, 27.37, 26.11, and 24.81. ESI-Q-TOF MS for the Fmoc-deprotected 4-DMNaa: theoretical mass calculated for [M + Na]+ 539.2265 and observed 539.2263. Synthesis of TPE-Acetic Acid and TPE-Hexanoic Acid. The TPE-acetic acid (TPE2) was prepared as reported previously.35 The synthetic scheme of TPE-hexanoic acid (TPE6) is shown in Scheme S3. To a solution of 4-(1,2,2triphenylvinyl)phenol (7 in Scheme S3) (1741 mg, 5.0 mmol, 5.0 equiv) and ethyl 6-bromohexanoate (222 mg, 1.0 mmol, 1.0 equiv) in acetonitrile (30.0 mL), NaHCO3 (1160 mg, 10.0 mmol, 10.0 equiv.) was added, and the resultant mixture was heated to reflux for 5 h. The NaHCO3 was filtered, the solvent was evaporated, and then the crude material was used for the next step without purification. The cured product was hydrolyzed in 1 N NaOH aqueous solution overnight. The solution was acidified with 6 N HCl to pH 2.0 and extracted with ethyl acetate (EA) (50 mL × 3). The organic phase was washed with water and brine and dried with anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography on silica gel (CH2Cl2:CH3OH, 100:1, v/v) to afford TPE-hexanoic acid (8 in Scheme S3) (385 mg, 0.83 mmol, yield 83%) as a white solid.

On the other hand, Tang et al. found the aggregationinduced emission (AIE) phenomenon: some nonemissive dyes can be induced to emit efficiently by the formation of aggregates.18 By using this phenomenon, biosensing probes have been fabricated for detecting various species in aqueous solutions, including DNA, proteins, and carbohydrates.19−22 Although the two strategies are promising, both require information about the ligand-protein interaction. Generally, it is difficult to find such fluorogenic probes that interact with arbitrary targets. Therefore, we have developed in vitro selection of fluorogenic peptides for arbitrary targets.23,24 The method was developed by combination of the in vitro translation system and synthesis of misacylated tRNA.25−28 We have introduced the environment-sensitive fluorophore, 7-nitro-2,1,3-benzoxadiazole (NBD),29 into a randomized peptide library and selected peptides that bind and emit fluorescence in response to conformational changes of calmodulin.23 One of the selected peptides, C5 (YWDKIKDBIGG-amide, B: NBD-modified 4amino-L-phenylalanine (NBDaa; Figure 1)) was soluble in aqueous buffer and bound specifically to Ca2+-loaded calmodulin (Ca2+/CaM) with modest signal enhancement.

Figure 1. Chemical structures of fluorescent compounds used in this study.

The environment-sensitive fluorophore of a probe was crucial for giving high fluorescence enhancement upon binding to a target protein. Here, to develop probes with improved signaling properties, we have synthesized new fluorogenic peptides by replacing NBD of C5 with three environmentsensitive dyes (Figure 1): Dansyl is a commercially available environment-sensitive fluorophore.30 4-N,N-Dimethylamino1,8-naphthalimide (4-DMN) is an excellent fluorogenic dye that exhibits higher fluorescence quantum yields in nonpolar solvents than in polar protic solvent.14,31 Tetraphenylethene (TPE)19−22 is a well-known AIE fluorogen. Here we found that the 4-DMN-incorporated peptide exhibits a fluorescent enhancement of over 100-fold in the presence of the target.

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EXPERIMENTAL SECTION Synthesis of Dansyl- and 4-DMN Modified 4-Amino-LPhenylalanine. Dansyl- and 4-DMN-modified 4-amino-Lphenylalanine (Dansylaa and 4-DMNaa, respectively) (Figure 1) were synthesized as shown in Schemes S1 and S2 and as reported previously.32−34 Dansylhexanoic acid (1 in Scheme S1)32 or 4-DMN-hexanoic acid (4 in Scheme S2)33 was activated using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The resulting activated esters (2 in Scheme S1 or 5 in Scheme S2) (3.0 mmol, 3.0 equiv) and Fmoc-4-amino-L-phenylalanine (1.0 mmol, 1.0 equiv) were dissolved in dimethylformamide (DMF; B

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Analytical Chemistry H NMR (400 MHz, DMSO-d6) δ (ppm): 7.17−7.07 (m, 9H), 6.99−6.93 (m, 6H), 6.84 (d, 2H, J = 8.8 Hz), 6.67 (d, 2H, J = 8.8 Hz), 3.85 (t, 2H, J = 6.4 Hz), 2.21 (t, 2h, J = 7.6 Hz), 1.69−1.62 (m, 2H), 1.57−1.49 (m, 2H), and 1.41−1.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.46, 157.18, 143.52, 143.47, 140.21, 139.60, 135.20, 132.06, 131.74, 130.82, 130.54, 127.93, 127.70, 127.58, 126.63, 126.57, 126.52, 126.27, 126.20, 126.16, 113.87, 113.48, 67.12, 33.62, 28.45, 25.16, and 24.28. ESI-Q-TOF MS for the Fmoc-deprotected TPE6: theoretical mass for [M + Na]+ 582.2251, observed 582.2249. Synthesis of Fluorenylmethyloxycarbonyl Polyoxyethylene Terminated with Carboxylic Acid. The synthetic scheme of fluorenylmethyloxycarbonyl polyoxyethylene terminated with carboxylic acid (Fmoc-PEG-COOH) is shown in Scheme S4. Tertiary butyloxycarbonyl polyoxyethylene terminated with carboxylic acid (9 in Scheme S4) (1.86 g, 4.3 mmol) was deprotected using 50% trifluoroacetic acid (TFA) in dichloromethane for 2 h. The solvent was evaporated under reduced pressure, and the crude material was placed under high vacuum overnight. The crude material was redissolved in NaHCO3 solution, and the pH was tested to ensure the solution was basic. A solution of N-(9-fluorenylmethoxycarbonyloxy) succinimide (1.74 g, 5.15 mmol) in dioxane (30 mL) was then added, and the resultant solution was stirred at room temperature for 2 h before concentrating to remove dioxane. The residue was redissolved in H2O (100 mL) and washed with diethyl ether to remove the excess Fmoc-OSu. The aqueous layer was acidified with 6 N HCl and then extracted with dichloromethane (3 × 100 mL). The combined organic layer was dried with anhydrous Na2SO4. The solvent was evaporated, and the product was purified by flash chromatography on silica gel (CH2Cl2:CH3OH, 50:1, v/v) to afford Fmoc-PEG-COOH (10 in Scheme S4) (1.93 g, yield 81%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 7.88 (d, 2H, J = 8.0 Hz), 7.54 (t, 1H, J = 6.4 Hz), 7.67 (d, 2H, J = 8.0 Hz), 7.4 (d, 2H, J = 7.6 Hz), 7.32 (t, 2H, J = 7.6 Hz), 7.24 (t, 1H, J = 6.4 Hz), 4.28 (d, 2H, J = 6.8 Hz), 4.19 (t, 1H, J = 6.8 Hz), 3.49− 3.45 (m, 12H), 3.08−3.00 (m, 4H), 2.17 (t, 2H, J = 7.2 Hz), 2.08−2.04 (m, 2H), and 1.72−1.57 (m, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm): 175.70, 172.52, 156.64, 143.97, 141.29, 129.00, 128.20, 127.64, 127.00, 125.03, 119.94, 70.78, 70.43, 70.30, 70.05, 69.98, 69.24, 66.45, 47.28, 38.84, 38.31, 35.22, 32.94, 29.32, 28.64, and 20.92. ESI-Q-TOF MS for Fmoc-PEG-COOH: theoretical mass for [M + Na]+ 579.2677, observed 579.2675. Synthesis of Fluorophore-Modified C5 Peptides. Dansyl-C5 and 4-DMN-C5 peptides were synthesized using standard Fmoc-SPPS at the RIKEN Brain Science Institute. The synthesized peptides were confirmed by MALDI-TOF mass analysis. TPE2-C5, TPE2-C5-PGE2, and TPE6-C5-PEG2 were synthesized by standard Fmoc-solid phase peptide synthesis on a Discover microwave peptide synthesizer (CEM) and postmodification, according to Scheme S5. Synthesis of peptides Ac-YWDKIKDXIGG-amide (X: 4amino-L-phenylalanine) and Ac-YWDKIKDXIGG-PEG-PEGamide were performed at a 50 μmol scale using 190 mg of Rink amide MBHA resin. The N-termini of the peptides were acetylated using acetic anhydride. The peptides were cleaved using 5.0 mL of 95% TFA, 2.5% thioanisole, and 2.5% H2O at 38 °C for 30 min. The cleavage solutions were dropped into cold diethyl ether. The resulting precipitates were collected by centrifugation, washed with cold diethyl ether (3 × 20 mL), 1

and dried under vacuum overnight. The peptides were dissolved in DMF (5 mL), TPE-OSu was added to the DMF solution, and this was followed by the addition of 5 M aqueous pyridine-HCl pH 5.0 (5 mL). The resulting solutions were stirred at 37 °C overnight. The peptides were purified using high-performance liquid chromatography at the RIKEN Brain Science Institute. The couplings between 4-amino-L-phenylalanine and TPE2-Osu or TPE6-OSu were confirmed using MALDI-MS/MS analysis (Figures S1−S3 and Tables S2−S7). Surface Plasmon Resonance (SPR) Analysis. SPR measurements were performed on a Biacore T100 instrument with a CM5 sensor chip (Biacore, Uppsala, Sweden). All experiments were performed at 25 °C with a constant flow rate of 30 μL/min. Calmodulin (CaM) (100 μg/mL) in 10 mM sodium acetate (pH 3.7) was immobilized on the chip surface using a standard amine coupling method (contact time 30 min). To determine the binding affinities of the synthesized peptides to immobilized CaM, 39, 78, 156, 312, 625, 1250, 2500, 5000, and 7500 nM solutions of different fluorophore modified C5 peptides in a running buffer (50 mM Tris-HOAc pH 7.5, 150 mM KCl, 5 mM CaCl2, 5% DMSO, 0.05% surfactant P20) were injected over the sensor chip surface for 3 min. Three hundred microliters of running buffer was then injected over the surface of the sensor chip to dissociate the peptides from the surface-bound CaM. The sensor chip was regenerated by injection of 15 μL of 50 mM ethylene glycol tetraacetic acid (pH 8.0) for 30 s, followed by flushing with the running buffer for 5 min to stabilize the baseline. Solvent correction (eight samples) was performed after every 10 cycles. The sensorgrams were analyzed using the Biacore T100 Evaluation Software (version 2.0.3, Biacore). A curve derived from a reference flow cell was subtracted (baseline) from the binding curves, and the data were fitted using the steady-state affinity model. Fluorescence Measurements. The fluorescence measurements were performed on a FP-6500 spectrofluorometer (Jasco, Hachioji, Japan) at 20 °C. The samples were each excited at the wavelength appropriate for the fluorescent amino acids, peptides, and fluorophores (NBDaa and NBD-C5:488 nm; 4-DMNaa and 4-DMN-C5:430 nm; Dansylaa and DansylC5:330 nm; TPE2-C5, TPE2-C5-PEG2, and TPE6-C5PEG2:330 nm). The ex/em slit-widths were set to 5/10 nm for NBDaa, NBD-C5, Dansylaa, and Dansyl-C5 and 10/10 nm for 4-DMNaa, 4-DMN-C5, TPE2-C5, TPE2-C5-PEG2, and TPE6-C5-PEG2. The data points were collected at 1 nm increments with a 0.5 s integration period. To determine whether the samples would dissolve in the buffer, the absorbance of each sample was recorded before and after centrifugation at 12,000 r.p.m for 10 min. The stock solutions of 10 mM NBD-C5 and Dansyl-C5 were prepared in ultrapure water, whereas 10 mM 4-DMN-C5, NBDaa, TPE2-C5, TPE2C5-PEG2, and TPE6-C5-PEG2, as well as 5 mM 4-DMNaa and Dansylaa, were prepared in DMSO. The fluorescence spectra of 200 μL of 5 μM NBDaa, NBD-C5, 4-DMNaa, 4-DMN-C5, Dansylaa, Dansyl-C5, TPE2-C5, TPE2-C5-PEG2, and TPE6C5-PEG2 were obtained in binding buffer (50 mM Tris-HOAc pH 7.5, 50 mM magnesium acetate, 150 mM KCl, 5 mM CaCl2, 1% DMSO) that contains different percentages of organic solvents (Table S1). We met failure to use over 60% dioxane and 40% acetonitrile in the binding buffer due to the poor solubility of tested compounds in the organic solvents. To investigate the fluorogenicity of the synthesized peptides upon binding to the target protein, the fluorescence of 5 μM peptide, C

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Analytical Chemistry 5 μM peptide, and 10 μM target protein (CaM or Toroponin C) in the presence of Ca2+ or Pb2+ were recorded, respectively. Fluorescence intensity changes (FIC) were calculated by FIC = (Fx−F0)/F0, where Fx and F0 are the maximum fluorescence intensity of the peptide with and without CaM, respectively. Cell Imaging. We performed a cell staining experiment with the 4-DMN-C5 peptide after ionomycin treatment to increase Ca2+ concentration inside culture cells. Cells were fixed with paraformaldehyde, treated with triton X-100 for enhanced permeability and blocking reagent for decreasing nonspecific binding, and finally stained with the 4-DMN-C5 peptide solution. We quantified the fluorescence intensity using ImageJ software.

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RESULTS AND DISCUSSION Peptide Synthesis. NBD-C5, 4-DMN-C5, and Dansyl-C5 were synthesized successfully by a conventional solid phase method, and the synthesized peptides were soluble in aqueous buffers and dioxane. Since the double bond of TPE is degraded during the solid phase process, we modified TPE to the 4amino-L-phenylalanine in the Ac-YWDKIKDXIGG-amide (X: 4-amino-L-phenylalanine). More precisely we deprotonated the 4-amino group of 4-amino-L-phenylalanine with holding the protonation on the amino of the Lys side chain in the C5 peptide by using 5 M aqueous pyridine-HCl pH 5.0 buffer.36 Then, we coupled the TPE-acetic acid or TPE-hexanoic acid to the 4-amino-L-phenylalanine. Although TPE2-C5 was soluble in aqueous buffers, TPE6-C5 was not soluble in aqueous solutions. To improve the solubility of TPE6-C5, PEG linkers were added to the C-terminus of the peptide according to a previous report.37 One and two PEG-COOHs were added to the TPE6-C5 peptide. Because the addition of two PEGs was found to assist the solubility of TPE6-C5 in aqueous buffers, we used the two PEGs-incorporated TPE6-C5 (TPE6-C5-PEG2) for further experiments. In order to examine the PEG effects on the fluorogenic property, we also synthesized TPE2-C5-PEG2 based on the same protocol as TPE6-C5-PEG2. Fluorescence Properties of Synthesized Chemicals. NBD, Dansyl, and 4-DMN are known to emit higher fluorescence in hydrophobic solvents than in aqueous buffers and exhibit blue shifts of the emission peak upon changing the solvent from a polar to nonpolar solvent.14 To confirm that the fluorogenic properties of the amino acids (NBDaa, 4-DMNaa, and Dansylaa) were held in the synthesized peptides (NBD-C5, 4-DMN-C5, and Dansyl-C5), we measured the emission spectra in various concentrations of dioxane (Figure 2) and acetonitrile (Figure S4) in the binding buffer. Although the fluorescence intensity of NBDaa, 4-DMNaa, and Dansylaa were slightly stronger in dioxane than in acetonitrile (Figure S5), we observed similar signal increases at higher ratio of the organic solvents and slight blue-shift of the emission peak (Table S1). Similar tendencies were observed for the corresponding peptides also (Figure 2 and Figure S4), implying that the fluorogenic properties of the amino acids were held even in the C5 peptides. In sharp contrast with these three fluorescent probes, the emission spectra of TPE2 and TPE6 did not effectively depend on the concentration of dioxane (Figures S5D and S6), which supports that emission spectra of an AIE type fluorophore is not affected by solvent polarity but affected by aggregation. Since TPE-coupled C5 peptides were not dissolved in dioxane and acetonitrile, the solvent-dependent fluorescence difference could not be compared.

Figure 2. Fluorescence spectra of the modified amino acids and peptides in the binding buffer with various concentrations of dioxane. A) Dansyl modified amino acid (Dansylaa); B) Dansyl modified C5 peptide (Dansyl-C5); C) 4-DMN modified amino acid (4-DMNaa); D) 4-DMN modified C5 peptide (4-DMN-C5); E) NBD modified amino acid (NBDaa); F) NBD modified C5 peptide (NBD-C5). All spectra were recorded at 20 °C and corrected for background.

Binding with Calmodulin. To investigate if the replacement of fluorogenic dyes affects binding affinity, we have determined the Kd (equilibrium dissociation constant) values between the synthetic peptides and Ca2+ loaded CaM (Ca2+/ CaM) complex using SPR (Figure 3). The Kd values were determined to be 1.2, 0.7, 4.5, and 3.5 μM for Dansyl-C5, 4DMN-C5, TPE2-C5, and TPE6-C5-PEG2, respectively (Table 1). The Kd between the NBD-C5 and Ca2+/CaM complex was previously determined to be ∼1 μM.23 The results indicate that the replacement of NBD in C5 with Dansyl, 4-DMN, or TPE did not significantly affect the binding affinity. In addition, the PEG linker and different spacer lengths in TPE2 and TPE6 did not affect the binding of the peptides to the target protein. Fluorogenicity by Interaction with Calmodulin. To test the fluorogenicity of our peptides upon binding to the target protein, we examined the fluorescence intensity changes of the synthesized peptides by mixing with Ca2+/CaM. NBD-C5 and Dansyl-C5 showed 8.6- and 5.4-fold fluorescence enhancements (Figure 4A and B) upon binding to the target Ca2+/ CaM. The fluorescence of 4-DMN-C5 was extremely weak in aqueous buffer, but this was dramatically enhanced (by 103fold) upon addition of Ca2+/CaM (Figure 4C). These fluorogenic tendencies were held for Pb2+ bound CaM (Pb2+/ CaM, Figure S7) and another Ca2+-binding protein, troponin C (Ca2+/TnC, Figure S8). All three peptides exhibited less signal increases upon the addition of Pb2+/CaM or Ca2+/TnC than Ca2+/CaM. Thus, we concluded that the 4-DMN-C5 is the greatest fluorogenic peptide for Ca2+/CaM. In contrast to NBD-C5 and Danysl-C5, 4-DMN-C5 exhibited a different behavior in 60% dioxane buffer when compared with that of the binding to Ca2+/CaM (Figure 5). D

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Figure 4. Fluorogenicity has observed for all C5 peptides. A) NBDC5; B) Dansyl-C5; C) 4-DMN-C5; D) TPE2-C5; E) TPE2-C5-PEG2; F)TPE6-C5-PEG2. Three separated conditions were examined for each peptide: green line; 5 μM peptide, red line; 5 μM peptide + 10 μM CaM, and blue line; 5 μM peptide + 10 μM CaM + 5 mM Ca2+ in a Tris buffer (50 mM Tris-HOAc pH 7.5, 50 mM magnesium acetate, 150 mM KCl, 1% DMSO).

Figure 3. Interaction of synthetic peptides with immobilized CaM. A, C, E, and G represent SPR sensorgrams using different concentrations of Dansyl-C5, 4-DMN-C5, TPE2-C5, and TPE6-C5-PEG2 against immobilized CaM, respectively. B, D, F, and H represent plots of response units against the concentration of Dansyl-C5, 4-DMN-C5, TPE2-C5, and TPE6-C5-PEG2 with immobilized CaM. The Kd values were determined using a steady-state affinity model.

Figure 5. Normalized fluorescence intensity of 10 μM Dansyl-C5 (red circle), 4-DMN-C5 (blue triangle), and NBD-C5 (black square) in binding buffer containing different dioxane percentages (solid line), titrated with CaM with 5 mM Ca2+(dash line). The normalized fluorescence intensity represents the maximum fluorescence intensity of each measurement divided by the fluorescent intensity of the corresponding peptide in aqueous buffer.

Table 1. Fluorescence Intensity Change (FIC) and Dissociation Constant for Synthetic Peptides in the Presence of Ca2+/CaM peptide

blue shift (nm)

FIC

Kd (μM)

NBD-C5 Dansyl-C5 4-DMN-C5 TPE2-C5 TPE6-C5-PEG2

9 20 23 no shift no shift

8.3 5.4 103.4 −7.0 5.1

0.85 1.2 0.7 4.5 3.5

corresponding fluorescence amino acids (Table S1), when we changed the solvent from the binding buffer to 60% dioxane. These results indicate that the blue-shifted emission peak and the fluorescence enhancement of NBD-C5, Dansyl-C5, and 4DMN-C5 upon binding with CaM are due to environment changes nearby the fluorophore. In order to examine the selectivity of the best fluorogenic peptide, we performed a cell staining experiment using 4DMN-C5 based on an immunostaining-like method. Cells which were treated with ionomycin to increase Ca 2+ concentration exhibited higher fluorescence intensity than nontreated cells (Figure S9). This observation indicates the selectivity was held even after the replacement of the fluorogenic dye from NBD to DMN. In this study we have found that the fluorogenicity can be tuned by the length of the spacer chain between the peptide and the fluorescent probe. The TPE modified C5 peptide with a short acetic acid linker (TPE2-C5) was compared with the longer hexanoic acid linker (TPE6-C5-PEG2). TPE6-C5-PEG2 emitted a very weak fluorescence signal, and the signal was enhanced ∼5-fold after mixing with Ca2+/CaM (Figure 4F). In contrast, TPE2-C5 showed a 7-fold decrease in the fluorescence

For NBD-C5 and Dansyl-C5, the fluorogenicities in dioxane are higher than that observed for binding CaM (Figure 5). In contrast, the fluorogenicity of 4-DMN-C5 in dioxane is lower than that observed when this peptide binds CaM (Figure 5). Usually the fluorogenicity in dioxane is higher than that observed when the ligand binds CaM. In the present case, 4DMN-C5 is more sensitive to an environment change upon binding CaM. These results demonstrate that the fluorogenicity can be tuned by the type of fluorophore used. The emission peak positions of NBD-C5, Dansyl-C5, and 4DMN-C5 were blue-shifted by ∼9, 20, and 23 nm in aqueous buffer after mixing with Ca2+/CaM (Table 1). The similar blue shifts were observed not only for the peptides but also the E

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Analytical Chemistry intensity after mixing with Ca2+/CaM (Figure 4D). Here we note that the addition of the PEG2 at the C-terminal of TPE2C5 did not effectively effect the fluorogenicity (6.0-fold decrease, Figure 4E). To investigate the remarkable difference in the fluorescence change due to the length of the TPE linkers, we explored the complex structure by the replacement of NBD with TPE6 or TPE2 in the reported complex of Ca2+/CaM and NBD-C523 (Figure 6). We found a remarkable difference in the

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CaM and Pb2+/CaM; fluorescence spectra of fluorophores modified peptides interact with troponin C; cell staining experiment using 4-DMN-C5; observed b and y ions of the three TPE peptides; emission peak shifts of fluorophore modified amino acids and peptide in dioxane (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-48-467-5809. Fax: +81-48-467-9300. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by JSPS KEKENHI (22220009 and 15H01810).

Figure 6. Molecular interaction images of binding modes of TPE6-C5PEG2 (A) and TPE2-C5 (B) peptides. The protein and atoms in TPE(s) are drawn as molecule surface and space filled models, respectively.

environment of the phenyl groups between the TPE6-C5PEG2 and TPE2-C5; the phenyl groups of TPE6-C5-PEG2 faced the molecular surface of Ca2+/CaM, whereas the phenyl groups of TPE2 did not effectively interact with Ca2+/CaM. This observation implies that the longer linker presumably allows TPE to interact with CaM. The interaction would restrict the rotation of phenyl groups, which would result in emission of fluorescence. On the other hand, the less interaction between TPE2 and Ca2+/CaM would allow the phenyl groups to freely rotate, which would result in less fluorescence even in the complex form. This result implies that the replacement of the fluorogenic group is not sufficient to obtain high fluorogenicity and that the precise location of the fluorophore is crucial in defining the fluorescence enhancement upon target protein binding.

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CONCLUSIONS An in vitro selected fluorogenic peptide can be further tuned by the choice of fluorophore. These types of fluorogenic peptides should be useful in developing probes that report target proteins in bioimaging studies.

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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.analchem.6b01032. Synthetic schemes of fluorophore modified amino acids, Fmoc-PEG-COOH and TPE modified peptides; MS/MS spectra of TPE2-C5, TPE2-C5-PEG2, and TPE6-C5PEG2; fluorescence spectra of modified amino acids and peptides in binding buffer with various concentrations of acetonitrile; fluorescence spectra of TPE2 and TPE6 amino acids in 50% and 60% dioxane; fluorescence spectra of fluorophores modified peptides interact with F

DOI: 10.1021/acs.analchem.6b01032 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.6b01032 Anal. Chem. XXXX, XXX, XXX−XXX