Mitochondria-targeting peptoids

Jiwon Seo, Ph.D. Email: [email protected]. Keywords. mitochondria; amphipathic peptoid; mitochondrial delivery; cell-penetrating peptoid. Page 1 of 26...
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Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Mitochondria-Targeting Peptoids Ho Yeon Nam,†,# Jong-Ah Hong,§,# Jieun Choi,† Seungheon Shin,‡ Steve K. Cho,∥,‡ Jiwon Seo,*,† and Jiyoun Lee*,§ †

Department of Chemistry, School of Physics and Chemistry, ‡School of Life Sciences, and ∥Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology, Buk-gu, Gwangju 61005, Republic of Korea § Department of Global Medical Science, Sungshin University, Kangbuk-gu, Seoul 01133, Republic of Korea S Supporting Information *

ABSTRACT: Mitochondria-specific delivery methods offer a valuable tool for studying mitochondria-related diseases and provide breakthroughs in therapeutic development. Although several small-molecule and peptide-based transporters have been developed, peptoids, proteolysis-resistant peptidomimetics, are a promising alternative to current approaches. We designed a series of amphipathic peptoids and evaluated their cellular uptake and mitochondrial localization. Two peptoids with cyclohexyl residues demonstrated highly efficient cell penetration and mitochondrial localization without significant adverse effects on the cells and mitochondria. These mitochondria-targeting peptoids could facilitate the selective and robust targeted delivery of bioactive compounds, such as drugs, antioxidants, and photosensitizers, with minimal off-target effects.

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INTRODUCTION Mitochondria are crucial in cellular physiology, regulating ATP production, calcium homeostasis, and signal transduction. Mitochondria are probably the most studied subcellular organelle, due to their multifaceted functions associated with various diseases,1 such as cancer,2−4 diabetes,5 and neurodegenerative diseases.6 However, interrogation of mitochondria-specific proteins and pathways is a challenging task, due to the presence of the two membrane layers that are maintained by an electrochemical potential, and which surround a matrix that encloses the mitochondrial DNA. The study of mitochondria depends mainly on subcellular fractionation and fluorescent labeling, using fluorescent proteins and mitochondria-staining dyes. While these methods can specifically isolate or label mitochondria in vitro, given the importance of mitochondria as a disease marker, targeted delivery methods are more desirable for practical applications. Lipophilic carbocations such as triphenylphosphonium (TPP, Figure S1, Supporting Information) ions have been widely used for mitochondria-specific delivery of small molecules.7 Because of the large mitochondrial membrane potential, TPP rapidly accumulates inside the mitochondria and successfully delivers various small-molecule drugs and nanoparticles in vitro and in vivo.8,9 Cationic peptides such as Szeto−Schiller (SS) peptide antioxidants10−12 and mitochondria-penetrating peptides (MPPs)13,14 have also been demonstrated to be useful for mitochondrial transport of anticancer agents and antioxidants (Figure S1). Notably, Kelley and coworkers have developed cationic amphipathic peptides consisting of unnatural amino acids, cyclohexylalanine, and Darginine.14−16 These MPPs have proven to be useful in studies © XXXX American Chemical Society

of mitochondrial pathways and drug-resistance mechanisms because of their synthetic versatility and biocompatibility. However, these cationic peptides are prone to proteolytic degradation, which limits their potential applications for in vivo delivery. Peptoids are proteolysis-resistant peptidomimetics containing poly-N-substituted glycines. Peptoids have similar physicochemical properties compared to peptides and can be readily synthesized by solid-phase synthesis techniques.17 Polyarginine and polylysine analogs of peptoids18−20 and peptide-peptoid hybrids21,22 have been developed as an alternative approach to cell-penetrating peptides (CPPs). Several recent studies also found that amphipathic peptoids containing hydrophobic and cationic residues are highly membrane permeable without causing severe toxicity.23,24 Based on these findings, we speculated that amphipathic peptoids could function as mitochondrial transporters, and set out to design potential candidates. In this work, we evaluated cellular uptake and mitochondrial localization of our peptoid transporters, and analyzed their conformation. In addition, we examined effects of the synthesized peptoids on mitochondrial metabolism and membrane potential as well as cytotoxicity to validate their potential utility as a delivery vehicle.

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RESULTS AND DISCUSSION Design and Synthesis of Mitochondria-Targeting Peptoids. To construct the cationic amphipathic peptoids, Received: February 28, 2018 Revised: March 21, 2018 Published: March 26, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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the mitochondrial toxicity and cell viability, we synthesized the peptoids without CF (11−15). As shown in Table 1, ten CF-labeled (1−10) and five unlabeled (11−15) compounds were synthesized. Both peptoid and peptide sequences were synthesized manually according to the solid-phase submonomer protocol17 and standard Fmoc/ tBu method,29 respectively (Scheme 1). CF was conjugated at the N-terminus of the sequences by activating the carboxylic acid of 5(6)-carboxyfluorescein with DIC/HOBt. The phenolic hydroxyl group of fluorescein can be acylated during the conjugation reaction and forms phenolic ester side products. Therefore, piperidine was treated to remove the ester-bound CF.30 After the removal of the Mmt protecting group, 1Hpyrazole-1-carboxamidine was added for guanidylation of the primary amines. For unlabeled peptoids, the N-terminal secondary amine was protected using a Boc group followed by Mmt deprotection and guanidylation. Initially, Fmoc protection was attempted using Fmoc-Cl or Fmoc-OSu, but Boc protection always showed superior conversion compared to Fmoc protection. After deprotection and resin cleavage, the purity of peptoids was determined by analytical high performance liquid chromatography (HPLC; >98%) and characterized by electrospray ionization mass spectrometry (ESI-MS; Supporting Information). Cellular Uptake and Mitochondrial Localization. To evaluate the cellular uptake of the synthesized compounds, we first determined the mean fluorescence intensity of HeLa cells treated with the CF-labeled compounds by flow cytometry (Figure 1). All the newly designed compounds (1−5) showed

we incorporated one lipophilic residue (marked in bold, Table 1) per two cationic residues, Nlys and Nbtg, to generate a Table 1. Mitochondria-Targeting Peptoidsa

a Nch (N-(1-cyclohexylmethyl)glycine); Nsch ((S)-N-(1cyclohexylethyl)glycine; Npm (N-(1-phenylmethyl)glycine; Nspe ((S)-N-(1-phenylethyl)glycine; Ndp (N-(2,2-diphenylethyl)glycine); Nlys (N-(4-aminobutyl)glycine); Nbtg (N-(4-butylguanidine)glycine).

group of three monomers, defined as one repeating unit (n = 1). Overall, we designed peptoids to have 12 monomeric residues (n = 4), which is the optimal chain length for efficient cell penetration.23,25 We anticipated that this alternating sequence of the lipophilic and cationic residues would resemble previously identified amphipathic transporters, and potentially serve as a platform to control the molecular secondary structure by placing chiral monomers.26,27 Given that many of the mitochondria-targeting sequences (MTS) naturally found in mitochondrial proteins have amphipathic helical structures,28 we hypothesized that the added helicity may promote mitochondrial penetration. To investigate cellular uptake and mitochondria-specific penetration, we conjugated a carboxyfluorescein (CF) at the N-terminus of each peptoid (1−5) and included three CF-labeled octaarginine and octalysine derivatives (6−8) for comparison. In addition, we synthesized two shorter chain analogs of compound 2 (9−10) to examine the length-dependency of cellular uptake and localization. To assess

Figure 1. Mean fluorescence intensity of HeLa cells treated with compounds 1−8 at 5 μM for 1 h. Data were compared with the CFtreated control by using a one-way analysis of variance (ANOVA) with Dunnett’s t test (*: p < 0.05, **: p < 0.01). (−): untreated cells; CF: fluorescein-treated cells.

approximately 10- to 100-fold greater cellular uptake than fluorescein alone, indicating that the attachment of the peptoids

Scheme 1. Representative Synthetic Scheme of the Mitochondria-Targeting Peptoids

B

DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry promoted cellular penetration. In particular, compound 5, which contains biphenyl residues, showed the greatest cellular uptake, exceeding that of the octaarginine peptide (6), probably because of its high lipophilicity. Compounds 1, 2, and 4 also demonstrated slightly better cellular uptake than the polycationic peptoid controls (7 and 8), suggesting that amphipathicity plays an important role in cell penetration.23 To examine the subcellular localization of the synthesized peptoids, we performed live-cell imaging experiments using confocal microscopy. We acquired the fluorescence images of HeLa cells treated with compounds 1−8 at 5 μM for 1 h, followed by the addition of Mitotracker mitochondrial fluorescence marker (Figure 2 and Figure S2, Supporting

Figure 3. (a) Fluorescence intensity measured by flow cytometry and (b) confocal imaging of HeLa cells treated with each compound at 5 μM for 1 h. Data were compared with the CF-treated control by using a one-way analysis of variance (ANOVA) with Dunnett’s t test (*: p < 0.05, **: p < 0.01).

However, when we examined mitochondrial localization by confocal microscopy (Figure 3b), the nonameric variant 9 displayed nearly the same extent of mitochondrial-specific localization as compound 2, having an r-value of 0.81. The hexameric analog 10 demonstrated a slightly lower degree of mitochondrial localization (r = 0.70) than compound 2. Given that 10 is only half the length of 2, we believe that the repeating Nlys-Nsch-Nbtg sequence conferred this mitochondrial specificity. It should be noted that we conjugated aCF fluorophore to determine the cellular uptake and subcellular localization of each compound. The quantitative assessment of all tested compounds 1−10 indicated that these characteristics are mainly dependent on the sequences and the physicochemical properties of the side chains in each peptoid, and the effect of CF appeared to be marginal. To further confirm our assumption, we synthesized a tetramethylrhodamine (TAMRA)-conjugated version of peptoid 2 (S1, Figure S3, Supporting Information) and performed live cell imaging experiments. The replacement of CF with TAMRA did not significantly affect the subcellular localization, exhibiting mitochondria-specific localization with the r-value of 0.61 (Figure S3). Conformational Analysis. Conformational information on unlabeled peptoids 11−15 was obtained by circular dichroism (CD) spectroscopy (Figure 4). The peptoids constructed with achiral monomers (11, 13, and 15) did not show a distinctive CD signal and are nonhelical. Peptoid 14 composed of aromatic helix-inducing residue, Nspe, exhibited a typical signature of a right-handed polyproline type-I (PPI) peptoid helix.32 Peptoid 12 that contained a chiral aliphatic monomer, Nsch, showed minimum absorption at 198 nm and maximum absorption at 222 nm in acetonitrile, which intensified and blue-shifted in water (Figure 4c). The spectral features displayed by 12 showed a similarity to previously reported CD signatures of nonaromatic peptoid helices with greater backbone flexibility.33,34 The CD spectra indicate that the helixlike secondary structure formation of compounds 12 and 14 in organic and aqueous media, supporting our hypothesis that the helical structure of these compounds may promote mitochondrial localization. Mitochondrial Toxicity and Cellular Toxicity. To further assess the utility of mitochondria-penetrating peptoids, we evaluated the mitochondrial toxicity of compounds 11−15 by performing a JC-1 assay. The mitochondrial membrane potential (ΔΨm) can be determined by measuring the red/ green fluorescence intensity ratio of the JC-1 dye,35 which accumulates in healthy mitochondria, forming J-aggregates that have an intense red fluorescence, while emitting low green fluorescence in the cytosol. We measured the red/green ratio of

Figure 2. Live cell images of HeLa cells treated with compounds 1−5 (5 μM) and the Pearson correlation coefficients (r).

Information). We quantified the mitochondrial localization of each compound by calculating the Pearson’s correlation coefficients31 (r) between the green fluorescence signals from each compound and the red fluorescence signals from Mitotracker. As demonstrated in Figure 2, compounds with cyclohexyl residues (1 and 2) exhibited strong green fluorescence that overlapped with the red fluorescence signals from Mitotracker. Their r-values were significantly high (0.69 for 1, 0.81 for 2), indicating that these compounds are selectively transported to the mitochondria. In contrast, compounds with benzyl residues (3 and 4) showed punctate staining patterns with relatively low r-values (0.16 for 3 and 0.28 for 4). The diphenyl-group-containing peptoid 5 showed green fluorescence signals evenly distributed inside cells due to its high cell permeability; however, these signals appeared to only partially overlap with the mitochondria (r = 0.37). Among the three compounds in the control group (6−8), polyarginine (6) and polyarginine-mimicking peptoid (7) also displayed punctate fluorescence signals with low r-values (r = 0.26 for 6 and 0.19 for 7; Figure S2, Supporting Information). The octalysine-mimicking peptoid 8 exhibited low green fluorescence, likely due to a low membrane permeability. Interestingly, based on the r-values, compounds with chiral submonomers, such as Nsch (2), Nspe (4), and Arg (6), demonstrated slightly better mitochondrial colocalization compared to their achiral counterparts. As discussed subsequently, this may be ascribed to the formation of amphipathic secondary structures. To examine the length-dependent cellular uptake and localization, we additionally tested compounds 9 and 10, which are nonameric and hexameric variants of compound 2. As depicted in Figure 3a, the flow cytometry data indicated that the cellular uptake is proportional to the length of the peptoid. C

DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. Circular dichroism (CD) spectra of 11−15 recorded (a) in acetonitrile and (b) in H2O (50 μM, 20 °C), and (c) comparison between 12 and 14.

HeLa cells treated with each peptoid at 10 μM for 1 h and calculated the percentage of ΔΨm by normalizing the ratio to that of the untreated cells. As summarized in Table 2, Table 2. Percentages of the Mitochondrial Membrane Potential (ΔΨm) and Cytotoxicity peptoid

ΔΨm (%)a

LC50 (μM)b

11 12 13 14 15

87 84 112 103 45

>100 >100 >100 >100 15.6

a

The values are reported as the average values of a duplicate. bThe MTS assay was performed by treating MRC-5 cells with each compound for 72 h.

Figure 5. Effects of compound 12 on the mitochondrial oxygen consumption rate (OCR). U87 cells seeded in an XF24 cell plate 1 day prior to the measurements were equilibrated for 1 h in the presence of varying doses of 12 dissolved in assay buffer. OCR measurements were made during baseline and after injection of oligomycin (2 μM), FCCP (0.5 μM), and rotenone/antimycin A (0.5 μM) in that order. Data are shown as mean ± SEM, n = 4.

compounds with cyclohexyl residues (11 and 12) induced a slight depolarization, decreasing the percentage of ΔΨm to 87% and 84% of normal ΔΨm, respectively, likely due to their mitochondrial localization. Compounds with phenyl side chains (13 and 14) have virtually no effect on ΔΨm (ΔΨm > 100%). The diphenyl containing compound 15 significantly impaired ΔΨm (45% of normal ΔΨm), probably because the compound is the most hydrophobic and highly membrane-permeable, thus affecting cell viability. Next, we performed an MTS assay in MRC-5 human lung fibroblasts to determine the cytotoxicity of each peptoid. Compounds 11−14 did not exhibit noticeable cytotoxicity at low micromolar concentrations (LC50 > 100 μM), while compound 15 appeared to be cytotoxic with an LC50 value of 15.6 μM. Finally, we decided to examine the effects of compound 12 on mitochondrial function, because it showed the most selective mitochondrial localization with high cellular uptake. We measured the mitochondrial oxygen consumption rate (OCR) of the cells treated with 12 at various concentrations by performing the mitochondrial stress tests. As shown in Figure 5, the cells treated with 12 at 10 μM (red line) maintained normal basal respiration, ATP production, and maximal respiration compared to the untreated control (blue line), indicating that the specific mitochondrial localization of 12 did not adversely affect mitochondrial function. Compound 12 appeared to reduce the maximal cellular respiration only at much higher concentrations (50 and 100 μM). Taken together, compounds 11 and 12, which avidly localize to mitochondria, exhibited relatively low mitochondrial toxicity and cytotoxicity, indicating that these compounds can serve as mitochondria-targeted transporters without affecting mitochondrial function.

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CONCLUSION In summary, we identified amphipathic peptoids that are membrane-permeable and specifically accumulate in the mitochondria. In particular, two peptoids with cyclohexyl side chains, 1 and 2, demonstrated highly efficient mitochondrial localization with r-values of 0.69 and 0.81, respectively. These amphipathic peptoids did not significantly affect mitochondrial function and were not cytotoxic at low μM concentrations. Although additional structural−activity relationship studies are needed to confirm the mechanism of mitochondrial penetration employed by these peptoids, it appears that the ordered conformation of the amphipathic chain may promote efficient transport, as compound 2 demonstrated the most selective mitochondrial localization. We think that the main advantage of our peptoid transporter is the highly efficient and rapid mitochondrial localization with minimal adverse effects, thus providing a promising alternative to currently studied delivery vehicles. For example, the most widely used mitochondrial targeting cation TPP conjugates produce severe cytotoxicity and disrupt mitochondrial function depending on their physicochemical features, such as valency and polarity,36−38 while most effective MPPs demonstrate moderate cytotoxicity (LC50 ≈ 20 μM).13 Considering the enormous potential of mitochondria-targeting therapeutics, minimizing any possible toxicity issues is crucial. We believe that our mitochondriatargeting peptoids provide a highly selective and robust delivery method that can facilitate therapeutic development. D

DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION General Methods. Solvents and reagents were purchased from commercial vendors and used without further purification. Rink amide MBHA resin and Fmoc-Arg(pbf)-OH were purchased from Novabiochem (San Diego, CA, USA). HATU was purchased from ChemImpex (Wood Dale, IL, USA). DIC, DIEA, and TIS were purchased from TCI (ChuoKu, Japan). TFA, DMF, and acetonitrile were purchased from Acros Organics (Franklin Lakes, NJ, USA). Nch, Nsch, and Ndp were purchased from Alfa Aesar (Ward Hill, MA, USA). NMP was purchased from Merck Millipore (Damstadt, Germany). All other reagents for the synthesis of peptides and peptoids were purchased from Sigma-Aldrich (St. Louis, MO, USA). For manual solid-phase peptide and peptoid synthesis, the SPE cartridges (Applied Separations, Allentown, PA, USA) assembled with the 20 μm filters (Applied Separations) were used for reaction vessels. The reaction was accelerated using a CEM MARS 230/60 microwave reaction system (CEM Corp., Matthews, NC, USA) consisting of a fiber-optic temperature probe and magnetic stirrer under atmospheric pressure. Analytical HPLC chromatograms were obtained using a Waters reversed-phase HPLC system (2489 UV/visible detector, 1525 Binary HPLC pump, 2707 Autosampler, and 5CH column oven) with a C18 column (SunFire C18, 4.6 × 250 mm, 5 μm). The column oven temperature was set at 40 °C. A binary mobile phase system (A: deionized water +0.1% TFA, B: CH3CN + 0.1% TFA) was used as follows: 2 min 5% of B, linear gradient to 100% of B for 30 min, with 100% B maintained for 5 min. The flow rate was 1 mL/min. The purity of the compound was monitored by measuring the absorbance at 220 and 254 nm. All the peptoids were purified on a Waters preparative HPLC system (2489 UV/visible detector, 2545 Quaternary HPLC pump, fraction collector III), with a C18 column (SunFire C18, 19 × 150 mm, 5 μm) at room temperature. The flow rate was set to 14 mL/ min. A binary mobile phase system (A: deionized water +0.1% TFA, B: CH3CN + 0.1% TFA) was employed using 5 min 5% of B, linear gradient to 100% B for 30 min, and 100% B maintained for 5 min. Sample elution was monitored by measuring the absorbance at 220 and 254 nm. The purity of each fraction (>98%) was confirmed by analytical HPLC. ESIMS analysis was performed using a model 1260 Infinity liquid chromatography system (Agilent, Santa Clara, CA, USA) with an Agilent 6120 single quadrupole mass spectrometer. Fractions containing the pure product were collected, lyophilized, and stored at −80 °C. High-resolution mass spectrometry (HR-MS) data were obtained using an Agilent QTOF 6520 with dual ESI source. Circular dichroism (CD) spectra were recorded on a model 810 spectropolarimeter (Jasco, Easton, MD, USA) in quartz cells with 10 mm path length. The response time was set to 2 s with 1.0 nm bandwidth for all spectra. For conformational examination, samples were prepared in acetonitrile and water. The spectra were acquired in the range of 190−260 nm with a 20 nm/min scanning speed at 20 °C. Spectra recorded three times were averaged. The spectra are expressed in terms of per-residue molar ellipticity (deg·cm2/ dmol) based on the chain length of the peptoids. Peptoid concentrations (50 μM) were determined using the dry weight of the lyophilized material, and are based on the TFA salt. General Procedure for Peptoid Synthesis. The microwave-assisted solid-phase submonomer protocol was used for synthesis of peptoids.17,39Fmoc-Rink amide MBHA resin (0.65

mmol/g, 769 mg, 0.50 mmol) was swelled in DMF (10.0 mL) for 20 min and treated with 20% piperidine in DMF (10.0 mL) at 80 °C in the microwave reactor (600 W max power, ramp 2 min, hold 2 min, stirring level 2). The Fmoc deprotection reaction was repeated twice. The resin was thoroughly washed with DMF (1×), CH2Cl2 (3×), DMF (2×), MeOH (2×), DMF (3×), and CH2Cl2 (3×). Bromoacylation was performed by the addition of bromoacetic acid (10.0 mmol, 8.3 mL of 1.2 M bromoacetic acid stock solution), followed by DIC (10.0 mmol, 1.6 mL, 1.3 g, d = 0.82 g/mL). The reaction mixture was stirred in the microwave reactor at 35 °C (300 W max power, ramp 30 s, hold 1 min, stirring level 2). The resin was then washed using the same washing sequence. Displacement reactions were performed by the addition of primary amine, Nch (3.8 mmol, 1.0 M in NMP, 3.8 mL), Nsch (3.8 mmol, 1.0 M in NMP, 3.8 mL), Npm (5.0 mmol, 1.0 M in NMP, 5.0 mL), Nspe (3.8 mmol, 1.0 M in NMP, 3.8 mL), Ndp (3.8 mmol, 1.0 M in NMP, 3.8 mL), Nlys(Boc) (5.0 mmol, 1.0 M in NMP, 5.0 mL), or Nlys(Mmt) (5.0 mmol, 1.0 M in NMP, 5.0 mL), followed by stirring at 80 °C in the microwave reactor (300 W max power, ramp 2 min, hold 90 s, stirring level 2). The resin was washed using the same washing sequence. Bromoacylation and displacement reactions were repeated until the desired sequence was obtained. After synthesis of the peptoid sequence, the resin-bound peptoid was reacted with 5(6)-carboxyfluorescein (2.5 mmol, 0.94 g), N,N′-diisopropyl carbodiimide (2.5 mmol, 0.32 g, 0.40 mL, d = 0.81 g/mL), and 1hydroxybenzotriazole hydrate (2.5 mmol, 0.34 g) in DMF (10.0 mL) for 16 h in the reaction vessel on a shaker at room temperature. Reactions were stopped by washing the resins as described above. Subsequently, the peptoids were treated with piperidine/DMF (1:4, v/v) to remove ester-bound carboxyfluorescein.30 After dye conjugation, the methoxytrityl protecting group was removed by multiple treatments with a 0.75% TFA cocktail (CH2Cl2:TFA = 99.25:0.75). The deprotected resin-bound peptoid was treated with DIEA (10.0 mmol, 1.8 mL, 1.3 g, d = 0.74 g/mL) and 1H-pyrazole-1-carboxamidine (10.0 mmol, 1.5 g) in DMF (10.0 mL), and heated at 50 °C for 24−48 h. After guanidinylation, the resin was washed and reacted for 1 h with a cleavage cocktail (TFA:CH2Cl2:TIS = 95:2.5:2.5). General Procedure of Peptide Synthesis. Standard Fmoc/tBu and microwave-assisted solid-phase peptide synthesis was used. Fmoc-Rink amide MBHA resin (0.65 mmol/g, 769 mg, 0.50 mmol) was swelled in DMF (10.0 mL) for 20 min, and Fmoc deprotection reaction was carried out following the procedure detailed above. For peptide coupling, FmocArg(pbf)-OH (0.8 mmol, 0.5 g, 0.08 M in DMF, 10.0 mL), HATU (0.8 mmol, 0.3 g), and DIEA (2.08 mmol, 0.4 mL, 0.3 g, d = 0.74 g/mL) were added, and the reaction mixture was stirred in the microwave reactor (300 W max power, ramp 2 min, hold 8 min, stirring level 2) at 75 °C. The peptide coupling and Fmoc deprotection reactions were repeated until the desired sequence was obtained. The carboxyfluorescein conjugation was performed following the procedure mentioned above. The resin was washed and reacted for 2 h with a cleavage cocktail (TFA:CH2Cl2:TIS = 95:2.5:2.5). Synthesis of Peptoid 11−15. After synthesis of the resinbound peptoid sequence, the resin in DMF (10.0 mL) was added with Boc2O (5.5 mmol, 1.2 g) and DMAP (catalytic amount). The reaction mixture was stirred in the microwave reactor (75 °C, 400 W maximum power, 75%, ramp 8 min, hold 52 min, stirring level 3), and washed with DMF (1×), E

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in medium and incubated for 24 h. Twenty microliters of the CellTiter 96 aqueous nonradioactive cell proliferation assay reagent (Promega, Madison, WI, USA), which contains the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was added to each well. The plate was incubated for 4 h at 37 °C to metabolize. The MTS-formazan products in the viable cells were measured at 490 nm using a microplate reader and percentages of cell viability (%) were measured and compared with untreated cells. The percentage of cell viability = A/(Acontrol) × 100, where A is the absorbance of the test well and Acontrol is the average absorbance of wells containing cells not treated with peptoids. Mitochondrial Oxygen Consumption Rate (OCR) Measurement. U87 cells were purchased from ATCC (Manassas, VA) and were maintained in high glucose DMEM (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany), 2 mM glutamine (Thermo Fisher Scientific), and 100 U/mL of penicillin-streptomycin (Thermo Fisher Scientific) in a 37 °C humidified incubator with 5% CO2. In each well, 40 000 cells were seeded in an XF24 cell plate (Agilent) 24 h prior to assay. After 24 h, the culture medium was replaced by the assay medium consisting of noncarbonated Seahorse XF Base Medium (Agilent) supplemented with 25 mM glucose, 6 mM glutamine, and 1 mM pyruvate. The cells were equilibrated for 1 h in a non-CO2 37 °C incubator. Subsequently, the assay was performed in accordance with the manufacturer’s instructions (Seahorse XF Cell Mito Stress Test Kit; Agilent). The analysis of OCR was performed in a Seahorse XFe24 analyzer, and the OCR values were obtained during baseline (prior to addition of any Mito Stress Test substances) and after the addition of oligomycin (2 μM final concentration), FCCP (0.5 μM final concentration), and rotenone/antimycin A (0.5 μM final concentration).

CH2Cl2 (3×), DMF (2×), MeOH (2×), DMF (3×), and CH2Cl2 (3×). After the N-terminal Boc protection, Mmt deprotection, guanidylation, and resin cleavage were performed as detailed above. Flow Cytometry. HeLa cells were seeded at a density of 350 000 cells in a six-well culture dish (SPL Life Sciences, Seoul, South Korea). After 24 h, cells were treated with each peptoid (5 μM) for 1 h and washed twice with phosphate buffered saline (PBS). The cells were trypsinized at 37 °C for 5 min and culture medium was added to deactivate the trypsin. The detached cells were spun down in a conical tube to remove the media and trypsin. After centrifugation, 600 μL of 2% fetal bovine serum (FBS)/PBS was added to each tube to resuspend the cell pellet. Cellular fluorescence signal was recorded using a SH800 flow cytometer (Sony, Tokyo, Japan) with a fluorescein excitation and emission wavelength of 488 and 530 nm, respectively. Measurements were repeated three times and data were expressed as mean ± SEM. Statistical analyses between groups were performed using Prism 7 software (GraphPad Software Inc., La Jolla, USA) Microscopy. HeLa cells were seeded at a density of 150 000 cells in a 35 mm confocal dish (SPL Life Sciences). After 24 h, the cells were treated with a CF-labeled peptoid (5 μM) for 1 h. After treating cells with 50 nM of MitoTracker red for 15 min, the cells were washed three times with PBS and 1 mL of OptiMEM was added. The cells were live-imaged using an inverted LSM 700 confocal microscope (Carl Zeiss, Jena, Germany) equipped with an oil immersion objective (63×) in the green and red channels. Fluorescence images were processed using ZEN software (Carl Zeiss), and the Pearson’s correlation coefficients (r) of the images were determined using ImageJ software,40 based on the fluorescence of individual cells using optical slices from 20 to 24 independent Z-stacks, each containing two to six fluorescently labeled cells. JC-1 Mitochondrial Membrane Potential Assay. HeLa cells were seeded at a density of 30000 cells in a clearbottomed, black, 96-well plate (BD FALCON) 1 day prior to assay. JC-1 (750 μM; Sigma-Aldrich) in dimethylsulfoxide stock solution was dissolved in phenol red-free Opti-MEM medium (GIBCO, Franklin Lakes, NJ, USA) to a final concentration of 7.5 μM JC-1 per well. The medium was removed from the plate and 100 μL per well of JC-1 was added. Plates were incubated for 75 min at 37 °C and washed twice with 100 μL of PBS per well. Subsequently, cells were treated with 25 μL of 10 μM of each compound in Opti-MEM and incubated at 37 °C for another hour. Fluorescence was measured at excitation and emission wavelengths of 530 and 580 nm, respectively (“red”) and 485 and 530 nm, respectively (“green”) on a Spectramax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). The ratio of red to green fluorescence was determined and the percentage ΔΨm from each compound was calculated and normalized using the percentage of the untreated cells as 100%. MTS Assay. MRC-5 was purchased from the Korean Cell Link Bank and were grown in DMEM media with 10% FBS at 37 °C with 5% CO2. The MTS cell-viability assay was carried out to determine the effects of peptoids on MRC-5 cell growth.41 Aliquots (100 μL) of medium containing 1.3 × 104 MRC-5 cells were distributed into each well of a 96-well plate (Eppendorf). The cells were grown in a humidified atmosphere of 5% CO2 in air at 37 °C and allowed to attach for 24 h. When the cell density reached approximately 70% confluency, the medium was replaced with serial dilutions of the peptoid stock

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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.8b00148. Confocal images of HeLa cells treated with compounds 1−10; detailed characterization data for compounds 1− 15 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Jiwon Seo: 0000-0002-5433-5071 Jiyoun Lee: 0000-0003-3819-5889 Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT F

DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

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(NRF-2015R1C1A2A01055457 to J.L.) and by the Ministry of Education (NRF-2017R1D1A1B03036089 to J.S.). J.S. also acknowledges a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1074).

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ABBREVIATIONS Boc2O, di-tert-butyl dicarbonate; Boc, tert-butoxycarbonyl; Fmoc, 9-fluorenylmethoxycarbonyl; TFA, trifluoroacetic acid; DMF, N,N-dimethylformamide; DIC, N,N-diisopropylcarbodiimide; TIS, triisopropylsilane; NMP, N-methyl-2-pyrrolidone; DIEA, N,N-diisopropylethylamine; DMAP, 4-(dimethylamino)pyridine; Nspe, (S)-(−)-α-methylbenzylamine; Npm, benzylamine; Nlys(Boc), N-tert-butoxycarbonyl-1,4-butanediamine; Nlys(Mmt), N-methoxytrityl-1,4-butanediamine; Nch, cyclohexanemethylamine; Nsch, (S)-(+)-1-cyclohexylethylamine; Ndp, 2,2-diphenylethylamine; pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

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DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.bioconjchem.8b00148 Bioconjugate Chem. XXXX, XXX, XXX−XXX