Comparison of Cell Permeability of Cyclic Peptoids and Linear Peptoids

Feb 26, 2018 - KEYWORDS: cyclic peptoids, linear peptoids, cell permeability, combinatorial library, protein ligands. □ INTRODUCTION ... systems as ...
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Research Article Cite This: ACS Comb. Sci. XXXX, XXX, XXX−XXX

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Comparison of Cell Permeability of Cyclic Peptoids and Linear Peptoids Min-Kyung Shin,† Yu-Jung Hyun,† Ji Hoon Lee,‡ and Hyun-Suk Lim*,† †

Department of Chemistry and Division of Advanced Material Science, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea ‡ New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu 41061, South Korea S Supporting Information *

ABSTRACT: Cyclic peptoids are emerging as an attractive class of peptidomimetics. Compared to their linear counterparts, cyclic peptoids should have increased conformational rigidity and preorganized structures, enabling them to bind more tightly to target proteins without major entropy penalty. Because cyclic peptoids lack the amide protons in their backbones like linear peptoids, it is perceived that cyclic peptoids are seemingly cell permeable as much as linear peptoids. However, no systematic investigation for cell permeability of cyclic peptoids has been reported yet. Here, we, for the first time, demonstrate that cyclic peptoids are far more cell permeable than linear counterparts irrespective of their size and side chains. This study highlights that cyclic peptoids, along with combinatorial library and high-throughput screening technologies, will serve as a rich source of protein binding molecules, particularly targeting intracellular proteins, given their excellent cell permeability in addition to their conformational rigidity and proteolytic stability. KEYWORDS: cyclic peptoids, linear peptoids, cell permeability, combinatorial library, protein ligands

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INTRODUCTION Peptoids are a class of peptidomimetics based on N-alkylated glycines and have several desirable features as peptidomimetics.1−3 They can be efficiently synthesized on solid support by a submonomer route.2,4 Like other unnatural peptidomimetics, peptoids are proteolytically stable.5 Notably, peptoids are known to have better membrane permeability than native peptides.6−8 Thus, peptoids hold great promise for many biological applications. Indeed, screening combinatorial libraries of peptoids has discovered various biologically active peptoids.2,3,9−12 However, the biological application of peptoids is often limited by their flexible nature. Owing to the lack of backbone chirality and amide protons, peptoids generally have relatively flexible structures, making it challenging to identify peptoid-based protein ligands with high affinity and specificity. There has been a great deal of efforts to develop conformationally restricted peptoids.12−14 One such strategy is to prepare macrocyclic systems.15−25 As demonstrated in many cyclic peptides,26,27 cyclic peptoids would have relatively rigid and preorganized structures compared to linear peptoids, allowing them to bind more tightly to target proteins without major entropy penalty. Thus, cyclic peptoids are of great interest as an important class of peptidomimetics Because cyclic peptoids do not possess amide protons in their backbones like linear peptoids, it is perceived that cyclic peptoids are seemingly cell permeable as much as linear peptoids. Recent studies showed that incorporation of one or © XXXX American Chemical Society

more peptoid residues into cyclic peptides increased cell permeability.28 It was also demonstrated that multiple backbone N-methylation of cyclic peptides, thereby increasing peptoid character, remarkably improved their cell permeability.29−32 Overall, these findings suggest that cyclic peptoids are relatively cell-permeable because of the lack of amide protons. However, no systematic investigation for cell permeability of cyclic peptoids has been reported yet. Herein, we report the evaluation of cell-permeability of triazine-bridged cyclic peptoids relative to their linear counterparts. Previously, we have developed triazine-bridged cyclic peptoid systems as conformationally rigid peptoid structures (Figure 1).17,19 We incorporated a triazine moiety into the peptoid backbone for efficient macrocyclization. Cyclization reaction under mild conditions gave exclusively monocyclic peptoids without producing unwanted byproducts, such as cyclic dimer, trimer, or oligomers, which are commonly found byproducts during macrocyclization reactions. Furthermore, the introduction of a triazine ring would be able to create additional structural rigidity in addition to the conformational restriction conferred by cyclization as demonstrated previously.33−36 As a result, triazine-bridged cyclic peptoids have great potential as protein-capture agents. Indeed, we recently successfully identified a cyclic peptoid-based inhibitor of Skp2 by highReceived: December 29, 2017 Published: February 26, 2018 A

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX

Research Article

ACS Combinatorial Science

Table 1. Sequence of Synthesized Cyclic C1-5 and Linear Peptoids L1-5 with a Fluorescein Tag (FL) or a Halotag (HT) compd FL-C1 or HT-C1 FL-C2 or HT-C2 FL-C3 or HT-C3 FL-C4 or HT-C4 FL-C5 or HT-C5 FL-L1 or HT-L1 FL-L2 or HT-L2 FL-L3 or HT-L3 FL-L4 or HT-L4 FL-L5 or HT-L5

Figure 1. Structures of peptides, peptoids, and triazine-bridged cyclic peptoids.

throughput affinity-based screening of a 116 000-member combinatorial library of triazine-bridged cyclic peptoids,37,38 demonstrating the utility of triazine-bridged cyclic peptoids.

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RESULTS AND DISCUSSION For the purpose of testing their cell permeability, we set out to synthesize a series of cyclic and linear peptoids as fluorescently labeled forms (Scheme S1). Initially we prepared cyclic FL-C15 and linear peptoids FL-L1-5 of various sizes containing alternating Nphe and Nhse residues (Figure 2 and Table 1).

chain length

sequencea

3

cyclo[Cys-Nphe-Nhse-Nphe‑T]-X

4

cyclo[Cys-Nphe-Nhse-Nphe-T]-X

5

cyclo[Cys-Nphe-Nhse-Nphe-Nhse-Nphe-T]-X

6

cyclo[Cys-Nphe-Nhse-Nphe-NhseNphe-Nhse-T]-X cyclo[Cys-Nphe-NhseNphe-Nhse-Nphe-Nhse-Nphe-T]-X Nphe-Nhse-Nphe-X Nphe-Nhse-Nphe-X Nphe-Nhse-Nphe-Nhse-Nphe-X Nphe-Nhse-Nphe-Nhse-Nphe-Nhse-X Nphe-Nhse-Nphe-Nhse-Nphe-Nhse-Nphe-X

7 3 4 5 6 7

a

T in the sequences indicates triazine ring. For FL series, X = linkerfluorescein. For HT series, X = chloroalkane tag.

To determine the cell penetration ability of the synthesized cyclic FL-C1-5 and linear peptoids FL-L1-5, we employed fluorescence-activated cell sorting (FACS) that is routinely used for sorting live cells (Figure 3 and Figure S2). HeLa cells

Figure 3. (A) FACS analysis of cellular uptake of cyclic and linear peptoids (FL-C1−5 and FL-L1−5) with alternating Nphe-Nhse determined by mean fluorescence intensity. Error bars represent s.d. from three independent experiments. (B) Fluorescence microscope images of HeLa cells treated with a linear peptoid FL-L5 and a cyclic peptoid FL-C5 for 4 h at 37 °C. Figure 2. General structures of cyclic and linear peptoids used in this study. FL and HT denote linker-fluorescein and halotag, respectively.

were incubated with each of the synthesized compounds at 37 °C for 4 h at 10 μM concentration and detached from culture surface by trypsinization. Cells were then treated with propidium iodide (PI), a dye to detect dead cells, and analyzed immediately by FACS. Dead cells stained with PI were excluded from analysis. Triazine-bridged cyclic peptoids FL-C1-5 exhibited far higher fluorescence intensity than the corresponding linear peptoids FL-L1-5, suggesting that cyclic peptoids are more cellpermeable than linear ones (Figure 3A and Figure S2). Next, we performed live cell confocal microscopy experiment to validate the cell permeability difference. To this end, FL-C5 and FL-L5 were chosen as a representative pair of cyclic and linear peptoids. HeLa cells were treated with cyclic peptoid FLC5 or linear peptoid FL-L5 at 10 μM, and their cellular uptake was visualized under a confocal microscope (Figure 3B). The microscopy experiments showed superficial punctate fluorescence staining, indicating that significant fractions of cyclic peptoids were entrapped inside membrane-bound lysosomes or endosomes rather than reaching the cytosol or nucleus. This is

Peptoids were prepared on Rink amide MBHA resin by wellknown submonomer route.4,39 After peptoid synthesis, the Nterminus of each peptoid was coupled with Abu linker followed by conjugation of 5,6-carboxyfluorescein to give desired linear peptoids tagged with fluorescein. For the preparation of cyclic peptoids, a cysteine was first loaded to resins. Following Fmocdeprotection, various peptoid residues ranging from 3-mer to 7mer were added. After the completion of peptoid synthesis, the N-terminal was capped with triazine by using cyanuric chloride. After removing Mmt protecting group on the cysteine, macrocyclization was achieved using DIPEA at room temperature.17 The remaining chloride on triazine was replaced with 1,4-diaminobutane as a spacer. Subsequently, the NH2 group of the spacer was coupled with 5,6-carboxyfluorescein to provide fluorescently labeled cyclic peptoids. The synthesized linear and cyclic peptoids were purified by reverse-phase HPLC, and their identity and purity were analyzed by LC/MS and MALDI-TOF (Figure S1). B

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science presumably due to the use of non-cell-permeable carboxyfluorescein as a probe, which could negatively affect the cell penetration ability of its conjugated molecules. To avoid the ambiguous results, we chose a different cell penetration assay that utilizes HaloTag technology. Kritzer and co-workers have recently developed an elegant cell permeability assay method called ChloroAlkane Penetration Assay (CAPA), which uses cells expressing a HaloTag.40 HaloTag is a modified haloalkane dehalogenase enzyme that covalently binds chloroalkane functional groups. If any molecules conjugated to the chloroalkane (halotagged-, or HT-molecules) reach the cytosol, they are covalently labeled to HaloTag. Given that HaloTag is a cytosolic protein, it is possible to evaluate the cell permeability of halotagged molecules by monitoring unreacted HaloTag. To assess the cell permeability using CAPA, we synthesized a series of cyclic HT-C1-5 and linear peptoids HT-L1-5 having alternating Nphe and Nhse residues as chloroalkane-conjugated forms through the similar synthetic procedures used for preparing fluorescently labeled derivatives (Scheme S2). HeLa cells stably expressing a GFP-HaloTag fusion were treated with each of the synthesized halotagged peptoids at 37 °C. After 4 h of incubation followed by washing, cells were exposed to a chloroalkane-conjugated red fluorescent dye, HT-TAMRA, for 30 min, and then analyzed by FACS. By monitoring red fluorescence, the amount of unreacted HaloTag during the incubation with HT-molecules was quantified, which is inversely proportional to the cell-penetration ability of the tested HT-molecules. DMSO and a chloroalkane-conjugated polyarginine peptide, HT-PolyR8, were used as a negative and a positive control, respectively. As shown in Figure 4, triazine-

Figure 5. Confocal microscopy images of HaLo-GFP-Mito HeLa cells treated with DMSO, HT-PolyR8 (a cell-penetrating peptide), a cyclic peptoid HT-C3, and a linear peptoid HT-L3. (A) Bright field images. (B) Haloenzyme-GFP (green) fluorescence images. (C) HT-TAMRA red fluorescence images. (D) Merged images.

further validating that cyclic peptoids reach the cytosol (Figure 5D). Because we used cyclic and linear peptoids having only two kinds of residues, Nphe and Nhse, the cell permeability observed from the above experiments might be a unique phenomenon to those series of peptoids. In order to generalize the findings that cyclic peptoids are more cell permeable than linear ones, we also synthesized other series of cyclic and linear peptoids with random sequences possessing a variety of side chains such as Ncha, Nala, Nphe, Nhse, Nval, Net, and Nleu. (Table 2, and Figure S1). Likewise, these cyclic peptoids (HTTable 2. Sequence of Halotagged Cyclic HT-C6-7 and Linear Peptoids HT-L6-7 with Random Sequences

Figure 4. (A) FACS analysis of halotagged cyclic HT-C1-5 and linear peptoids HT-L1-5 with alternating Nphe-Nhse. HaLo-GFP-Mito HeLa cells were incubated with 10 μM compounds for 4 h at 37 °C. Mean red fluorescence for each sample was normalized by the fluorescence intensity for vehicle control (DMSO + HT-TMR) (100%) and for vehicle with no HT-TMR (0%). Error bars represent standard deviations from three independent experiments. (B) FACS analysis for representative cyclic peptoid (HT-C3) and linear peptoid (HT-L3).

a

compd

chain length

HT-C6 HT-C7

6 8

HT-L6 HT-L7

6 8

sequencea cyclo[Cys-Nval-Nphe-Nhse-Nala-Ncha-Nleu-T]-HT cyclo[Cys-Nval-Nphe-Nhse-Nala-NchaNleu-Nala-Net-T]-HT Nval-Nphe-Nhse-Nala-Ncha-Nleu-HT Nval-Nphe-Nhse-Nala-Ncha-Nleu-Nala-Net-HT

T in the sequences indicates triazine ring.

C8 and HT-C9) with random sequences also showed remarkably reduced red fluorescence intensity in comparison with the corresponding linear peptoids (HT-L8 and HT-L9) in both FACS (Figure 6 and Figure S4) and fluorescence microscopy (Figure 7 and Figure S5). Taken together, these results demonstrate that cyclic peptoids enter the cytosol far more effectively than linear peptoids irrespective of side chains and ring sizes. Next, we examined whether cellular uptake of bridged cyclic peptoids is energy dependent. HeLa cells expressing GFPHaloTag were incubated with cyclic peptoids (HT-C5 or HTC7) in the presence or absence of NaN3, which is known to block energy-dependent cellular uptake by depleting ATP production. As shown in Figure S6, the cellular uptake of cyclic

bridged cyclic peptoids HT-C1-5 significantly suppressed TAMRA fluorescence intensity by up to 90% regardless of their ring sizes, whereas reduction of TAMRA signal by linear peptoids HT-L1-5 was relatively moderate, This finding suggests that cyclic peptoids are more cell permeable than their linear counterparts. The result was confirmed by confocal microscopy experiments (Figures 5 and S3). In good agreement with the flow cytometry results, fluorescence microscopy showed that preincubation with cyclic peptoids HT-C1-5 remarkably decreased red fluorescence in comparison with linear peptoids HT-L1-5. Note that the spatial overlap between red fluorescence and GFP was observed, indicating colocalization of HT-TAMRA and cytosolically located GFP-HaloTag, C

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science

combinatorial library of such cyclic peptoids by employing structurally diverse building blocks.

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EXPERIMENTAL PROCEDURES Synthesis of Halotag-Labeled Linear Peptoids. Rink amide MBHA resins (100 mg, 75 μmol) were swollen with DMF (2 mL) in a 5 mL fritted syringe for 2 h. The Fmoc protecting group was removed by treating with 20% piperidine in DMF (2 × 10 min). Next, peptoid residues were coupled to resin by a standard submonomer route. Briefly, NH2-functionalized beads were treated with 1 M bromoacetic acid (BAA) (20 equiv) and 1 M N,N-diisopropylcarbodiimide (DIC) (20 equiv) in DMF. After shaking for 20 min, the reaction mixture was drained and the resins were washed with DMF (3×), CH2Cl2 (2×), MeOH (2×), and DMF (3×). Unless otherwise noted, this washing cycle was used for each reaction step. Beads were then treated with a primary amine (2 M in DMF) for 2 h for amine displacement reaction. This bromoacetylation/ amination process was repeated until desired sequences of peptoids were obtained (n = 3−8). A haloalkane residue was introduced on the N-terminus of peptoids by treating bromoacetlated peptoids with 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine (20 equiv in DMF) for the 2 h. For cleavage, the resins were treated with a solution of trifluoroacetic acid (TFA), water, triisopropylsilane (TIS) (95:2.5:2.5, v/v) for 2 h. The released crude peptoids were purified by HPLC and analyzed by LC/MS and MALDI-TOF mass spectrometry (Figure S2). Synthesis of Halotag-Labeled Cyclic Peptoids. Cyclic peptoids were synthesized by the similar procedure described above except for the introduction of cysteine and triazine moiety for cyclization reaction. In this case, prior to peptoid synthesis, cysteine residue was first loaded on Rink amide MBHA resins. After Fmoc deprotection followed by peptoid synthesis, N-terminus of peptoids was capped with triazine ring by reacting with cyanuric chloride (5 equiv) and DIPEA (6 equiv) in THF overnight at rt. After washing, the Mmt protecting group on the cysteine was removed by treatment with 2% TFA and 5% TIS in CH2Cl2 (8 × 2 min). For macrocyclization, the beads were treated with 2 M DIPEA (20 equiv) in DMF overnight at rt. The remaining chloride group on cyclic peptoids was displaced with 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine (20 equiv) in the presence of DIPEA (20 equiv) in N-methyl-2-pyrrolidone overnight at 60 °C. Finally, cyclic peptoids were cleaved from the beads by treating with 95% TFA cleavage cocktail and purified by HPLC. Their identity and purity were analyzed by LC/MS and MALDI-TOF mass spectrometry (Figure S2). Cell Culture and FACS Analysis. HaLo-GFP-Mito HeLa cells were kindly provided by the Kritzer laboratory. HaLoGFP-Mito HeLa cells were maintained in medium consisting of Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1 μg/mL puromycin at 37 °C and 5% CO2. 1.0 × 105 cells/well were placed in a 24-well plate and incubated overnight. Cells were then treated with 0.5 mL of 10 μM compounds solution in acidified phenol red-free Opti-MEM (0.15% 6 N HCl). After 4 h incubation, cells were washed with phenol red-free OptiMEM for 30 min. Then, cells were treated with 5 μM HTTAMRA (Promega) in phenol red-free Opti-MEM. After 30 min, cells were washed with phenol red-free DMEM (Hyclone), 10% FBS, and 1% pen/strep for 15 min. Cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS)

Figure 6. (A) FACS analysis of halotagged cyclic peptoids HT-C6−7 and linear peptoids HT-L6−7 with random sequences. HaLo-GFPMito HeLa cells were incubated with 10 μM compounds for 4 h at 37 °C. Mean red fluorescence for each sample was normalized by the fluorescence intensity for vehicle control (DMSO + HT-TMR) (100%) and for vehicle with no HT-TMR (0%). Error bars represent standard deviations from three independent experiments. (B) FACS analysis for a cyclic peptoid HT-C6 and a linear peptoid HT-L6.

Figure 7. Confocal microscopy images of HaLo-GFP-Mito HeLa cells treated with a cyclic peptoid HT-C6 and a linear peptoid HT-L6 with random sequences. (A) Bright field images. (B) Haloenzyme-GFP (green) fluorescence images. (C) HT-TAMRA red fluorescence images. (D) Merged images.

peptoids was not significantly reduced by preincubation of NaN3, suggesting that triazine-bridged cyclic peptoids may cross cellular membrane through an energy-independent process. In addition, we tested the cell-penetrating ability of cyclic peptoids at 4 °C that is known to prevent not only energy-dependent uptake but also the passive uptake by increasing the plasma membrane rigidity. At 4 °C, red fluorescence intensity was enhanced, indicating that cellpenetration of cyclic peptoids was reduced. Taken together, these results suggest that a fraction of cyclic peptoids may enter the cytosol by passive diffusion. Further studies to elucidate the mechanism should be needed. In summary, we have synthesized a series of halotagged cyclic peptoids of various ring sizes and side chains as well as the corresponding linear peptoids. Using FACS analysis and confocal fluorescence microscopy, we have systematically evaluated the cell permeability of cyclic peptoids relative to their linear counterparts. We, for the first time, show that cyclic peptoids are far more cell-permeable than linear peptoids irrespective of their size and side chains. The improved cell permeability of cyclic peptoids may be due to its restricted flexibility just as cyclic peptides are generally more cellpermeable than linear ones.41,42 Given their excellent cell permeability, along with conformational rigidity and proteolytic stability, triazine-bridged cyclic peptoids will serve as an excellent source of protein binding molecules, particularly targeting intracellular proteins. We are currently creating a large D

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science buffer (1 mL × 3). After collecting by trypsinization, cells were resuspended in cold DPBS and placed on ice. Next, cells were analyzed immediately by flow cytometer. The data presented are the mean fluorescence intensity for 10 000 cells collected. For energy-depletion analysis, the assays were conducted as mentioned above except that HaLo-GFP-Mito HeLa cells were preincubated with 10 mM sodium azide in phenol red-free Opti-MEM for 1 h at 37 °C before treating compounds. Confocal Microscopy. Two ×104 HaLo-GFP-Mito HeLa cells per well were seeded in a Lab-Tek chambered coverglass (8 well). After incubation for 24 h, cells were treated with 250 μL of peptoid solution (10 μM in phenol red-free Opti-MEM with 0.15% 6 N HCl). After incubation at 37 °C for 4 h, cells were washed with phenol red-free Opti-MEM for 30 min. Then, cells were treated with 5 μM HT-TAMRA (Promega) in phenol red-free Opti-MEM. After 30 min, cells were washed with phenol red-free DMEM (Hyclone), 10% FBS and 1% pen/strep for 15 min. Cells were washed with ice-cold DPBS five times and then imaged using confocal microscope.

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(6) Yu, P.; Liu, B.; Kodadek, T. A high-throughput assay for assessing the cell permeability of combinatorial libraries. Nat. Biotechnol. 2005, 23, 746−751. (7) Kwon, Y. U.; Kodadek, T. Quantitative evaluation of the relative cell permeability of peptoids and peptides. J. Am. Chem. Soc. 2007, 129, 1508−1509. (8) Tan, N. C.; Yu, P.; Kwon, Y. U.; Kodadek, T. High-throughput evaluation of relative cell permeability between peptoids and peptides. Bioorg. Med. Chem. 2008, 16, 5853−5861. (9) Lim, H. S.; Muralidhar Reddy, M.; Xiao, X.; Wilson, J.; Wilson, R.; Connell, S.; Kodadek, T. Rapid identification of improved protein ligands using peptoid microarrays. Bioorg. Med. Chem. Lett. 2009, 19, 3866−3869. (10) Oh, M.; Lee, J. H.; Wang, W.; Lee, H. S.; Lee, W. S.; Burlak, C.; Im, W.; Hoang, Q. Q.; Lim, H. S. Potential pharmacological chaperones targeting cancer-associated MCL-1 and Parkinson disease-associated alpha-synuclein. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 11007−11012. (11) Sanii, B.; Kudirka, R.; Cho, A.; Venkateswaran, N.; Olivier, G. K.; Olson, A. M.; Tran, H.; Harada, R. M.; Tan, L.; Zuckermann, R. N. Shaken, Not Stirred: Collapsing a Peptoid Monolayer To Produce Free-Floating, Stable Nanosheets. J. Am. Chem. Soc. 2011, 133, 20808−20815. (12) Yoo, B.; Kirshenbaum, K. Peptoid architectures: elaboration, actuation, and application. Curr. Opin. Chem. Biol. 2008, 12, 714−721. (13) Horne, W. S. Peptide and peptoid foldamers in medicinal chemistry. Expert Opin. Drug Discovery 2011, 6, 1247−1262. (14) Lee, K. J.; Lee, W. S.; Yun, H.; Hyun, Y. J.; Seo, C. D.; Lee, C. W.; Lim, H. S. Oligomers of N-Substituted beta(2)-Homoalanines: Peptoids with Backbone Chirality. Org. Lett. 2016, 18, 3678−3681. (15) Shin, S. B.; Yoo, B.; Todaro, L. J.; Kirshenbaum, K. Cyclic peptoids. J. Am. Chem. Soc. 2007, 129, 3218−3225. (16) Maulucci, N.; Izzo, I.; Bifulco, G.; Aliberti, A.; De Cola, C.; Comegna, D.; Gaeta, C.; Napolitano, A.; Pizza, C.; Tedesco, C.; Flot, D.; De Riccardis, F. Synthesis, structures, and properties of nine-, twelve-, and eighteen-membered N-benzyloxyethyl cyclic alphapeptoids. Chem. Commun. (Cambridge, U. K.) 2008, 3927−3929. (17) Lee, J. H.; Meyer, A. M.; Lim, H. S. A simple strategy for the construction of combinatorial cyclic peptoid libraries. Chem. Commun. (Cambridge, U. K.) 2010, 46, 8615−8617. (18) Comegna, D.; Benincasa, M.; Gennaro, R.; Izzo, I.; De Riccardis, F. Design, synthesis and antimicrobial properties of non-hemolytic cationic alpha-cyclopeptoids. Bioorg. Med. Chem. 2010, 18, 2010− 2018. (19) Lee, J. H.; Kim, H. S.; Lim, H. S. Design and facile solid-phase synthesis of conformationally constrained bicyclic peptoids. Org. Lett. 2011, 13, 5012−5015. (20) Khan, S. N.; Kim, A.; Grubbs, R. H.; Kwon, Y. U. Ring-closing metathesis approaches for the solid-phase synthesis of cyclic peptoids. Org. Lett. 2011, 13, 1582−1585. (21) Simpson, L. S.; Kodadek, T. A Cleavable Scaffold Strategy for the Synthesis of One-Bead One-Compound Cyclic Peptoid Libraries That Can Be Sequenced By Tandem Mass Spectrometry. Tetrahedron Lett. 2012, 53, 2341−2344. (22) Lee, K. J.; Lim, H. S. Facile method to sequence cyclic peptides/ peptoids via one-pot ring-opening/cleavage reaction. Org. Lett. 2014, 16, 5710−5713. (23) Culf, A. S.; Cuperlovic-Culf, M.; Leger, D. A.; Decken, A. Small head-to-tail macrocyclic alpha-peptoids. Org. Lett. 2014, 16, 2780− 2783. (24) Kaniraj, P. J.; Maayan, G. A Facile Strategy for the Construction of Cyclic Peptoids under Microwave Irradiation through a Simple Substitution Reaction. Org. Lett. 2015, 17, 2110−2113. (25) Meli, A.; Macedi, E.; De Riccardis, F.; Smith, V. J.; Barbour, L. J.; Izzo, I.; Tedesco, C. Solid-State Conformational Flexibility at Work: Zipping and Unzipping within a Cyclic Peptoid Single Crystal. Angew. Chem., Int. Ed. 2016, 55, 4679−4682.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.7b00194. Experimental details, synthesis, and characterization (LC and MS spectra) of all compounds; experimental procedures for FACS analysis and confocal microscopy in HeLa cells; and FACS analysis data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hyun-Suk Lim: 0000-0003-4083-2998 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Joshua A. Kritzer (Tufts University) for helpful discussion and HeLa cells stably expressing GFP-Halo Tag. This work was supported by the National Research Foundation of Korea (NRF-2017R1A2B3004941).

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REFERENCES

(1) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; et al. Peptoids: a modular approach to drug discovery. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 9367−9371. (2) Lim, H. S.; Archer, C. T.; Kim, Y. C.; Hutchens, T.; Kodadek, T. Rapid identification of the pharmacophore in a peptoid inhibitor of the proteasome regulatory particle. Chem. Commun. 2008, 1064−1066. (3) Zuckermann, R. N.; Kodadek, T. Peptoids as potential therapeutics. Curr. Opin. Mol. Ther. 2009, 11, 299−307. (4) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. Efficient Method for Preparation of Peptoids [Oligo(N-substituted glycines)] by Submonomer Solid-Phase Synthesis. J. Am. Chem. Soc. 1992, 114, 10646. (5) Wang, Y.; Lin, H.; Tullman, R.; Jewell, C. F., Jr.; Weetall, M. L.; Tse, F. L. Absorption and disposition of a tripeptoid and a tetrapeptide in the rat. Biopharm. Drug Dispos. 1999, 20, 69−75. E

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX

Research Article

ACS Combinatorial Science (26) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The exploration of macrocycles for drug discovery−an underexploited structural class. Nat. Rev. Drug Discovery 2008, 7, 608−624. (27) White, C. J.; Yudin, A. K. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011, 3, 509−524. (28) Schwochert, J.; Turner, R.; Thang, M.; Berkeley, R. F.; Ponkey, A. R.; Rodriguez, K. M.; Leung, S. S.; Khunte, B.; Goetz, G.; Limberakis, C.; Kalgutkar, A. S.; Eng, H.; Shapiro, M. J.; Mathiowetz, A. M.; Price, D. A.; Liras, S.; Jacobson, M. P.; Lokey, R. S. Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides. Org. Lett. 2015, 17, 2928−2931. (29) Biron, E.; Chatterjee, J.; Ovadia, O.; Langenegger, D.; Brueggen, J.; Hoyer, D.; Schmid, H. A.; Jelinek, R.; Gilon, C.; Hoffman, A.; Kessler, H. Improving oral bioavailability of peptides by multiple Nmethylation: somatostatin analogues. Angew. Chem., Int. Ed. 2008, 47, 2595−2599. (30) Doedens, L.; Opperer, F.; Cai, M.; Beck, J. G.; Dedek, M.; Palmer, E.; Hruby, V. J.; Kessler, H. Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: pharmacological and conformational studies. J. Am. Chem. Soc. 2010, 132, 8115−8128. (31) Ovadia, O.; Greenberg, S.; Chatterjee, J.; Laufer, B.; Opperer, F.; Kessler, H.; Gilon, C.; Hoffman, A. The effect of multiple Nmethylation on intestinal permeability of cyclic hexapeptides. Mol. Pharmaceutics 2011, 8, 479−487. (32) White, T. R.; Renzelman, C. M.; Rand, A. C.; Rezai, T.; McEwen, C. M.; Gelev, V. M.; Turner, R. A.; Linington, R. G.; Leung, S. S.; Kalgutkar, A. S.; Bauman, J. N.; Zhang, Y.; Liras, S.; Price, D. A.; Mathiowetz, A. M.; Jacobson, M. P.; Lokey, R. S. On-resin Nmethylation of cyclic peptides for discovery of orally bioavailable scaffolds. Nat. Chem. Biol. 2011, 7, 810−817. (33) Scharn, D.; Germeroth, L.; Schneider-Mergener, J.; Wenschuh, H. Sequential nucleophilic substitution on halogenated triazines, pyrimidines, and purines: a novel approach to cyclic peptidomimetics. J. Org. Chem. 2001, 66, 507−513. (34) Jiang, L.; Burgess, K. Oxidative induction of beta-turn conformations in cyclic peptidomimetics: conformational analyses as indicators of configuration. J. Am. Chem. Soc. 2002, 124, 9028−9029. (35) Grieco, P.; Cai, M.; Liu, L.; Mayorov, A.; Chandler, K.; Trivedi, D.; Lin, G.; Campiglia, P.; Novellino, E.; Hruby, V. J. Design and microwave-assisted synthesis of novel macrocyclic peptides active at melanocortin receptors: discovery of potent and selective hMC5R receptor antagonists. J. Med. Chem. 2008, 51, 2701−2707. (36) Horne, W. S.; Olsen, C. A.; Beierle, J. M.; Montero, A.; Ghadiri, M. R. Probing the bioactive conformation of an archetypal natural product HDAC inhibitor with conformationally homogeneous triazole-modified cyclic tetrapeptides. Angew. Chem., Int. Ed. 2009, 48, 4718−4724. (37) Lee, Y.; Lim, H. S. Skp2 Inhibitors: Novel Anticancer Strategies. Curr. Med. Chem. 2016, 23, 2363−2379. (38) Oh, M.; Lee, J. H.; Moon, H.; Hyun, Y. J.; Lim, H. S. A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53Mediated Apoptosis. Angew. Chem., Int. Ed. 2016, 55, 602−606. (39) Figliozzi, G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.; Zuckermann, R. N. Synthesis of N-substituted glycine peptoid libraries. Methods Enzymol. 1996, 267, 437−447. (40) Peraro, L.; Zou, Z.; Makwana, K. M.; Cummings, A. E.; Ball, H. L.; Yu, H.; Lin, Y.-S.; Levine, B.; Kritzer, J. A. Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy. J. Am. Chem. Soc. 2017, 139, 7792−7802. (41) Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lokey, R. S.; Jacobson, M. P. Conformational Flexibility, Internal Hydrogen Bonding, and Passive Membrane Permeability: Successful in Silico Prediction of the Relative Permeabilities of Cyclic Peptides. J. Am. Chem. Soc. 2006, 128, 14073−14080. (42) Rezai, T.; Yu, B.; Millhauser, G. L.; Jacobson, M. P.; Lokey, R. S. Testing the Conformational Hypothesis of Passive Membrane Permeability Using Synthetic Cyclic Peptide Diastereomers. J. Am. Chem. Soc. 2006, 128, 2510−2511. F

DOI: 10.1021/acscombsci.7b00194 ACS Comb. Sci. XXXX, XXX, XXX−XXX