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Article

Conformational Analysis and Organocatalytic Activity of Helical Stapled Peptides Containing α-Carbocyclic α,α-Disubstituted α-Amino Acids

by
Akihiro Iyoshi
1,
Atsushi Ueda
1,*,
Tomohiro Umeno
1,
Takuma Kato
2,
Kazuhiro Hirayama
1,
Mitsunobu Doi
2 and
Masakazu Tanaka
1,*
1
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
2
Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Osaka 569-1094, Japan
*
Authors to whom correspondence should be addressed.
Molecules 2024, 29(18), 4340; https://doi.org/10.3390/molecules29184340
Submission received: 25 July 2024 / Revised: 10 September 2024 / Accepted: 11 September 2024 / Published: 12 September 2024
(This article belongs to the Section Organic Chemistry)
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Abstract

:
Conformational freedom-restricted peptides, such as stapled peptides, play a crucial role in the advancement of functional peptide development. We synthesized stapled octapeptides using α-carbocyclic α,α-disubstituted α-amino acids, particularly 3-allyloxy-1-aminocyclopentane-1-carboxylic acid, as the crosslink motifs. The organocatalytic capabilities of the synthesized stapled peptides were assessed in an asymmetric nucleophilic epoxidation reaction because the catalytic activities are known to be proportional to α-helicity. Despite incorporating side-chain crosslinks, the enantioselectivities of the epoxidation reaction catalyzed by stapled octapeptides were found to be comparable to those obtained using unstapled peptides. Interestingly, the stapled peptides using α-carbocyclic α,α-disubstituted α-amino acids demonstrated higher reactivities and stereoselectivities (up to 99% ee) compared to stapled peptides derived from (S)-α-(4-pentenyl)alanine, a commonly used motif for stapled peptides. These differences could be attributed to the increased α-helicity of the former stapled peptide in contrast to the latter, as evidenced by the X-ray crystallographic structures of their N-tert-butoxycarbonyl derivatives.

1. Introduction

Conformationally restricted peptides typically adopt specific secondary structures, such as α-helices, β-turns, or β-sheets, which are advantageous for the development of peptide foldamers intended for use as organocatalysts [1,2,3,4]. For example, helical peptides demonstrate efficacy as organocatalysts for asymmetric reactions such as epoxidation [5,6,7,8,9,10,11,12,13,14,15,16,17], Michael addition [18,19,20,21,22,23,24], and transphosphorylation [25,26]. Similarly, β-turn peptides can serve as organocatalysts in asymmetric reactions, including kinetic resolution [27], conjugate addition [28,29], and epoxidation [30,31]. Hence, synthesizing peptides with stable secondary structures holds paramount importance in the field of peptide catalyst development. The Juliá–Colonna epoxidation is an asymmetric nucleophilic epoxidation of chalcones catalyzed by poly(L-α-amino acids) under triphasic and biphasic conditions [5,6,7]. The stereoselectivity observed in the Juliá–Colonna epoxidation correlates directly with the α-helical content of the catalysts [6,9,11]. Incorporating α,α-disubstituted α-amino acids (dAAs) into peptides not only enhances the helicity of the peptide secondary structure but also augments their activity [32,33,34,35,36,37,38,39,40]. Takagi and Ohkata et al. developed oligo-L-leucine helical peptide catalysts containing 2-aminoisobutyric acid (Aib), rendering them soluble in organic solvents [10]. Consequently, in prior work, we achieved the enantioselective Juliá–Colonna-type epoxidation of α,β-unsaturated ketones catalyzed by 5 mol % L-leucine-based nonapeptides incorporating α-carbocyclic dAA, (1S,3S)-1-amino-3-methoxycyclopentane-1-carboxylic acid [Ac5cOM], yielding 96–98% ee of epoxides [14]. Additionally, introducing a crosslink on the peptide side-chain presents another option to stabilize secondary structures. All-hydrocarbon stapling, as developed by Verdine et al., can enhance the α-helicity of peptides through ring-closing metathesis reactions involving (S)-α-(4-pentenyl)alanine residues at the i, i + 4 or i, i + 7 positions [41]. Hence, stapled peptides offer potential as organocatalysts. For example, Demizu et al. illustrated that a hydrocarbon-stapled peptide linking O-allyl-L-homoserine residues at the i, i + 4 position could enhance the enantioselectivity of the Juliá–Colonna-type epoxidation reaction [15,16]. Moreover, a side-chain tethered α-helical peptide linking cis-4-allyloxy-L-proline and O-allyl-L-serine at the i, i + 1 position served as an organocatalyst for the Michael addition of 1-methylindole to the α,β-unsaturated aldehyde, with improved conversion observed following the introduction of the side-chain staple [42,43]. These findings motivated us to explore the development of helical peptides containing α-carbocyclic dAAs incorporating side-chain hydrocarbon stapling motifs [44,45]. Herein, we report the synthesis of α-helical peptides containing α-carbocyclic dAAs, specifically 3-allyloxy-1-aminocyclopentane-1-carboxylic acids (Ac5cOAll), along with their secondary structures and organocatalytic activities in the asymmetric epoxidation of α,β-unsaturated ketones. This report also aimed to compare the structural and catalytic features of the stapled peptides incorporating Ac5cOAll and (S)-α-(4-pentenyl)alanine, a commonly used peptide stapling motif [46,47].

2. Results and Discussion

Because N-terminal-free peptide catalysts demonstrate higher catalytic activity than N-protected peptide catalysts in the nucleophilic epoxidation reactions [14], we chose N-terminal-free peptides 1a′–1e′ and 2a′–2e′ as catalysts in this study (Scheme 1). The synthesis of unstapled peptide catalysts 1a′–1d′ involved the tert-butoxycarbonyl (Boc) deprotection of previously reported octapeptides 1a–1d under acidic conditions [45]. The ring-closing metathesis reaction of peptide 1a, utilizing a second-generation Grubbs catalyst, followed by hydrogenation of the resultant double bond, yielded stapled peptide 2a in 93% yield in two steps. Similarly, stapled peptides 2b–2d, characterized by different stereochemical patterns of dAAs, were also synthesized from 1b to 1d, in 75–30% yields over two steps, with the recovery of starting materials 1b–1d (24–58%) in the first step. Finally, the stapled peptide catalysts 2a′–2d′ were obtained after Boc deprotection in yields ranging from 96% to quantitative yields. Stapled peptide 2e, derived from 1e [45], which contains acyclic dAAs of (S)-α-(4-pentenyl)alanine, was synthesized in a manner similar to that described for the synthesis of 2a, in 43% yield over two steps. Boc deprotection of peptides 1e and 2e yielded peptide catalysts 1e′ and 2e′, respectively, in quantitative yield.
Next, the synthesized peptide catalysts, denoted as 1a′–1e′ and 2a′–2e′, underwent assessment in the asymmetric epoxidation of chalcone 3a (Table 1). The reaction utilized a 5 mol % peptide catalyst with a 1.1 equivalent of urea hydrogen peroxide and 5.6 equivalent of DBU. All reactions, except for those involving peptide 2e′, progressed quantitatively, yielding trans-epoxide 4a. Enantioselectivities of the reactions were comparable despite the presence or absence of the side-chain crosslink in the catalysts (entries 2, 4, 6, 8, 10 vs. entries 1, 3, 5, 7, 9). Notably, reactions catalyzed by the acyclic dAA-containing peptide 1e′ and its stapled derivative 2e′ resulted in reduced enantiomeric excesses of 89% ee and 90% ee, respectively (entries 9 and 10). These results indicate that the introduction of dAAs to peptide catalysts significantly influences the stereoselectivity compared to the introduction of the side-chain crosslink. Moreover, peptides containing α-carbocyclic dAAs demonstrate greater suitability than peptides incorporating acyclic dAAs for this reaction.
To investigate the impact of α-carbocyclic dAAs in comparison to acyclic dAAs on the catalytic activities, various α,β-unsaturated ketones were subjected to asymmetric epoxidation reactions in the presence of catalysts 2a′ or 2e′ (Figure 1). Substituting the α′-phenyl group with a 2-furyl group in the substrate resulted in a slight enhancement of enantioselectivities to 98% ee with 2a′ and 94% ee with 2e′, yielding epoxide 4b. Despite the tert-butyl substitution in substrate 3c leading to moderate conversion compared to other substrates, the enantioselectivity of the reaction with 2a′ (99% ee) remained higher than that with 2e′ (93% ee). Introducing a 4-chloro group on the β-phenyl group of the chalcone reduced the ee values of product 4d (88% ee with 2a′ and 65% ee with 2e′). Conversely, incorporating a 4-methoxy group yielded epoxide 4e with exceptional enantioselectivities of 99% ee with 2a′ and 97% ee with 2e′. Overall, the α-carbocyclic dAAs-containing peptide 2a′ demonstrated higher yields and enantioselectivities across all substrates 3a–3e compared to the acyclic dAAs-containing peptide 2e′.
Given the significant influence of peptide catalysts’ secondary structure on the stereochemical outcomes of epoxidation reactions, a thorough conformational investigation of the peptides was conducted. Specifically, X-ray crystallographic analysis was employed to examine peptides 2a and 2e following recrystallization through slow evaporation of solvents (MeOH/H2O for 2a and MeOH/EtOAc/n-hexane for 2e) at room temperature. Figure 2 provides an overview of the X-ray crystallographic structures of peptides 2a and 2e. The crystal structure of peptide 2a was resolved in the space group of P21, while that of peptide 2e was resolved in the space group of P212121, containing three crystallographically independent molecules labeled A, B, and C in the asymmetric unit (Figures S1–S3, Tables S1–S6) [48]. Peptide 2a demonstrated the presence of four consecutive intramolecular hydrogen bonds [N(n + 4)H···O=C(n)] of the i ← i + 4 type, indicating the existence of an α-helical structure (Table S2), while peptide 2e (Mol A) exhibited two sets of consecutive intramolecular hydrogen bonds [N(n + 4)H···O=C(n)] of the i ← i + 4 type at the N-terminus and the i ← i + 3 type at the C-terminus. This suggests a preference for an α-helical structure at the N-terminus and a 310-helical structure at the C-terminus. Additionally, the torsion angles provided insights into the secondary structure of the peptides. The ideal values of the torsion angles for the right-handed α-helix are ϕ = −63° and ψ = −42°, while for the right-handed 310-helix, they are typically ϕ = −57° and ψ = −30° [48,49]. The average torsion angles observed in peptide 2e were calculated as follows: avg. (ϕ1–ϕ7) = −74.3° (mol A), −72.9° (mol B), and −73.5° (mol C) and avg. (ψ1–ψ7) = −31.8° (mol A), −27.8° (mol B), and −32.3° (mol C), indicating a preference for the right-handed 310-helical structure in peptide 2e. Conversely, peptide 2a demonstrated a higher preference for the right-handed α-helical structure, with averaged torsion angles of avg. (ϕ1–ϕ7) = −68.2° and avg. (ψ1–ψ7) = −35.9. In the epoxidation reactions detailed in Figure 1, the α-carbocyclic dAAs-containing peptide 2a′ exhibited higher enantioselectivities compared to the acyclic dAAs-containing peptide 2e′. This difference in enantioselectivities could be attributed to their respective preferred peptide secondary structures. In particular, the α-helical structure of the α-carbocyclic dAAs-containing peptide 2a′ is more effective in asymmetric induction compared to the 310-helical structure of the acyclic dAAs-containing peptide 2e′ [6,9,11].
The circular dichroism (CD) spectra of unstapled peptides 1a–1e and stapled peptides 2a–2e were analyzed in an aqueous solution containing 10% MeOH (Figure 3 and Figure S4). The CD spectra indicated that all stapled peptides 2a–2e displayed an increased right-handed helicity, accompanied by the presence of α- and 310-helices, in contrast to the corresponding unstapled peptides 1a–1e. Consequently, the introduction of stapled helical peptide organocatalysts could offer advantages for reactions in aqueous media by stabilizing the helical secondary structures of the peptides.
Based on the Roberts model [50] and X-ray crystallographic analysis of peptide 2a, a plausible mechanism for the asymmetric epoxidation of chalcone 3a catalyzed by peptide catalyst 2a′ is illustrated in Figure 4. Initially, a reversible nucleophilic addition of hydroperoxide to chalcone 3a leads to the formation of (R)-configured peroxyenolate. The peroxyenolate can then bind to free amidic protons at N(2)H and N(3)H with enolate oxygen (red) and at N(4)H with hydroperoxide oxygen (blue). Finally, the enolate attacks the hydroperoxide moiety, releasing (2R,3S)-epoxide 4a.

3. Materials and Methods

3.1. General Procedure and Method

The melting points were measured using an AS ONE melting point apparatus ATM-01 (AS ONE Corporation, Osaka, Japan) and were uncorrected. Optical rotations were measured using CHCl3 or MeOH as solvents on a JASCO DIP-370 polarimeter (JASCO Corporation, Tokyo, Japan). 1H NMR and 13C{1H} NMR spectra were recorded on JEOL JNM-AL-400 (400 MHz and 100 MHz; JEOL Ltd., Tokyo, Japan), Varian NMR System 500PS SN (500 MHz and 125 MHz; Agilent Inc., Santa Clara, USA), or Varian Gemini 300 (300 MHz; Agilent Inc., Santa Clara, USA) spectrometers. Chemical shifts (δ) are reported in parts per million (ppm). For 1H NMR spectra (CDCl3), tetramethylsilane was used as the internal reference (0.00 ppm), while the central solvent peak was used as the reference (77.0 ppm in CDCl3) for 13C{1H} NMR spectra. Infrared (IR) spectra were acquired using a Shimadzu IRAffinity-1 FT-IR spectrophotometer (Shimadzu Corporation, Kyoto, Japan). High-resolution mass spectra (HRMS) were obtained on a JEOL JMS-T100TD (JEOL Ltd., Tokyo, Japan) using electrospray ionization (ESI) or direct analysis in real-time (DART) ionization in time-of-flight (TOF) mode. Circular dichroism (CD) spectra were measured with a JASCO J-725N spectropolarimeter (JASCO Corporation, Tokyo, Japan) using a 1.0 mm path length cell. Analytical and semi-preparative thin layer chromatography (TLC) was performed with Merck Millipore pre-coated TLC plates (MilliporeSigma, Burlington, United States), silica gel 60 F254, with layer thicknesses of 0.25 and 0.50 mm, respectively. The compounds were observed under UV light at 254 nm and then visualized by staining with iodine, p-anisaldehyde, or phosphomolybdic acid. Flash and gravity column chromatography separations were performed on Kanto Chemical (Kanto Chemical Co.,Inc., Tokyo, Japan) silica gel 60N and spherical neutral particles with sizes of 40–50 μm and 63–210 μm, respectively. Analytical high-performance liquid chromatography (HPLC) was carried out with a UV spectrophotometric detector (254 nm), to which a 4.6 × 250 mm size chiral column (Daicel Chiralpak AD-H or IB N-5, Daicel Corporation, Osaka, Japan) was attached. All moisture-sensitive reactions were carried out in an inert atmosphere. The reagents and solvents were of commercial grade and were used as supplied, unless otherwise noted. Structures of the second-generation Grubbs catalyst and olefin-tethered α-carbocyclic and acyclic dAAs are shown in Figure 5. Peptides 1a–1e were prepared by liquid-phase peptide synthesis according to a previous report [45]. The α,β-unsaturated ketones 4a–4e were prepared according to a previous report [14].

3.2. General Procedure for Peptide-Catalyzed Asymmetric Epoxidation

A mixture of the peptide catalyst (5.23 mg for 1a′–1d′, 4.95 mg for 1e′, 5.10 mg for 2a′–2d′, 4.82 mg for 2e′, 5.00 μmol) and chalcone (0.100 mmol) was dissolved in THF (0.50 mL). Urea–hydrogen peroxide (10.3 mg, 0.110 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 0.0836 mL, 0.560 mmol) were added to the mixture at 0 °C, and the mixture was gradually warmed to room temperature. After stirring for 24 h, the reaction mixture was diluted with EtOAc and washed with 10% aqueous Na2S2O3. The organic layer was concentrated under vacuum to obtain a residue, which was purified by flash column chromatography on silica gel to yield the desired epoxide.

3.3. Synthesis of Stapled Peptides 2a–2e

Boc-(L-Leu)3-(1S,3S)-Ac5c3OT-(L-Leu)3-(1R,3S)-Ac5c3OT′-OMe (2a, T,T′ = butyl tether): to a solution of octapeptide 1a (102 mg, 89.1 μmol) in CH2Cl2 (17 mL), the second-generation Grubbs catalyst (14.9 mg, 17.5 μmol) was added at room temperature. The resultant mixture was stirred at room temperature for 4 h before being passed through a short plug of silica gel and eluted with EtOAc. Evaporation of the solvent gave a residue, which was used for the next step without further purification. The crude product was dissolved in THF (10 mL), and 5% palladium on carbon (25 mg) was added under a nitrogen atmosphere. The reaction mixture was vigorously stirred overnight at room temperature under a hydrogen atmosphere. The resulting black suspension was filtered through a Celite pad and washed with THF. After removing the solvent under vacuum, the residue was purified by flash column chromatography on silica gel (90% EtOAc in n-hexane) to yield the desired product 2a (92.4 mg, 93% in two steps) as a white amorphous solid. Mp: 248–250 °C. [α]22D: +6.3 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.67 (d, J = 8.4 Hz, 1H), 7.58 (br s, 1H), 7.53 (br s, 1H), 7.50 (d, J = 5.8 Hz, 1H), 7.27 (d, J = 6.1 Hz, 1H), 7.25 (d, J = 4.5 Hz, 1H), 7.17 (d, J = 2.5 Hz, 1H), 6.07 (br s, 1H), 4.46 (ddd, J = 12.0, 8.4, 3.5 Hz, 1H), 4.24 (ddd, J = 11.0, 6.0, 3.3 Hz, 1H), 4.17–4.02 (m, 3H), 4.02–3.95 (m, 1H), 3.95–3.86 (m, 2H), 3.68 (s, 3H), 3.44–3.32 (m, 1H), 3.32–3.23 (m, 1H), 3.22–3.04 (m, 2H), 2.70 (s, 1H), 2.63 (dd, J = 14.5, 6.0 Hz, 1H), 2.56 (dd, J = 13.5, 6.1 Hz, 1H), 2.32–2.24 (m, 2H), 2.08–1.56 (m, 26H), 1.49 (s, 9H), 1.47–1.33 (m, 3H), 1.02–0.85 (m, 36H). 13C{1H} NMR (125 MHz, CDCl3) δ: 175.2, 175.1, 174.93, 174.90, 174.1, 173.7, 173.5, 173.3, 157.3, 81.1, 79.9, 78.6, 68.3, 67.3, 65.2, 64.9, 55.5, 54.9, 54.6, 54.4, 54.1, 52.3, 51.6, 43.7, 40.6, 40.1, 40.0, 39.8, 39.7, 39.4 (2C), 35.5, 33.2, 32.4, 31.9, 28.2 (3C), 27.5, 26.2, 25.1, 24.9, 24.8 (2C), 24.7, 24.5, 23.5, 23.4, 23.3, 22.8, 22.7, 22.5, 21.8 (2C), 21.2, 21.0 (2C), 20.8. IR (KBr): 3329, 2959, 1655, 1535 cm–1. HRMS (ESI) m/z: [M + Na]+ calcd for C58H102N8O13Na, 1141.7459; found, 1141.7466. CCDC 2341454 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/ (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected]).
Boc-(L-Leu)3-(1R,3R)-Ac5c3OT-(L-Leu)3-(1R,3S)-Ac5c3OT′-OMe (2b, T,T′ = butyl tether): compound 2b (76.7 mg) was synthesized from octapeptide 1b (101 mg, 88.2 μmol) in 75% yield in two steps under the same conditions as described for the synthesis of compound 2a. The starting material 1b (24.2 mg, 24%rsm) was recovered after the first step. Eluent for column: 90% EtOAc in n-hexane. White solid. Mp: 250–252 °C. [α]24D: +21.5 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.59 (br s, 1H), 7.46 (br s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.33–7.27 (m, 2H), 7.25 (d, J = 5.1 Hz, 1H), 7.23 (d, J = 6.0 Hz, 1H), 6.23 (br s, 1H), 4.58–4.47 (m, 1H), 4.29 (ddd, J = 10.8, 6.4, 4.3 Hz, 1H), 4.15–3.94 (m, 5H), 3.90 (td, J = 7.7, 2.9 Hz, 1H), 3.68 (s, 3H), 3.51–3.43 (m, 1H), 3.43–3.25 (m, 3H), 3.17 (d, J = 15.0 Hz, 1H), 2.92 (dd, J = 14.4, 6.9 Hz, 1H), 2.66 (br s, 1H), 2.48 (dt, J = 13.3, 7.6 Hz, 1H), 2.41–2.30 (m, 1H), 2.13 (dd, J = 14.5, 3.6 Hz, 1H), 2.10–2.01 (m, 2H), 1.99–1.56 (m, 26H), 1.49 (s, 9H), 1.03–0.83 (m, 36H). 13C{1H} NMR (125 MHz, CDCl3) δ: 175.2, 175.1, 174.8, 174.1 (2C), 174.0, 173.6, 173.5, 157.5, 81.0, 79.6, 79.4, 66.8, 66.5, 66.0, 64.7, 55.6, 55.2, 54.9, 54.7, 53.7, 52.4, 52.1, 41.2, 41.1, 40.5, 40.14, 40.11, 40.0, 39.7, 39.6, 35.9, 35.4, 32.8, 30.5, 28.3 (3C), 27.0, 26.7, 24.91, 24.85, 24.78, 24.72, 24.6, 24.5, 23.3, 23.2, 23.1, 22.5, 22.31, 22.27, 22.25, 22.0, 21.5, 21.3, 21.2, 21.0. IR (KBr): 3341, 2959, 1655, 1535 cm–1. HRMS (ESI) m/z: [M + Na]+ calcd for C58H102N8O13Na, 1141.7459; found, 1141.7466.
Boc-(L-Leu)3-(1S,3S)-Ac5c3OT-(L-Leu)3-(1S,3R)-Ac5c3OT′-OMe (2c, T,T′ = butyl tether): compound 2c (48.4 mg) was synthesized from octapeptide 1c (102 mg, 89.0 μmol) in 49% yield in two steps under the same conditions as described for the synthesis of compound 2a. The starting material 1c (42.7 mg, 42%rsm) was recovered after the first step. Eluent for column: 90% EtOAc in n-hexane. White solid. Mp: 133–135 °C. [α]23D: +0.4 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.57 (br s, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.46 (br s, 1H), 7.40 (d, J = 6.9 Hz, 1H), 7.23 (d, J = 5.9 Hz, 1H), 7.18 (d, J = 4.8 Hz, 1H), 7.09 (d, J = 3.9 Hz, 1H), 5.96 (d, J = 2.9 Hz, 1H), 4.40 (ddd, J = 10.6, 8.3, 5.0 Hz, 1H), 4.28 (ddd, J = 11.0, 6.7, 3.6 Hz, 1H), 4.17–4.09 (m, 1H), 4.08–4.02 (m, 2H), 3.97–3.87 (m, 3H), 3.70 (s, 3H), 3.39–3.32 (m, 2H), 3.26–3.20 (m, 1H), 3.16 (td, J = 8.5, 3.8 Hz, 1H), 3.03 (dt, J = 13.0, 8.5 Hz, 1H), 2.75 (ddd, J = 12.6, 8.1, 3.8 Hz, 1H), 2.53 (d, J = 14.5 Hz, 1H), 2.35 (dd, J = 14.7, 5.4 Hz, 1H), 2.33–2.22 (m, 2H), 2.13–2.03 (m, 2H), 2.01–1.92 (m, 2H), 1.86–1.73 (m, 13H), 1.70–1.60 (m, 11H), 1.49 (s, 9H), 0.99–0.88 (m, 36H). 13C{1H} NMR (125 MHz, CDCl3) δ: 175.7, 175.0, 174.9, 174.1, 174.0, 173.7, 173.5, 173.3, 157.3, 81.1, 79.8, 78.2, 67.8, 66.9, 65.5, 64.4, 55.5, 54.72, 54.69, 54.4, 53.7, 53.0, 52.4, 44.32, 44.26, 40.8, 40.10, 40.07, 39.7, 39.61, 39.56, 35.5, 32.9, 31.7 (2C), 28.2 (3C), 27.7, 26.5, 25.2, 24.95, 24.86, 24.77, 24.71, 24.6, 23.5, 23.3, 23.1, 22.8, 22.6, 22.5, 22.0, 21.9, 21.5, 21.3, 21.1, 20.9. IR (KBr): 3337, 2959, 1659, 1535 cm–1. HRMS (ESI) m/z: [M + Na]+ calcd for C58H102N8O13Na, 1141.7459; found, 1141.7468.
Boc-(L-Leu)3-(1R,3R)-Ac5c3OT-(L-Leu)3-(1S,3R)-Ac5c3OT′-OMe (2d, T,T′ = butyl tether): compound 2d (35.3 mg) was synthesized from octapeptide 1d (116 mg, 101 μmol) in 30% yield in two steps under the same conditions as described for the synthesis of compound 2a. The starting material 1d (67.8 mg, 58%rsm) was recovered after the first step. Eluent for column: 85% EtOAc in n-hexane. White solid. Mp: 252–254 °C dec. [α]22D: +12.4 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.78 (br s, 1H), 7.51 (br s, 1H), 7.36 (d, J = 6.1 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.31–7.27 (m, 2H), 7.23 (d, J = 8.0 Hz, 1H), 6.17 (br s, 1H), 4.47–4.40 (m, 2H), 4.04–3.94 (m, 4H), 3.92–3.84 (m, 2H), 3.75 (s, 3H), 3.63–3.51 (m, 2H), 3.37–3.29 (m, 2H), 3.27–3.21 (m, 1H), 3.08 (d, J = 14.9 Hz, 1H), 2.73–2.66 (m, 1H), 2.35 (d, J = 14.5 Hz, 2H), 2.20 (br s, 1H), 2.16–2.10 (m, 2H), 2.05–1.97 (m, 3H), 1.92–1.87 (m, 4H), 1.81–1.72 (m, 12H), 1.66–1.61 (m, 7H), 1.49 (s, 9H), 0.96–0.88 (m, 36H). 13C{1H} NMR (125 MHz, CDCl3) δ: 175.7, 175.5, 175.1, 174.4, 174.0, 173.0, 172.7, 172.6, 157.4, 81.0, 80.2, 79.7, 67.7, 67.6, 66.4, 64.6, 55.6, 54.9, 54.6, 54.4, 53.5, 52.8, 52.6, 43.2, 41.6, 41.2, 40.2, 40.1 (2C), 39.6, 39.2, 36.0, 35.9, 33.2, 32.8, 28.3 (3C), 27.3, 26.5, 25.0, 24.9, 24.81, 24.76, 24.74, 24.6, 23.4, 23.3, 23.0, 22.7, 22.5, 22.4, 22.2, 22.0, 21.8, 21.4, 21.2, 20.8. IR (KBr): 3341, 2959, 1655, 1535 cm–1. HRMS (ESI) m/z: [M + Na]+ calcd for C58H102N8O13Na, 1141.7459; found, 1141.7468.
Boc-(L-Leu)3-(S)-Ala(4-BteT)-(L-Leu)3-(S)-Ala(4-BteT′)-OMe (2e: T,T′ = tether): to a solution of octapeptide 1a (147 mg, 0.135 mmol) in CH2Cl2 (27 mL), the second-generation Grubbs catalyst (22.9 mg, 27.0 μmol) was added at room temperature, and the resultant solution was stirred at room temperature for 24 h. The reaction mixture was passed through a short plug of silica gel, eluted with CHCl3 (discarded), followed by EtOAc (collected), and concentrated. The 1H NMR spectrum of the crude product revealed 49% conversion with a 44:56 ratio of E and Z isomers [E isomer: Rf = 0.43 (80% EtOAc in n-hexane); Z isomer: Rf = 0.30 (80% EtOAc in n-hexane)]. The residue was purified by flash column chromatography on silica gel (60–80% EtOAc in n-hexane) to produce crude product S-1, which was used for the next step without further purification. A suspension of the above octapeptide S-1 and 10% Pd/C (36 mg) in MeOH (1 mL) was stirred at room temperature for 2 d under a hydrogen atmosphere. After filtration through a Celite pad and concentration under vacuum, the obtained residue was purified by flash column chromatography on silica gel (70% EtOAc in n-hexane) to yield 2e (62.4 mg, 43% in two steps) as a white solid. Rf = 0.37 (80% EtOAc in n-hexane). Mp: 241–243 °C. [α]25D: –12.0 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.49 (d, J = 7.2 Hz, 1H), 7.45 (d, J = 4.8 Hz, 1H), 7.42 (d, J = 9.4 Hz, 1H), 7.38 (br s, 1H), 7.29 (d, J = 5.4 Hz, 1H), 7.22 (br s, 1H), 6.91 (d, J = 4.2 Hz, 1H), 5.77 (d, J = 2.8 Hz, 1H), 4.55 (td, J = 9.3, 6.0 Hz, 1H), 4.34 (ddd, J = 11.3, 7.2, 3.6 Hz, 1H), 4.09–4.03 (m, 1H), 4.03–3.96 (m, 2H), 3.94 (ddd, J = 8.9, 6.1, 2.8 Hz, 1H), 3.73 (s, 3H), 1.95–1.54 (m, 20H), 1.61 (s, 6H), 1.49 (s, 9H), 1.49–1.16 (m, 14H), 1.02–0.93 (m, 21H), 0.93–0.84 (m, 15H). 13C{1H} NMR (125 MHz, CDCl3) δ: 176.4, 175.2, 174.3, 173.6, 173.42, 173.39, 173.34, 172.7, 157.2, 81.3, 60.0, 59.6, 55.3, 54.8, 54.7, 54.4, 53.4, 52.9, 52.4, 41.4, 40.2, 40.1, 39.79, 39.76, 39.6, 38.8, 38.7, 28.2 (3C), 26.8, 26.6, 26.5, 26.4, 25.00, 24.97, 24.95, 24.89, 24.7, 24.6, 23.55, 23.50, 23.0, 22.88, 22.85, 22.77, 22.5, 22.4, 21.84, 21.77, 21.5, 21.4, 21.2, 21.1, 20.8, 20.6. IR (KBr): 3325, 2957, 1755, 1647 cm–1. HRMS (ESI) m/z: [M + Na]+ calcd for C56H102N8O11Na, 1085.7566; found, 1085.7547. CCDC 2341476 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/ (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected]).

3.4. Synthesis of Unstapled Peptide Catalysts 1a′–1e′ and Stapled Peptide Catalysts 2a′–2e′

H-(L-Leu)3-(1S,3S)-Ac5c3OAll-(L-Leu)3-(1R,3S)-Ac5c3OAll-OMe (1a′): to a solution of octapeptide 1a (20.0 mg, 17.5 μmol) in CH2Cl2 (0.175 mL), trifluoroacetic acid (17.5 μL) was added at room temperature and stirred at the same temperature for 19 h. Additional trifluoroacetic acid (17.5 μL) was added to the reaction mixture, which was then stirred for 7 h. The reaction was quenched by adding sat. NaHCO3 aq (1.5 mL), and the mixture was extracted with EtOAc (2 mL × 5). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel (5% MeOH in CHCl3) to give the title compound 1a′ (17.5 mg) in 96% yield as a white solid. 1H NMR (500 MHz, CDCl3) δ: 8.23 (br s, 1H), 7.73 (s, 1H), 7.55 (s, 1H), 7.48 (d, J = 6.4 Hz, 1H), 7.39 (d, J = 5.1 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 3.5 Hz, 1H), 5.93–5.83 (m, 2H), 5.26 (dq, J = 15.1, 1.7 Hz, 1H), 5.23 (dq, J = 15.1, 1.7 Hz, 1H), 5.14 (dq, J = 10.4, 1.3 Hz, 1H), 5.10 (dq, J = 10.4, 1.3 Hz, 1H), 4.48–4.40 (m, 1H), 4.25–4.20 (m, 1H), 4.19–4.14 (m, 1H), 4.12–4.03 (m, 2H), 4.03–3.92 (m, 5H), 3.88 (ddt, J = 12.8, 5.7, 1.5 Hz, 1H), 3.67 (s, 3H), 3.48 (dd, J = 9.7, 4.1 Hz, 1H), 2.85–2.75 (m, 2H), 2.63 (dd, J = 13.8, 6.4 Hz, 1H), 2.40–2.33 (m, 1H), 2.16–2.09 (m, 1H), 2.02 (dd, J = 14.1, 5.3 Hz, 1H), 1.99–1.85 (m, 7H), 1.85–1.73 (m, 10H), 1.73–1.63 (m, 6H), 1.61 (dd, J = 8.3, 5.4 Hz, 1H), 1.57–1.49 (m, 1H), 1.40 (ddd, J = 14.2, 9.7, 4.6 Hz, 1H), 1.01 (d, J = 6.2 Hz, 3H), 0.99–0.93 (m, 18H), 0.92–0.89 (m, 9H), 0.88 (d, J = 6.7 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) m/z: [M + Na]+ calcd for C55H96N8O11Na, 1067.7091; found, 1067.7089.
H-(L-Leu)3-(1R,3R)-Ac5c3OAll-(L-Leu)3-(1R,3S)-Ac5c3OAll-OMe (1b′): compound 1b′ (18.2 mg) was synthesized from octapeptide 1b (20.0 mg, 17.5 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 5% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.22 (br s, 1H), 7.65 (s, 1H), 7.56 (s, 1H), 7.49 (d, J = 6.1 Hz, 1H), 7.37 (d, J = 5.3 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 2.8 Hz, 1H), 5.92–5.81 (m, 2H), 5.27–5.19 (m, 2H), 5.15–5.07 (m, 2H), 4.47–4.39 (m, 1H), 4.25–4.18 (m, 1H), 4.12–4.03 (m, 2H), 4.00–3.91 (m, 5H), 3.91–3.85 (m, 2H), 3.67 (s, 3H), 3.46 (dd, J = 8.9, 4.4 Hz, 1H), 2.80 (dd, J = 14.1, 6.7 Hz, 1H), 2.64 (dd, J = 13.9, 7.1 Hz, 1H), 2.41–2.29 (m, 2H), 2.20–2.04 (m, 4H), 2.04–1.93 (m, 4H), 1.92–1.66 (m, 18H), 1.62–1.51 (m, 1H), 1.38 (ddd, J = 14.1, 9.4, 5.0 Hz, 1H), 1.01 (d, J = 6.4 Hz, 3H), 0.99–0.93 (m, 21H), 0.92–0.86 (m, 12H). HRMS (ESI) m/z: [M + Na]+ calcd for C55H96N8O11Na, 1067.7091; found, 1067.7097.
H-(L-Leu)3-(1S,3S)-Ac5c3OAll-(L-Leu)3-(1S,3R)-Ac5c3OAll-OMe (1c′): compound 1c′ (18.2 mg) was synthesized from octapeptide 1c (20.0 mg, 17.5 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 5% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.28 (br s, 1H), 7.76 (s, 1H), 7.66 (s, 1H), 7.50 (d, J = 6.4 Hz, 1H), 7.45 (d, J = 5.3 Hz, 1H), 7.34 (d, J = 6.8 Hz, 1H), 7.13 (br s, 1H), 5.94–5.84 (m, 2H), 5.29–5.21 (m, 2H), 5.17–5.10 (m, 2H), 4.45–4.36 (m, 1H), 4.25–4.14 (m, 2H), 4.11–4.04 (m, 2H), 4.01–3.93 (m, 6H), 3.66 (s, 3H), 3.49 (dd, J = 9.4, 4.5 Hz, 1H), 2.87–2.78 (m, 2H), 2.65 (dd, J = 13.8, 6.7 Hz, 1H), 2.32–2.23 (m, 2H), 2.21–2.12 (m, 3H), 2.03 (dd, J = 14.1, 6.0 Hz, 1H), 1.98–1.86 (m, 4H), 1.85–1.75 (m, 8H), 1.74–1.66 (m, 8H), 1.61–1.55 (m, 2H), 1.46–1.37 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H), 0.98–0.92 (m, 18H), 0.91–0.83 (m, 15H). HRMS (ESI) m/z: [M + Na]+ calcd for C55H96N8O11Na, 1067.7091; found, 1067.7083.
H-(L-Leu)3-(1R,3R)-Ac5c3OAll-(L-Leu)3-(1S,3R)-Ac5c3OAll-OMe (1d′): compound 1d′ (15.8 mg) was synthesized from octapeptide 1d (20.0 mg, 17.5 μmol) in 86% yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 5% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.23 (br s, 1H), 7.67 (s, 1H), 7.62 (s, 1H), 7.47 (d, J = 6.1 Hz, 1H), 7.40 (d, J = 5.1 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.07 (br s, 1H), 5.94–5.81 (m, 2H), 5.25 (dq, J = 17.2, 1.7 Hz, 1H), 5.23 (dq, J = 17.2, 1.7 Hz, 1H), 5.12 (dt, J = 10.5, 1.6 Hz, 2H), 4.45–4.37 (m, 1H), 4.25–4.18 (m, 1H), 4.11–4.04 (m, 2H), 4.00–3.92 (m, 5H), 3.88 (ddt, J = 12.7, 5.6, 1.5 Hz, 1H), 3.65 (s, 3H), 3.46 (dd, J = 9.1, 4.7 Hz, 1H), 2.82 (dd, J = 14.1, 7.0 Hz, 1H), 2.69 (dd, J = 14.1, 6.6 Hz, 1H), 2.33–2.23 (m, 2H), 2.23–2.13 (m, 3H), 2.14–1.99 (m, 6H), 1.97–1.90 (m, 1H), 1.90–1.67 (m, 16H), 1.62–1.55 (m, 2H), 1.42–1.35 (m, 1H), 1.00 (d, J = 6.2 Hz, 3H), 0.98–0.92 (m, 21H), 0.92–0.85 (m, 12H). HRMS (ESI) m/z: [M + Na]+ calcd for C55H96N8O11Na, 1067.7091; found, 1067.7090.
H-(L-Leu)3-(S)-Ala(4-Pte)-(L-Leu)3-(S)-Ala(4-Pte)-OMe (1e′): compound 1e′ (18.2 mg) was synthesized from octapeptide 1e (20.0 mg, 18.4 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 5% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.11 (br s, 1H), 7.65 (d, J = 6.5 Hz, 1H), 7.37 (d, J = 5.3 Hz, 1H), 7.31 (br s, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.19 (br s, 1H), 7.00 (br s, 1H), 5.87–5.69 (m, 2H), 5.02–4.89 (m, 4H), 4.48–4.39 (m, 1H), 4.26–4.18 (m, 1H), 4.08–3.98 (m, 2H), 3.97–3.90 (m, 1H), 3.69 (s, 3H), 3.51 (dd, J = 9.5, 4.2 Hz, 1H), 2.38–2.12 (m, 2H), 2.09–2.01 (m, 4H), 2.00–1.86 (m, 2H), 1.86–1.78 (m, 4H), 1.77–1.66 (m, 11H), 1.65–1.51 (m, 3H), 1.48 (s, 3H), 1.47 (s, 3H), 1.44–1.36 (m, 4H), 1.03–0.95 (m, 15H), 0.95–0.92 (m, 6H), 0.91–0.85 (m, 15H). HRMS (ESI) m/z: [M + Na]+ calcd for C53H96N8O9Na, 1011.7192; found, 1011.7193.
H-(L-Leu)3-(1S,3S)-Ac5c3OT-(L-Leu)3-(1R,3S)-Ac5c3OT′-OMe (2a′, T,T′ = butyl tether): compound 2a′ (17.6 mg) was synthesized from octapeptide 2a (20.0 mg, 17.9 μmol) in 96% yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 8% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.34 (br s, 1H), 7.77 (br s, 1H), 7.72–7.47 (m, 3H), 7.46–7.28 (m, 1H), 7.15 (br s, 1H), 4.42–4.27 (m, 1H), 4.24–4.14 (m, 1H), 4.12–3.95 (m, 4H), 3.90 (br s, 1H), 3.70 (s, 3H), 3.62–3.50 (m, 1H), 3.39–3.31 (m, 1H), 3.30–3.24 (m, 1H), 3.20–3.13 (m, 1H), 3.03 (q, J = 10.3 Hz, 1H), 2.64 (dd, J = 14.5, 5.6 Hz, 1H), 2.54–2.46 (m, 1H), 2.43–2.17 (m, 5H), 2.02–1.91 (m, 3H), 1.89–1.80 (m, 5H), 1.79–1.52 (m, 18H), 1.45 (s, 3H), 1.01–0.97 (m, 6H), 0.97–0.91 (m, 18H), 0.91–0.87 (m, 9H), 0.86–0.84 (m, 3H). HRMS (ESI) m/z: [M + Na]+ calcd for C53H94N8O11Na, 1041.6934; found, 1041.6933.
H-(L-Leu)3-(1R,3R)-Ac5c3OT-(L-Leu)3-(1R,3S)-Ac5c3OT′-OMe (2b′, T,T′ = butyl tether): compound 2b′ (18.2 mg) was synthesized from octapeptide 2b (20.0 mg, 17.9 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 8% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.46 (br s, 1H), 8.25–7.75 (m, 5H), 7.66 (br s, 1H), 4.32–4.24 (m, 1H), 4.22–4.14 (m, 1H), 4.08 (s, 1H), 4.00–3.93 (m, 4H), 3.76 (s, 3H), 3.57 (dd, J = 8.9, 5.2 Hz, 1H), 3.48–3.38 (m, 2H), 3.35–3.30 (m, 2H), 3.11 (d, J = 16.1 Hz, 1H), 2.88 (dd, J = 14.6, 7.0 Hz, 1H), 2.46 (dt, J = 13.8, 7.8 Hz, 1H), 2.30–2.24 (m, 1H), 2.10–2.01 (m, 3H), 1.94–1.89 (m, 2H), 1.86–1.79 (m, 7H), 1.77–1.72 (m, 5H), 1.70–1.60 (m, 8H), 1.58–1.51 (m, 4H), 1.44–1.30 (m, 3H), 0.99 (d, J = 6.1 Hz, 3H), 0.95–0.93 (m, 6H), 0.93–0.91 (m, 9H), 0.90–0.88 (m, 9H), 0.88–0.85 (m, 9H). HRMS (ESI) m/z: [M + Na]+ calcd for C53H94N8O11Na, 1041.6934; found, 1041.6936.
H-(L-Leu)3-(1S,3S)-Ac5c3OT-(L-Leu)3-(1S,3R)-Ac5c3OT′-OMe (2c′, T,T′ = butyl tether): compound 2c′ (18.2 mg) was synthesized from octapeptide 2c (20.0 mg, 17.9 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 8% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.44 (1H, br s), 8.16–7.74 (m, 5H), 7.57 (br s, 1H), 4.27–4.19 (m, 1H), 4.19–4.10 (m, 2H), 4.04–3.98 (m, 2H), 3.96–3.91 (m, 2H), 3.77 (s, 3H), 3.70–3.63 (m, 1H), 3.62–3.56 (m, 1H), 3.42–3.33 (m, 2H), 3.28–3.15 (m, 2H), 2.92 (s, 1H), 2.71–2.64 (m, 1H), 2.55 (d, J = 13.9 Hz, 1H), 2.26 (s, 1H), 2.15–2.08 (m, 1H), 2.04–1.99 (m, 1H), 1.92–1.83 (m, 6H), 1.80–1.74 (m, 6H), 1.71–1.64 (m, 6H), 1.63–1.56 (m, 6H), 1.53–1.48 (m, 2H), 1.44–1.35 (m, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.96 (d, J = 6.6 Hz, 6H), 0.94–0.92 (m, 12H), 0.91–0.90 (m, 3H), 0.89–0.86 (m, 12H). HRMS (ESI) m/z: [M + Na]+ calcd for C53H94N8O11Na, 1041.6934; found, 1041.6939.
H-(L-Leu)3-(1R,3R)-Ac5c3OT-(L-Leu)3-(1S,3R)-Ac5c3OT′-OMe (2d′, T,T′ = butyl tether): compound 2d′ (17.9 mg) was synthesized from octapeptide 2d (20.0 mg, 17.9 μmol) in 98% yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 8% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.43 (br s, 1H), 8.19–7.74 (m, 5H), 7.59 (br s, 1H), 4.36–4.11 (m, 3H), 4.04–3.89 (m, 6H), 3.80 (s, 3H), 3.69–3.65 (m, 1H), 3.57–3.50 (m, 2H), 3.35–3.26 (m, 2H), 3.26–3.18 (m, 1H), 3.11 (d, J = 13.6 Hz, 1H), 2.68–2.61 (m, 1H), 2.55–2.40 (m, 1H), 2.32–2.13 (m, 3H), 2.07–1.99 (m, 3H), 1.95–1.90 (m, 2H), 1.81–1.67 (m, 16H), 1.60–1.52 (m, 6H), 0.98 (d, J = 6.1 Hz, 3H), 0.95–0.93 (m, 9H), 0.92–0.90 (m, 12H), 0.89–0.87 (m, 12H). HRMS (ESI) m/z: [M + Na]+ calcd for C53H94N8O11Na, 1041.6934; found, 1041.6939.
H-(L-Leu)3-(S)-Ala(4-BteT)-(L-Leu)3-(S)-Ala(4-BteT′)-OMe (2e′: T,T′ = tether): compound 2e′ (18.1 mg) was synthesized from octapeptide 2e (20.0 mg, 18.8 μmol) in quantitative yield under the same conditions as described for the synthesis of compound 1a′. Eluent for column: 8% MeOH in CHCl3. White solid. 1H NMR (500 MHz, CDCl3) δ: 8.28 (br s, 1H), 8.07–7.68 (m, 3H), 7.67–7.30 (m, 3H), 4.39 (s, 1H), 4.31–4.21 (m, 1H), 4.06–3.89 (m, 3H), 3.76 (s, 3H), 3.53 (dd, J = 9.0, 4.8 Hz, 1H), 2.49–2.15 (m, 3H), 1.99–1.82 (m, 5H), 1.81–1.60 (m, 15H), 1.58 (s, 3H), 1.47 (s, 3H), 1.44–1.17 (m, 13H), 0.99 (d, J = 6.2 Hz, 3H), 0.98–0.91 (m, 18H), 0.91–0.85 (m, 15H). HRMS (ESI) m/z: [M + Na]+ calcd for C51H94N8O9Na, 985.7036; found, 985.7052.

3.5. Synthesis Outlined in Figure 1

(2R,3S)-trans-Epoxy-3-phenyl-1-phenylpropan-1-one (4a): according to the general procedure for peptide-catalyzed asymmetric epoxidation, the reaction of 3a (20.8 mg, 0.100 mmol) with catalyst 2a′ (5.10 mg, 5.00 μmol) yielded 4a (20.9 mg, 93%) as colorless crystals. Eluent for column: 5% EtOAc in n-hexane. Rf = 0.31 (10% EtOAc in n-hexane). [α]19D –191.9 (c 1.00, CHCl3) [Lit., for the antipode, +182.2 (c 1.14, CHCl3)] [51]. 1H NMR (500 MHz, CDCl3) δ: 8.15–7.96 (2H, m), 7.70–7.60 (1H, m), 7.57–7.47 (2H, m), 7.47–7.35 (5H, m), 4.31 (1H, d, J = 2.0 Hz), 4.08 (1H, d, J = 2.0 Hz). HPLC (Chiralpak AD-H, 10% EtOH in n-hexane, flow rate = 1.0 mL/min): tR = 23.4 min (major), tR = 30.0 min (minor), ee = 96%.
(2R,3S)-trans-Epoxy-3-phenyl-1-(2-furyl)propan-1-one (4b): according to the general procedure for peptide-catalyzed asymmetric epoxidation, the reaction of 3b (19.8 mg, 0.100 mmol) with catalyst 2a′ (5.10 mg, 5.00 μmol) yielded 4b (20.7 mg, 97%) as colorless crystals. Eluent for column: 20% EtOAc in n-hexane. Rf = 0.10 (10% EtOAc in n-hexane). [α]19D –201.4 (c 1.00, CHCl3) [Lit., –200 (c 1.00, CHCl3)] [51]. 1H NMR (300 MHz, CDCl3) δ: 7.68 (1H, dd, J = 1.7, 0.7 Hz), 7.46 (1H, dd, J = 3.7, 0.7 Hz), 7.44–7.31 (5H, m), 6.60 (1H, dd, J = 3.7, 1.7 Hz), 4.15 (1H, d, J = 2.1 Hz), 4.15 (1H, d, J = 2.1 Hz). HPLC (Chiralpak IB N-5, 5% EtOH in n-hexane, flow rate = 1.0 mL/min): tR = 17.5 min (major), tR = 16.1 min (minor), ee = 98%.
(1S,2R)-trans-1,2-Epoxy-4,4-dimethyl-1-phenylpentan-3-one (4c): according to the general procedure for peptide-catalyzed asymmetric epoxidation, the reaction of 3c (18.8 mg, 0.100 mmol) with catalyst 2a′ (5.10 mg, 5.00 μmol) yielded 4c (14.1 mg, 69%) as colorless crystals. Eluent for column: 5% EtOAc in n-hexane. Rf = 0.28 (10% EtOAc in n-hexane). [α]19D –183.9 (c 1.00, CHCl3) [Lit., –194 (c 1.00, CHCl3)] [52]. 1H NMR (400 MHz, CDCl3) δ: 7.43–7.35 (3H, m), 7.34–7.29 (2H, m), 3.87 (1H, d, J = 1.9 Hz), 3.86 (1H, d, J = 1.9 Hz), 1.24 (9H, s). HPLC (Chiralpak AD-H, 5% EtOH in n-hexane, flow rate = 0.7 mL/min): tR = 17.3 min (major), tR = 21.4 min (minor), ee = 99%.
(2R,3S)-trans-Epoxy-3-(4-chlorophenyl)-1-phenylpropan-1-one (4d): according to the general procedure for peptide-catalyzed asymmetric epoxidation, the reaction of 3d (24.3 mg, 0.100 mmol) with catalyst 2a′ (5.10 mg, 5.00 μmol) yielded 4d (24.4 mg, 95%) as yellow crystals. Eluent for column: 5% EtOAc in n-hexane. Rf = 0.35 (10% EtOAc in n-hexane). [α]19D –176.1 (c 1.00, CHCl3) [Lit., –233 (c 1.00, CH2Cl2)] [53]. 1H NMR (500 MHz, CDCl3) δ: 8.08–7.97 (2H, m), 7.69–7.59 (1H, m), 7.56–7.46 (2H, m), 7.44–7.36 (2H, m), 7.35–7.29 (2H, m), 4.26 (1H, d, J = 1.8 Hz), 4.06 (1H, d, J = 1.8 Hz). HPLC (Chiralpak IB N-5, 2% i-propanol in n-hexane, flow rate = 1.0 mL/min): tR = 25.3 min (major), tR = 24.1 min (minor), ee = 88%.
(2R,3S)-trans-Epoxy-3-(4-methoxyphenyl)-1-phenylpropan-1-one (4e): according to the general procedure for peptide-catalyzed asymmetric epoxidation, the reaction of 3e (23.8 mg, 0.100 mmol) with catalyst 2a′ (5.10 mg, 5.00 μmol) yielded 4e (21.1 mg, 83%) as yellow crystals. Eluent for column: 5% EtOAc in n-hexane. Rf = 0.28 (10% EtOAc in n-hexane). [α]19D –185.6 (c 1.00, CHCl3) [Lit., for the antipode, +131 (c 0.70, CHCl3)] [54]. 1H NMR (500 MHz, CDCl3) δ: 8.07–7.99 (2H, m), 7.67–7.59 (1H, m), 7.54–7.46 (2H, m), 7.34–7.27 (2H, m), 6.99–6.91 (2H, m), 4.30 (1H, d, J = 2.0 Hz), 4.03 (1H, d, J = 2.0 Hz), 3.83 (3H, s). HPLC (Chiralpak IB N-5, 2% i-propanol in n-hexane, flow rate = 1.0 mL/min): tR = 33.6 min (major), tR = 31.1 min (minor), ee = 99%.

4. Conclusions

Peptide foldamer catalysts, featuring a side-chain crosslink, were synthesized via a ring-closing metathesis reaction involving allyloxy groups on the α-carbocyclic dAAs. Stapled and unstapled octapeptide catalysts were prepared and tested via the asymmetric epoxidation of chalcones. However, the enantioselectivities observed in the reactions involving stapled peptides were similar to those of unstapled peptides. Notably, the stapled peptide 2a′ containing α-carbocyclic dAAs exhibited higher yields and enantioselectivities across all substrates 3a–3e than the acyclic dAAs-containing stapled peptide 2e′. These results could be attributed to the α-helicities of the peptides. X-ray crystallographic analysis revealed that α-carbocyclic dAAs-containing peptide 2a favored a right-handed α-helical structure, while acyclic dAAs-containing peptide 2e formed a mixture of α- and 310-helices. CD spectra of stapled peptides 2a–2e in H2O/MeOH (9:1) indicated enhanced right-handed helicities compared to unstapled peptides 1a–1e, potentially advantageous for developing peptide foldamer catalysts in aqueous media. Ongoing research in our laboratory includes further investigations into peptide catalysis in aqueous media and their potential applications in functional peptides.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29184340/s1, Figure S1–S3, Table S1–S6, X-ray crystallographic data of compounds 2a and 2e; CD spectra of peptides 1a–1e and 2a–2e; 1H and 13C NMR spectra of compounds 2a–2e, 1a′–1e′, 2a′–2e′, and 4a–4e; HPLC chart of compounds 4a–4e.

Author Contributions

Conceptualization, A.U. and M.T.; methodology, A.U. and M.T.; validation, A.I., A.U. and M.T.; formal analysis, A.I., A.U., T.U., T.K., K.H., M.D. and M.T.; investigation, A.I., A.U., T.U., T.K. and K.H.; writing—original draft preparation, A.U. and M.T.; writing—review and editing, A.I., A.U., T.U., T.K., K.H., M.D. and M.T.; visualization, A.U. and M.T.; supervision, A.U. and M.T.; project administration, A.U.; funding acquisition, A.U. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by JSPS KAKENHI Grant Numbers JP23K06050 (A.U.), JP20K06967 (A.U.), Takahashi Industrial and Economic Research Foundation (A.U.). This work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting introduction of the new sharing system) Grant Number JPMXS0422500320.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article or Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 29 04340 sch001
Scheme 1. Synthesis of the peptide catalysts 1a′–1e′ and 2a′–2e′ with hydrocarbon stapling at i (brown) and i + 4 (green) positions.
Scheme 1. Synthesis of the peptide catalysts 1a′–1e′ and 2a′–2e′ with hydrocarbon stapling at i (brown) and i + 4 (green) positions.
Molecules 29 04340 sch001
Molecules 29 04340 g001
Figure 1. Comparison of the catalytic activities of peptides 2a′ and 2e′ with various α,β-unsaturated ketones. Isolated yields are shown. Ee was determined by HPLC. * Conversion was >99% determined by 1H NMR analysis.
Figure 1. Comparison of the catalytic activities of peptides 2a′ and 2e′ with various α,β-unsaturated ketones. Isolated yields are shown. Ee was determined by HPLC. * Conversion was >99% determined by 1H NMR analysis.
Molecules 29 04340 g001
Molecules 29 04340 g002
Figure 2. X-ray crystallographic structures of peptides 2a and 2e (Mol A). (A) A view perpendicular to the helical axis, (B) a view along the helical axis, and (C) superimposed structures of 2a (magenta) and 2e (cyan).
Figure 2. X-ray crystallographic structures of peptides 2a and 2e (Mol A). (A) A view perpendicular to the helical axis, (B) a view along the helical axis, and (C) superimposed structures of 2a (magenta) and 2e (cyan).
Molecules 29 04340 g002
Molecules 29 04340 g003
Figure 3. CD spectra of (A) unstapled peptides 1a–1e and (B) stapled peptides 2a–2e. Conditions: 0.050 mM concentration in 10% MeOH in H2O.
Figure 3. CD spectra of (A) unstapled peptides 1a–1e and (B) stapled peptides 2a–2e. Conditions: 0.050 mM concentration in 10% MeOH in H2O.
Molecules 29 04340 g003
Molecules 29 04340 g004
Figure 4. Plausible mechanism for the asymmetric epoxidation of 3a catalyzed by peptide 2a′.
Figure 4. Plausible mechanism for the asymmetric epoxidation of 3a catalyzed by peptide 2a′.
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Molecules 29 04340 g005
Figure 5. Structures of the second-generation Grubbs catalyst and olefin-tethered α-carbocyclic and acyclic dAAs used in this study.
Figure 5. Structures of the second-generation Grubbs catalyst and olefin-tethered α-carbocyclic and acyclic dAAs used in this study.
Molecules 29 04340 g005
Table 1. Peptide-catalyzed asymmetric epoxidation.
Table 1. Peptide-catalyzed asymmetric epoxidation.
Molecules 29 04340 i001
EntryPeptideConv (%) 1Ee (%) 2
11a′>9997
22a′>9997
31b′>9998
42b′>9998
51c′>9997
62c′>9998
71d′>9998
82d′>9997
91e′>9989
102e′9290
11none710
1 Conversion was determined by 1H NMR analysis. 2 Ee was determined by HPLC.
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Iyoshi, A.; Ueda, A.; Umeno, T.; Kato, T.; Hirayama, K.; Doi, M.; Tanaka, M. Conformational Analysis and Organocatalytic Activity of Helical Stapled Peptides Containing α-Carbocyclic α,α-Disubstituted α-Amino Acids. Molecules 2024, 29, 4340. https://doi.org/10.3390/molecules29184340

AMA Style

Iyoshi A, Ueda A, Umeno T, Kato T, Hirayama K, Doi M, Tanaka M. Conformational Analysis and Organocatalytic Activity of Helical Stapled Peptides Containing α-Carbocyclic α,α-Disubstituted α-Amino Acids. Molecules. 2024; 29(18):4340. https://doi.org/10.3390/molecules29184340

Chicago/Turabian Style

Iyoshi, Akihiro, Atsushi Ueda, Tomohiro Umeno, Takuma Kato, Kazuhiro Hirayama, Mitsunobu Doi, and Masakazu Tanaka. 2024. "Conformational Analysis and Organocatalytic Activity of Helical Stapled Peptides Containing α-Carbocyclic α,α-Disubstituted α-Amino Acids" Molecules 29, no. 18: 4340. https://doi.org/10.3390/molecules29184340

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