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Article

Design and Synthesis of Helical N-Terminal l-Prolyl Oligopeptides Possessing Hydrocarbon Stapling

by
Atsushi Ueda
*,
Mei Higuchi
,
Kazuki Sato
,
Tomohiro Umeno
and
Masakazu Tanaka
*
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
*
Authors to whom correspondence should be addressed.
Molecules 2020, 25(20), 4667; https://doi.org/10.3390/molecules25204667
Submission received: 30 September 2020 / Revised: 10 October 2020 / Accepted: 12 October 2020 / Published: 13 October 2020
(This article belongs to the Special Issue Advances in Research of Short Peptides)
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Abstract

:
We designed and synthesized helical short oligopeptides with an l-proline on the N-terminus and hydrocarbon stapling on the side chain. Side-chain stapling is a frequently used method for the development of biologically active peptides. Side-chain stapling can stabilize the secondary structures of peptides, and, therefore, stapled peptides may be applicable to peptide-based organocatalysts. Olefin-tethered cis-4-hydroxy-l-proline 1 and l-serine 2 and 8, and (R)-α-allyl-proline 18 were used as cross-linking motifs and incorporated into helical peptide sequences. The Z- and E-selectivities were observed for the ring-closing metathesis reactions of peptides 3 and 11 (i,i+1 series), respectively, while no E/Z-selectivity was observed for that of 19 (i,i+3 series). The stapled peptide B’ catalyzed the Michael addition reaction of 1-methylindole to α,β-unsaturated aldehyde, which was seven times faster than that of unstapled peptide B. Furthermore, the high catalytic activity was retained even at lower catalyst loadings (5 mol %) and lower temperatures (0 °C). The circular dichroism spectra of stapled peptide B’ showed a right-handed helix with a higher intensity than that of unstapled peptide B. These results indicate that the introduction of side-chain stapling is beneficial for enhancing the catalytic activity of short oligopeptide catalysts.

1. Introduction

Hydrocarbon stapling is one of the most commonly used methods to stabilize the secondary structure of peptides as a way to provide enhanced functionality [1,2,3]. This powerful tool is especially important for short oligopeptides due to their flexible secondary structure. Grubbs et al. reported the synthesis of 310-helical heptapeptides stabilized by hydrocarbon stapling at the i,i+4 positions using ring-closing metathesis [4,5]. In 2000, Verdine’s group reported all-hydrocarbon stapling using α-olefin-tethered alanine at i,i+4 as well as at i,i+7 positions, and the introduction of these staples highly induced α-helicities as well as the metabolic stabilities of the peptides [6]. Nowadays, the all-hydrocarbon staplings at the i,i+4 and i,i+7 positions are widely used in the medicinal chemistry of peptides [7,8,9].
Short peptides are also attractive compounds in the field of organocatalysis [10,11,12,13]. These peptide catalysts can be categorized into several classes based on their secondary structure, those including β-turn [14,15], helix [16,17,18,19,20], turn-helix types [21,22,23], and so forth. Therefore, hydrocarbon stapling can be an effective tool for the development of peptide-based organocatalysts by controlling their secondary structure. However, there are few examples of stapled peptide-catalyzed asymmetric organocatalytic reactions. Demizu and co-workers reported an enantioselective Juliá–Colonna epoxidation of chalcone catalyzed by a helical peptide-based primary amino catalyst possessing a crosslink between two l-homoserines at the i,i+4 positions (Figure 1) [24,25]. Likewise, secondary amino catalysts are powerful catalysts with a broad range of applicable reactions [26,27]. Moreover, the peptide hydrocarbon staplings at the i,i+1 and i,i+3 [28,29] positions are rarely examined compared to i,i+4 series but have potential as constrained cyclic peptides for organocatalysis, material science, drug discovery, and so forth [30]. From this point of view, the introduction of allyl tethered cis-4-hydroxy-l-proline or (R)-α-allyl-proline can be suitable for this purpose. Secondary structures, as well as helical screw directions, can be controlled by introducing 1-aminocycloalkane-1-carboxylic acid in homopeptides [31,32,33,34,35,36,37,38,39,40] and heteropeptides [41,42,43], and these constrained peptides catalyze asymmetric 1,4-addition reactions [44,45,46]. Therefore, poly l-leucine-incorporating 1-aminocyclopentane-1-carboxylic acid was used as an α-helix-inducing motif. The stapling efficiency was evaluated by comparing the catalytic activities of stapled and unstapled peptides in Friedel–Crafts type 1,4-addition reactions [47,48]. Herein, we report the synthesis of helical N-terminal prolyl oligopeptides with hydrocarbon stapling at i,i+1 as well as i,i+3 positions and the enhancements of their catalytic activity for the Michael addition of 1-methylindole to α,β-unsaturated aldehyde.

2. Results and Discussion

The synthesis of the unstapled peptides A and B and stapled peptides of the i,i+1 series A’ and B’ began from allyl tethered cis-4-hydroxy-l-proline 1 [49,50] and l-serine 2, as illustrated in Scheme 1. The coupling of 1 and 2 produced dipeptide 3, which was successfully introduced to a helix-inducing motif, H-(l-Leu-l-Leu-Ac5c)2-OMe [41,44]. The deprotection of the Boc-protecting groups of 3 and 4 produced N-terminal free peptides A and B in quantitative yields. On the other hand, the ring-closing metathesis of dipeptide 3 was performed with 20 mol % of the second-generation Grubbs catalyst to give i,i+1-stapled dipeptide 5. In this reaction, the Z-configured product was obtained as a major product (E/Z = 1.0:5.6), possibly due to the medium ring size of 5 (13-membered ring) along with the rigid 4-hydroxyproline part. The hydrogenation of 5 provided dipeptide 6, which was coupled with H-(l-Leu-l-Leu-Ac5c)2-OMe to afford stapled octapeptide 7. The N-Terminal free peptides A’ and B’ were obtained by the Boc-deprotection of 6 and 7, respectively. It should be noted that neither the ring-closing metathesis of octapeptide 4 nor that of the trans-4-hydroxy-l-proline derivatives of 3 produced the desired cyclization products. This poor reactivity may be caused by a ring strain of the 13-membered ring product, which resulted in a preference for the Z-configured isomer of 5.
Stapled peptide C’ possessing tethered side chains at the i,i+1 positions with a 15-membered macrocyclic ring was also synthesized from 4-pentenyl tethered l-Ser 8 [51] by a similar manner as described in Scheme 1 (Scheme 2). In contrast to the reaction of 4 to 7, unstapled peptide 11 underwent the ring-closing metathesis reaction smoothly to provide stapled peptide 12 in a 93% yield with a preference for E-isomer over Z-isomer (E/Z = 5.5:1.0). This smooth reaction implies the released macrocyclic ring strain of the product, which resulted in a preference for thermodynamically favored E-olefin isomer.
The peptide stapling at the i,i+3 positions could be another choice to restrict the conformational freedom of the N-terminus. We designed spirocyclic-stapled peptide D’ possessing tethered crosslinks at the i,i+3 positions by introducing (R)-α-allyl-proline 18 [52,53] (Scheme 3). The synthesis of D’ began from O-(4-pentenyl)-l-serine 8, which was sequentially coupled with H-l-Leu-l-Leu-Ac5c-OMe [44] on C-terminus and with Boc-l-Leu-l-Leu-OH followed by 18 on N-terminus to produce heptapeptide 19. The ring-closing metathesis of 19 proceeded at high yield, but no E/Z-selectivity was observed (E/Z = 1.1:1.0). The poor selectivity may be caused by sterically congested (R)-α-allyl-proline. N-terminal-free heptapeptides D and D’ were synthesized by the hydrogenolysis of 19 and 20, respectively.
Next, we examined the Michael addition reaction of 1-methylindole (22) and α,β-unsaturated aldehyde 21 using 20 mol % of unstapled peptides A–D and stapled peptides A’–D’ to compare their catalytic activities (Table 1). The reaction with stapled octapeptide B’ showed a faster reaction rate than the reaction with stapled dipeptide A’ (entries 2 and 4, 46% conversion after 6 d vs. 83% conversion after 1 d). This result suggests that the helical motif -[l-Leu-l-Leu-Ac5c]2- of stapled peptide B’ is important to catalytic activity. Furthermore, stapled peptide B’ is more active than unstapled peptide B (entries 3 and 4, 83% conversion with a 76% isolated yield vs. 12% conversion). Similar trends were observed for other stapled peptides A’, C’, and D’. Therefore, the introduction of side-chain stapling plays a key role in enhancing catalytic activity. Moderate ee values could be improved by peptide sequence screening. The absolute configuration of 23 was determined by comparisons of the chiral HPLC chart and the specific rotation with those in the literature [47,48].
The reaction with reduced catalyst loading (10 or 5 mol %) of stapled peptide B’ displayed almost the same results as the reaction with 20 mol % (Table 2, entries 4 and 6), while that of unstapled peptide B resulted in further decreases in both the conversion yield and ee value (entries 3 and 5). Lowering the reaction temperature to 0 °C contributed to the deactivation of unstapled peptide B (entry 7), but the reaction with stapled peptide B’ retained a high conversion, with increased ee values (entry 8). These results also support that side-chain hydrocarbon stapling enhances the catalytic activities in the reaction.
Circular dichroism (CD) spectra were measured for all peptide catalysts to obtain their secondary structure information (Figure 2). The CD spectra of octapeptides B, B’, C, and C’ and heptapeptides D and D’ showed right-handed helical structures, while those of dipeptides A and A’ showed β-turn structures. The helicity of stapled peptide B’, which gave the best conversion in Table 1, was higher than that of unstapled peptide B. These results suggest that reinforcement of helicity via side-chain stapling presumably increased the catalytic activity of the stapled peptide B’. Interestingly, the Michael reactions catalyzed by unstapled peptide B and stapled peptide B’ produced opposite ee values, whereas both peptides showed right-handed helical structures. Therefore, the introduction of side-chain stapling to peptide catalysts can possibly reverse the enantioselectivities after the fine-tuning of the peptide sequence.
Based on the right-handed helical structure, the plausible reaction mechanism catalyzed by stapled peptide B’ and unstapled peptide B is shown in Figure 3. For the reaction catalyzed by stapled peptide B’, the reactive iminium ion was formed inside the helical pipe with a rigid conformation caused by the hydrocarbon stapling, which accelerated the Friedel–Crafts type attack of 1-methylindole from the si face. On the other hand, the iminium species formed by unstapled peptide B exists outside the helical pipe with a flexible conformation, which decreased the reaction rate and enabled 1-methylindole to be accessed from both faces. Although the pioneering organocatalyst in this transformation, MacMillan’s imidazolidinone catalyst showed high enantioselectivities [54]; its derivatization as such a covalent immobilization to polymer supports as a recyclable catalyst is difficult and resulted in decreasing yield and enantioselectivities [55]. On the other hand, peptide catalysts are easier to modify and can be reused by immobilization in the resin at C-terminus, which does not affect the reactive site of N-terminus [21,22,23].
In summary, we have developed synthetic routes to the N-terminal l-prolyl oligopeptides A’–D’ possessing side-chain hydrocarbon stapling. The ring-closing metathesis reactions of peptides A and C selectively produced Z- and E-configured stapled peptides, respectively, while no E/Z-selectivity was observed for the ring-closing metathesis of D. The stapled peptide B’ catalyzed the Michael addition of 1-methylindole to α,β-unsaturated aldehyde, which was seven times faster than that of unstapled peptide B. Since the reactions with B’ at lower catalyst loadings or lower temperatures retained conversion yields comparable to those of B, the introduction of side-chain hydrocarbon stapling is effective in enhancing the catalytic activity of peptides. These results provide useful information related to the recent progress of the E/Z-selective ring-closing metathesis of peptides [56,57], l-prolyl catalysts [58,59,60], and peptide foldamer [61,62,63]. Further studies including enantioselectivity improvement, an expansion of the reaction scope using catalyst B’, as well as applications to cell-penetrating peptides [64,65,66] are ongoing in our laboratory.

3. Materials and Methods

3.1. General Procedure and Method

Melting points were taken on an AS ONE melting point apparatus ATM-01 (AS ONE Corporation, Osaka, Japan) and were uncorrected. Optical rotations were measured on a JASCO DIP-370 polarimeter (JASCO Corporation, Tokyo, Japan) using CHCl3 as a solvent. 1H NMR and 13C NMR spectra were recorded on the JEOL JNM-AL-400 (400 MHz), a Varian NMR System 500PS SN (500 MHz and 125 MHz) spectrometer (Agilent Inc., Santa Clara, CA, USA). Chemical shifts (δ) are reported in parts per million (ppm). For the 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 the 13C NMR spectra. The IR spectra were recorded on a Shimadzu IRAffinity-1 FT-IR spectrophotometer (Shimadzu Corporation, Kyoto, Japan). High-resolution mass spectra (HRMS) were obtained on a JEOL JMS-T100TD using electrospray ionization (ESI) (JEOL Ltd., Tokyo, Japan) or direct analysis in the 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, NJ, USA), silica gel 60 F254, and layer thicknesses of 0.25 and 0.50 mm, respectively. Compounds were observed in UV light at 254 nm and then visualized by staining with iodine, p-anisaldehyde, or phosphomolybdic acid stain. Flash and gravity column chromatography separations were performed on Kanto Chemical silica gel 60N, spherical neutral, with particle sizes of 63–210 μm and 40–50 μm, respectively. Analytical high-performance liquid chromatography (HPLC) was carried out with JASCO PU-2089 on a UV spectrophotometric detector (254 nm, JASCO UV-2075, JASCO Corporation, Tokyo, Japan), to which a 4.6 × 250 mm size chiral column (Daicel Chiralpak AD-H, Daicel Corporation, Osaka, Japan) was attached. All moisture-sensitive reactions were conducted under an inert atmosphere. Reagents and solvents were of commercial grade and were used as supplied, unless otherwise noted. Compounds 1 [49,50], 2 [24,25], 8 [51], 18 [52,53], H-(l-Leu-l-Leu-Ac5c)2-OMe [44], and H-l-Leu-l-Leu-Ac5c-OMe [44] were prepared according to the reported procedures. Copies of NMR Spectra are given in the Supplementary Materials.

3.2. Synthesis of Unstapled Peptides A and B and Stapled Peptides A’ and B’

Boc-l-HypOAll-l-SerOAll-OMe (3): To a solution of Boc-l-HypOAll-OH (1 [49,50]; 4.22 g, 15.5 mmol) in CH2Cl2 (52 mL) were added 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate (HBTU; 6.48 g, 17.1 mmol) and 1-hydroxybenzotriazole hydrate (HOBt·H2O; 2.62 g, 17.1 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of HCl·H-l-SerOAll-OMe (2 [24,25].; 2.47 g, 15.5 mmol) in CH2Cl2 (52 mL) and N,N-diisopropylethylamine (DIPEA; 5.41 mL, 31.1 mmol) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring overnight, the CH2Cl2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (40% EtOAc in n-hexane) to give 3 (3.89 g, 61%) as a yellow oil. Rf = 0.69 (EtOAc). [ α ] D 30 −3.2 (c 1.02, CHCl3). 1H NMR (500 MHz, CDCl3, VT = 50 °C) δ: 5.98–5.77 (m, 2H), 5.28–5.11 (m, 4H), 4.77–4.66 (m, 1H), 4.33 (d, J = 8.8 Hz, 1H), 4.07–4.04 (m, 1H), 4.00–3.86 (m, 4H), 3.82 (dd, J = 9.7, 3.5, Hz, 1H), 3.78–3.70 (m, 1H), 3.74 (s, 3H), 3.61–3.49 (m, 3H), 2.56–2.44 (m, 1H), 2.23–2.09 (m, 1H), 1.48 (s, 9H). 13C NMR (125 MHz, CDCl3, VT = 50 °C) δ: 172.0, 170.5, 156.0, 134.6, 134.3, 117.0, 116.9, 81.0, 76.4, 72.2, 69.9, 69.6, 58.6, 53.0, 52.8, 52.2, 37.0, 28.3 (3C). IR (film): 3304, 2978, 2933, 1751, 1701 cm−1. HRMS (DART) m/z: [M + H]+ calcd for C20H33N2O7, 413.2288; found, 413.2282.
H-l-HypOAll-l-SerOAll-OMe (A): To a solution of Boc-protected dipeptide 3 (100 mg, 0.242 mmol) in CH2Cl2 (2.4 mL) was added trifluoroacetic acid (0.24 mL) dropwise at room temperature, and the reaction mixture was stirred for 2 days at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3 and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give amine A (75.2 mg, quant) as an amorphous solid. Rf = 0.29 (EtOAc). Mp 81–83 °C. [ α ] D 28 −39.4 (c 1.00, CHCl3). 1H NMR (400 MHz, CDCl3) δ: 8.27 (d, J = 8.6 Hz, 1H), 5.93–5.78 (m, 2H), 5.29–5.21 (m, 2H), 5.21–5.11 (m, 2H), 4.72 (dt, J = 8.3, 3.5 Hz, 1H), 4.08–4.00 (m, 1H), 4.00–3.96 (m, 2H), 3.94 (dt, J = 5.4, 1.5 Hz, 1H), 3.92–3.85 (m, 2H), 3.82 (dd, J = 8.3, 4.9 Hz, 1H), 3.76 (s, 3H), 3.58 (dd, J = 9.5, 3.7 Hz, 1H), 3.17 (dd, J = 11.2, 5.4 Hz, 1H), 3.05 (dd, J = 11.2, 2.9 Hz, 1H), 2.28–2.16 (m, 2H). 13C NMR (125 MHz, CDCl3) δ: 168.4, 163.9, 134.1, 133.6, 118.0, 117.4, 74.6, 72.3, 70.1, 68.5, 57.2, 54.1, 52.5, 50.7, 33.6. IR (KBr): 3215, 2874, 1692, 1450 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C15H24N2O5Na, 335.1583; found, 335.1573.
Boc-l-HypOAll-l-SerOAll-[(l-Leu)2-Ac5c]2-OMe (4): To a solution of dipeptide 3 (50.0 mg, 0.121 mmol) in MeOH (1.2 mL) was added 1 M of aqueous NaOH (0.121 mL, 0.121 mmol) at room temperature, and the mixture was stirred overnight at the same temperature. The solution was acidified with 1 M of aqueous HCl and the MeOH was removed in vacuo. The resulting aqueous solution was extracted with EtOAc three times. The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated in vacuo to give a carboxylic acid (44.7 mg, 93%). To a solution of the acid (206 mg, 0.500 mmol) in CH2Cl2 (2.5 mL) was added EDCI·HCl (96.0 mg, 0.500 mmol) and HOBt·H2O (92.0 mg, 0.600 mmol) at 0 °C, and the mixture was stirred at the same temperature for 30 min. Then, a solution of H-[(l-Leu)2-Ac5c]2-OMe (354 mg, 0.500 mmol) in CH2Cl2 (2.5 mL) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred overnight. After the removal of CH2Cl2, the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give crude product, which was purified by flash column chromatography on silica gel (70% EtOAc in n-hexane) to give 4 (269 mg, 49%) as a white solid. Rf = 0.20 (60% EtOAc in n-hexane). Mp 76–79 °C. [ α ] D 27 +2.2 (c 1.02, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.49 (d, J = 5.6 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 4.9 Hz, 1H), 7.28–7.23 (m, 4H), 5.96–5.74 (m, 2H), 5.37–5.16 (m, 4H), 4.34 (t, J = 8.6 Hz, 1H), 4.25–4.12 (m, 5H), 4.05–3.90 (m, 5H), 3.84–3.73 (m, 2H), 3.72–3.65 (m, 1H), 3.67 (s, 3H), 3.48 (dd, J = 12.0, 3.4 Hz, 1H), 2.70–2.60 (m, 1H), 2.39–2.22 (m, 3H), 2.22–2.11 (m, 3H), 2.11–2.02 (m, 1H), 1.96–1.57 (m, 22H), 1.50 (s, 9H), 1.02–0.83 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.6, 175.2, 174.6, 174.2, 174.1, 173.2, 173.1, 171.8, 155.7, 133.9, 133.5, 117.8, 117.5, 81.7, 72.2, 69.6, 67.6, 66.7, 65.7, 60.1, 56.2, 54.8, 54.1, 54.0, 53.3, 52.23, 52.22, 52.1, 39.6, 39.4, 39.1, 38.3, 37.3, 36.74, 36.70, 35.5, 34.4, 28.32, 28.26 (3C), 25.2, 25.0, 24.73, 24.65, 24.54, 24.53, 24.46, 23.5, 23.4, 22.99, 22.97, 21.1, 21.0, 20.90, 20.87. IR (CDCl3): 3325, 2961, 1732, 1661, 1530 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C56H94N8O13Na, 1109.6838; found, 1109.6808.
H-l-HypOAll-l-SerOAll-[(l-Leu)2-Ac5c]2-OMe (B): To a solution of Boc-protected peptide 4 (135 mg, 0.124 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic acid (0.12 mL) dropwise at room temperature, and the reaction mixture was stirred overnight at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3 and the aqueous phase was extracted with CHCl3 four times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under vacuum to give amine product B (124 mg, quant). Rf = 0.10 (80% EtOAc in n-hexane). Mp 107–108 °C. [ α ] D 30 −1.9 (c 0.99, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 9.04 (br s, 1H), 8.06 (br s, 1H), 7.64 (d, J = 6.8 Hz, 1H), 7.57 (br s, 1H), 7.52–7.42 (m, 2H), 7.35 (d, J = 4.9 Hz, 1H), 5.92–5.75 (m, 2H), 5.32–5.14 (m, 4H), 4.60 (br s, 1H), 4.35 (d, J = 4.2 Hz, 1H), 4.29 (br s, 1H), 4.21–4.04 (m, 3H), 4.04–3.95 (m, 4H), 3.95–3.88 (m, 1H), 3.86–3.74 (m, 2H), 3.68 (s, 3H), 3.60 (d, J = 11.7 Hz, 1H), 3.47 (d, J = 8.8 Hz, 1H), 2.65–2.44 (m, 3H), 2.29–2.16 (m, 2H), 2.12 (br s, 2H), 2.03 (dd, J = 11.7, 6.1 Hz, 1H), 1.92–1.53 (m, 22H), 1.03–0.78 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.7, 175.5, 175.4, 174.7, 174.4, 174.3, 173.9, 171.8, 133.8, 133.5, 118.0, 117.9, 76.1, 72.3, 70.0, 68.1, 66.7, 66.0, 59.3, 56.4, 55.0, 54.9, 54.4, 53.3, 52.4, 51.3, 40.1, 39.7, 39.4, 39.2, 37.9, 36.89, 36.85, 35.2, 35.0, 29.7, 25.0, 24.78, 24.76, 24.58, 24.56, 24.4, 24.3, 24.23, 24.17, 23.2, 23.1, 22.5, 21.8, 21.6, 21.2. IR (KBr): 3329, 2959, 1736, 1655, 1535 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C51H86N8O11Na, 1009.6314; found, 1009.6288.
Stapled Boc-l-Hyp-l-Ser-OMe (6): Under an argon atmosphere, to a solution of 3 (90.0 mg, 0.218 mmol) in CH2Cl2 (11 mL) was added second-generation Grubbs catalyst (37.0 mg, 0.0436 mmol) at room temperature, and the mixture was stirred for 2 h at the same temperature. The reaction mixture was filtered through a short pad of silica gel (60% EtOAc in n-hexane) and concentrated. The crude material was purified by flash chromatography on silica gel (60% EtOAc in n-hexane) to provide a stapled peptide 5 (46.2 mg, 55%) as a mixture of E- and Z-isomers (E/Z = 1.0:5.6). Rf = 0.30 (EtOAc). Next, to a solution of stapled peptides 5 (46.2 mg, 0.120 mmol) in MeOH (12 mL) was added 10% Pd-C (23 mg, 50 wt %) under a nitrogen atmosphere. After being vigorously stirred under a hydrogen atmosphere for 19 h at room temperature, the reaction mixture was passed through a short plug of Celite. The filtrate was concentrated under vacuum to give a crude product, which was purified by flash column chromatography on silica gel (70% EtOAc in n-hexane) to give 6 (35.5 mg, 77%) as an amber oil. Rf = 0.29 (EtOAc). [ α ] D 29 −3.5 (c 1.07, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.27 (br s, 1H), 4.76–4.56 (m, 1H), 4.39–4.21 (m, 1H), 3.97 (br s, 2H), 3.87–3.78 (m, 1H), 3.76 (s, 3H), 3.59 (br s, 3H), 3.54–3.39 (m, 2H), 3.38–3.29 (m, 1H), 2.40–2.17 (m, 2H), 1.92–1.77 (m, 2H), 1.77–1.54 (m, 2H), 1.54–1.38 (m, 9H). 13C NMR (125 MHz, CDCl3) δ: 172.5, 170.3, 154.9, 80.8, 70.8, 69.7, 69.5, 69.1, 60.6, 53.0, 52.4, 52.0, 37.2, 28.1 (3C), 26.9, 25.5. IR (film): 3422, 2934, 1751, 1697 cm−1. HRMS (DART) m/z: [M + H]+ calcd for C18H31N2O7, 387.2131; found, 387.2130.
Stapled H-l-Hyp-l-Ser-OMe (A’): To a solution of Boc-protected dipeptide 6 (45.0 mg, 0.116 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic acid (0.2 mL) dropwise at room temperature, and the reaction mixture was stirred for 24 h at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under vacuum to give crude product A’ (20.5 mg, 62%) as an amber oil, which was used for the next step without further purification. Rf = 0.30 (EtOAc). [ α ] D 28 +11.6 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.52 (d, J = 7.6 Hz, 1H), 4.69–4.63 (m, 1H), 3.95 (t, J = 3.8 Hz, 1H), 3.91–3.82 (m, 2H), 3.78 (s, 3H), 3.79–3.74 (m, 1H), 3.61 (dt, J = 9.8, 5.9 Hz, 1H), 3.53–3.43 (m, 2H), 3.43–3.31 (m, 2H), 2.99 (dd, J = 10.5, 3.2 Hz, 1H), 2.27 (d, J = 14.2 Hz, 1H), 2.15 (ddd, J = 14.2, 11.0, 4.2 Hz, 1H), 1.78–1.46 (m, 4H). 13C NMR (125 MHz, CDCl3) δ: 175.1, 171.0, 78.6, 71.1, 69.0, 68.3, 58.9, 53.4, 52.5, 51.3, 37.0, 26.5, 26.1. IR (KBr): 3345, 2920, 2868, 1748, 1658, 1526, 1441 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C13H22N2O5Na, 309.1426; found, 309.1428.
Stapled Boc-l-Hyp-l-Ser-[(l-Leu)2-Ac5c]2-OMe (7): To a solution of stapled dipeptide 6 (104 mg, 0.269 mmol) in MeOH (3 mL) was added 1 M of aqueous NaOH (0.270 mL, 0.270 mmol) at room temperature, and the mixture was stirred overnight at the same temperature. The solution was acidified with 1 M of aqueous HCl and the MeOH was removed in vacuo. The resulting aqueous solution was extracted with EtOAc three times. The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated in vacuo to give a crude product (95.0 mg, 95%), which was used for the next step without further purification. Rf = 0.27 (EtOAc). To a solution of the crude acid (85.6 mg, 0.230 mmol) in CH2Cl2 (2.3 mL) was added N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI·HCl; 44.0 mg, 0.230 mmol) and HOBt·H2O (42.0 mg, 0.276 mmol) at 0 °C, and the mixture was stirred at the same temperature for 30 min. Then, a solution of H-[(l-Leu)2-Ac5c]2-OMe [44] (163 mg, 0.230 mmol) in CH2Cl2 (1 mL) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 2 days. After the removal of CH2Cl2, the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (80% EtOAc in n-hexane) to give 7 (178 mg, 73%) as a yellow oil. Rf = 0.46 (EtOAc). [ α ] D 28 −4.1 (c 1.07, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.75 (br s, 1H), 7.54–7.41 (m, 2H), 7.31 (s, 1H), 7.28–7.25 (m, 2H), 7.22 (d, J = 6.1 Hz, 1H), 4.37–4.30 (m, 1H), 4.28 (dd, J = 10.5, 4.2 Hz, 1H), 4.24–4.16 (m, 2H), 4.14 (d, J = 10.3 Hz, 1H), 4.08–4.03 (m, 1H), 3.98 (dd, J = 11.0, 4.9 Hz, 1H), 3.96–3.90 (m, 1H), 3.84 (d, J = 11.7 Hz, 1H), 3.75–3.69 (m, 1H), 3.67 (s, 3H), 3.65–3.50 (m, 3H), 3.46–3.36 (m, 2H), 2.70–2.60 (m, 1H), 2.39 (ddd, J = 14.6, 10.9, 4.0 Hz, 1H), 2.31–2.03 (m, 8H), 1.94–1.59 (m, 24H), 1.52 (s, 9H), 1.01–0.84 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.4, 175.2 (2C), 174.4, 174.2, 173.2, 173.1, 171.0, 155.4, 81.4, 78.9, 70.8, 68.5, 68.4, 66.7, 65.7, 60.4, 54.9, 54.7, 54.0, 53.9, 53.4, 52.2, 52.0, 40.0, 39.6, 39.4, 39.2, 38.2, 37.2, 36.7, 35.4, 35.3, 28.3 (3C), 27.4, 26.3, 25.0, 24.82, 24.81, 24.6, 24.50 (2C), 24.48, 24.4, 23.5, 23.4, 22.9, 22.6, 21.5, 21.2, 21.0, 20.9. IR (KBr): 3329, 2957, 1736, 1647, 1522 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C54H92N8O13Na, 1083.6682; found, 1083.6685.
Stapled H-l-Hyp-l-Ser-[(l-Leu)2-Ac5c]2-OMe (B’): To a solution of Boc-protected peptide 7 (120 mg, 0.113 mmol) in CH2Cl2 (2 mL) was added trifluoroacetic acid (0.113 mL) dropwise at room temperature, and the reaction mixture was stirred overnight at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product B’ (96.9 mg, 89%), which was used for the next step without further purification. Rf = 0.25 (EtOAc). Mp 117–118 °C. [ α ] D 26 −5.2 (c 0.95, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.83 (d, J = 5.4 Hz, 1H), 7.58 (d, J = 4.6 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.32–7.24 (m, 3H), 4.37–4.27 (m, 2H), 4.23–4.16 (m, 1H), 4.11 (br s, 2H), 4.02 (dd, J = 10.9, 5.0 Hz, 1H), 3.94 (dt, J = 9.7, 4.8 Hz, 1H), 3.89 (dd, J = 8.8, 3.4 Hz, 1H), 3.78 (dd, J = 10.9, 3.1 Hz, 1H), 3.67 (s, 3H), 3.66–3.61 (m, 1H), 3.60–3.50 (m, 3H), 3.34 (d, J = 10.5 Hz, 1H), 3.06 (dd, J = 10.5, 2.4 Hz, 1H), 2.69–2.60 (m, 1H), 2.36 (br s, 3H), 2.30–2.21 (m, 4H), 2.20–2.03 (m, 3H), 1.97–1.55 (m, 24H), 1.03–0.80 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 178.1, 175.6, 175.1, 174.2, 173.9, 173.2, 173.1, 172.4, 80.0, 70.3, 69.8, 68.7, 66.8, 65.7, 59.8, 55.2, 54.8, 54.1, 54.0, 52.3, 52.1, 51.5, 39.6, 39.3, 38.2, 37.2, 36.7, 36.3, 35.4, 29.6, 28.4, 27.2, 26.5, 25.2, 25.12, 25.07, 24.7, 24.50, 24.49, 24.4 (2C), 23.5, 23.4, 23.1, 22.8, 21.3, 21.1, 21.0, 20.8. IR (CDCl3): 3325, 2958, 1655, 1526 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C49H84N8O11Na, 983.6157; found, 983.6142.

3.3. Synthesis of Unstapled Peptide C and Stapled Peptide C’

Boc-l-SerOPte-[(l-Leu)2-Ac5c]2-OMe (9): To a solution of Boc-l-SerOPte-OH 8 [51] (193 mg, 0.707 mmol) in CH2Cl2 (2.5 mL) were added EDCI·HCl (136 mg, 0.707 mmol) and HOBt·H2O (130 mg, 0.848 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of H-[(l-Leu)2-Ac5c]2-OMe [44] (500 mg, 0.707 mmol) in CH2Cl2 (2.5 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring overnight, CH2Cl2 was removed, and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (60% EtOAc in n-hexane) to give 9 (478 mg, 70%) as a white solid. Rf = 0.66 (EtOAc). Mp 109–115 °C. [ α ] D 26 −4.3 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.42 (d, J = 8.1 Hz, 1H), 7.33 (s, 1H), 7.29 (d, J = 4.4 Hz, 1H), 7.25–7.19 (m, 2H), 6.60 (d, J = 3.7 Hz, 1H), 5.80 (ddt, J = 17.0, 10.3, 6.6 Hz, 1H), 5.50 (d, J = 2.4 Hz, 1H), 5.08–4.96 (m, 2H), 4.39–4.32 (m, 1H), 4.20 (dd, J = 11.2, 6.1 Hz, 1H), 4.14–4.07 (m, 1H), 4.04 (q, J = 3.7 Hz, 1H), 3.98 (dt, J = 9.7, 5.0, Hz, 1H), 3.77–3.70 (m, 2H), 3.67 (s, 3H), 3.55–3.44 (m, 2H), 2.70–2.60 (m, 1H), 2.32–2.23 (m, 1H), 2.23–2.02 (m, 6H), 1.96–1.54 (m, 24H), 1.50 (s, 9H), 1.05–0.82 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.5, 175.1, 173.6, 173.4, 173.0, 172.9, 172.0, 156.9, 137.7, 115.1, 81.7, 71.0, 68.8, 66.8, 65.7, 56.9, 54.7, 54.1, 54.0, 52.2, 52.1, 40.1, 39.8, 39.6, 39.4, 38.1, 37.3, 36.7, 35.5, 30.2, 28.5, 28.1 (3C), 25.3, 25.1, 24.8, 24.7, 24.53, 24.51, 24.43, 24.40, 23.5, 23.4, 23.0, 22.9, 21.4, 21.3, 21.1, 20.9. IR (KBr): 3329, 2957, 1701, 1632, 1524 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C50H87N7O11Na, 984.6361; found, 984.6386.
Boc-l-HypOAll-l-SerOPte-[(l-Leu)2-Ac5c]2-OMe (11): To a solution of Boc-protected peptide 9 (480 mg, 0.499 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic acid (0.749 mL) dropwise at room temperature, and the reaction mixture was stirred overnight at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product 10 (391 mg, 91%), which was used for the next step without further purification. Rf = 0.20 (60% EtOAc in n-hexane). To a solution of Boc-l-HypOAll-OH (123 mg, 0.454 mmol) in CH2Cl2 (2.5 mL) were added EDCI·HCl (87.0 mg, 0.454 mmol) and HOBt·H2O (84.0 mg, 0.545 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of amine 10 (391 mg, 0.454 mmol) in CH2Cl2 (2.5 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring overnight, the CH2Cl2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give crude product, which was purified by flash column chromatography on silica gel (70% EtOAc in n-hexane) to give 11 (248 mg, 49%) as a white solid. Rf = 0.56 (EtOAc). Mp 75–85 °C. [ α ] D 20 −3.2 (c 1.03, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.49–7.41 (m, 2H), 7.37 (d, J = 4.9 Hz, 1H), 7.26–7.20 (m, 4H), 5.93–5.72 (m, 2H), 5.33–5.19 (m, 2H), 5.06–4.95 (m, 2H), 4.39–4.30 (m, 1H), 4.25–4.09 (m, 5H), 4.05–3.90 (m, 3H), 3.83–3.72 (m, 2H), 3.67 (s, 3H), 3.67–3.63 (m, 1H), 3.52–3.38 (m, 3H), 2.70–2.61 (m, 1H), 2.36–2.32 (m, 1H), 2.31–2.11 (m, 4H), 2.10–2.02 (m, 3H), 1.96–1.66 (m, 21 H), 1.66–1.56 (m, 4H), 1.51 (s, 9H), 1.03–0.80 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.6, 175.2, 174.6, 174.2, 174.1, 173.1, 173.0, 171.9, 155.7, 137.5, 133.9, 117.5, 115.2, 81.6, 77.2, 70.8, 69.6, 68.3, 66.8, 65.7, 60.2, 56.2, 54.8, 54.1, 54.0, 53.4, 52.2, 52.1, 39.7, 39.6, 39.4, 39.2, 38.3, 37.3, 36.7, 35.5, 34.3, 30.0, 28.4, 28.3 (3C), 25.2, 25.1, 24.8, 24.7, 24.57, 24.55 (2C), 24.47, 23.5, 23.4, 23.00, 22.97, 21.12, 21.06, 21.0, 20.9. IR (CDCl3): 3325, 2959, 2872, 1732, 1661, 1530 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C58H98N8O13Na, 1137.7151; found, 1137.7201.
H-l-HypOAll-l-SerOPte-[(l-Leu)2-Ac5c]2-OMe (C): To a solution of Boc-protected peptide 11 (30.0 mg, 0.0269 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic acid (0.03 mL) dropwise at room temperature, and the reaction mixture was stirred overnight at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product C (23.5 mg, 86%), which was used for the next step without further purification. Rf = 0.31 (EtOAc). Mp 79–81 °C. [ α ] D 28 −7.8 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.45 (br s, 1H), 7.65 (d, J = 5.1 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H), 7.26–7.21 (m, 2H), 6.59 (br s, 1H), 5.90–5.74 (m, 2H), 5.27–5.12 (m, 2H), 5.08–4.95 (m, 2H), 4.37–4.26 (m, 1H), 4.25–4.15 (m, 1H), 4.15–4.08 (m, 2H), 4.04 (br s, 1H), 3.98–3.83 (m, 4H), 3.76 (dd, J = 10.0, 4.0 Hz, 1H), 3.70 (dd, J = 10.0, 3.9 Hz, 1H), 3.67 (s, 3H), 3.55–3.43 (m, 2H), 3.20 (dd, J = 11.3, 4.2 Hz, 1H), 3.13 (d, J = 11.3 Hz, 1H), 2.64 (dt, J = 13.6, 8.4 Hz, 1H), 2.37 (d, J = 13.7 Hz, 1H), 2.30–2.02 (m, 8H), 1.98–1.50 (m, 25H), 1.03–0.81 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 178.3, 175.6, 175.2, 174.2, 174.1, 173.3, 173.2, 172.1, 137.7, 134.6, 116.8, 115.1, 78.7, 70.7, 69.6, 68.8, 66.8, 65.7, 59.2, 56.5, 54.8, 54.1 (2C), 52.4, 52.3, 52.1, 39.64, 39.62, 39.4, 39.1, 38.2, 37.2, 36.7, 35.7, 35.3, 30.1, 28.6, 25.2, 25.0 (2C), 24.7, 24.52, 24.49, 24.4, 24.3, 23.45, 23.36, 22.99, 22.96, 21.13, 21.08 (2C), 20.9. IR (KBr): 3343, 2957, 1639, 1547 cm−1. HRMS (ESI) m/z: [M + H]+ calcd for C53H91N8O11, 1015.6807; found, 1015.6843.
Stapled Boc-l-Hyp-l-Ser-[(l-Leu)2-Ac5c]2-OMe (13): Under an argon atmosphere, to a solution of 11 (70.0 mg, 0.0628 mmol) in CH2Cl2 (3 mL) was added second-generation Grubbs catalyst (10.7 mg, 0.0126 mmol) at room temperature, and the mixture was stirred for 2 h at the same temperature. The reaction mixture was filtered through short pad of silica gel (EtOAc) and concentrated. The crude material was purified by flash chromatography on silica gel (70% EtOAc in n-hexane) to provide a stapled peptide 12 (63.2 mg, 93%) as a mixture of E- and Z-isomers (E/Z = 5.5:1). Rf = 0.43 (EtOAc). Next, to a solution of stapled peptides 12 (52.9 mg, 0.0486 mmol) in MeOH (4 mL) was added 10% Pd-C (26 mg, 50 wt %) under a nitrogen atmosphere. After being vigorously stirred under a hydrogen atmosphere for 23 h at room temperature, the reaction mixture was passed through a short plug of Celite. The filtrate was concentrated under vacuum to give a crude product, which was purified by flash column chromatography on silica gel (4% MeOH in CHCl3) to give 13 (46.9 mg, 89%) as a colorless oil. Rf = 0.13 (3% MeOH in CHCl3). [ α ] D 25 −7.8 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.58 (d, J = 5.9 Hz, 1H), 7.50 (d, J = 4.9 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.27–7.22 (m, 2H), 7.17 (d, J = 2.0 Hz, 1H), 4.37–4.30 (m, 2H), 4.23–4.13 (m, 3H), 4.02 (t, J = 3.4 Hz, 1H), 3.97–3.91 (m, 1H), 3.83–3.76 (m, 2H), 3.67 (s, 3H), 3.63–3.51 (m, 4H), 3.50–3.44 (m, 1H), 3.33 (dd, J = 12.0, 2.9 Hz, 1H), 2.65 (dt, J = 13.6, 8.2 Hz, 1H), 2.46–2.37 (m, 1H), 2.31–2.03 (m, 6H), 2.00 (br s, 1H), 1.95–1.54 (m, 26H), 1.52 (s, 9H), 1.51–1.42 (m, 3H), 1.01–0.82 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.6, 175.2, 174.5, 174.4, 174.1, 173.1, 173.0, 171.1, 155.8, 81.6, 78.0, 71.8, 69.7, 69.4, 66.7, 65.7, 60.5, 56.2, 54.8, 54.2, 54.0, 52.7, 52.2, 52.0, 39.7, 39.6, 39.4, 39.1, 38.3, 37.3, 36.7, 35.6, 35.4, 29.1, 28.2 (3C), 27.0, 26.9, 25.4, 25.1, 25.0, 24.8, 24.7, 24.54, 24.52 (2C), 24.4, 23.5, 23.4, 23.0, 22.8, 21.14, 21.05, 21.0, 20.9. IR (CDCl3): 3321, 2959, 1732, 1661, 1530 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C56H96N8O13Na, 1111.6995; found, 1111.7016.
Stapled H-l-Hyp-l-Ser-[(l-Leu)2-Ac5c]2-OMe (C’): To a solution of Boc-protected peptide 13 (11.5 mg, 0.0110 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic acid (0.0110 mL) dropwise at room temperature, and the reaction mixture was stirred for 2 days at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product C’ (11.6 mg, quant), which was used for the next step without further purification. Rf = 0.20 (EtOAc). Mp 105–107 °C. [ α ] D 27 −8.7 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.27 (d, J = 5.1 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 4.6 Hz, 1H), 7.26–7.21 (m, 3H), 7.00 (d, J = 4.2 Hz, 1H), 4.37–4.27 (m, 2H), 4.19 (dd, J = 10.8, 5.9 Hz, 1H), 4.13–4.04 (m, 2H), 3.97–3.86 (m, 2H), 3.84 (dd, J = 10.5, 5.9 Hz, 1H), 3.70 (dd, J = 10.6, 2.8 Hz, 1H), 3.67 (s, 3H), 3.64–3.59 (m, 1H), 3.59–3.53 (m, 1H), 3.48 (t, J = 8.9 Hz, 1H), 3.43–3.37 (m, 1H), 3.35 (d, J = 10.5 Hz, 1H), 3.00 (dd, J = 10.5, 2.9 Hz, 1H), 2.69–2.60 (m, 1H), 2.37–2.03 (m, 7H), 1.97–1.49 (m, 30H), 1.03–0.78 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.7, 175.3, 174.2, 174.0, 173.23, 173.20 (2C), 172.0, 79.5, 71.5, 69.3, 69.0, 66.8, 65.7, 59.6, 55.9, 54.8, 54.1 (2C), 52.4, 52.2, 51.0, 39.6, 39.5, 39.4, 39.2, 38.3, 37.2, 36.7, 36.0, 35.4, 28.9, 28.3, 27.5, 26.4, 25.23, 25.20, 25.1, 24.8, 24.6, 24.52, 24.49, 24.40, 23.5, 23.4, 23.1, 22.9, 21.3, 21.1, 21.0, 20.9. IR (KBr): 3337, 2957, 1736, 1655, 1535 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C51H88N8O11Na, 1011.6470; found, 1011.6467.

3.4. Synthesis of Unstapled Peptide D and Stapled Peptide D’

Boc-l-SerOPte-(l-Leu)2-Ac5c-OMe (14): To a solution of Boc-l-SerOPte-OH 8 [51] (200 mg, 0.732 mmol) in CH2Cl2 (2.5 mL) were added EDCI·HCl (140 mg, 0.732 mmol) and HOBt·H2O (135 g, 0.878 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of H-(l-Leu)2-Ac5c-OMe [44] (270 mg, 0.732 mmol) in CH2Cl2 (2.5 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring overnight, the CH2Cl2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (50% EtOAc in n-hexane) to give 14 (307 mg, 67%) as a white solid. Rf = 0.52 (60% EtOAc in n-hexane). Mp 109–115 °C. [ α ] D 26 −40.4 (c 0.995, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 6.84 (br s, 2H), 6.57 (d, J = 4.9 Hz, 1H), 5.79 (ddt, J = 17.0, 10.3, 6.6 Hz, 1H), 5.38 (br s, 1H), 5.06–4.95 (m, 2H), 4.44 (td, J = 9.2, 4.9 Hz, 1H), 4.37–4.29 (m, 1H), 4.12 (q, J = 4.9 Hz, 1H), 3.76 (dd, J = 9.5, 4.6 Hz, 1H), 3.68 (s, 3H), 3.62 (dd, J = 9.2, 5.3 Hz, 1H), 3.53–3.44 (m, 2H), 2.25 (dt, J = 13.2, 7.7 Hz, 1H), 2.17 (dt, J = 13.1, 7.8 Hz, 1H), 2.10 (q, J = 7.3 Hz, 2H), 2.07–1.95 (m, 2H), 1.87–1.63 (m, 9H), 1.63–1.49 (m, 3H), 1.47 (s, 9H), 1.00–0.85 (m, 12H). 13C NMR (125 MHz, CDCl3) δ: 174.6, 171.6, 171.5, 171.1, 156.3, 137.8, 115.1, 81.1, 71.0, 69.4, 65.9, 55.6, 53.0, 52.3, 51.5, 40.4, 39.9, 37.2, 36.9, 30.2, 28.6, 28.2 (3C), 25.1, 24.8, 24.5, 24.4, 23.1, 23.0, 21.6, 21.4. IR (KBr): 3277, 2957, 1719, 1670, 1560 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C32H56N4O8Na, 647.3996; found, 647.3991.
Boc-(l-Leu)2-l-SerOPte-(l-Leu)2-Ac5c-OMe (16): To a solution of Boc-protected peptide 14 (307 mg, 0.491 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic acid (0.982 mL) dropwise at room temperature, and the reaction mixture was stirred overnight at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 four times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product 15 (295 mg, quant), which was used for the next step without further purification. Rf = 0.20 (60% EtOAc in n-hexane). To a solution of Boc-(l-Leu)2-OH (194 mg, 0.562 mmol) in CH2Cl2 (2 mL) were added EDCI·HCl (108 mg, 0.562 mmol) and HOBt·H2O (103 mg, 0.674 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of amine 15 (295 mg, 0.562 mmol) in CH2Cl2 (2 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring for 5 days, the CH2Cl2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (60% EtOAc in n-hexane) to give 16 (300 mg, 63%) as a white solid. Rf = 0.30 (60% EtOAc in n-hexane). Mp 243–246 °C. [ α ] D 25 −43.2 (c 1.02, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.41 (d, J = 4.4 Hz, 1H), 7.30 (d, J = 6.6 Hz, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.02 (s, 1H), 6.84 (d, J = 3.7 Hz, 1H), 5.76 (ddt, J = 17.1, 10.3, 6.6 Hz, 1H), 5.05–4.92 (m, 2H), 5.00 (s, 1H), 4.42–4.34 (m, 1H), 4.34–4.27 (m, 1H), 4.25–4.20 (m, 1H), 4.06–3.96 (m, 2H), 3.86 (dd, J = 10.0, 5.4 Hz, 1H), 3.67 (s, 3H), 3.67–3.64 (m, 1H), 3.50–3.38 (m, 2H), 2.27–2.00 (m, 6H), 1.87–1.51 (m, 18H), 1.48 (s, 9H), 1.02–0.83 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 174.9, 174.3, 173.4, 172.47, 172.45, 170.9, 156.6, 137.8, 114.9, 81.5, 70.5, 68.7, 65.7, 56.3, 54.3, 54.0, 53.4, 52.2, 52.0, 40.2, 39.62 (2C), 39.59, 39.4, 37.2, 36.8, 30.1, 28.7, 28.22 (3C), 28.17 (2C), 25.0, 24.9, 24.8, 24.7, 24.4, 24.3, 23.4, 22.9, 21.5, 21.2, 20.7. IR (KBr): 3277, 2957, 1719, 1630, 1560 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C44H78N6O10Na, 873.5677; found, 873.5658.
Cbz-l-ProαAll-(l-Leu)2-l-SerOPte-(l-Leu)2-Ac5c-OMe (19): To a solution of Boc-protected peptide 16 (112 mg, 0.132 mmol) in CH2Cl2 (1.3 mL) was added trifluoroacetic acid (0.264 mL) dropwise at room temperature, and the reaction mixture was stirred for 2 days at the same temperature. The reaction mixture was neutralized by adding sat. aq NaHCO3, and the aqueous phase was extracted with CHCl3 three times. The combined organic extracts were dried over anhydrous MgSO4 and concentrated under a vacuum to give crude product 17 (85.8 mg, 87%), which was used for the next step without further purification. Rf = 0.37 (EtOAc). To a solution of Cbz-l-ProαAll-OH (18 [52,53]; 27.2 mg, 0.0939 mmol) in CH2Cl2 (1 mL) were added EDCI·HCl (18.0 mg, 0.0939 mmol) and HOBt·H2O (17.3 mg, 0.113 mmol) at 0 °C, and the solution was stirred for 30 min. Then, a solution of amine 17 (70.5 mg, 0.0939 mmol) in CH2Cl2 (1 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring for 23 h, the CH2Cl2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (50% EtOAc in n-hexane) to give 19 (77.0 mg, 80%) as a colorless oil. Rf = 0.60 (60% EtOAc in n-hexane). [ α ] D 25 −8.6 (c 2.13, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.65 (br s, 1H), 7.51 (br s, 1H), 7.42–7.35 (m, 3H), 7.35–7.31 (m, 2H), 7.29 (d, J = 6.6 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.33 (br s, 1H), 5.82–5.63 (m, 2H), 5.18 (s, 2H), 5.17–5.05 (m, 2H), 5.00–4.89 (m, 2H), 4.37 (q, J = 7.5 Hz, 1H), 4.32–4.24 (m, 2H), 4.10–3.98 (m, 2H), 3.92–3.85 (m, 1H), 3.83–3.71 (m, 2H), 3.67 (s, 3H), 3.54–3.47 (m, 1H), 3.45 (t, J = 6.6 Hz, 2H), 2.96 (dd, J = 14.2, 7.3 Hz, 1H), 2.76 (dd, J = 14.2, 7.6 Hz, 1H), 2.28–2.11 (m, 5H), 2.11–1.99 (m, 5H), 1.91–1.54 (m, 17H), 1.46–1.38 (m, 1H), 1.05–0.81 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 175.3, 175.0, 174.6, 174.1, 172.62, 172.60, 171.0, 155.2, 138.1, 136.0, 132.0, 128.7 (2C), 128.5, 127.4 (2C), 120.5, 114.6, 70.2, 69.0, 68.9, 67.5, 65.7, 56.7, 54.6, 54.2, 53.5, 52.12, 52.11, 48.6, 39.7 (2C), 39.4, 39.3, 37.6, 37.1, 36.8, 35.7, 30.2, 28.7, 25.3, 25.0, 24.9, 24.8, 24.4 (2C), 24.3, 23.4 (2C), 23.3 (2C), 23.1, 22.9, 21.3, 20.9. IR (CDCl3): 3323, 2959, 1663, 1531 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C55H87N7O11Na, 1044.6361; found, 1044.6360.
H-l-ProαAll-(l-Leu)2-l-SerOPte-(l-Leu)2-Ac5c-OMe (D): To a solution of Cbz-protected peptide 19 (21.4 mg, 0.0209 mmol) in MeOH (2 mL) was added 10% Pd-C (10 mg, 50 wt %) under a nitrogen atmosphere. After being vigorously stirred under a hydrogen atmosphere for 2 days at room temperature, the reaction mixture was passed through a short plug of Celite. The filtrate was concentrated under a vacuum to give a crude product, which was purified by flash column chromatography on silica gel (70% EtOAc in n-hexane) to give D (14.5 mg, 78%) as a white solid. Rf = 0.38 (EtOAc). [ α ] D 25 −54.3 (c 1.60, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.45 (d, J = 4.4 Hz, 1H), 7.32 (d, J = 5.1 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.19–7.10 (m, 2H), 6.99 (s, 1H), 4.43–4.29 (m, 2H), 4.22–4.14 (m, 2H), 4.00–3.92 (m, 1H), 3.85 (dd, J = 9.9, 4.8 Hz, 1H), 3.70 (dd, J = 9.8, 3.4 Hz, 1H), 3.67 (s, 3H), 3.48–3.37 (m, 2H), 3.16–3.07 (m, 1H), 2.84 (dt, J = 10.5, 5.4 Hz, 1H), 2.27–2.12 (m, 3H), 2.12–1.94 (m, 3H), 1.86 (d, J = 12.5 Hz, 2H), 1.83–1.61 (m, 18H), 1.59–1.46 (m, 4H), 1.41–1.22 (m, 4H), 1.04–0.81 (m, 30H). 13C NMR (125 MHz, CDCl3) δ: 179.4, 174.9, 173.9, 173.4, 172.5, 172.4, 171.0, 71.5, 70.0, 68.6, 65.7, 56.2, 54.5, 53.3, 52.3, 52.2, 52.0, 47.4, 41.3, 40.4, 39.6, 39.3, 38.5, 37.2, 37.0, 36.8, 29.3, 28.0, 26.3, 24.9, 24.8 (3C), 24.4, 24.3, 23.6, 23.4, 23.2, 22.9, 22.5, 21.3, 21.2, 21.1, 20.7, 18.6, 14.4, 14.0. IR (CDCl3): 3327, 2961, 1734, 1663, 1530 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C47H85N7O9Na, 914.6306; found, 914.6267.
Stapled H-l-Pro-(l-Leu)2-l-Ser-(l-Leu)2-Ac5c-OMe (D’): Under an argon atmosphere, to a solution of 19 (22.3 mg, 0.0218 mmol) in CH2Cl2 (1 mL) was added second-generation Grubbs catalyst (3.7 mg, 4.4 μmol) at room temperature, and the mixture was stirred for 2 h at the same temperature. The reaction mixture was filtered through a short pad of silica gel (EtOAc) and concentrated. The crude material was purified by flash chromatography on silica gel (80% EtOAc in n-hexane) to provide stapled peptide 20 (21.0 mg, 96%) as a mixture of E- and Z-isomers (E/Z = 1.1:1). Rf (E-form) = 0.35 (50% EtOAc in n-hexane), Rf (Z-form) = 0.26 (50% EtOAc in n-hexane). Next, to a solution of stapled peptides 20 (28.1 mg, 0.0283 mmol) in MeOH (1.5 mL) was added 10% Pd-C (15 mg, 50 wt %) under a nitrogen atmosphere. After being vigorously stirred under a hydrogen atmosphere for 21 h at room temperature, the reaction mixture was passed through a short plug of Celite. The filtrate was concentrated under a vacuum to give a crude product, which was purified by flash column chromatography on silica gel (5% MeOH in CHCl3) to give D’ (13.6 mg, 71%) as a white solid. Rf = 0.16 (3% MeOH in CHCl3). [ α ] D 23 −30.2 (c 1.38, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.49 (br s, 1H), 7.27 (d, J = 7.3 Hz, 1H), 7.23 (d, J = 6.1 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 6.96 (s, 1H), 6.38 (br s, 1H), 4.48 (ddd, J = 10.0, 6.1, 3.4 Hz, 1H), 4.40–4.27 (m, 2H), 4.03–3.93 (m, 2H), 3.90 (dd, J = 9.8, 3.4 Hz, 1H), 3.77–3.68 (m, 1H), 3.68 (s, 3H), 3.54 (dt, J = 9.2, 4.5 Hz, 1H), 3.50–3.43 (m, 1H), 3.14–3.06 (m, 1H), 2.86–2.78 (m, 1H), 2.31–2.20 (m, 2H), 2.19–2.12 (m, 1H), 2.11–1.99 (m, 2H), 1.99–1.91 (m, 1H), 1.90–1.58 (m, 21H), 1.57–1.36 (m, 7H), 1.05–0.82 (m, 24H). 13C NMR (125 MHz, CDCl3) δ: 174.9, 174.2, 173.39, 173.38, 172.5, 172.4, 170.4, 69.0, 68.8, 65.8, 56.0, 54.5, 54.3, 53.5, 52.2, 52.0, 46.6, 40.3, 40.1, 39.68, 39.66, 38.7, 37.2, 36.9, 36.8, 28.2, 27.4, 26.4, 25.5, 25.2, 25.1, 24.9, 24.7, 24.40, 24.35 (2C), 24.2, 23.3 (2C), 23.0, 22.7, 21.8, 21.2, 21.1, 21.0. IR (KBr): 3296, 3109, 2953, 1641, 1530 cm−1. HRMS (ESI) m/z: [M + Na]+ calcd for C45H79N7O9Na, 884.5837; found, 884.5874.

3.5. General Procedure for Peptide-Catalyzed Michael Addition of 1-Methylindole to α,β-Unsaturated Aldehyde

Michael adduct 23: To a mixture of (E)-4-nitrocinnamaldehyde (21; 8.9 mg, 0.050 mmol), peptide catalyst (0.010 mmol), and benzoic acid (1.2 mg, 0.010 mmol) in THF (0.25 mL) was added 1-methylindole 22 (0.0187 mL, 0.150 mmol) at room temperature, and the mixture was stirred for the given time. To the reaction mixture were added NaBH4 (9.5 mg, 0.25 mmol) and EtOH (0.25 mL) at the same temperature, and additionally stirred for 30 min. After filtration via a short plug of silica gel (60% EtOAc in n-hexane), the filtrate was concentrated under a vacuum to give crude product 23 as a white solid. Rf = 0.14 (50% EtOAc in n-hexane). 1H NMR (500 MHz, CDCl3) δ: 8.12 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.96 (s, 1H), 4.55 (t, J = 7.7 Hz, 1H), 3.79 (s, 3H), 3.74–3.61 (m, 2H), 2.52–2.45 (m, 1H), 2.31–2.24 (m, 1H). HPLC (Chiralpak AD-H, 10% i-propanol in n-hexane, flow rate = 1.0 mL/min): tR = 26.0 min (minor), tR = 35.2 min (major), ee = 47%. HPLC chart is given in the Supplementary Materials.

Supplementary Materials

The following are available online, 1H and 13C NMR spectra of compounds 3, A, 4, B, 6, A’, 7, B’, 9, 11, C, 13, C’, 14, 16, 19, D, and D’; HPLC chart of compound 23.

Author Contributions

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

Funding

This research was funded by JSPS KAKENHI Grant Numbers JP17H03998 (M.T.), JP18K14870 (A.U.), and JP20K06967 (A.U.), the Ube Industries Foundation (A.U.), and Shionogi Award in Synthetic Organic Chemistry, Japan (A.U.).

Acknowledgments

T.U. is grateful for a JSPS fellowship Grant Number JP17J05108. The authors would like to thank Enago (www.enago.jp) for the English language review. This work was the result of using research equipment shared in MEXT Project for promoting the public utilization of advanced research infrastructure (program for supporting introduction of the new sharing system), grant number JPMXS0422500320.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds are not available from the authors.
Molecules 25 04667 g001 550
Figure 1. Hydrocarbon-stapled peptides used in the organocatalytic reaction. (a) Helical peptide-based primary amino catalyst with stapling at i,i+4 and (b) helical peptide-based secondary amino catalysts with stapling at i,i+1 and i,i+3 (This work).
Figure 1. Hydrocarbon-stapled peptides used in the organocatalytic reaction. (a) Helical peptide-based primary amino catalyst with stapling at i,i+4 and (b) helical peptide-based secondary amino catalysts with stapling at i,i+1 and i,i+3 (This work).
Molecules 25 04667 g001
Molecules 25 04667 sch001 550
Scheme 1. Synthesis of unstapled peptides A and B and stapled peptides A’ and B’ (tethering at i, i+1 positions with a 13-membered ring).
Scheme 1. Synthesis of unstapled peptides A and B and stapled peptides A’ and B’ (tethering at i, i+1 positions with a 13-membered ring).
Molecules 25 04667 sch001
Molecules 25 04667 sch002 550
Scheme 2. Synthesis of unstapled peptide C and stapled peptide C’ (tethering at i, i+1 positions with a 15-membered ring).
Scheme 2. Synthesis of unstapled peptide C and stapled peptide C’ (tethering at i, i+1 positions with a 15-membered ring).
Molecules 25 04667 sch002
Molecules 25 04667 sch003 550
Scheme 3. Synthesis of unstapled peptide D and stapled peptide D’ (tethering at i, i+3 positions with an 18-membered ring).
Scheme 3. Synthesis of unstapled peptide D and stapled peptide D’ (tethering at i, i+3 positions with an 18-membered ring).
Molecules 25 04667 sch003
Molecules 25 04667 g002 550
Figure 2. Circular dichroism (CD) spectra of peptide catalysts A–D and A’–D’. (0.5 mM in 2,2,2-trifluoroethanol).
Figure 2. Circular dichroism (CD) spectra of peptide catalysts A–D and A’–D’. (0.5 mM in 2,2,2-trifluoroethanol).
Molecules 25 04667 g002
Molecules 25 04667 g003 550
Figure 3. A plausible reaction mechanism catalyzed by (a) stapled peptide B’ and (b) unstapled peptide B.
Figure 3. A plausible reaction mechanism catalyzed by (a) stapled peptide B’ and (b) unstapled peptide B.
Molecules 25 04667 g003
Table
Table 1. Catalytic activities of unstapled peptides A–D and stapled peptides A’–D’ in the Michael addition reaction of 21 and 22.
Table 1. Catalytic activities of unstapled peptides A–D and stapled peptides A’–D’ in the Michael addition reaction of 21 and 22.
Molecules 25 04667 i001
EntryPeptideTime (d)Conv. (%) 1Ee (%) 2
1A627−1
2A’6466
3B112−29
4B’18336
5C451−11
6C’481−5
7D250−47
8D’269−56
9none19N.D. 3
1 Conversion was determined by 1H NMR analysis. 2 Ee was determined by HPLC. 3 Not determined.
Table
Table 2. Effect of the catalyst loading and temperature in the Michael addition reaction.
Table 2. Effect of the catalyst loading and temperature in the Michael addition reaction.
Molecules 25 04667 i002
EntryPeptide (mol %)Temp. (°C)Conv. (%) 1Ee (%) 2
1B (20)rt 312−29
2B’ (20)rt 38336
3B (10)rt 36−13
4B’ (10)rt 38136
5B (5)rt 3traceN.D. 4
6B’ (5)rt 36630
7B (20)0traceN.D. 4
8B’ (20)07847
1 Conversion was determined by 1H NMR analysis. 2 Ee was determined by HPLC. 3 Room temperature. 4 Not determined.

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Ueda, A.; Higuchi, M.; Sato, K.; Umeno, T.; Tanaka, M. Design and Synthesis of Helical N-Terminal l-Prolyl Oligopeptides Possessing Hydrocarbon Stapling. Molecules 2020, 25, 4667. https://doi.org/10.3390/molecules25204667

AMA Style

Ueda A, Higuchi M, Sato K, Umeno T, Tanaka M. Design and Synthesis of Helical N-Terminal l-Prolyl Oligopeptides Possessing Hydrocarbon Stapling. Molecules. 2020; 25(20):4667. https://doi.org/10.3390/molecules25204667

Chicago/Turabian Style

Ueda, Atsushi, Mei Higuchi, Kazuki Sato, Tomohiro Umeno, and Masakazu Tanaka. 2020. "Design and Synthesis of Helical N-Terminal l-Prolyl Oligopeptides Possessing Hydrocarbon Stapling" Molecules 25, no. 20: 4667. https://doi.org/10.3390/molecules25204667

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