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

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.


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 3 10 -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].

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 helixinducing 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.
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-Ac 5 c-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-Ac 5 c] 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  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'. 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.  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'.

Entry
Peptide (mol %) 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.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 18 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 1methylindole 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]  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 Zand 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.
caused by the hydrocarbon stapling, which accelerated the Friedel-Crafts type attack of 1methylindole 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]

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 CHCl 3 as a solvent. 1 H NMR and 13 C 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 1 H NMR spectra (CDCl 3 ), tetramethylsilane was used as the internal reference (0.00 ppm), while the central solvent peak was used as the reference (77.0 ppm in CDCl 3 ) for the 13 C 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 F 254 , 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-Ac 5 c) 2 -OMe [44], and H-l-Leu-l-Leu-Ac 5 c-OMe [44] were prepared according to the reported procedures. Copies of NMR Spectra are given in the Supplementary Materials.

Synthesis of Unstapled Peptides A and B and Stapled Peptides A' and B'
Boc-l-Hyp OAll -l-Ser OAll -OMe (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 CH 2 Cl 2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous MgSO 4 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. 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 Na 2 SO 4 , 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 CH 2 Cl 2 (2.5 mL) was added EDCI·HCl (96.0 mg, 0.500 mmol) and HOBt·H 2 O (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 -Ac 5 c] 2 -OMe (354 mg, 0.500 mmol) in CH 2 Cl 2 (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 CH 2 Cl 2 , the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous MgSO 4 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.  H-l-Hyp OAll -l-Ser OAll -[(l-Leu) 2 -Ac 5 c] 2 -OMe (B): To a solution of Boc-protected peptide 4 (135 mg, 0.124 mmol) in CH 2 Cl 2 (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 NaHCO 3 and the aqueous phase was extracted with CHCl 3 four times. The combined organic extracts were dried over anhydrous MgSO 4 13  Stapled Boc-l-Hyp-l-Ser-OMe (6): Under an argon atmosphere, to a solution of 3 (90.0 mg, 0.218 mmol) in CH 2 Cl 2 (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 Eand Z-isomers (E/Z = 1.0:5.6). R f = 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. Stapled H-l-Hyp-l-Ser-OMe (A'): To a solution of Boc-protected dipeptide 6 (45.0 mg, 0.116 mmol) in CH 2 Cl 2 (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 NaHCO 3 , and the aqueous phase was extracted with CHCl 3 three times. The combined organic extracts were dried over anhydrous MgSO 4 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. R f = 0.30 (EtOAc).  [44] (163 mg, 0.230 mmol) in CH 2 Cl 2 (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 CH 2 Cl 2 , the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous MgSO 4 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.  Stapled H-l-Hyp-l-Ser-[(l-Leu) 2 -Ac 5 c] 2 -OMe (B'): To a solution of Boc-protected peptide 7 (120 mg, 0.113 mmol) in CH 2 Cl 2 (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 NaHCO 3 , and the aqueous phase was extracted with CHCl 3 three times. The combined organic extracts were dried over anhydrous MgSO 4 and concentrated under a vacuum to give crude product B' (96.9 mg, 89%), which was used for the next step without further purification.

Synthesis of Unstapled Peptide C and Stapled Peptide C'
Boc-l-Ser OPte -[(l-Leu) 2 -Ac 5 c] 2 -OMe (9): To a solution of Boc-l-Ser OPte -OH 8 [51] (193 mg, 0.707 mmol) in CH 2 Cl 2 (2.5 mL) were added EDCI·HCl (136 mg, 0.707 mmol) and HOBt·H 2 O (130 mg, 0.848 mmol) at 0 • C, and the solution was stirred for 30 min. Then, a solution of H-[(l-Leu) 2 -Ac 5 c] 2 -OMe [44] (500 mg, 0.707 mmol) in CH 2 Cl 2 (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, CH 2 Cl 2 was removed, and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous MgSO 4 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. R f = 0. 66  Boc-l-Hyp OAll -l-Ser OPte -[(l-Leu) 2 -Ac 5 c] 2 -OMe (11): To a solution of Boc-protected peptide 9 (480 mg, 0.499 mmol) in CH 2 Cl 2 (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 NaHCO 3 , and the aqueous phase was extracted with CHCl 3 three times. The combined organic extracts were dried over anhydrous MgSO 4 and concentrated under a vacuum to give crude product 10 (391 mg, 91%), which was used for the next step without further purification. R f = 0.20 (60% EtOAc in n-hexane). To a solution of Boc-l-Hyp OAll -OH (123 mg, 0.454 mmol) in CH 2 Cl 2 (2.5 mL) were added EDCI·HCl (87.0 mg, 0.454 mmol) and HOBt·H 2 O (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 CH 2 Cl 2 (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 CH 2 Cl 2 was removed and the residue was diluted with EtOAc. The solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous MgSO 4 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.  13  H-l-Hyp OAll -l-Ser OPte -[(l-Leu) 2 -Ac 5 c] 2 -OMe (C): To a solution of Boc-protected peptide 11 (30.0 mg, 0.0269 mmol) in CH 2 Cl 2 (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 NaHCO 3 , and the aqueous phase was extracted with CHCl 3 three times. The combined organic extracts were dried over anhydrous MgSO 4 and concentrated under a vacuum to give crude product C (23.5 mg, 86%), which was used for the next step without further purification.