X-ray Crystallographic Structure of α-Helical Peptide Stabilized by Hydrocarbon Stapling at i,i + 1 Positions

Hydrocarbon stapling is a useful tool for stabilizing the secondary structure of peptides. Among several methods, hydrocarbon stapling at i,i + 1 positions was not extensively studied, and their secondary structures are not clarified. In this study, we investigate i,i + 1 hydrocarbon stapling between cis-4-allyloxy-l-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled side chains and structure of the olefin-tethered amino acids, E- or Z-selectivities were observed during the ring-closing metathesis reaction (E/Z was up to 8.5:1 for 17–14-membered rings and up to 1:20 for 13-membered rings). We performed X-ray crystallographic analysis of hydrocarbon stapled peptide at i,i + 1 positions. The X-ray crystallographic structure suggested that the i,i + 1 staple stabilizes the peptide secondary structure to the right-handed α-helix. These findings are especially important for short oligopeptides because the employed stapling method uses two minimal amino acid residues adjacent to each other.


Introduction
Introducing hydrocarbon stapling on the side chains of peptides is a promising technique for stabilizing the secondary structure of peptides and enhancing their functionalities [1][2][3][4][5]. Hydrocarbon stapling can be easily obtained by ring-closing metathesis reactions between olefin-bearing amino acid residues using Ru catalysts [6,7]. After the report on αhelicity-inducing all-hydrocarbon stapled peptides at i,i + 4 and i,i + 7 positions by Verdine et al. [8], several studies focused on the approach (as illustrated in Figure 1a) [9][10][11]. Currently, all-hydrocarbon stapled peptides are very important in drug development targeting protein-protein interactions because the pharmacophores interact via α-helical motifs [12]. Hydrocarbon stapling at i,i + 3 positions are reported in the literature [13][14][15]. For example, O'Leary et al. reported E-selective ring-closing metathesis between O-allyl-tethered L-serines at i,i + 3 positions to produce 3 10 -helical peptides [13]. Other hydrocarbon staples, such as i,i + 1 and i,i + 2, were not well researched, and their 3D structures are unknown (as illustrated in Figure 1b) [16][17][18][19]. In general, hydrocarbon stapling sacrifices two amino acid residues for the crosslinking motif, and those residues should not include essential residues for their biological activities. Based on this, the development of a large variety of hydrocarbon stapling at different positions can be achieved. Herein, we report hydrocarbon stapling of peptides at i,i + 1 positions by ring-closing metathesis reactions and the X-ray crystallographic structure of the right-handed α-helical octapeptide stabilized by i,i + 1 stapling.

Results and Discussion
Our previous report suggests the usefulness of cis-4-hydroxy-L-proline as an olefinbearing amino acid for peptide stapling [19]. Thus, in this study, we started by optimizing the reaction conditions for i,i + 1 peptide stapling using cis-4-hydroxy-L-proline. We screened the ring-closing metathesis reaction at i,i + 1 positions using dipeptide 1 as the cyclization precursor (as illustrated in Table 1). The reaction catalyzed by 20 mol% of second-generation Grubbs catalyst in CH2Cl2 (20 mM) produced the desired 1′ in 55% yield as a mixture of E/Z-isomers (E/Z = 1.0:5.6; Entry 1). A comparable result was obtained using the first-generation Grubbs catalyst (Entry 2). Replacing the reaction solvents, such as toluene, 1,2-dichloroethane (DCE) and tetrahydrofuran (THF), decreased the yields and Z-selectivities (Entries 3-5). The reaction under diluted condition (5 mM in CH2Cl2) afforded the best yield at 76% (Entry 6). The reactions in refluxing CH2Cl2 resulted in insufficient yields due to the degradation of the desired product (Entries 8 and 9). Grubbs 2nd (20) CH2Cl2 (5) reflux 2 28 1.0:4.9 9 Grubbs 2nd (20) CH2Cl2 (

Results and Discussion
Our previous report suggests the usefulness of cis-4-hydroxy-L-proline as an olefinbearing amino acid for peptide stapling [19]. Thus, in this study, we started by optimizing the reaction conditions for i,i + 1 peptide stapling using cis-4-hydroxy-L-proline. We screened the ring-closing metathesis reaction at i,i + 1 positions using dipeptide 1 as the cyclization precursor (as illustrated in Table 1). The reaction catalyzed by 20 mol% of second-generation Grubbs catalyst in CH 2 Cl 2 (20 mM) produced the desired 1 in 55% yield as a mixture of E/Z-isomers (E/Z = 1.0:5.6; Entry 1). A comparable result was obtained using the first-generation Grubbs catalyst (Entry 2). Replacing the reaction solvents, such as toluene, 1,2-dichloroethane (DCE) and tetrahydrofuran (THF), decreased the yields and Z-selectivities (Entries 3-5). The reaction under diluted condition (5 mM in CH 2 Cl 2 ) afforded the best yield at 76% (Entry 6). The reactions in refluxing CH 2 Cl 2 resulted in insufficient yields due to the degradation of the desired product (Entries 8 and 9). closing metathesis reactions and the X-ray crystallographic structure of the right-handed α-helical octapeptide stabilized by i,i + 1 stapling.

Results and Discussion
Our previous report suggests the usefulness of cis-4-hydroxy-L-proline as an olefinbearing amino acid for peptide stapling [19]. Thus, in this study, we started by optimizing the reaction conditions for i,i + 1 peptide stapling using cis-4-hydroxy-L-proline. We screened the ring-closing metathesis reaction at i,i + 1 positions using dipeptide 1 as the cyclization precursor (as illustrated in Table 1). The reaction catalyzed by 20 mol% of second-generation Grubbs catalyst in CH2Cl2 (20 mM) produced the desired 1′ in 55% yield as a mixture of E/Z-isomers (E/Z = 1.0:5.6; Entry 1). A comparable result was obtained using the first-generation Grubbs catalyst (Entry 2). Replacing the reaction solvents, such as toluene, 1,2-dichloroethane (DCE) and tetrahydrofuran (THF), decreased the yields and Z-selectivities (Entries 3-5). The reaction under diluted condition (5 mM in CH2Cl2) afforded the best yield at 76% (Entry 6). The reactions in refluxing CH2Cl2 resulted in insufficient yields due to the degradation of the desired product (Entries 8 and 9). Further, we investigated the substrate scope for the ring-closing metathesis of peptides at i,i + 1 positions using the optimized reaction conditions (as illustrated in Scheme 1). As the ring size of the stapled peptides increased from 13-to 15-membered rings, the yields and E-selectivities increased (Entries 1-3). L-Tyrosine and D-serine-derived unstapled peptides 4 and 5 produced the desired stapled peptides 4 and 5 in 23% and 21% yields, respectively, with large amounts of unreacted starting material (Entries 4 and 5). Surprisingly, high Z-selectivities were observed for the reaction of dipeptides 6 and 7, which were composed of either O-allyl-tethered L-threonine or (S)-α-(4-pentenyl)alanine (Entries 6 and 7; E/Z = 1: >20 for 6 and 1:14 for 7 ). These results suggest that α-methyl or β-methyl groups of i + 1 residue strongly affect the transition state of the ring-closing metathesis to yield Z-isomers. Further, we investigated the substrate scope for the ring-closing metathesis of peptides at i,i + 1 positions using the optimized reaction conditions (as illustrated in Scheme 1). As the ring size of the stapled peptides increased from 13-to 15-membered rings, the yields and E-selectivities increased (Entries 1-3). L-Tyrosine and D-serinederived unstapled peptides 4 and 5 produced the desired stapled peptides 4′ and 5′ in 23% and 21% yields, respectively, with large amounts of unreacted starting material (Entries 4 and 5). Surprisingly, high Z-selectivities were observed for the reaction of dipeptides 6 and 7, which were composed of either O-allyl-tethered L-threonine or (S)-α-(4pentenyl)alanine (Entries 6 and 7; E/Z = 1: > 20 for 6′ and 1:14 for 7′). These results suggest that α-methyl or β-methyl groups of i + 1 residue strongly affect the transition state of the ring-closing metathesis to yield Z-isomers.  The i,i + 1 hydrocarbon-stapling reaction of octapeptide 8, in possession of 1-aminocycl oalkane-1-carboxylic acid [20][21][22][23][24][25][26][27][28][29][30][31][32][33], was investigated under the optimized reaction conditions for the ring-closing metathesis (Scheme 2). In contrast with the moderate Z-selectivity The i,i + 1 hydrocarbon-stapling reaction of octapeptide 8, in possession of 1-aminocycloalkane-1-carboxylic acid [20][21][22][23][24][25][26][27][28][29][30][31][32][33], was investigated under the optimized reaction conditions for the ring-closing metathesis (Scheme 2). In contrast with the moderate Zselectivity of 1 (E/Z = 1:5), much higher Z-selectivity was observed for the ring-closing metathesis reaction of 8 (E/Z = 1: > 20). The Z-selectivity could be influenced by their secondary structure. Hydrogenation of 9 afforded saturated stapled peptide 10 in high yield. The high Z-selectivities (E/Z was up to 1: > 20) of the i,i + 1 hydrocarbon stapling is advantageous for peptide staples compared to those reported for i,i + 4 and i,i + 7 hydrocarbon stapling (E/Z was up to 1: > 9) [15]. Crystals suitable for X-ray crystallographic analyses were successfully obtained by slow evaporation of the solution of 10 in N,N-dimethylformamide (DMF)/water at room temperature (20-30 °C) [34]. The structure was solved in the orthorhombic P212121 space group to give an α-helical structure with a DMF molecule in the asymmetric unit (as illustrated in Figures 2 and S1 and Tables 2, 3, and S1). To the best of our knowledge, this is the first X-ray crystallographic structure of α-helical stapled peptides at i and i + 1 positions. In the crystal state of the (i,i + 1)-stapled peptide 10, four consecutive intramolecular hydrogen bonds of the i←i +  [30,36,37]. On the other hand, no intermolecular hydrogen bonds between peptides were observed in the packing mode ( Figure S2). These results suggest that packing contacts have a small or no influence on the secondary structure of right-handed α-helix in this case. Thus, introducing hydrocarbon stapling at i,i + 1 positions using cis-4-hydroxyproline could be used for the stabilization of α-helical peptides likewise i,i + 4 and i,i + 7 staples. In our previous study, we reported asymmetric Crystals suitable for X-ray crystallographic analyses were successfully obtained by slow evaporation of the solution of 10 in N,N-dimethylformamide (DMF)/water at room temperature (20-30 • C) [34]. The structure was solved in the orthorhombic P2 1 2 1 2 1 space group to give an α-helical structure with a DMF molecule in the asymmetric unit (as illustrated in Figure 2 and Figure S1 and Tables 2 and 3, and Table S1). To the best of our knowledge, this is the first X-ray crystallographic structure of α-helical stapled peptides at  [35]. Therefore, the crosslinkage of the i,i + 1 staples at the N-terminus could affect the stabilization of the α-helical structure of 10. On the C-terminus, weak intramolecular hydrogen bonds of the i←i + 3 type were observed, N(7)H···O = C(4) (N···O, 3.37 Å; N-H···O, 136.7 • ) and N(8)H···O = C(5) (N···O, 3.40 Å; N-H···O, 162.7 • ), while the N(8)-H···O(4) angle of i←i + 4 type was too small for a hydrogen bond. These bifurcated hydrogen bonds suggest that the conformation of the C-terminus exists as a mixture of αand 3 10 -helix. Another intramolecular hydrogen bond between the N(2)-H of the main chain and ethereal oxygen of cis-4-hydroxyproline, N(2)H···O = C(Hyp 4 ) (N···O, 2.93 Å; N-H···O, 137.6 • ), was observed. Such hydrogen bond stabilizes the secondary structures of peptides [30,36,37]. On the other hand, no intermolecular hydrogen bonds between peptides were observed in the packing mode ( Figure S2). These results suggest that packing contacts have a small or no influence on the secondary structure of righthanded α-helix in this case. Thus, introducing hydrocarbon stapling at i,i + 1 positions using cis-4-hydroxyproline could be used for the stabilization of α-helical peptides likewise i,i + 4 and i,i + 7 staples. In our previous study, we reported asymmetric Michael addition of 1-methylindole to α,β-unsaturated aldehydes catalyzed by Boc-deprotected 10 [19]. We hypothesized that the reactive iminium ion intermediate between cis-4-hydroxy-L-proline and α,β-unsaturated aldehyde was formed inside the helical pipe with a rigid conformation caused by i,i + 1 staple. The X-ray crystallographic structure of 10 supports this observed conformation of the intermediate. Michael addition of 1-methylindole to α,β-unsaturated aldehydes catalyzed by Bocdeprotected 10 [19]. We hypothesized that the reactive iminium ion intermediate between cis-4-hydroxy-L-proline and α,β-unsaturated aldehyde was formed inside the helical pipe with a rigid conformation caused by i,i + 1 staple. The X-ray crystallographic structure of 10 supports this observed conformation of the intermediate.      In summary, we developed i,i + 1 peptide stapling between cis-4-allyloxy-L-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled peptides, Eor Z-selectivities were observed. The E-configured stapled product was preferred when the product was greater than a 14-membered ring, whereas the Z-configured isomer was preferred when the product was a 13-membered ring. The α-or β-methyl substituent of the i + 1 residue improved the Z-selectivities of the ring-closing metathesis (E:Z = 1: >20). X-ray crystallographic analysis of the octapeptide 10 revealed a stabilized α-helical structure. These results are useful for developing peptide-based organocatalysts [38][39][40] (i.e., considering mechanistic insights and structural modification of peptide catalysts based on the X-ray crystal structure), fluorinated peptides [41] (e.g., stabilization effects of using intramolecular hydrogen bonds beside main chain hydrogen bonds), and peptide-based drug delivery systems [42][43][44][45][46] (e.g., introducing i,i + 1 hydrocarbon stapling with essential residues for their biological activities remained intact).

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 realtime (DART) ionization in time-of-flight TOF mode. Analytical and semipreparative thin layer chromatography (TLC) was performed with Merck Millipore precoated TLC plates (MilliporeSigma, Burlington, VT, 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. 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, 8 [19], S-1 [47,48], S-2 [49,50], S-3 [51], and S-5 [52] were prepared according to the reported procedures. Copies of NMR Spectra are given in the Supplementary Materials. 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 three days, 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 Na2SO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (40% EtOAc in nhexane) to give 2 (72.1 mg, 58%)  (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 three days, CH 2 Cl 2 was removed, and the residue was diluted with EtOAc. The solution was washed succes-sively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous Na 2 SO 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 2 (72.1 mg, 58%) as a pale yellow oil. R f = 0.58 (EtOAc were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI·HCl, 67.9 mg, 0.354 mmol) and 1-hydroxybenzotriazole hydrate (HOBt·H2O; 54.2 mg, 0.354 mmol) at 0 °C, and the solution was stirred for 30 min at 0 °C. Then, a solution of O-allyl-L-homoserine methyl ester (H-L-Hse OAll -OMe, S-2 [49,50], 51.1 mg, 0.295 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 three days, 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 Na2SO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (40% EtOAc in nhexane) to give 2 (72.1 mg, 58%) as a pale yellow oil. Rf = 0.58 (EtOAc  (3): to a solution of carboxylic acid S-3 [51] (135 mg, 0.495 mmol) in MeOH (5 mL), thionyl chloride (0.143 mL, 1.98 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 h and was concentrated to give H-L-Ser OPte -OMe·HCl (S-4, Rf = 0.57 with 0.5% AcOH in EtOAc), which was used for the next step without further purification. To a mixture of H-L-Ser OPte -OMe·HCl (S-4, 0.495 mmol) and Boc-L-Hyp OAll -OH (S-1, 148 mg, 0.545 mmol) in CH2Cl2 (5 mL) were added EDCI·HCl (114 mg, 0.594 mmol), HOBt·H2O (91.0 mg, 0.594 mmol), and DIPEA (0.253 mL, 1.49 mmol) at 0 °C, and the mixture was gradually warmed to room temperature. After stirring for 17 h, CH2Cl2 was removed under vacuum, and the residue was diluted with EtOAc. The resultant solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 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 (90.1 mg, 41% in 2 steps) as a pale yellow oil. Rf = 0.71 (EtOAc).  13 [52], 386 mg, 1.64 mmol) in CH 2 Cl 2 (3 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring for 35 h, CH 2 Cl 2 was removed in vacuo, and the residue was diluted with EtOAc. The resultant solution was washed successively with 1 M of HCl, water, sat. aq NaHCO 3 , and brine. The organic layer was dried over anhydrous Na 2 SO 4 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 4 (562 mg, 70%) as a pale yellow oil. R f = 0.75 (EtOAc).  171.1, 171.0, 157.6, 155.4, 154.5, 134.3, 134.1, 133.22, 133.18, 130.5, 130.2, 128.0, 127.8, 117.6,  117.5, 117.3, 117.2, 114.7, 114.6, 114.4, 81.0, 76.1, 72.0, 69.5, 68.69, 68.67, 65.9, 60.1, 59.3 in CH2Cl2 (8 mL) were added EDCI·HCl (314 mg, 1.64 mmol) and HOBt·H2O (301 mg, 1.97 mmol) at 0 °C, and the reaction mixture was stirred for 30 min at 0 °C. Then, a solution of O-allyl-L-tyrosine methyl ester (H-L-Tyr OAll -OMe, S-5 [52], 386 mg, 1.64 mmol) in CH2Cl2 (3 mL) was added to the reaction mixture at the same temperature, and the resultant mixture was gradually warmed to room temperature. After stirring for 35 h, CH2Cl2 was removed in vacuo, and the residue was diluted with EtOAc. The resultant solution was washed successively with 1 M of HCl, water, sat. aq NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to give a crude product, which was purified by flash column chromatography on silica gel (50% EtOAc in nhexane) to give 4 (562 mg, 70%) as a pale yellow oil. Rf = 0.75 (EtOAc).