Total Synthesis of the Highly N-Methylated Peptide Jahanyne - The

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Total Synthesis of the Highly N-Methylated Peptide Jahanyne Baijun Ye, Peng Jiang, Tingrong Zhang, Yahui Ding, Yuanjun Sun, Xin Hao, Lanshu Li, Liang Wang, and Yue Chen J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00503 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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The Journal of Organic Chemistry

Total Synthesis of the Highly N-Methylated Peptide Jahanyne Baijun Ye, Peng Jiang, Tingrong Zhang, Yahui Ding, Yuanjun Sun, Xin Hao, Lanshu Li, Liang Wang,*, and Yue Chen*, The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300350, People's Republic of China. Supporting Information Placeholder

ABSTRACT: Total synthesis of jahanyne (1) was achieved from commercially available materials in 38 mg scale. Boc-N-Me-L-Val-OH fragment along with the HATU/DIPEA coupling condition, were applied to avoid diketopiperazine side reaction in solution phase synthesis.

Secondary metabolites from marine microorganisms constitute a large reservoir of valuable pharmaceuticals ranging from antibiotics to anti-cancer agents.1-3 Cyanobacteria (blue-green algae) are widely distributed throughout the world4 and produce a large variety of secondary metabolites containing cyclic/acyclic peptides.5 Jahanyne, an acetylene-containing lipopeptide, was first discovered in 2015 as a secondary metabolite of cyanobacterium Lyngbya sp. by Suenaga et al.6 Structurally characterized with multiple N-methylated amino acids and two rare partial structures, C-terminal 2-(1-oxo-ethyl)-pyrrolidine moiety (Oep) and N-terminal 2,4-dimethyldec-9-ynoic acid moiety (fatty acid), jahanyne showed good inhibitory activities against HL-60 and Hela cancer cell line. Due to the scarceness of jahanyne, its medicinal chemistry as well as mechanism of action haven’t been explored until the recent work reported by Chandrasekhar et al.7 They completed the total synthesis of desmethyl jahanyne and discovered 3 lipo-tetrapeptides derived from the skeleton of jahanyne, which exhibited comparable bioactivity towards Hela cell line via inducing the apoptosis of cancer cells. Further research on the synthesis of jahanyne and and its analogues may provide new leads for drug discovery. Very recently, Brimble et al. reported the first total synthesis of jahanyne featured with a modified Fmoc solid-phase synthetic strategy to effectively couple hindered N-methylated animo acids without

epimerization.8 Herein, we report our total synthesis of jahanyne. Scheme 1. Retrosynthetic analysis of jahanyne (1)

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Scheme 2. Synthesis of fatty acid 4

Retrosynthetically, disconnected at Oep-L-Pro1 peptide bond, we envisaged that the direct coupling of 2 with Oep moiety may be difficult due to the decomposition of Oep moiety in regular coupling condition.9 So the more stable 3 was chosen as the latest introduced building block to replace the Oep moiety. Further disconnection of 2 at the N-terminal gave rise to two building blocks, including the fatty acid 4 and the peptide 5 (Scheme 1). The fatty acid 4 could be prepared in seven steps from commercially available starting materials and the peptide 5 could be prepared by sequential coupling reactions starting from L-Pro1 to N-Me-L-Ala. The forward synthesis commenced with the synthesis of 4. Treated with n-BuLi, 7 was then subjected to the reaction with 6 to afford 8 in 88% yield.10 Acylation of 8 using oxalyl dichloride followed by amidation with Evans auxiliary 9 provided 10. Asymmetric methylation of the sodium enolate generated from 10 proceeded smoothly to afford 11 in 66% yield with d.r. > 20:1 according to 1H-NMR.11,12 Reduction of 11 with LiAlH4 followed by Appel reaction13 or treatment with Tf2O afforded iodide 13a and 13b in 75% and 65% yields respectively.14 The asymmetric alkylation between 13b and 14b failed to give desired product. To our delight, employing

modified Evans conditions,15 iodide 13a was subjected to asymmetric alkylation with the enolate generated from 14a and gave rise to 15a in 61% yield (d.r. > 20:1 according to 1 H-NMR), installing the required 2,4-anti-dimethyl stereochemistry. Treatment of the resulting 15a with 1 N HCl led to the hydrolysis of both the TMS group and the amide bond, which provided 4 in 57% yield (1.2 g scale, one batch) (Scheme 2). The compound 3 was obtained via reduction of commercially available N-Boc-Oep followed by TFA treatment.16 Scheme 3. Synthesis of compound 3


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Scheme 4. Preparation of peptide chain 5

Scheme 5. Total synthesis of jahanyne (1)

Next, union of N-methylated amino acids was pursued (Scheme 4). The coupling reaction between 16 and 17 under the conventional coupling condition (EDCI/HOBt) led to rapid formation of the condensation product 18 in 75% yield.17 Then we moved on to investigate on the optimal choice of coupling reagents to efficiently couple three sequential sterically hindered N-Me-L-Val-OHs. In addition to the steric hindrance imposed by the additional methyl group, the challenges in the coupling reaction lie in the undesired diketopiperazine (DKP) formation and problematic epimerization.18,19 Brimble selected Fmoc-N-Me-L-Val as the building block in the solid phase synthetic strategy to avoid DKP and fragmentation caused by Boc-N-Me-L-Val. Herein we chose Boc-N-Me-L-Val-OH as the building block. Deprotection of 18 using TFA followed by condensation with 19 under EDCI/HOBt led to trace amount

of product 20, presumably due to steric hindrance. Using the coupling method (HATU/DIPEA) discovered by Louis A. Carpino,20 we were pleased to obtain the condensation product 20 in 60% yield. Under this condition, tetrapeptide 21 and pentapeptide 22 were achieved in 50% and 50% yields respectively. The dropped yields was attributed to the growing hindrance in the N-terminal of 18, 20 and 21. It was noteworthy that during these sequential transformations, no diketopiperazine (DKP) product and 4.1%, 4.5%, 1.6% epimer of 20, 21, 22 were observed respectively according to LCMS analysis. (see Figures S5-S7 in the Supporting Information). Using this coupling condition, 23 (Boc-N-L-pro2-OH) and 25 (Boc-N-Me-L-Ala-OH) were also introduced, provided hexapeptide 24 and heptapeptide 5 in 66% (2.1% epimer) and 62% (1.3% epimer) yields respectively (1.1 g scale, one



batch) (see Figures S8-S9 in the Supporting Information) . All the starting materials in the synthesis of the peptide chain 5 were commercially available. With the key intermediates 3, 4 and 5 in hand, we fulfilled the total synthesis of jahanyne as shown in Scheme 5. Coupling conditions between fatty acid 4 and peptide chain 5 were investigated. The COMU/DIPEA and BOP-Cl/DIPEA conditions21,22 only resulted in low yields (10-20%) of product 26, while the condition using HATU/DIPEA provided 26 in moderate yield with 2.4% epimer was observed. (see Figures S10-S11 in the Supporting Information). For model study, careful hydrolysis of methyl ester of 21 followed by direct condensation (HATU/DIPEA) of Oep moiety led to the rapid consumption of Oep moiety while only trace amount of condensation product was observed. Thus the reduction form of Oep (3) was employed to couple with the acid obtained from hydrolysis of methyl ester of 26. Under HATU/DIPEA condition, compound 27 was obtained in 62% yield (4.2% epimer). Late stage oxidation of 27 using DMP9 followed by subsequent purification of the crude reaction mixture by column chromatography afforded jahanyne (1) (76% yield, 38 mg scale). The characterization data obtained for our synthetic 1 were fully accord with those reported data for the natural product, as well as the synthetic jahanyne reported by Brimble. In summary, total synthesis of jahanyne (1) was achieved from commercially available materials in 38 mg scale. Boc-N-Me-L-Val-OH fragment along with the HATU/DIPEA coupling condition, were applied to avoid diketopiperazine side reaction and less than 5% epimer in each coupling reaction was observed.. Based on this established route, synthesis and biological evaluation of the structural analogs of jahanyne are currently in progress and will be reported in due course.

EXPERIMENT SECTION General Information. Reagents were purchased from commercial suppliers and used without purification unless otherwise stated. Hexamethylphosphoric triamide (HMPA), Ethyl acetate (EtOAc), N,N-Dimethylformamide (DMF), Lithium diisopropylamide (LDA), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCl), 1-Hydroxybenzotriazole (HOBt), Dichloromethane (DCM), N,N-Diisopropy-lethylamine (DIPEA), trifluoroacetic acid (TFA), 1-[Bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), Dess-Martin periodinane (DMP), (Benzotriazol-1yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methanaminiumhexaflu orophosphate (COMU). All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Tetrahydrofuran (THF) was distilled immediately before use from sodium-benzophenone ketyl; Diisopropylamine was distilled from calcium hydride. Solvents for chromatography were used as supplied by Tianjin Reagents chemical. Reactions were monitored by thin layer chromatography (TLC) carried out on silica gel plates using UV light as visualizing agent and aqueous phosphomolybdic acid or basic aqueous potassium permanganate as developing agent. 200-300 mesh silica gel

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was used for column chromatography. 1H NMR, 13C NMR and 2D NMR were recorded on Bruker AV 400 and calibrated by using internal references and solvent signals CDCl3 (δH = 7.26 ppm, δC =77.16 ppm) and CD3OD (δH =3.31 ppm, δC = 49.0 ppm). 1H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants and integration. IR spectra were recorded on a Bruker Tensor II instrument. High-resolution mass spectra (HRMS) were detected on an IonSpec FT-ICR mass spectrometer by Varian 7.0T FTMS. Optical rotations were recorded on an Insmark IP 120 digital polarimeter. LCMS spectra were performed on a Shimadzu LCMS-2020 Quadrupole mass spectrometer (Ion polarity positive, ESI-MS) using a PDA detector at 210 nm. An analytical column (Waters XBridge C18, 3.5 µm; 4.6 × 50 mm) was used at room temperature. Solvent A was 0.1% ammonia solution (25%) in H2O and B was acetonitrile, the linear gradient was 5% B to 95% B over 35 min at a flow rate of 1.0 mL min-1. Preparation of 8. To the solution of Trimethylethynylsilane (70.0 g, 0.712 mol) in THF (500 mL) was added n-BuLi (2.5 M, 295 mL, 0.738 mol) dropwise at -78 oC under argon, the mixture was stirred for 1 h at -78 oC. A solution of compound 6 (51.1 g, 0.262 mol) in THF (400 mL) was slowly added dropwise before HMPA (200 mL) was introduced. After addition, stirring was continued for 2 h at -78 oC and then at -20 oC for 4 h. The reaction was quenched with saturated aqueous NH4Cl (500 mL). EtOAc (500 mL) was added and the layers were separated, the organic layer was washed with brine (3 × 300 mL), dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 100/1) to afford compound 8 (49.2 g, 88%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 2.37 (t, J = 7.4 Hz, 2H), 2.23 (t, J = 6.9 Hz, 2H), 1.70 – 1.62 (m, 2H), 1.58 – 1.43 (m, 4H), 0.14 (s, 9H). Spectroscopic data are in agreement with those reported in literature.10 Preparation of 10. To the solution of compound 8 (49.0 g, 0.231 mol) and DMF (0.50 mL) in DCM (490 mL) was added Oxalyl dichloride (58.6 g, 0.462 mol) dropwise at -5 oC under argon, the mixture was warmed to room temperature and stirred for 3 h. The mixture was concentrated, then THF (50 mL) was added and concentrated again to afford the crude acyl chloride and used directly. To the solution of compound 9 (53.2 g, 0.299 mol) in THF (490 mL) was added n-BuLi (2.5 M, 129.2 mL, 0.32 mol) dropwise at -78 oC under argon. After stirring for 2 h at -78 oC, the crude acyl chloride in THF (245 mL) was added dropwise at -78 oC. After stirring for 2 h at -78 o C, the reaction was quenched with saturated aqueous NH4Cl (200 mL). EtOAc (400 mL) was added and the layers were separated, the organic layer was washed with brine (3 × 300 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 100/1 – 50/1) to afford compound 10 (76.7 g, 89%) as an off white solid. [α]D19 = + 38.5o (c = 1.0, CHCl3); IR (neat) νmax: 2958, 2931, 2172, 1778, 1698, 1243, 1056, 758, 644 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.36 – 7.20 (m, 5H), 4.69 – 4.65 (m, 1H), 4.22 – 4.16 (m, 2H), 3.30 (d, J = 13.3 Hz, 1H), 3.03 – 2.87 (m, 2H), 2.77 (dd, J = 12.9, 10.0 Hz, 1H), 2.25 (t, J = 6.9 Hz, 2H), 1.75 – 1.68 (m, 2H), 1.60 – 1.54 (m, 2H), 1.52 – 1.45 (m, 2H), 0.15


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(s, 9H); 13C NMR (100 MHz, CDCl3) δ 173.3, 153.6, 135.4, 129.5, 129.1, 127.5, 107.4, 84.7, 66.3, 55.23, 38.1, 35.5, 28.5, 28.4, 23.8, 19.8, 0.3; HRMS (ESI) calculated for [M + Na]+ C21H29NNaO3Si+: 394.1809, found 394.1813. Preparation of 11. To the solution of compound 10 (76.5 g, 0.206 mol) in THF (760 mL) was added NaHMDS (2 M, 133.8 mL, 0.267 mol) dropwise at -78 oC under argon. After stirring for 1 h at -78 oC, CH3I (87.7 g, 0.618 mol) was added dropwise at -78 oC. After stirring for 3 h at -78 oC, the reaction was quenched with saturated aqueous NH4Cl (400 mL). EtOAc (500 mL) was added and the layers were separated, the organic layer was washed with brine (3 × 300 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 100/1 – 50/1) to afford compound 11 (52.5 g, 66%) as a colorless oil. [α]D18 = +51.5o (c = 1.0, CHCl3); IR (neat) νmax: 2936, 2861, 2172, 1777, 1696, 1208, 759, 639 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.36 – 7.19 (m, 5H), 4.67 (qd, J = 6.8, 3.1 Hz, 1H), 4.23 – 4.16 (m, 2H), 3.73 – 3.67 (m, 1H), 3.27 (dd, J = 13.3, 3.2 Hz, 1H), 2.77 (dd, J = 13.3, 9.6 Hz, 1H), 2.22 (t, J = 7.0 Hz, 2H), 1.77 – 1.74 (m, 1H), 1.55 – 1.39 (m, 5H), 1.23 (d, J = 6.8 Hz, 3H), 0.14 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 177.3, 153.2, 135.4, 129.6, 129.1, 127.5, 107.4, 84.7, 66.2, 55.5, 38.0, 37.7, 32.9, 28.6, 26.5, 19.8, 17.4, 0.3; HRMS (ESI) calculated for [M + Na]+ C22H31NNaO3Si+ 408.1965, found 408.1968. Preparation of 12. To the solution of compound 11 (52.5 g, 0.136 mol) in THF (520 mL) was added LiAlH4 (6.21 g, 0.163 mol) batchwise at 0 oC under argon. After stirring for 2 h at 0 o C, the reaction was quenched with EtOAc (10 mL). Water (6 mL) was added followed by Na2SO4 (60 g), the mixture was stirred for 0.2 h at room temperature and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 80/1 – 60/1) to afford compound 12 (21.0 g, 73%) as a colorless oil. [α]D18 = – 5.7o (c = 1.0, CHCl3); IR (neat) νmax: 3332, 2956, 2934, 2174, 1248, 1039, 757, 639 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.57 – 3.39 (m, 2H), 2.23 (t, J = 6.6 Hz, 2H), 1.67 – 1.61 (m, 1H), 1.56 – 1.33 (m, 5H), 1.27 (t, J = 5.5 Hz, 1H), 1.20 – 1.06 (m, 1H), 0.93 (d, J = 6.7 Hz, 3H), 0.15 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 107.7, 84.6, 68.4, 35.8, 32.6, 28.9, 26.1, 19.9, 16.6, 0.3; HRMS (ESI) calculated for [M + Na]+ C12H24NaOSi+ 235.1489, compound 12 was not observed by HRMS analysis due to complete fragmentation. Preparation of 13a. To the solution of PPh3 (14.5 g, 55.3 mmol) in DCM (90 mL) was added I2 (14 g, 57.1 mmol) and Imidazole (3.62 g, 53.2 mmol) at 0 oC under argon. After stirring for 15 min at 0 oC, compound 12 (9.0 g, 42.4 mmol) in DCM (15 mL) was added dropwise at 0 oC. After addition, stirring was continued for 2 h at room temperature, then diluted with DCM (65 mL), washed with Na2S2O3 saturated aqueous (3 × 20 mL) and brine (3 × 20 mL), the organic layer was washed with brine (3 × 300 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether) to afford compound 13a (10.2 g, 75%) as a colorless oil. [α]D19= + 2.5o (c = 1.0, CHCl3); IR(neat) νmax: 2958, 2933, 2174, 1248, 1194, 758 638 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.26 – 3.11 (m, 2H), 2.23 (t, J = 6.1 Hz, 2H), 1.54 – 1.31 (m, 6H), 1.29 – 1.17 (m, 1H), 0.98 (d, J = 6.2 Hz, 3H), 0.15 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 107.4, 84.7, 36.0, 34.7, 28.6, 26.1, 20.7, 19.9, 17.9,

0.3; HRMS (ESI) calculated for [M + Na]+ C12H23INaSi+ 345.0506, compound 13a was not observed by HRMS analysis due to complete fragmentation. Preparation of 15a. To the solution of Diisopropylamine (5.92 g, 58.5 mmol) in THF (30 mL) was added n-BuLi (2.5 M, 22.4 mL, 56.0 mmol) dropwise at -10 oC under argon. After addition, the reaction was warmed to room temperature and stirred for 40 min, then cooled to -10 oC, compound 14a (4.00 g, 25.5 mmol) was added dropwise. After stirring for 1 h, HMPA (10.5 g, 58.4 mmol) was introduced. The reaction was cooled to -78 oC, compound 13a (7.80 g, 24.2 mmol) in THF (10 mL) was added dropwise at -78 oC. After stirring for 4 h at -50 oC, the reaction was quenched with saturated aqueous NH4Cl (30 mL). EtOAc (50 mL) was added and the layers were separated, the organic layer was washed with brine (3 × 20 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 5/1 – 3/1) to afford compound 15a (5.20 g, 61%) as a colorless oil. [α]D20= – 29.7o (c = 1.0, CHCl3); IR(neat) νmax: 3386, 2957, 2934, 2173, 1615, 1462, 1248, 1049, 758, 639, cm-1; 1H NMR (400 MHz, CDCl3) δ 4.22 (br, 2H), 3.70 – 3.45 (m, 4H), 2.64 – 2.56 (m, 1H), 2.21 (t, J = 6.8 Hz, 2H), 2.09 – 1.83 (m, 3H), 1.61 – 1.20 (m, 10H), 1.13 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 5.6 Hz, 3H), 0.14 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ 178.8, 107.6, 84.6, 68.0, 61.3, 48.0, 40.7, 36.9, 35.8, 30.4, 28.9, 28.4, 26.1, 24.6, 19.9, 19.5, 17.4, 0.3; HRMS (ESI) calculated for [M + Na]+ C20H37NNaO2Si+ 374.2486, found 374.2490. Preparation of 4. The solution of compound 15a (3.80 g, 10.8 mmol) in HCl (1 N, 40 mL) was refluxed for 4 h, then the reaction was extracted with EtOAc (3 × 50 mL), the organic layer was washed with brine (3 × 30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 50/1 – 30/1) to afford compound 4 (1.20 g, 57%) as a colorless oil. [α]D20= – 5.8o (c = 1.0, CHCl3); IR(neat) νmax: 3308, 2935, 2861, 2118, 1702, 1291, 628 cm-1; 1H NMR (400 MHz, CDCl3) δ 2.59 – 2.50 (m, 1H), 2.19 (td, J = 6.9, 2.6 Hz, 2H), 1.94 (t, J = 2.6 Hz, 1H), 1.59 – 1.30 (m, 9H), 1.16 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 184.2, 84.7, 68.3, 40.8, 37.3, 36.5, 30.4, 28.8, 26.1, 19.3, 18.5, 16.9; HRMS (ESI) calculated for [M – H]– C12H19O2– 195.1391, found 195.1388. Preparation of 18. To the solution of compound 16 (25.0 g, 0.151 mol), compound 17 (32.4 g, 0.116 mol), HOBt (18.8 g, 0.139 mmol) and EDCI (26.7 g, 0.139 mmol) in DCM (750 mL) was added DIPEA (45.0 g, 0.348 mol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with DCM (300 mL), the whole mixture was washed with 10% citric acid (2 × 100 mL), 5% aqueous NaHCO3 (2 × 100 mL), and brine (3 × 100 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 20/1 – 5/1) to afford compound 18 (34.0 g, 75%) as a colorless oil. 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.27 – 7.15 (m, 5H), 5.28, 5.14 (dd, J = 9.1, 6.3 Hz, 0.5H), 4.92, 4.80 (dd, J = 10.4, 4.5 Hz, 0.5H), 4.49 – 4.44 (m, 1H), 3.75, 3.70, 3.67 (s, 3H), 3.55 – 3.34(m, 2H), 3.15, 3.05 (dd, J = 14.4, 5.9 Hz, 1H), 2.97, 2.91 (dd, J = 14.2, 10.6 Hz, 1H), 2.82, 2.80, 2.69, 2.66 (s, 3H),



2.16–1.92 (m, 4 H), 1.32, 1.16 (s, 9 H). Spectroscopic data are in agreement with those reported in literature.17 Preparation of 20. To the solution of compound 18 (26.0 g, 66.7 mmol) in DCM (200 mL) was added TFA (50 mL). After stirring for 7 h at room temperature, toluene (200 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by water (200 mL) and extracted with petroleum ether (3 × 50 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 150 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 19 (16.9 g, 73.2 mmol) and HATU (30.4 g, 79.9 mmol) in DMF (85 mL) was added DIPEA (25.8 g, 200 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (350 mL), the whole mixture was washed with 1% HCl (2 × 50 mL), 5% aqueous NaHCO3 (2 × 50 mL), and brine (2 × 50 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 20/1 – 5/1) to afford compound 20 (20.0 g, 60%) as a colorless oil. [α]D21 = – 169.9o (c = 1.0, CHCl3); IR (neat) νmax: 2983, 2961, 1752, 1682, 1641, 1433, 1170, 741, 558 cm-1; 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.29 – 7.10 (m, 5H), 5.78 (dd, J = 9.9, 5.7 Hz, 1H), 4.57, 4.33 (d, J = 10.7 Hz, 1H), 4.45 (dd, J = 8.5, 3.8 Hz, 1H), 3.72 (s, 3H), 3.69 – 3.53 (m, 2H), 3.22 – 3.12 (m, 1H), 3.07, 3.06 (s, 3H), 3.03 – 2.94 (m, 1H), 2.24, 2.15 (s, 3H), 2.23 – 2.11 (m, 2H), 2.08 – 1.84 (m, 3H), 1.44 (s, 9H), 0.81 – 0.76 (m, 6H); 13C NMR (101 MHz, CDCl3) mixture of rotamers δ 172.7, 172.6, 170.8, 170.5, 169.6, 169.3, 156.0, 154.9, 137.1, 136.7, 129.3, 129.1, 128.7, 128.5, 126.9, 126.6, 80.3, 79.8, 61.4, 59.8, 59.1, 54.8, 54.8, 52.4, 47.2, 35.0, 34.9, 30.7, 30.6, 29.2, 28.7, 28.5, 28.5, 28.4, 27.4, 27.0, 25.0, 20.2, 19.9, 18.3, 17.9; HRMS (ESI) calculated for [M + Na]+ C27H41N3NaO6+ 526.2888, found 526.2891. Preparation of 21. To the solution of compound 20 (17.0 g, 27.5 mmol) in DCM (136 mL) was added TFA (34 mL). After stirring for 6 h at room temperature, toluene (150 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by water (200 mL) and extracted with petroleum ether (3 × 50 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 150 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 19 (10.2 g, 43.9 mmol) and HATU (19.3 g, 50.7 mmol) in DMF (85 mL) was added DIPEA (6.5 g, 50.7 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (300 mL), the whole mixture was washed with 1% HCl (3 × 50 mL), 5% aqueous NaHCO3 (3 × 50 mL), and brine (3 × 50 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 20/1 – 5/1) to afford compound 21 (10.3 g, 50%) as a colorless oil. [α]D19 = – 246.6o (c = 1.0, CHCl3); IR(neat) νmax: 2965, 2931, 1749, 1698, 1641, 1431, 1170, 743, 558 cm-1; 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.33 – 7.12 (m, 5H), 5.91, 5.86 (dd, J =

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10.9, 5.3 Hz, 1H), 5.03 (dd, J = 10.6, 2.1 Hz, 1H), 4.56 – 4.11 (m, 2H), 3.74, 3.73 (s, 3H), 3.71 – 3.45 (m, 2H), 3.21 – 3.01 (m, 2H), 2.99, 2.97 (s, 3H), 2.69, 2.66 (s, 3H), 2.37, 2.21 (s, 3H), 2.35 – 1.88 (m, 6H), 1.49, 1.43 (s, 9H), 0.87 – 0.63 (m, 12H); 13C NMR (101 MHz, CDCl3) mixture of rotamers δ 172.7, 170.6, 170.2, 170.1, 169.5, 169.3, 169.0, 156.3, 155.3, 137.1, 136.7, 129.5, 129.2, 128.6, 128.4, 127.0, 126.7, 80.5, 80.0, 61.5, 59.6, 59.2, 59.1, 58.5, 54.5, 54.4, 52.4, 52.4, 47.3, 47.2, 34.7, 30.7, 30.6, 29.6, 29.5, 29.2, 29.0, 29.0, 28.5, 28.4, 27.2, 27.1, 27.1, 25.0, 20.2, 20.1, 20.0, 19.8, 18.2, 18.1, 17.8, 17.5; HRMS (ESI) calculated for [M + Na]+ C33H52N4NaO7+ 639.3728, found 639.3732. Preparation of 22. To the solution of compound 21 (4.30 g, 7.00 mmol) in DCM (32 mL) was added TFA (8 mL). After stirring for 6 h at room temperature, toluene (20 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by Water (40 mL) and extracted with petroleum ether (3 × 10 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 50 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 19 (4.80 g, 2.10 mmol) and HATU (8.00 g, 2.10 mmol) in DMF (25 mL) was added DIPEA (2.80 g, 2.21 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (100 mL), the whole mixture was washed with 1% HCl (3 × 20 mL), 5% aqueous NaHCO3 (3 × 20 mL), and brine (3 × 20 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 20/1 – 3/1) to afford compound 22 (2.50 g, 50%) as a colorless oil. [α]D19 = – 253.3o (c = 1.0, CHCl3); IR (neat) νmax: 2967, 2936, 1749, 1689, 1640, 1432, 1365, 1047,701, 574 cm-1; 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.30 – 7.14 (m, 5H), 5.85 (dd, J = 10.9, 5.1 Hz, 1H), 5.04, (d, J = 10.6 Hz, 1H), 4.98, 4.95 (d, J = 10.7 Hz, 1H), 4.61, 4.35 (d, J = 10.8 Hz, 1H), 4.45 (dd, J = 8.6, 3.8 Hz, 1H) 3.72 (s, 3H), 3.68 – 3.49 (m, 2H), 3.22 – 3.02 (m, 2H), 2.99, 2.98 (s, 3H), 2.95, 2.94 (s, 3H), 2.73 (s, 3H), 2.34, 2.29 (s, 3H), 2.26 – 1.83 (m, 7H), 1.44, 1.43 (s, 9H), 0.88 – 0.54 (m, 18H); 13C NMR (100 MHz, CDCl3) mixture of rotamers δ 172.7, 171.0, 170.3, 170.1, 170.0, 169.8, 169.3, 169.2, 156.3, 155.3, 136.8, 136.7, 129.2, 129.2, 128.6, 128.6, 127.1, 127.0, 80.6, 80.2, 61.6, 60.1, 59.1, 58.5, 58.1, 58.1, 54.5, 52.4, 47.2, 34.7, 30.7, 30.7, 30.2, 30.0, 29.7, 29.6, 29.5, 29.1, 29.0, 28.5, 28.4, 27.3, 27.2, 27.2, 27.1, 25.0, 20.0, 20.0, 19.9, 19.8, 19.7, 18.2, 18.1, 18.0, 17.7, 17.4; HRMS (ESI) calculated for [M + Na]+ C39H63N5NaO8+ 752.4569, found 752.4573. Preparation of 24. To the solution of compound 22 (2.40 g, 3.29 mmol) in DCM (19.2 mL) was added TFA (4.8 mL). After stirring for 6 h at room temperature, toluene (10 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by water (25 mL) and extracted with petroleum ether (3 × 5 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 30 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 23 (2.12 g, 9.86


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mmol) and HATU (3.75 g, 9.86 mmol) in DMF (12 mL) was added DIPEA (1.32 g, 10.2 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (50 mL), the whole mixture was washed with 1% HCl (3 × 15 mL), 5% aqueous NaHCO3 (3 × 15 mL), and brine (3 × 15 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 5/1 – 1/1) to afford compound 24 (1.80 g, 66%) as a colorless oil. [α]D17 = – 306.0o (c = 1.0, CHCl3); IR(neat) νmax: 2963, 2935, 1747, 1696, 1635, 1454, 1171, 749, 633 cm-1; 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.31 – 7.18 (m, 5H), 5.84 (dd, J = 10.5, 4.9 Hz, 1H), 5.10 (dd, J = 9.5, 6.6 Hz, 1H), 5.02 (d, J = 9.7 Hz, 1H), 4.95 (d, J = 10.7 Hz, 1H), 4.61 (dd, J = 21.3, 7.0 Hz, 1H), 4.45 (dd, J = 8.2, 3.0 Hz, 1H), 3.71 (s, 3H), 3.68 – 3.35 (m, 4H), 3.19 – 3.08 (m, 2H), 3.06 – 2.99 (m, 6H), 2.94, 2.90 (s, 3H), 2.31, 2.29 (s, 3H), 2.24 – 1.75 (m, 10H), 1.71 – 1.63 (m, 1H), 1.41, 1.39 (s, 9H), 0.88 (d, J = 5.6 Hz, 3H), 0.84 – 0.75 (m, 9H), 0.71 (d, J = 6.4 Hz, 3H), 0.61 (d, J = 5.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) mixture of rotamers δ 173.6, 172.3, 172.6, 170.9, 170.8, 170.0, 169.7, 169.2, 169.2, 154.4, 153.9, 136.8, 129.2, 128.6, 127.0, 79.8, 79.6, 59.1, 58.6, 58.5, 58.5, 58.3, 58.0, 57.4, 56.7, 54.5, 52.4, 47.2, 46.8, 46.7, 34.7, 30.6, 30.4, 30.3, 30.2, 30.0, 29.6, 29.1, 28.5, 27.1, 27.1, 27.0, 25.0, 24.3, 22.8, 20.0, 19.9, 19.8, 19.8, 19.6, 19.3, 18.9, 18.3, 17.8, 17.7, 17.5, 17.4; HRMS (ESI) calculated for [M + Na]+ C44H70N6NaO9+ 849.5096, found 849.5099. Preparation of 5. To the solution of compound 24 (1.60 g, 1.93 mmol) in DCM (16 mL) was added TFA (4 mL). After stirring for 6 h at room temperature, toluene (10 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by water (20 mL) and extracted with petroleum ether (3 × 5 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 25 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 25 (1.38 g, 6.77 mmol) and HATU (2.61 g, 6.87 mmol) in DMF (7 mL) was added DIPEA (900 mg, 6.97 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (30 mL), the whole mixture was washed with 1% HCl (3 × 6 mL), 5% aqueous NaHCO3 (3 × 6 mL), and brine (3 × 6 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 2/1 – 1/1) to afford compound 5 (1.10 g, 62%) as a colorless oil. [α]D17 = – 28.8o (c = 0.09, CHCl3); IR(neat) νmax: 2964, 2936, 1749, 1691, 1637, 1454, 1197, 743, 572 cm-1; 1H NMR (400 MHz, CDCl3) mixture of rotamers δ 7.32 – 7.11 (m, 5H), 5.84 (dd, J = 10.7, 5.0 Hz, 1H), 5.06 (d, J = 10.9 Hz, 1H), 5.01, 4.78 (d, J = 10.6 Hz, 3H), 4.94 (d, J = 10.6 Hz, 1H), 4.44 (dd, J = 8.5, 3.6 Hz, 1H), 3.71 (s, 3H), 3.68 – 3.40 (m, 4H), 3.18 – 3.11 (m, 1H), 3.08, 3.06 (s, 3H), 3.04 – 2.98 (m, 1H), 2.98 (s, 3H), 2.93, 2.91 (s, 3H), 2.77, 2.72 (s, 3H), 2.29 (s, 3H), 2.27 – 1.83 (m, 10H), 1.76 – 1.65 (m, 1H), 1.43 (s, 9H), 1.25 (d, J = 5.9 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.83 – 0.73 (m, 9H), 0.70 (d, J = 6.6 Hz, 3H), 0.60 (d, J = 6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3) mixture of rotamers δ 172.7, 172.6, 170.9, 170.9, 170.4, 170.0,

169.6, 169.5, 169.2, 155.7, 155.1, 136.7, 129.2, 128.5, 127.0, 80.3, 80.0, 59.1, 58.5, 58.0, 57.2, 56.9, 54.4, 53.8, 52.4, 51.6, 47.2, 47.0, 46.6, 34.7, 30.6, 30.3, 29.7, 29.6, 29.2, 29.1, 28.8, 28.7, 28.5, 27.1, 25.0, 24.9, 24.9, 19.9, 19.8, 19.5, 18.4, 18.4, 17.7, 17.4, 14.3; HRMS (ESI) calculated for [M + Na]+ C48H77N7NaO10+ 934.5624, found 934.5628. Preparation of 26. To the solution of compound 5 (600 mg, 0.658 mmol) in DCM (4 mL) was added TFA (1 mL). After stirring for 3 h at room temperature, toluene (3 mL) was added and then the whole mixture was concentrated under reduced pressure. The residue was dissolved by water (10 mL) and extracted with petroleum ether (3 × 3 mL). The aqueous phase was adjusted pH to 8 – 9 by Na2CO3 and extracted with DCM (3 × 15 mL), the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to afford a free amine as a colorless oil and used for next step directly. To the solution of the free amine, compound 4 (258 mg, 1.32 mmol) and HATU (500 mg, 1.32 mmol) in DMF (2.5 mL) was added DIPEA (179 mg, 1.38 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (15 mL), the whole mixture was washed with 1% HCl (3 × 3 mL), 5% aqueous NaHCO3 (3 × 3 mL), and brine (3 × 3 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 2/1 – 1/3) to afford compound 26 (296 mg, 45%) as a colorless oil. [α]D17 = – 284.22o (c = 0.1, CHCl3); IR(neat) νmax: 2961, 2933, 2116, 1747, 1634, 1435, 1096, 699, 633 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.28 – 7.15 (m, 5H), 5.85 (dd, J = 10.9, 5.1 Hz, 1H), 5.43 (q, J = 6.8 Hz, 1H), 5.07 (d, J = 10.8 Hz, 1H), 5.02 (d, J = 10.6 Hz, 1H), 4.95 (d, J = 10.7 Hz, 1H), 4.79 (dd, J = 7.5, 3.6 Hz, 1H), 4.45 (dd, J = 8.7, 3.8 Hz, 1H), 3.72 (s, 3H), 3.69 – 3.58 (m, 4H), 3.18 – 3.07 (m, 2H), 3.05 (s, 3H), 3.01 (s, 3H), 2.98 (s, 3H), 2.91 (s, 3H), 2.82 – 2.75 (m, 1H), 2.30 (s, 3H), 2.21 – 1.89 (m, 13H), 1.74 – 1.67 (m, 1H), 1.48 – 1.34 (m, 8H), 1.28 (d, J = 7.0 Hz, 3H), 1.17 – 1.09 (m, 1H), 1.06 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 5.4 Hz, 3H), 0.81 – 0.77, (m, 9H), 0.71 (d, J = 6.7 Hz, 3H), 0.60 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 177.2, 172.7, 172.6, 170.8, 170.4, 170.0, 169.6, 169.2, 136.7, 129.2, 128.6, 127.0, 84.7, 68.3, 59.1, 58.5, 58.0, 56.9, 54.5, 52.4, 50.0, 47.2, 47.1, 41.2, 36.8, 34.7, 33.6, 30.7, 30.6, 30.5, 30.3, 30.3, 29.6, 29.1, 28.8, 28.8, 27.1, 27.1, 27.0, 26.1, 25.0, 24.9, 20.0, 19.8, 19.6, 19.5, 18.5, 18.4, 17.7, 17.4, 17.2, 14.3; HRMS (ESI) calculated for [M + Na]+ C55H87N7NaO9+ 1012.6457, found 1012.6461. Preparation of 29. To the solution of compound 28 (5.00 g, 9.38 mmol) in MeOH (20 mL) was added NaBH4 (358 mg, 9.42 mmol) batchwise at 0 oC under argon. After stirring for 2 h at room temperature, the reaction was quenched with water (6 mL), EtOAc (100 mL) was added and the layers were separated, the organic layer was washed with brine (3 × 30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc = 50/1 – 40/1) to afford compound 29 (1.10 g, 54%) as a white solid and isomer tert-butyl (S)-2-((R)-1-hydroxyethyl) yrrolidine-1-carboxylate (0.410 g, 20%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.20 (br, 1H), 3.76 – 3.61 (m, 2H), 3.53 – 3.43 (m, 1H), 3.31 – 3.21 (m, 1H), 2.00 – 1.87 (m, 1H), 1.84 – 1.70 (m, 2H), 1.68



– 1.54 (m, 1H), 1.47 (s, 9H), 1.13 (d, 3H, J = 6.0 Hz). Spectroscopic data are in agreement with those reported in literature.16 Preparation of 27. To the solution of compound 26 (120 mg, 0.121 mmol) in MeOH-THF-H2O (3:1:1, 1.5 mL) was added LiOH·H2O (15.3 mg, 0.364 mmol). After stirring for 4 h at room temperature, the mixture was adjusted pH to 2 – 3 by 1 N HCl and extracted with EtOAc (3 × 5 mL), the combined organic layer was washed with brine (3 × 3 mL), dried over Na2SO4 and filtered. The filtrate was concentrated to afford an acid as a colorless oil and used for next step directly. To the solution of compound 29 (64.6 mg, 0.300 mmol) in DCM (0.8 mL) was added TFA (0.2 mL). After stirring for 3 h at room temperature, toluene (1 mL) was added and then the whole mixture was concentrated under reduced pressure to afford compound 3 (TFA salt) as a colorless oil and used for next step directly. To the solution of the acid, compound 3 and HATU (59.9 mg, 0.158 mmol) in DMF (0.6 mL) was added DIPEA (62.7 mg, 0.485 mmol) at 0 oC under argon, then the mixture was warmed to room temperature and stirred overnight. After diluted with EtOAc (10 mL), the whole mixture was washed with 1% HCl (3 × 2 mL), 5% aqueous NaHCO3 (3 × 2 mL), and brine (3 × 2 mL), the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (DCM/MeOH = 80/1 – 40/1) to afford compound 27 (80.1 mg, 62%) as a colorless oil. [α]D17 = – 228.0o (c = 0.1, CHCl3); IR(neat) νmax: 3422, 2962, 2932, 2116, 1632, 1432, 699, 632 cm-1; 1H NMR (400 MHz, CD3OD) mixture of rotamers δ 7.34 – 7.21 (m, 5H), 5.89 (dd, J = 11.6, 4.2 Hz, 1H), 5.30 (q, J = 6.7 Hz, 1H), 5.06 (d, J = 11.1 Hz, 1H), 5.02 (d, J = 10.7 Hz, 1H), 4.93 (d, J = 10.9 Hz, 1H), 4.84 (dd, J = 8.5, 3.9 Hz, 1H), 4.74 (dd, J = 8.5, 3.9 Hz, 1H), 4.22 – 4.15 (m, 1H), 4.10 – 4.02 (m, 1H), 3.91 – 3.84 (m, 1H), 3.79 – 3.73 (m, 1H), 3.69 – 3.52 (m, 4H), 3.15 – 2.95 (m, 3H), 3.10 (s, 3H), 3.03 (s, 3H), 2.96 (s, 3H), 2.93 (s, 3H), 2.35 – 2.25 (m, 5H), 2.21 – 2.15 (m, 4H), 2.13, 2.11 (s, 3H), 2.04 – 1.91 (m, 8H), 1.75 – 1.68 (m, 1H), 1.53 – 1.42 (m, 4H), 1.35 – 1.30 (m, 4H), 1.27, 1.21 (d, J = 7.1 Hz, 3H), 1.11 – 1.04 (m, 7H), 0.93 – 0.88 (m, 9H), 0.83 (d, J = 6.3 Hz, 6H), 0.76 (d, J = 6.7 Hz, 3H), 0.65 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 179.3, 174.7, 174.2, 172.1, 171.8, 171.1, 170.1, 170.5, 138.3, 130.5, 129.6, 128.1, 85.0, 69.7, 69.6, 64.1, 60.1, 60.0, 59.8, 58.8, 56.6, 52.6, 42.5, 37.5, 35.1, 34.8, 31.8, 31.4, 31.3, 30.9, 30.8, 30.2, 30.0, 29.9, 28.2, 28.1, 27.1, 26.8, 25.8, 25.8, 25.7, 20.4, 20.2, 20.1, 19.9, 19.2, 18.9, 18.8, 18.3, 18.0, 17.8, 14.2; HRMS (ESI) calculated for [M + Na]+ C60H96N8NaO9+ 1095.7192, found 1095.7195. Preparation of 1. To a solution of 27 (50.0 mg, 0.047 mmol) in DCM (0.5 mL) was added DMP (29.6 mg, 0.070 mmol). After stirring for 3 h at room temperature, the reaction mixture was quenched with saturated aqueous Na2S2O3 (1 mL) and saturated aqueous NaHCO3 (1 mL), DCM (3 mL) was added and the layers were separated, the aqueous layer was extracted with DCM (2 × 3 mL). The combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (DCM/MeOH = 80/1 – 40/1) to afford a colorless oil, n-pentane (1.5 mL) was added and then the mixture was stirred for 1 h at room temperature and filtered to afford compound 1 (38.0 mg, 76%) as a white powder. [α]D17 = – 310.7o (c = 0.1, CHCl3); IR(neat)

νmax: 2961, 2931, 2114 1723, 1633, 1430, 1096, 699, 632 cm-1; HRMS (ESI) calculated for [M + Na]+ C60H94N8NaO9+ 1093.7036, found 1093.7040. 1H and 13C NMR data in supporting information.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. NMR comparison of natural and synthetic jahanyne (1), 1 H and 13C NMR spectra of all new compounds (PDF)

AUTHOR INFORMATION INFORMATION Corresponding Author *E-mail: [email protected] [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (NSFC) (NO. 81573282 to Y.C.; NO. 81703350 to L.W.), the National Science Fund for Distinguished Young Scholars (NO. 81625021) to Y.C., the Fundamental Research Funds for the Central Universities and Hundred Young Academic Leaders Program of Nankai University.

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Total Synthesis of the Highly N-Methylated Peptide Jahanyne - The

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