Natural products from ocean serve as a rich source of bioactive substances^[1],for instance,alkaloids^[2],cyclic peptides^[3]and cyclic depsipeptides^[4].Marine fungi and cyanobacteria,predominantly with structural skeletons as modified peptides,depsipeptides,polyketides and peptide-polyketide hybrids,have emerged as a valuable source of marine natural products with remarkable biological activities^{[5, 6]}.As a class of promising compounds in drug discovery,most of these secondary metabolites display a variety of physiological activities,including antimicrobial,antimalarial,cytotoxic and neorotoxic properties^[7].Although a few of them were investigated for the application in potential cancer therapeutics^{[4, 8]},most secondary metabolites are not well studied,thus require further research on either their structural modifications or modes of actions^[9].

Emericellamides A and B (Fig.1) are two secondary metabolites of marine cyclic depsipeptides,isolated from the co-culture of the marine-derived fungus Emericella sp.and actinomycete Salinispora arenicola by Fenical and his co-workers^[10].Their structures were determined based on chemical and spectroscopic methods,and the absolute configurations were assigned by Marfey's method^[11]and the modified Mosher method^[12].Emericellamides A and B exhibit antimicrobial activity against methicillin-resistant Staphylococcus aureus (MIC 3.8 mmol/L and 6.0 mmol/L,respectively),as well as cytotoxicity against HCT-116 human colon carcinoma cell line (IC₅₀ 23 mmol/L and 40 mmol/L,respectively).The biosynthetic pathway for emericellamides was identified in 2008^[13].

Fig. 1

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Fig. 1.The structures of emericellamides A (1) and B (2).

Due to the attractive bioactivities and intriguing structure and their analogous,considerable efforts have been contributed to their synthesis,and several enantioselective synthetic methods were reported^{[14, 15, 16, 17, 18]}.As a continuation of our interest in pursuing diverse synthesis of alkaloids^{[19, 20]},and investigating their structure-activity relationship,we synthesized several secondary metabolites^{[16, 21, 22]}including emericellamide A^[16].Herein we present a general and efficient method for total synthesis of emericellamide B.

THF was distilled from sodium/benzophenone.Reactions were monitored by thin layer chromatography (TLC) on glass plates coated with silica gel with fluorescent indicator.Flash chromatography was performed on silica gel (300-400) with petroleum ether/EtOAc as eluent.Optical rotations were measured on a polarimeter with a sodium lamp.HRMS were measured on a LCMSIT-TOF apparatus.IR spectra were recorded using film on a Fourier Transform Infrared Spectrometer.NMR spectra were recorded at 400 MHz or 500 MHz,and chemical shifts are reported in δ (ppm) referenced to an internal TMS standard for ¹H NMR and CDCl₃(77.0 ppm) for ¹³C NMR.¹H NMR and ¹³C NMR spectra of compounds 5-10,12-16,18 and emericellamide B are put in Supporting information.

To a suspension of NaH (1.16 g,31.37 mmol) in dry THF (60 mL) was added dropwise a solution of triethyl phosphonoacetate (6.6 mL,32.80 mmol) at 0 ℃ under argon atmosphere.After being stirred for 30min,the mixture was cooled to-78 ℃ and a solution of aldehyde 4(5.0 g,14.26mmol) in THF (10 mL) was added drop wise.The resulting mixture was stirred for 1 h at 0 ℃,then a phosphate buffer solution (20 mL) and Et₂O (100mL) were added.The mixture was allowed to warm to room temperature,and the organic layer was separated,washed with saturated NH₄Cl solution and brine,dried over anhydrous Na₂SO₄,filtered and concentrated.The residue was purified by chromatography on silica gel to give 5(5.78 g,yield 98%) as a colorless oil.[α]_D²⁵ -28.6(c 0.44,CHCl₃);IR (film,cm^-1):υ_max 2957,2929,1720,1651,1464,1366,1259;¹H NMR (400 MHz,CDCl₃):δ 7.36-7.27(m,5H),6.98(dd,1H,J=7.82,15.85 Hz),5.78(dd,1H,J=1.17,15.85 Hz),4.48(d,1H,J=12.13 Hz),4.45(d,1H,J=12.13 Hz),4.18(m,2H),3.62(dd,1H,J=4.69,5.29 Hz),3.50(dd,1H,J=5.29,9.39 Hz),3.29(dd,1H,J=7.43,9.00 Hz),2.58-2.51(m,1H),1.97(m,1H),1.28(t,3H,J=7.05 Hz),1.04(d,3H,J=6.65 Hz),0.98(d,3H,J=6.65 Hz),0.88(s,9H),0.03(s,3H);¹³CNMR (125 MHz,CDCl₃):δ 166.7,152.8,138.6,128.3(2C),127.5(2C),127.4,120.5,72.9,72.2,60.1,40.3,38.1,26.0(3C),14.9,14.3,14.2,-3.9,-4.1;MS (ESI):443.2(M+Na⁺);HRMS (ESI) calcd.for (C₂₄H₄₀O₄Si+Na⁺):443.2595,found:443.2629.

To a solution of compound 5(5.50 g,13.07 mmol) in MeOH (100 mL) and THF (30 mL) was added NiCl₂·6H₂O (1.56 g,6.54 mmol) at 0 ℃ under argon atmosphere,then NaBH₄(1.00 g,26.15 mmol) was added in several portions.After being stirred for 1 h,the mixture was concentrated,filtered with celite,and washed with Et₂O (100 mL).The organic phase was concentrated,and the residue was purified by chromatography on silica gel to give 6(5.39 g,yield 97%) as a colorless oil.[α]_D²⁵ -14.7(c 0.82,CHCl₃);IR (film,cm^-1):υ_max 2957,2856,1736,1471,1254,1178,1093;¹H NMR (400 MHz,CDCl₃):δ 7.36-7.25(m,5H),4.50(d,1H,J=12.13 Hz),4.46(d,1H,J=12.13 Hz),4.12(m,2H),3.53(dd,2H,J=4.30,9.00 Hz),3.49(dd,1H,J=2.54,6.46 Hz),3.28(dd,1H,J=7.64,9.00 Hz),2.36-2.25(m,2H),1.98-1.92(m,1H),1.74-1.66(m,1H),1.53-1.47(m,2H),1.24(t,3H,J=7.04 Hz),0.95(d,3H,J=6.72 Hz),0.91-0.84(m,12H,CH₃),0.02(s,3H),-0.52(s,3H);¹³C NMR (125 MHz,CDCl₃):δ 173.9,138.7,128.3(2C),127.5(2C),127.3,76.8,73.0,72.9,60.2,38.0,35.8,32.7,29.9,26.1(3C),18.4,15.1,14.2,13.8,-3.8,
-4.1;MS (ESI):445.2(M+Na⁺);HRMS (ESI) calcd.for (C₂₄H₄₂O4Si+Na⁺):445.2752,found:445.2749.

To a solution of 6(5.20 g,12.36 mmol) in THF/H₂O (150mL,v/v=3/1) was added LiOH·H₂O (5.20 g,123.60 mmol) at room temperature.After being stirred for 6 h at 60 ℃,the mixture was quenched with 1 mol/L HCl and extracted with EtOAc (150 mL × 3).The combined organic layers were dried over anhydrous Na₂SO₄,filtered,and concentrated to yield 7(4.78 g,yield 98%),which was used in next step without further purification.[α]_D²⁵ -11.3(c 5.05,CHCl₃);IR (film,cm^-1):υ_max 3030,2928,1709,1454,1383,1253,1093;¹H NMR (400 MHz,CDCl₃):δ 7.37-7.26(m,5H),4.51(d,1H,J=12.13 Hz),4.45(d,1H,J=12.13 Hz),3.55-3.49(m,1H),3.28(dd,1H,J=7.62,9.00 Hz),2.43-2.25(m,2H),1.99-1.92(m,1H),1.76-1.70(m,1H),1.66-1.57(m,1H),1.55-1.49(m,1H),0.96(d,3H,J=7.04 Hz),0.89-0.84(m,12H),0.03(s,3H),0.01(s,3H);¹³C NMR (125 MHz,CDCl₃):δ 180.1,138.7,128.3(2C),127.6(2C),127.4,76.2,73.0,72.9,37.9,35.8,32.3,29.5,26.1(3C),18.4,15.1,13.9,-3.8,-4.1;MS (ESI):417.2(M+Na⁺);HRMS (ESI) calcd.for C₂₂H₃₈O₄Si:394.2539(C₂₂H₃₈O₄Si+Na⁺):417.2439,found 417.2459.

To a solution of crude acid 7(5.30 g,13.43 mmol) and Et₃N (3.7 mL,26.86 mmol) in dry THF (70 mL) was added pivaloyl chloride (1.95 mL,16.12 mmol) dropwise at -78 ℃ under argon atmosphere.After being stirred at this temperature for 2 h,oxazolidinone (2.40 g,13.70 mmol) and LiCl (1.70 g,40.29 mmol) were added in one portion,then the mixture was slowly warmed to room temperature and stirred overnight.The resulting mixture was diluted with water (100 mL) and ether (100 mL),the organic phase was separated and the aqueous layer was extracted with ether (100 mL × 3).The combined organic layers were washed with brine,dried and concentrated,and the residue was purified by chromatography on silica gel to give 8(6.44 g,yield 87%) as a colorless oil.[α]^D₂₅ -24.2(c 5.34,CHCl₃);IR (film,cm^-1):υ_max 2956,2927,1784,1702,1384,1096;¹H NMR (400 MHz,CDCl₃):δ 7.36-7.20(m,10H),4.66(ddd,1H,J=3.32,6.12,13.10 Hz),4.51(d,1H,J=11.94 Hz),4.48(d,1H,J=11.94 Hz),4.21-4.13(m,2H),3.59-3.53(m,2H),3.33-3.28(m,2H),3.07-2.99(m,1H),2.89-2.81(m,1H),2.72(dd,1H,J=9.80,13.31 Hz),2.02-1.95(m,1H),1.81-1.73(m,1H),1.70-1.66(m,1H),1.63-1.55(m,1H),0.98(d,3H,J=6.85 Hz),0.92-0.87(m,12H,CH₃),0.07(s,3H),0.04(s,3H);¹³C NMR (125 MHz,CDCl₃):δ 173.3,153.4,138.8,135.3,129.4(2C),128.9(2C),128.3(2C),127.5(2C),127.4,127.3,76.8,73.0,72.9,66.1,55.1,38.0,37.9,
35.8,33.9,29.3,26.1(3C),18.4,15.1,13.9,-.3.6,-4.1;MS (ESI):554.3(M+H⁺);HRMS (ESI) calcd.for (C₃₂H₄₇NO₅Si+Na⁺):576.3123,found:576.3152.

To a solution of amide 8(4.50 g,8.13 mmol) in THF (45 mL) was added a solution of NaHMDS (8.9 mL,1 mol/L in THF,8.94 mmol) dropwise at -78 ℃ under argon atmosphere,the mixture was stirred for 30 min,then MeI (1.50 mL,24.38 mmol) was added.After being stirred at this temperature for 3 h,the mixture was warmed to room temperature and stirred overnight.The reaction was quenched with H₂O (50 mL) and extracted with Et₂O (50 mL × 3).The combined organic extracts were washed with brine,dried and concentrated,and the residue was purified by chromatography on silica gel to give 9(3.40 g,yield 74%) as a colorless oil.[α]_D²⁵ -50.7(c 1.15,CHCl₃);IR (film,cm^-1):υ_max 2928,1782,1698,1454,1385,1249,1208,1099;¹H NMR (600 MHz,CDCl₃):δ 7.33-7.17(m,10H),4.63-4.58(m,1H),4.48(d,1H,J=12.06 Hz),4.45(d,1H,J=12.06 Hz),4.11(d,2H,J=5.11 Hz),3.82(ddd,1H,J=4.74,7.02,11.52 Hz),3.55(dd,1H,J=4.32,9.00 Hz),3.45(dd,1H,J=2.82,6.48 Hz),3.26(dd,1H,J=8.04,9.00 Hz),3.21(dd,1H,J=3.30,13.36,Hz),2.73(dd,1H,J=9.60,13.36 Hz),1.95(ddd,1H,J=1.80,6.96,14.10 Hz),1.86(ddd,1H,J=3.96,10.18,14.05 Hz),1.57-1.53(m,1H),1.27(ddd,1H,J=4.62,9.72,14.05 Hz),1.20(d,3H,J=6.90 Hz),0.94(d,3H,J=6.90 Hz),0.86(s,9H),0.83(d,3H,J=6.72 Hz),0.01(s,3H),0.00(s,3H);¹³C NMR (150 MHz,CDCl₃):δ 177.1,153.0,138.9,135.3,129.5(2C),128.9(2C),128.3(2C),127.5(2C),127.4(2C),77.9,73.1,73.0,66.0,55.3,39.1,38.2,
37.8,35.5,34.0,26.2(3C),19.0,18.5,15.1,13.9,-3.5,-4.0;MS (ESI):590.3(M+Na⁺);HRMS (ESI) calcd.for (C₃₃H₄₉NO₅Si+Na⁺):590.3280,found:590.3305.

To a solution of amide 9(3.30 g,5.81 mmol) in dry THF (30 mL) was added a solution of LiBH₄(8.7 mL,2.0 mol/L solution in THF,17.44 mmol) at 0 ℃ under argon atmosphere,then dry methanol (0.6 mL,17.44 mmol) was slowly added.After being stirred for 45 min at 0 ℃ and 1 h at room temperature,the mixture was cooled back to 0 ℃ and quenched carefully with a 1.0 mol/L aqueous solution of NaOH (30 mL).The mixture was extracted with Et₂O (50 mL × 3),and the combined organic layers were washed with brine,dried and concentrated.The residue was purified by chromatography on silica gel to give alcohol 10(2.13 g,yield 93%) as a colorless oil.[α]_D²⁵ -11.9(c 3.0,CHCl₃);IR (film,cm^-1):υ_max 3377,2957,2928,2856,1462,1381,1252,1069;¹H NMR (400 MHz,CDCl₃)δ 7.37-7.26(m,5H),4.50(d,1H,J=12.13 Hz),4.46(d,1H,J=12.13 Hz),3.56(dd,1H,J=5.09,9.00 Hz),3.50(dd,1H,J=4.11,10.47 Hz),3.44(dd,1H,J=3.32,5.67 Hz),3.36(dd,1H,J=6.65,10.47 Hz),3.25(dd,1H,J=7.04,9.00 Hz),2.03-1.96(m,1H),1.77-1.62(m,2H),1.49-1.36(m,2H),0.95(d,3H,J=6.85 Hz),0.93(d,3H,J=6.65 Hz),0.89-0.85(m,12H),0.03(s,3H),0.01(s,3H);¹³C NMR (125 MHz,CDCl₃)δ 138.6,128.3(2C),127.6(2C),127.4,76.8,73.0,72.9,67.6,38.1,37.8,33.4,33.0,26.1(3C),18.5,17.8,15.5,15.3,-3.6,-4.0;MS (ESI):417.3(M+Na⁺);HRMS (ESI) calcd.for (C₂₃H₄₂O₃Si+Na⁺):417.2803,found:417.2772.

To a solution of oxalyl chloride (0.94 mL,11.15 mmol) in dry CH₂Cl₂(30 mL) was added DMSO (1.60 mL,22.30 mmol) over 15 min at -78 ℃ under argon atmosphere.After being stirred for 30 min,a solution of the alcohol 10(2.20 g,5.57 mmol) in dry CH₂Cl₂(5.0 mL) was added slowly and the mixture was stirred for another 1 h,then Et₃N (3.9 mL,27.85 mmol) was added drop wise and the mixture was allowed to warm to room temperature.The reaction was quenched with aqueous NaHSO₄ solution (1 mol/L,30 mL),then the organic layer was separated,and the aqueous phase was extracted with CH₂Cl₂.The combined organic layerswere washed with saturated aqueous solution of NaHSO₄,saturated aqueous NaHCO₃,brine,and dried.The solvents were concentrated under reduced pressure to give aldehyde 11(2.0 g,yield 92%) as a colorless oil,which was used in next step without further purification.To a suspension of pentane-triphenylphosphonium bromine (11.17 g,27.02 mmol) in dry THF (50 mL) was added n-BuLi (16.8 mL,1.6 mol/L in hexane,26.8 mmol) drop wise at room temperature under argon atmosphere.After the red mixture was stirred for 30 min,a solution of the above crude aldehyde 11(2.10 g,5.35 mmol) in dry THF (5 mL) was added,and the resulting mixture was stirred for 2 h.The mixture was diluted with ethyl acetate and washed with saturated NaHCO₃ aqueous,brine,dried over anhydrous Na₂SO₄.The solvents were concentrated,and the residue was purified by chromatography on silica gel to give 12(2.30 g,yield 97%) as a colorless oil.[α]_D²⁵ -5.07(c 4.48,CHCl₃);IR (film,cm^-1):υ_max 2956,2856,1454,1379,1252;¹H NMR (400 MHz,CDCl₃)δ 7.35-7.25(m,5H),5.29(ddd,1H,J=7.24,11.15,14.47 Hz),4.99(dd,1H,J=9.97,11.15 Hz),4.50(d,1H,J=11.93 Hz),4.46(d,1H,J=11.93 Hz),3.55(dd,1H,J=4.30,8.90 Hz),3.40(dd,1H,J=3.13,6.06 Hz),3.26(dd,1H,J=7.63,8.43 Hz),2.53-2.46(m,1H),2.05-1.97(m,2H),1.97-1.89(m,1H),1.62-1.54(m,1H),1.36-1.27(m,5H),1.20(m,2H),0.96(d,3H,J=6.84 Hz),0.91-0.85(m,15H),0.82(d,3H,J=6.8 Hz),0.01(s,3H),0.00(s,3H);¹³C NMR (125 MHz,CDCl₃)δ 138.8,135.9,128.9,128.3(2C),127.5(2C),127.3,78.6,73.1,72.9,42.7,37.7,34.2,32.2,29.3,27.3,26.1(3C),22.4(2C),18.4,
15.5,14.1,14.0,-3.6,-4.1;MS (ESI):469(M+Na⁺);HRMS (ESI) calcd.for (C₂₈H₅₀O₂Si+Na⁺):469.3480,found:469.3464.

To a solution of 12(2.0 g,4.48 mmol) in MeOH (15 mL) was added a solution of HCl/MeOH (2 mol/L,5 mL) at room temperature,and the resulting mixture was stirred for 3 h.Then the mixture was concentrated in vacuo,the residue was diluted with water (20 mL) and Et₂O (30 mL).The resulting mixture was separated,the aqueous phase was extracted with Et₂O (30 mL × 3),and the combined organic layers were washed with saturated aqueous NaHCO₃,brine,and dried over anhydrous Na₂SO₄.The solvents were concentrated,and the residue was purified by chromatography on silica gel to give 13(1.48 g,yield 100%) as a colorless oil.[α]_D²⁵+26.0(c 0.4,CHCl₃);IR (film,cm^-1):υ_max 3507,2958,2926,2857,1455,1378,1095;1HNMR (600 MHz,CDCl₃)δ 7.36-7.27(m,5H),5.30(ddd,1H,J=7.32,9.72,10.92 Hz),5.05(dd,1H,J=9.89,10.80 Hz),4.52(s,2H),3.60(dd,1H,J=4.14,8.94 Hz),3.48(dd,1H,J=8.22,8.94 Hz),3.38(d,1H,J=2.82 Hz),3.36-3.32(m,1H),2.57-2.51(m,1H),2.07-2.01(m,2H),1.98-1.91(m,1H),1.59-1.55(m,1H),1.38-1.29(m,5H),1.23(m,1H),0.94(d,3H,J=6.62 Hz),0.90(t,3H,J=7.12 Hz),0.85(d,3H,J=6.71 Hz),0.82(d,3H,J=7.03 Hz);¹³C NMR (150 MHz,CDCl₃)δ 137.8,136.1,128.9,128.5(2C),127.8(2C),127.7,80.4,76.3,73.6,42.5,36.0,33.0,32.2,29.3,27.3,22.4,22.2,14.0,13.7,
12.0;MS (ESI):355.2(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₂₂H₃₆O₂+Na⁺):355.2613,found:355.2609.

To a solution of alcohol 13(1.0 g,3.01 mmol),EDCI (2.91 g,152.0 mmol) in dry DCM (25 mL) was added N-Boc-Ala-OH (2.85 g,15.05 mmol) at 0 ℃ under argon atmosphere,and the mixture was stirred for 0.5 h,then DMAP (183 mg,1.50 mmol) was added in one portion.After being stirred for 3 h,the mixture was allowed to warm to room temperature and stirred for additional 6 h.The resulting mixture was diluted with CH₂Cl₂(30 mL),the organic layer was separated and the aqueous layer was extracted with CH₂Cl₂(30 mL × 2).The combined organic layers were washed with an aqueous solution of HCl (5%),saturated aqueous NaHCO₃ solution and brine,dried and concentrated.The residue was purified by chromatography on silica gel to give 14(0.90 g,yield 60%;90% based on the recovery of starting material) as a light yellow oil.[α]_D²⁵ -11.3(c 0.56,CHCl₃);IR (film,cm^-1):υ_max 3993,2962,2928,2871,1716,1497,1454,1366,1167;¹H NMR (600 MHz,CDCl₃)δ 7.34-7.27(m,5H),5.29(m;1H),5.06(d,1H,J=7.26 Hz),4.98(dd,1H,J=10.28,10.77 Hz),4.87-4.84(m,1H),4.45(s,2H),4.26(dd,1H,J=6.92,7.72 Hz),3.41(dd,1H,J=3.87,8.93 Hz),3.22(dd,1H,J=7.00,8.93 Hz),2.54-2.48(m,1H),2.07-1.94(m,2H),1.79-1.75(m,1H),1.46-1.42(m,9H),1.36(d,3H,J=7.14 Hz),1.39-1.25(m,4H),1.17(m,1H),1.06(m,1H),0.96(d,3H,J=6.84 Hz),0.92-0.88(m,6H),0.83(d,3H,J=6.72 Hz);¹³C NMR (150 MHz,CDCl₃)δ 173.1,155.1,138.6,135.3,129.2,128.3(2C),127.7(2C),127.5,80.0,79.6,73.2,72.1,49.5,41.7,35.5,32.2,31.2,29.0,
28.4(3C),27.2,22.4(2C),22.0,14.4,14.0(2C);MS (ESI):526.4(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₃₀H₄₉NO₅+Na⁺):526.3508,found:526.3507.

To a suspension of Pd/C (10%,80 mg) in CH₃OH (10 mL) was added a solution of 14(800 mg,1.589 mmol) in CH₃OH (2 mL).After the resulting mixture was stirred at room temperature under H₂ atmosphere (1 atm) for 4 h,the catalyst was filtered off and the filtrate was concentrated in vacuo to give the crude alcohol,which was purified by chromatography on silica gel to give 15(658 mg,quantitative yield) as a light colorless oil.[α]_D²⁵ -28.5(c 0.85,CHCl₃);IR (film,cm^-1):υ_max 3438,3368,2962,2927,1715,1682,1502,1456,1367,1215,1169,1068;¹H NMR (600MHz,CDCl₃)δ 5.01(d,1H,J=7.40 Hz),4.85(d,1H,J=10.26 Hz),4.30-4.24(m,1H),3.47-3.42(m,1H),3.46-3.43(m,1H),2.47(bs,1H),1.92-1.86(m,1H),1.86-1.81(m,1H),1.46-1.42(m,11H),1.40(d,3H,J=7.30Hz),1.30-1.16(m,9H),1.00(d,3H,J=6.96 Hz),0.90(d,3H,J=6.84 Hz),0.87(t,3H,J=7.10 Hz),0.80(d,3H,J=6.60Hz);¹³C NMR (150 MHz,CDCl₃,25 ℃,TMS)δ 174.8,155.4,80.1,77.6,64.1,49.6,41.6,37.2,36.9,31.9,30.6,29.6,29.5,28.3(3C),26.9,22.7,19.8,18.4,14.1,14.0,13.6;MS (ESI):438.2(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₂₃H₄₅NO₅+Na⁺):438.3195,found:438.3183.

To a stirred solution of 15(200 mg,0.482 mmol) in CCl₄(5 mL) were added CH₃CN (5 mL) and water (7.5 mL),then NaIO₄(413 mg,1.93 mmol) and RuCl₃·xH₂O (5.0 mg,0.04 mmol) were added sequentially at room temperature.After being stirred for 4 h,the reaction was diluted with Et₂O (20 mL) and stirred for 20 min to precipitate black RuO₂.Then the mixture was dried and filtered through celite,the solid residue was washed with ether,and the combined organic phases were concentrated in vacuo to give the carboxylic acid (200 mg),which was used in next step without further purification.The above crude carboxylic acid (200 mg,0.466 mmol) and HOBt (69 mg,0.513 mmol) were added in dry DMF (4 mL) and stirred for 15 min at room temperature,then the reaction mixture was cooled to -15 ℃,EDCI (98 mg,0.513 mmol) was added in one portion and stirred for another 1.5 h at this temperature,H-Gly-OBn (85 mg,0.513 mmol) was added in one portion.After being stirred overnight,the mixture was quenched with H₂O (10 mL),and extracted with Et₂O (20 mL × 3).The combined organic layers were washed with water,saturatedaqueous NaHCO₃ and brine,dried and concentrated.The residue was purified by chromatography on silica gel to give 16(238 mg,yield 88% over three steps) as a light colorless oil.[α]_D²⁵ -13.2(c 1.61,CHCl₃);IR (film,cm^-1):υ_max 3325,2959,2927,1730,1717,1659,1525,1456,1366,1174;¹H NMR (600 MHz,CDCl₃)δ 7.37-7.31(m,5H),6.30(bs,1H),5.20-5.14(m,3H),5.06(m,1H),4.31-4.28(m,1H),4.06(d,2H,J=3.84 Hz),2.65(m,1H),1.93-1.87(m,1H),1.58-1.51(m,1H),1.42-1.39(m,10H),1.37(d,3H,J=7.20 Hz),1.30-1.18(m,9H),1.15(d,3H,J=7.01 Hz),1.04-0.98(m,1H),0.93-0.86(m,7H),0.83(d,3H,J=6.60 Hz);¹³C NMR (150 MHz,CDCl₃)δ 173.7,172.6,170.1,155.2,135.2,128.6(2C),128.5(2C),128.3,79.7,78.0,67.2,49.4,43.6,41.4,41.1,36.5,31.9,31.4,29.7,
29.6,28.3(3C),26.7,22.7,20.1,18.4,14.6,14.1,13.9;MS (ESI):599.4(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₃₂H₅₂N₂O₇+Na⁺):599.3672,found:599.3659.

To a solution of 16(200 mg,0.347 mmol) in CH₂Cl₂(3 mL) was added TFA (3 mL) and stirred for 2 h at room temperature,then the solvent was evaporated in vacuo.The residue was dissolved in 1,2-dichloroethane (3 mL),the solvent was removed in vacuo and this process was repeated three times to give crude 17.HOBt (52 mg,0.347 mmol) was added to a solution of N-Cbz-Val-Leu-Ala-OH (137 mg,0.315 mmol) in dry DMF (5 mL) at -15 ℃.After 30 min of stirring,the resulting cold solution was treated with EDCI (73 mg,0.347 mmol) and stirred at -15 ℃ for 1.5 h,then NMM (76 mL,0.694 mmol) and a solution of amide 17 in DMF provided above were added before being warmed to room temperature overnight.The reaction was quenched with H₂O (15 mL) and extracted with EtOAc (30 mL × 3).The combined organic extracts were washed with water,saturated aqueous NaHCO₃ and brine,dried and concentrated.The residue was purified by chromatography on silica gel to give 18(201 mg,yield 65% over two steps) as a white solid.[α]_D²⁵ -34.7(c 0.36,CHCl₃);IR (film,cm^-1):υ_max 3280,2958,2928,1741,1697,1634,1541,1454,1384,1210;¹H NMR (600 MHz,DMSO-d₆)δ 8.25(t,1H,J=5.80 Hz),8.01(d,1H,J=7.41 Hz),7.91-7.87(m,2H),7.38-7.28(m,11H),5.11(d,1H,J=13.14 Hz),5.09(d,1H,J=13.14 Hz),5.03(d,1H,J=13.08 Hz),5.00(d,1H,J=13.08 Hz),4.91(dd,1H,J=1.82,9.62 Hz),4.33-4.24(m,3H),3.89-3.82(m,2H),3.76(dd,1H,J=5.64,17.52 Hz),2.69-2.63(m,1H),1.97-1.91(m,1H),1.85-1.79(m,1H),1.63-1.57(m,1H),1.55-1.50(m,1H),1.44-1.40(m,2H),1.28-1.14(m,17H),1.02-0.97(m,1H),0.96(d,3H,J=7.02 Hz),0.87-0.77(m,21H);¹³C NMR (150 MHz,DMSO-d₆)δ 173.9,171.9,171.5,171.4,170.3,156.6,137.6,136.4,128.9(2C),128.8(2C),128.5(2C),128.4(2C),128.2,128.1,79.7,
77.0,66.3,65.8,60.7,51.2,48.2,48.0,42.2,41.3,41.1,40.6,36.8,31.8,30.8,30.7,29.5,29.4,26.5,24.5,23.5,22.6,27.0,
20.2,19.7,18.7,15.6,17.9,14.6,14.4,14.1;MS (ESI):916.7(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₄₉H₇₅N₅O₁₀+Na⁺):916.5412,found:916.5426.

To a suspension of Pd/C (10%,10 mg) in CH₃OH was added a solution of 18(100 mg,0.123 mmol) in CH₃OH (3 mL).After the resulting mixture was stirred at room temperature under H₂ atmosphere (1 atm) for 3 h,the catalyst was filtered off and the filtrate was concentrated in vacuo to give the crude deprotected product (74 mg,yield 99%),which was used in next step without further purification.The crude deprotected product was suspended in anhydrous CH₃CN (120 mL,1.0 × 10^-3 mol/L) and cooled to 0 ℃,pentafluorophenyldiphenylphosphinate (FDPP) reagent (85 mg,0.221 mmol) was added in one portion and stirred for 20 min at the same temperature.Then DIPEA (76 μL,0.435 mmol) was slowly added over a period of 30 min.After the addition was over,the mixture was allowed to warm to room temperature and stirred for 26 h.The mixture was concentrated in vacuo,and the residue was purified by RP C18 HPLC with 70% aqueous CH₃CN (Sepax-tech Amethyst C18 semipreparative column,10 mm × 150 mm,2 mL/min,refractive index detection) to give emericellamide B (39 mg,yield 55% over two steps).[α]_D²⁵ -43.6(c 0.11,MeOH){lit.[α]_D²⁵ -34(c 0.076,MeOH)^[10];[α]_D²⁵ -31(c 0.066,MeOH)^[15];[α]_D³⁰-34.1(c 0.12,MeOH)^[18]};IR (film,cm^-1):υ_max 3309,2931,1756,1632,1554;¹H NMR (600 MHz,DMSO-d₆)δ 8.27-7.98(m,2H),7.87(d,1H,J=7.51 Hz),7.49(bs,1H),7.34(d,1H,J=6.72 Hz),4.90(dd,1H,J=2.40,9.90 Hz),4.30(dd,1H,J=5.58,17.40 Hz),4.10^-3.94(m,4H),3.63(dd,1H,J=5.58,17.40 Hz),2.87(dq,1H,J=7.13,10.24 Hz),1.89-1.84(m,1H),1.83-1.79(m,1H),1.62-1.50(m,4H),1.24(d,J=7.32 Hz,3H),1.21(d,3H,J=6.72 Hz),1.24-1.08(m,10H),1.06-0.98(m,1H),0.92-0.75(m,25H);¹³C NMR (150 MHz,DMSO-d₆)δ 173.4,171.7,171.6,171.5,171.2,169.2,76.3,60.5,55.4,52.2,48.6,47.7,42.9,41.5,41.2,37.3,31.8,30.7,30.6,29.5,29.2,
26.6,25.0,23.6,22.6,21.3,19.9,19.5,19.2,18.8,16.8,14.8,14.5,14.1;MS (ESI):674.5(M+Na⁺);HRMS (MALDI/DHB) calcd.for (C₃₄H₆₁N₅O₇+Na⁺):674.4469,found:674.4474.

The retrosynthetic analysis of emericellamide B is shown in Scheme 1.Due to a highlymethylated (2R,3R,4S,6S)-3-hydroxy-2,4,6-trimethyldodecanoic acid (HTMD) unit in its structure the most critical step is the connection of the sterically hindered units (HTMD),with the other peptide-amino acid moiety.Yamaguchi protocol^{[23, 24]}and condensation reaction between C-22 and C-1 were found to be ineffective^{[14, 15, 16, 17, 18]}.The goal of initial synthetic efforts is to achieve a general method for preparation of HTMD using cheapest material,and the esterification of small amino-acid with HTMD is the key task.In addition,convenient removal of the protecting groups in all these units would also have to be taken into account.

Scheme. 1

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Scheme. 1.Strategic bond disconnections of emericellamide B (2).

Scheme 2 shows the synthesis of the HTMD moiety of emericellamide B.The aldehyde 4 could be conveniently achieved from vinylacetic acid 3 using our previous method^[16],and the (2R,3R,4S,6S)-3-hydroxy-2,4,6-trimethyldodecanoic acid (HTMD) was prepared using the following sequence.The Horner-Wadsworth-Emmons (HWE) reaction^{[25, 26]}of aldehyde 4 with triethylphosphonoacetate in the presence of sodiumhydride predominantly afforded olefin 5 in E-form (The ratio of Z/E was 2:98) in 98% combined yield,and the minor Z-isomer could be easily separated by chromatography on silica gel.Reduction of 5 with sodium borohydride in the presence of nickel chloride^[27]gave the ester 6 in 97% yield{[α]_D²⁵-14.7(c 0.82,CHCl₃)}.In order to stereoselectively introduce methyl group into the α-position of carboxylic acid 7,Evans'methodwas chosen in our case.After the ester 6 was hydrolyzed to carboxylic acid 7 in excellent yield in the presence of LiOH.The reaction with (R)-4-benzyloxazolidin-2-one through the activated anhydride with pivaloyl chloride at -78 ℃ led to the amide 8{[α]_D²⁵ -24.2(c 5.34,CHCl₃)}in 87% yield.The subsequent asymmetric methylation afforded 9 as the only desirable diastereomer in 74% yield^{[28, 29]}{[α]_D²⁵ -50.7(c 1.15,CHCl₃)}.The optimal conditions entailed the slow addition of 3 equiv.of methyl iodide over 30 min,and the reaction temperature was kept at -78 ℃ for 3 h to avoid the formation of a-dimethylated side-product.Removal of the Evan's auxiliary^[30](LiBH₄,MeOH) and Swern oxidation^{[31, 32]}(DMSO,(COCl)₂) produced aldehyde 11,which was subjected to treatment with pentyltriphenylphosphonium bromide in the presence of n-BuLi to afford benzylic ether 12{[α]_D²⁵ -5.07(c 4.48,CHCl₃)}in three steps with 83% yield.

Scheme. 2

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Scheme. 2.Synthesis of benzylic ether 12.Reagents and conditions:(a) triethylphosphonoacetate,NaH,THF,0 ℃ 1 h,98%;(b) NiCl2·6H2O,NaBH4,MeOH,THF,0 ℃,1 h,97%;(c) LiOH·H2O,THF/H2O,60 ℃,98%;(d)(R)-4-benzyloxazolidin-2-one,PivCl,Et3N,LiCl,THF,-78 ℃ to r.t.,overnight,87%;(e) NaHMDS,MeI,THF,-78 ℃ to r.t.,overnight,74%;(f) LiBH4,MeOH,THF,93%;(g) DMSO,(COCl)2,-78 ℃,30 min,then 10,-78 ℃ to -40 ℃,3 h;Et3N,-78 ℃ to r.t.,1 h,92%;(h) pentyltriphenylphosphonium bromide,n-BuLi,THF,r.t.,3 h,97%.

It was found that the Yamaguchi method for the macrocylization of emericellamide A was inefficient^[14],most likely due to the steric hindrance in ex-macrocylization product.Furthermore,we have demonstrated that the macrolactonization of C-2 and C-4 was also inefficient in total synthesis of emericellamide A^[16].Therefore,the esterification of less hindered secondary hydroxyl in 13 with N-Boc-Ala-OH was considered to form first in our synthetic strategy (Scheme 3).The coupling reaction of alcohol 13 with N-Boc-Ala-OH in the presence of EDC and DMAP in CH₂Cl₂ at 0 ℃ gave ester 14 in 60% yield,with partial epimerization at C-2 of product 14(13:1 ratio)^[33],while the starting material could be recovered.It was worth mentioning that the minor epimer of 14 could be easily removed by chromatography on silica gel to give enatiopure 14{[α]_D²⁵ -11.3(c 0.56,CHCl₃)}.Hydrogenation (Pd/C) of the ester 14 gave the alcohol 15 in quantitative yield.The crude acid,which was obtained through oxidation (RuCl₃-NaIO₄) of 15^[34]was condensed with H-Gly-OBn in the presence of EDC/HOBt to produce 16 in 88% yield (three steps).Deprotection of N-Boc group (TFA,CH₂Cl₂) of 16 gave salt 17,which was coupled with N-Cbz-Val-Leu-Ala-OH under the same conditions as above to give polypeptide 18 in 65% yield (two steps).Removal of both Bn and Cbz-protecting groups (Pd/C,MeOH) led to free acid and amine in one pot,which was subjected to macrolactamization by treatmentwith pentafluorophenyl diphenylphoophinate (FDPP) and DIPEA under highly diluted solution (10^-3 mol/L) of the substrate in CH₃CN at room temperature to give emericellamide B in 55% yield (two steps).The crude emericellamide B was further purified by RP C18 HPLC with 70% aqueous CH₃CN (Sepax-tech Amethyst C18 semipreparative column,10 mm × 150 mm,2 mL/min,refractive index detection){[α]_D²⁵ -43.6(c 0.11,MeOH);lit.[α]_D²⁵ -34(c 0.076,MeOH)^[10];[α]_D²⁵ -31(c 0.066,MeOH)^[15];^[a]D 30 -34.1(c 0.12,MeOH)^[18]}.In spite of the slightly different optical rotation of our synthetic emericellamide B,the spectroscopic data for our synthetic sample agreed well with that of the natural product^[10].

Scheme. 3

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Scheme. 3.Synthesis of emericellamide B (2).Reagents and conditions:(a) MeOH/HCl,0 ℃ to r.t.,3 h,quantitative;(b) N-Boc-Ala-OH,EDCI,DMAP,CH2Cl2,0 ℃ to r.t.,6 h,60%,90%(starting material was recovered);(c) Pd/C (10%),H2,MeOH,r.t.,4 h;(d)(i) RuCl3·xH2O (cat.),NaIO4;(ii) HOBt,EDCI,-15 ℃,1.5 h;then H-Gly-OBn,DMF,-15 ℃ to r.t.,overnight,88%(three steps);(e) TFA,CH2Cl2,r.t.,2 h;(f) HOBt,N-Cbz-Val-Ile-Ala-OH,EDCI,-15 ℃,1.5 h;then 17,DMF,-15 ℃ to r.t.,overnight,65%(two steps);(g)(i) Pd/C (10%),H2,MeOH,r.t.,3 h;(ii) FDPP,DIPEA,CH3CN,0 ℃ to r.t.,26 h,55%(two steps).

In conclusion,we have achieved a convenient method for total synthesis of emericellamide B.Our strategy demonstrated the preparation of the nonribosomal moieties of emericellamide B starting from cheap vinylacetic acid 3.Therefore,a general and efficient method for total synthesis of emericellamides and their analogues was established.Additionally,the concise synthesis could be beneficial for studying their structure-activity-relationship and their effects on human health,as well as providing enough material for exploring their possible antimicrobial mechanism and pharmacology. Acknowledgments

We thank the National Natural Science Foundation of China (Nos.21472022,21272041,21072034),Key Laboratory for Chemical Biology of Fujian Province for financial support.

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.07.023.