First total synthesis of hoshinoamide A

Hoshinoamides A, B and C, linear lipopeptides, were isolated from the marine cyanobacterium Caldora penicillata, with potent antiplasmodial activity against chloroquine-sensitive Plasmodium falciparum. Herein, we describe the first total synthesis of hoshinoamide A by the combination of liquid-phase and solid-phase peptide synthesis. Liquid-phase synthesis is to improve the coupling yield of ʟ-Val3 and N-Me-ᴅ-Phe2. Connecting other amino acids efficiency and convergence is achieved by solid-state synthesis. Our synthetic strategy could synthesize the target peptide in high yield with good purity

Fmoc-Aha-OH was synthesized using a modified procedure reported by aminocaproic acid [3] . All data for known compounds are consistent with those reported in literature.

Scheme S1: Synthesis of Fmoc-N-Me-D-Phe.
Boc-D-Phe-OH (5.00 g, 18.9 mmol) and MeI (1.8 mL, 28.4 mmol)were dissolved in DMF (50 mL). The reaction mixture was stirred at 0 °C for 45 min, then NaH (2.27 g, 56.7 mmol, 60%) was slowly added to the reaction mixture. The reaction mixture was stirred at room temperature for an additional 3 h, then quenched with water (150 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (100 mL) and brine (100 mL). The organic phase was dried over Na2SO4, filtered and concentrated to afford Boc-N-Me-D-Phe-OH which was used without purification.
Boc-N-Me-D-Phe-OH was dissolved in 10% HCl-dioxanne (50 mL). The mixture was stirred at room temperature for 4 h, then concentrated to give a brown oil. The resulting crude oil was azeotroped with toluene (3 × 10 mL) and concentrated in vacuo to remove any residual HCl. The concentrated crude material was then dissolved in a mixture of THF (25 mL), H2O (25 mL) and NaHCO3 (3.2 g, 37.8 mmol), Fmoc-OSu (9.5 g, 28.4 mmol) was added to this mixture and the reaction mixture was stirred at room temperature for 8 h. Then added with water (150 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (100 mL) and brine (100 mL). The mixture was concentrated to give a S3 crude white foam which was purified by flash chromatography (n-hexanes/EA = 2:1) to afford Fmoc-N-Me-D-Phe-OH (5.6 g, 14.0 mmol, 74%) as a white foam. 1

Scheme S2: Synthesis of Fmoc-N-Me-D-Val-OH.
Boc-D-Val-OH (5.0 g, 23.0 mmol) and MeI (2.2 mL, 28.4 mmol)were dissolved in DMF (50 mL). The reaction mixture was stirred at 0 o C for 45 min, then NaH (2.7 g, 68.0 mmol, 60%) was slowly added to the reaction mixture. The reaction mixture was stirred at room temperature for an additional 5 h, then quenched with water (150 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (100 mL) and brine (100 mL). The organic phase was dried over Na2SO4, filtered and concentrated to afford Boc-N-Me-D-Val-OH which was used without purification.
Boc-N-Me-D-Val-OH was dissolved in 10% HCl-dioxanne (50 mL). The mixture was stirred at room temperature for 3 h, then concentrated to give a brown oil. The resulting crude oil was azeotroped with toluene (3 × 10 mL) and concentrated in vacuo to remove any residual HCl. The concentrated crude material was then dissolved in a mixture of THF (25 mL), H2O (25 mL) and NaHCO3 (3.8 g, 45.4 mmol), Fmoc-OSu (11.4 g, 34.1 mmol) was added to this mixture and the reaction mixture was stirred at room temperature for 8 h. Then added with water (150 mL) and S4 extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (100 mL) and brine (100 mL). The mixture was concentrated to give a crude white foam which was purified by flash chromatography (DCM/MeOH = 100:1) to afford Fmoc-N-Me-D-Val-OH (6.6 g, 18.6 mmol, 81%) as a white foam. 1
Then added with water (150 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (100 mL) and brine (100 mL). The mixture was concentrated to give a crude white foam which was purified by flash chromatography (DCM:MeOH = 50:1) to afford Fmoc-Aha-OH (4.8 g, 13.7 mmol, 90%) as a white foam.   The resin-bound peptide was added to a mixture of 20% piperidine in DMF (20 mL), and the mixture was shaken to for 30 minutes. Then the mixture was filtered, the resin was washed with MeOH (3 × 20 mL) and DCM (3 × 20 mL). Fmoc-N-Me-D-Phe-OH (1000 mg, 2.5 mmol), HATU (950 mg, 2.5 mmol) and DIPEA (871 µL, 5.0 mmol) in DMF were added on the resin and the reactor was shaken for 1h at room temperature.
Then the mixture was filtered, the resin was washed with MeOH (3 × 20 mL) and DCM (3 × 20 mL) to afford the resin-bound dipeptide. The resin-bound dipeptide was added to a mixture of 20% piperidine in DMF (20 mL), and the mixture was shaken to for 30 minutes. Then the mixture was filtered, the resin was washed with MeOH (3 × 20 mL) and DCM (3 × 20 mL). Fmoc-Val-OH (848 mg, 2.5 mmol), HATU (950 mg, 2.5 mmol) and DIPEA (871 µL, 5.0 mmol) in DMF were added on the resin and the reactor was shaken for 1h at room temperature. The resulting tripeptide 3 was analysed on a Thermo Scientific MSQ instrument, and few product was observed.  To a stirred solution of dipeptide 6 (118 mg, 0.20 mmol) was added 20% Et2NH in Coupling reagents was added to the solution and the mixture was stirred at room temperature for 3 h. This mixture poured onto water (10 mL) and extracted with CH2Cl2 (3 × 10 mL). Then washed by 1.0 M HCl (10 mL), aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried with anhydrous Na2SO4 and concentrated in vacuo. The crude residue was purified by flash column S7 chromatography (n-hexanes/EA = 2:1) to afford tripeptide 7. 1  Comparison of the effects of different coupling reagents on the reaction yield (Table   S1).  Hba 9 are coupled with HATU and DIPEA.) 0.5% TFA in DCM (20 mL) were added on the resin and the mixture was shaken for 2h to cleavage the peptide from the resin.

Synthesis of hoshinoamide A
The mixture was filtered and the filtrate was concentrated in vacuo to give a white foam. The peptide was re-dissolved in a mixture of TFA:Et3SiH:H2O (10 mL, 50/50/50 v/v/v). The reaction mixture was stirred for 3 h, and then concentrated in vacuo. The crude peptide was precipitated using cold Et2O and centrifuged at 7000 rpm to give a white solid. This solid was further purified by RP-HPLC using protocols described in the general method. Fractions were collected, concentrated and S9 lyophilized to give nanopeptide 10 as a white solid. Nanopetide 10 was dissolved in dry DMF (5 mL). K2CO3 (3.1 mg, 0.022 mmol) and MeI (3.13 mg, 0.022 mmol) was added to this solution. The reaction mixture was stirred for 3 h. This mixture poured onto water (5 mL) and extracted with CH2Cl2 (3 × 5 mL). Then washed by 1.0 M HCl (10 mL), aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried with anhydrous Na2SO4 and concentrated in vacuoto give brown oil . This oil was further purified by RP-HPLC using protocols described in the general method.