First Total Synthesis of Hoshinoamides A

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Introduction
Malaria is an insect-borne infectious disease caused by parasites of the genus Plasmodium, which seriously threatens human life and health [1] . Half of the world's population is at the risk of malaria, with about 200 million new infections and killing hundreds of thousands of people each year [2] . Current medicines for malaria include quinolone [3,4] , folic acid antagonist [5,6] and artemisinin derivatives [7] . The emergence of drug resistance makes the efficacy of these drugs decline year by year, forcing scientists to constantly search for new antimalarial drugs [8][9][10] .
In recent years, Iwasaki and co-workers reported three novel linear lipopeptides natural products, Hoshinoamides A, B [11] and C [12] , from a microbial metabolite of marine cyanobacterium Caldora penicillata (Figure 1). Hoshinoamides A and B showed potent activities against chloroquine-sensitive Plasmodium falciparum 3D7 with IC50 values of 0.52 and 1.0 μM, respectively. Hoshinoamides C inhibited the growth of the malaria parasites (IC50 3 0.96 μM ) and African sleeping sickness (IC50 2.9 μM ). Both Hoshinoamides A and B are highly methylated polypeptides containing three N-methyl amino acids: N-Me-L-Leu7, N-Me-D-Val5 and N-Me-D/L-Phe2. Hoshinoamides C includes two N-methyl amino acids: N-Me-D-Phe2 and N-Me-D-IIe5. The C-terminal is Pro methyl ester while the N-terminal polypeptide is linked to long alkyl chain amino acid Aha8/Ana8/Ama7 and p-hydroxybenzoic acid Hba9/Hba8. Hoshinoamides have a relatively simple structure and therefore make an attractive target for further medicinal chemistry studies. To enable these new SAR studies, we would first need to develop efficient synthetic method to provide sufficient material. Hoshinoamides A shows better antimalarial activity and less cytotoxicity compared to Hoshinoamides B.
Herein, we report the initial progress on the total synthesis of Hoshinomaides A.
The key challenges for the total synthesis of Hoshinoamides A are the coupling of highly methylated amino acids and the purification of hydrophobic peptides.

Results and Discussion
As shown in Scheme 1, we initially tested Fmoc Solid-phase peptide synthesis (SPPS) to get 2-chlorotrityl resin-bound Pro1-(N-Me)Phe2 dipeptide 2 under the condition of HCTU and DIPEA. Unfortunately, the N-Me coupling proceeded in low yield (< 10%).

Scheme 1. Synthesis of resin-bound tripeptide 3 by SPPS
In order to improve the coupling yield of hindered peptide, we tried to condensation of Val3 with dipeptide in solution phase. Pro-Bn 5 was first coupled with Fmoc-N-Me-D-Phe-OH by the treatment of HATU and DIPEA, giving the dipeptide 6 in 83% yield ( Table 1). We envisioned a sequential deprotection of Fmoc of dipeptide 6 and then coupling with Fmoc-Val-OH will deliver tripeptide 7. With this in mind, we next screened a series of coupling reagents. As shown in Table 1, the coupling reagents have a significant effect on the efficiencies of the reactions. While most of coupling reagents could give the tripeptide 7, the combination of HATU/DIPEA shown the best result, delivering 7 in 78% isolated yield (Table 1, entry 2). Only trace product could be detected while EEDQ was used as the coupling reagent. The purity of tripeptide 7 was determined by HPLC and no racemization was observed, ensuring the smooth progress of the total synthesis of Hoshinoamides A With the Tripeptide 7 in hand, we went on to construct the peptide scaffold (Scheme 2).
Deprotection of the Bn groups by Pd(OH)2-catalyzed hydrogenolysis gave the tripeptide 8. Hoshinoamides A in 2% yield (10 mg). The spectroscopic data of synthetic Hoshinoamides A were in excellent agreement with the data previously reported for the natural product. NaHCO3 (20 mL) and brine (20 mL). The organic phase was dried with anhydrous Na2SO4

Scheme 2. Synthesis of Hoshinoamides
and concentrated in vacuo. The crude residue was purified by flash column chromatography (n-hexanes: EA=2:1) to afford dipeptide 6 (4.9 g, 83%). 1  group was removed following the general procedure and the remain amino acids were successively coupled using the standard SPPS method. 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.
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 lyophilized to give nanopeptide 10 as a white solid. Nanopetide 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 x 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. Fractions were collected, concentrated and lyophilized to give Hoshinoamides A as a white solid.(10 mg, 2% yield).
The 1 H NMR and 13 C NMR spectra of synthetic product were fully consistent with the data of isolated samples reported in the literature. 4