Solid-phase total synthesis and structural confirmation of antimicrobial longicatenamide A

Longicatenamides A–D are cyclic hexapeptides isolated from the combined culture of Streptomyces sp. KUSC_F05 and Tsukamurella pulmonis TP-B0596. Because these peptides are not detected in the monoculture broth of the actinomycete, they are key tools for understanding chemical communication in the microbial world. Herein, we report the solid-phase total synthesis and structural confirmation of longicatenamide A. First, commercially unavailable building blocks were chemically synthesized with stereocontrol. Second, the peptide chain was elongated via Fmoc-based solid-phase peptide synthesis. Third, the peptide chain was cyclized in the solution phase, followed by simultaneous cleavage of all protecting groups to afford longicatenamide A. Chromatographic analysis corroborated the chemical structure of longicatenamide A. Furthermore, the antimicrobial activity of synthesized longicatenamide A was confirmed. The developed solid-phase synthesis is expected to facilitate the rapid synthesis of diverse synthetic analogues.


1
H and 13 C NMR spectra were recorded on a JEOL ECZ600 (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) spectrometer. Chemical shifts are denoted in δ (ppm) relative to residual solvent peaks as internal standard (CDCl3, 1 H δ 7.25, 13 C δ 77.2, DMSO-d6, 1 H δ 2.50, 13 C δ 39.5). ESIMS and LC-MS experiments were recorded on a Shimadzu LCMS-IT-TOF. Optical rotations were recorded on a JASCO P-2200 polarimeter. High performance liquid chromatography (HPLC) experiments were performed with a SHIMADZU HPLC system equipped with an LC-20AD intelligent pump. All reactions sensitive to air and/or moisture were conducted under nitrogen atmosphere using dry, freshly distilled solvents, unless otherwise noted. All reagents were used as supplied unless otherwise stated. Analytical thin-layer chromatography (TLC) was performed using Merck silica gel 60 F254 pre-coated plates. Silica gel column chromatography was performed using 40-50 μm silica gel 60N (Kanto Chemical Co., Inc.).

Synthesis and compound characterization
To a solution of compound 14 (2.66 g, 6.44 mmol) in THF (10 mL) was added NaHMDS (3.1 mL, 1.9 M in THF, 5.9 mmol) at 0 ℃. After being stirred at 0 ℃ for 1 h, compound 13 (439 mg, 1.92 mmol) in THF (10 mL) was added dropwise to the solution. The reaction mixture was stirred at room temperature for 16 h, and then quenched with saturated aqueous NH4Cl. The resulting solution was extracted with hexane/EtOAc 9:1 (three times). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was roughly purified by silica gel column chromatography (hexane/EtOAc 97:3 to 90:10) to afford crude 15, which was used in the next reaction without further purification.
To a solution of the above compound 15 in CH2Cl2 (13 mL) were added TFA (6 mL) and H2O (1 mL) at room temperature. After being stirred for 1 h, the mixture was concentrated and azeotroped with toluene (three times) to afford crude 16, which was used in the next reaction without further purification.
To a solution of the above compound 16 in THF/H2O 2:1 (15 mL) were added NaHCO3 (386 mg, 4.59 mmol) and FmocCl (594 mg, 2.30 mmol) at 0 ℃. After being stirred at room temperature for 3 h, the reaction mixture was treated with saturated aqueous NH4Cl. The resulting mixture was extracted with EtOAc (three times). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was roughly purified by silica gel column chromatography (hexane/EtOAc 75:25 to 55:45) to afford crude 17, which was used in the next reaction without further purification.
To a solution of compound 18 (227 mg, 0.617 mmol) in CH2Cl2 (7 mL) was added Dess-Martin periodinane (392 mg, 0.924 mmol) at room temperature. After being stirred at room temperature for 1.5 h, to the reaction mixture were added saturated aqueous solutions of Na2S2O3 and NaHCO3. The resulting mixture was extracted with EtOAc (three times). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo to afford crude 19, which was used in the next reaction without further purification.

Procedures for the solid-phase peptide synthesis (SPPS).
Step 1: To the solution of compound 6-11 (3 equiv) were added DIC (3 equiv, 0.50 M in NMP) and Oxyma (3 equiv, 0.50 M in DMF). After approximately 2-3 min of preactivation, the mixture was injected to the reaction vessel. The resulting mixture was stirred at 40 °C for 20 min, under microwave irradiation.
Step 3: Fmoc group of the solid supported peptide was removed with 20% piperidine/DMF solution (8 min, room temperature).
Amino acids were condensed onto the solid support by repeating steps 1-4. The resin-bound D-Trp 28 was subjected to the SPPS protocol (steps 3 and 4), and then 5 cycles of the SPPS S7 protocol to afford resin-bound peptide 5.
To resin-bound peptide 5 was added TFA/CH2Cl2 1:99 (0.5 mL). After being stirred for 30 min, the reaction mixture was filtered. This procedure was repeated twice. The filtrate was concentrated in vacuo and azeotroped with toluene (× 3) to afford crude peptide 30, which was used in the next reaction without further purification.