Semi preparative hplc system

Chromatography columns were carried out on silica gel 60 (70–230 mesh, Merck) and Sephadex LH-20 (Pharmacia). while TLC was conducted on precoated silica gel aluminum sheets (60F254, 0.20 mm, Merck). The NMR spectra, in MeOD-d₄, were run on a Bruker Avance DPX-300 spectrometer and the data were processed using the Academic TopSpin software by Bruker. A Shimadzu-UFLC semi-preparative HPLC system, equipped with ternary pumps and diode array SPD-M20A UV/VIS detector, was used for high-performance liquid chromatography (HPLC). The sponge Petrosia sp. was collected in March 2014, at the municipality of Trairí (3° 13′ 7,29″ S-39° 15′ 48,40″) in the state of Ceará, Brazil. A voucher specimen (MNRJ 17832) was deposited in the Porifera collection of the Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

The crude peptides were purified by semi-preparative RP-HPLC on a Shimadzu semi-preparative HPLC system with LC-20AD pumps, SPD-20A UV-Vis detector, using a solvent system of eluent–A 0.1% TFA/H₂O; eluent-B: 0.1% TFA, 20% H₂O/acetonitrile on a Phenomenex Luna^® 10 μm C18(2) 100 Å column (10 × 250 mm). Gradient elution with the following profile was used to elute peptides from the column: 0% to 30% B over 60 minutes at a flow rate of 2.0 ml/min. Peptide signals were detected at 220 nm. Purity of the peptides was proved by analytical RP-HPLC on a Phenomenex Luna^® 5 μm C18(2) 100 Å column (4.6 × 250 mm).

Peptides were synthesized by a Liberty Microwave Peptide synthesizer (CEM Co.). To generate amidated peptides at carboxyl-termini, Rink Amide ProTide Resin (0.58 mmol/g) was used. Fmoc deprotection was completed using 20% piperidine in N,N-Dimethylformamide (DMF). Coupling of each Fmoc-amino acid was achieved in the presence of diisopropyl carbodiimide and Oxyma in DMF. To generate N-terminal fluorescently labeled peptides, Flamma675-carboxylic acid was added to the peptide-bound resin. The protecting groups and peptides on the resin were removed using trifluoroacetic acid(TFA)/diH₂O/triisopropylsilane (95: 2.5: 2.5, v/v/v) for 40 min at 40 °C. The filtrated peptide solution was precipitated and washed in ice-cold diethyl ether. The solid powder was isolated by centrifugation and then dried under a vacuum. The purification of crude peptides was done on a C18 column (Zorbax, 21.2 × 250 mm, 300Å, 7-μm) on a Shimadzu semi-preparative HPLC system, using a 10–90% acetonitrile gradient in water with 0.05% TFA for 80 min. The purity and molecular masses of the isolated peptide were measured on an analytical HPLC system and a matrix-assisted laser desorption ionization (MALDI) mass spectrometer (Kratos Analytical Ins.), respectively.

LA (2 g) was dissolved in 50 mL of methanol. Rose bengal (0.5 mg) was added, and the solution was exposed to light-emitting diode irradiation (50,000 lux, >30 °C) for 5 h. Rose bengal was removed by Sep-Pak Vac QMA (3 cc, 500 mg, Waters, MA, USA), and the solution was evaporated under a nitrogen gas stream. The residue was dissolved in 50 mL of hexane, and the sample (1 mL) was injected into a semipreparative HPLC system (Shimadzu, Kyoto, Japan) to isolate the HpODE isomers. Inertsil SIL-100A (5 μm, 10 × 250 mm, GL Sciences Inc., Tokyo, Japan) was eluted with hexane/2-propanol/acetic acid (100:1:0.1, v/v/v) at 20 mL/min.^{43 (link)} The column temperature was maintained at 40 °C, and the HpODE isomers were detected by UV absorbance at 210 nm. To refine the purity of the references, the obtained HpODE isomers were subjected once more to semipreparative HPLC under the same conditions.