Pack pro c18

Melting point was measured on a XT4 microscopic melting-point apparatus (Shanghai Jingke Instruments Company, Shanghai, China). Optical rotation was measured with a 241 polarimeter (Perkin-Elmer, Waltham, MA, USA). Electrospray ionization mass spectroscopy (ESIMS) data were recorded with a Micro TOF II spectrometer (Bruker, Bremen, Germany). High-resolution ESIMS data were recorded with an APEX II HR-TOF spectrometer (Bruker). NMR spectra were obtained in DMSO-d₆ on a Bruker Avance DRX 400-MHz spectrometer at 400 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR. Precoated silica gel GF₂₅₄ plates (Merck, Darmstadt, Germany) were used for TLC. For column chromatography, SiO₂ (100–200 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Rp-C18 (ODS-A, 50 μm, YMC, Yantai, China) were used. HPLC purifications were performed on HPLC columns (YMC-Pack Pro C18, 5 μm, 250 mm × 4.6 mm and 250 mm × 10 mm, YMC, Kyoto, Japan) with a L-2000 HPLC system (Hitachi, Tokyo, Japan).

Optical rotations were determined on a PerkinElmer 343 polarimeter (Waltham, MA, USA). IR spectra were recorded using an Equinox 55 spectrophotometer (Bruker, Bremen, Germany). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III 700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 and 176.04 MHz, respectively, with chemical shifts referenced to the respective residual solvent signal (δ_H 7.21/δ_C 123.5 for C₅D₅N). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); the samples were dissolved in MeOH (at 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a differential refractometer; the columns used were as follows: Diasfer-110-C18 (10 µm, 250 × 15 mm, Biochemmack, Moscow, Russia), Discovery C₁₈ (5 µm, 250 × 4 mm, Supelco, North Harrison, PA, USA), and YMC-Pack Pro C18 (5 μm, 250 × 4.6 mm, YMC Co., Ltd., Kyoto, Japan). Low-pressure liquid column chromatography was carried out on Polychrome 1 (powdered Teflon, 0.25−0.50 mm; Biolar, Olaine, Latvia), Florisil (60–100 µm, Sigma-Aldrich Co., St. Louis, MO, USA), and Si gel KSK (50–160 µm, Sorbpolimer, Krasnodar, Russia) columns. Sorbfil Si gel plates (4.5 × 6.0 cm, 5–17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography (TLC).

Optical rotations, Perkin-Elmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). NMR spectra, Bruker Avance III 700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 MHz (¹H)/176.04 MHz (¹³C), internal standard CD₃OD at δ_H 3.30/δ_C 49.0. HRESIMS spectra, Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); sample concentration in MeOH 0.001 mg/mL. HPLC, Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA USA) with a differential refractometer; columns Discovery C18 (5 µm, 10.0 × 250 mm, Supelco, Bellefonte, PA, USA) and YMC-Pack Pro C18 (5 µm, 10.0 × 250 mm and 4.6 × 250 mm, YMC Co., Ltd., Kyoto, Japan). LPLC, column sorbents Polychrom 1 (powdered Teflon, 0.25–0.50 mm, Biolar, Olaine, Latvia), Si gel KSK (50–160 µm, Sorbpolimer, Krasnodar, Russia), and Florisil (60–100 µm, Sigma-Aldrich, Co., St. Louis, MO, USA).

The NMR experiments were performed using a JNM-ECA 500 MHz NMR instrument (JEOL Ltd., Tokyo, Japan). HRESIMS was carried out on a JMS-700 MStation Mass Spectrometer (JEOL Ltd., Tokyo, Japan). UV spectra were recorded on an Evolution 260 Bio UV–Visible spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). Thin-layer chromatographic (TLC) analysis was performed on Kieselgel 60 F254 (Merck, Darmstadt, Germany) and Kieselgel 60 RP-18 F254S (Merck), with visualization performed under UV light (254 and 365 nm) and 10% (v/v) sulfuric acid spray followed by heating (200 °C, 2 min). YMC Gel ODS-A (12 nm, S-150 μm; YMC Co., Kyoto, Japan) was used for column chromatography (CC). Preparative HPLC was performed using a Gilson Preparative HPLC system (Gilson Inc., Middleton, WI, USA) equipped with YMC Pack Pro C18 (5 μm, 250 × 20 mm, YMC Co.). Analytical HPLC was performed using an Agilent 1100 series system (Agilent Technologies, Palo Alto, CA, USA) equipped with YMC-Triart C18 (5 μm, 250 × 4.6 mm, YMC Co.). A [⁶⁰Co] γ-irradiator (150 TBq capacity; AECL, Ottawa, Canada) was used for gamma irradiation. All other chemicals and solvents used in this study were of analytical grade.

The dried algal sample (250 g) was extracted and separated as described in a previous paper [12 (link)]. Omaezol (1, 3.2 mg) and intricatriol (2, 17.0 mg) were isolated by HPLC (YMC-Pack Pro C18 (YMC, Kyoto, Japan) with CH₃CN and H₂O) from the omaezallene containing silica-gel fraction.
1: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −67.4 (c 0.17, CHCl₃); IR (neat), ν_max 3428, 1259, 1185, 1088, 1065, 954, 801 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.
2: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −45.2 (c 0.39, CHCl₃) ; IR (neat), ν_max 3414, 2972, 1451, 1369, 1214, 1101, 756 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

The air-dried branches and leaves of T. ramosissima (5.0 kg) were smashed and extracted under refluxing condition with 70% EtOH/H₂O (3 × 40 L). The successive extracts were combined and evaporated under reduced pressure to afford a crude extract (412 g), which was partitioned in 1 L H₂O and extracted with petroleum ether (PE), EtOAc, and n-BuOH successively (4 × 1 L). Then these combined extracts were evaporated under reduced pressure separately to give three different crude extracts. The EtOAc extract (43 g) was subjected to a silica gel column with gradient elution (CH₂Cl₂-MeOH) to give 9 fractions (Fr.1 to Fr.9). Fr.6 (2.6 g), which was eluted with 10% MeOH in CH₂Cl₂, was fractionated on a Rp-C18 column using MeOH-H₂O gradient eluent, affording 6 subfractions (Fr.6-1 to Fr.6-6). Fr.6-3 (110 mg) was further purified by recrystallization from MeOH to yield compound 1 (15 mg). Fr.4 (1.4 g), which was eluted with 5% MeOH in CH₂Cl₂, was fractionated on a silica gel column using PE-acetone gradient eluent, affording 5 subfractions (Fr.4-1 to Fr.4-5). Fr.4-5 (78 mg) was further purified on HPLC (YMC-Pack Pro C18, 5 μm, 250 mm × 10 mm, YMC, Kyoto, Japan), using 45% MeOH/H₂O at a flow rate of 3.2 mL/min and UV detection at 220 nm, to yield compound 2 (7 mg) and 3 (18 mg).