Agilent dd2 500 mhz nmr spectrometer

FTIR and NMR assays were carried out according to previous methods [80 (link),81 (link)]. For FTIR spectra, KSC and its oligosaccharides (2.0 mg) were mixed with KBr (200 mg) powder, ground and pressed, and then measured on a Nicolet Nexus 470 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For NMR spectra, KSC (50 mg) was dissolved in 500 μL 99% of the D₂O, freeze-dried and repeated 3 times. The sample was then redissolved in 500 μL D₂O and transferred to an NMR tube. Finally, ¹H-NMR/¹³C-NMR with Agilent DD₂ 500 MHz NMR spectrometer was performed with acetone as the internal standard.

Due to the small-scale fermentation, only two compounds were obtained from each extract. The pretreated crude extract of P18 (described in Section 3.4) was first subjected to octadecylsilyl silica gel (ODS) CC (RP18, 40–63 μm; Merck, Billerica, MA, USA), followed by step-gradient elution using MeOH-H₂O (30–50%) and purification via Sephadex LH-20 CC (GE Healthcare, Pittsburgh, PA, USA) using CH₂Cl₂-MeOH (v/v = 1:1) to yield 1 (4.4 mg) and 2 (2.1 mg). The EtOAc extract of P8 (described in Section 3.4, 20 bottles) was first subjected to ODS CC and eluted with 30–50% MeOH-H₂O, followed by separation by Sephadex LH-20 CC (CH₂Cl₂-MeOH; v/v = 1:1) and HPLC purification with 25% MeCN-H₂O to yield 3 (4.1 mg) and 4 (3.6 mg).
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Inc., Easton, MD, USA), and UV spectra were recorded on a Techcomp UV2310ΙΙ spectrophotometer (Techcomp, Ltd., Shanghai, China). ECD spectra were obtained on a Jasco J-815-150S circular dichroism spectrometer (Jasco, Inc., Tokyo, Japan). NMR spectra were recorded on an Agilent DD2 500 MHz NMR spectrometer (500 MHz for ¹H and 125 MHz for ¹³C; Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane as an internal standard. HRESIMS spectra were obtained from a Thermo Scientific LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

The UV spectra were measured using a Shimadzu UV-2700 spectrometer (Shimadzu Co., Ltd., Kyoto, Japan). The optical rotations were measured using a Jasco P-1020 automatic polarimeter (JASCO, Tokyo, Japan). The NMR spectra were recorded on an Agilent DD2 500 MHz NMR spectrometer (Agilent Technologies, Waldbronn, Germany) with tetramethylsilane (TMS) as an internal standard. The HRESIMS data were obtained in the positive ion mode on a Waters Xevo G2-XS QTof mass spectrometer (Waters, Milford, MA, USA). Commercially available silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Lobar LiChroprep RP-18 (40‒60 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (Merck) were used for open column chromatography.