Av 3 600 nmr spectrometer

IR spectra of ASP, ASP-SA, and cASP were recorded with FT- IR spectrometer (Bruker Vertex 70; Karlsruhe, Baden-Württemberg, Germany) in the region of 4000–400 cm⁻¹ for detecting functional groups. ¹H NMR spectra was recorded with a Bruker AVIII-600 NMR spectrometer (Bruker Corporation; Karlsruhe, Baden-Württemberg, Germany). 10 mg ASP, 10 mg ASP-SA, 10 mg cASP, and 10 mg SA were dissolved in 99.9% D₂O, and then lyophilized. The operation described above would be done in triplicate to ensure complete heavy water exchange in the samples. Thereafter, the samples were dissolved in 0.5 mL of 99.9% D₂O, respectively. Meanwhile, 10 mg Cy5.5 was dissolved in 99.9% DMSO-d₆ (Cy5.5 was insoluble in D₂O) for ¹H NMR spectra analysis.

Optical rotation data were measured with a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). UV and IR data spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers (Shimadzu, Kyoto, Japan). NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer with chemical shift values presented as δ values having TMS (Tetramethylsilane) as the internal standard. HRESIMS was performed using an LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, USA). Column–chromatography (CC) was performed using silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical Plant, Qingdao, China), Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Precoated silica gel GF₂₅₄ plates (Zhi Fu Huang Wu Pilot Plant of silica gel Development, Yantai, China) were used for TLC. All solvent used was of analytical grade (Beijing Chemical Plant, Beijing, China).

All chemical reagents and solvents were obtained from Sigma Aldrich or other commercial source and used without further purification. ¹H and ¹³C NMR spectra were measured in pyridine-d₅ on Bruker AV III 600 NMR spectrometer (Bruker, Billerica, German) with TMS as internal standard. HRESIMS spectra were performed on an LTQ Obitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, USA). Melting points were determined on an X-4B apparatus (Shanghai Precision Instruments Co., Ltd., Shanghai, China) and are uncorrected. Optical rotations were obtained on an Anton Paar MCP200 polarimeter (Anton Paar, Graz, Austria). Semi-preparative HPLC on a CXTH LC3050N equipped with two pumps of P3000 and a UV3000 detector (Beijing ChuangXinTongHeng Science and Technology Co., Ltd., Beijing, China), and a ZORBAX Eclipse XDB-C18 (9.4 × 250 mm, 5 μm, Agilent, Santa Clara, CA, USA) column. Precoated silica gel GF₂₅₄ plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China) and Merck precoated silica gel 60 F254 plates were used for TLC and spots visualized by heating plates sprayed with 10% H₂SO₄ in EtOH. Flash chromatograph was carried out on a Biotage Isolera Four system with a Buchi FlashPure Select Silica gel cartridge (15 μm, spherical, 4 g).