Nmr spectrometer

All reagents and solvents used in this synthesis were used without further purification. Melting points were obtained by the Gallenkamp apparatus. The IR spectra (KBr) were determined on a Thermo Scientific Nicolet iS10 FTIR spectrometer (Faculty of Science, Mansoura University). The ¹H NMR spectra was recorded in DMSO-d₆ using a JEOL’s NMR spectrometer (500 MHz) at Faculty of Science, Mansoura University. The mass analysis (molecular ion peaks) was measured using a Thermo Scientific GC/MS model ISQ and/or Agilent LC-MSD IQ Infinity II 1260 (Al-Azhar University, Egypt). Elemental analyses (C, H, and N) were performed with the Perkin-Elmer 2400 instrument (Microanalytically Unit, Cairo University). The anticancer activity evaluation was carried out at the bioassay-cell Culture Laboratory, National Research Centre, El-Tahrir Street, Dokki, Cairo, 12622, Egypt.

The Egyptian Red Sea soft coral Nephthea sp. (voucher specimen: 399RS-9100-X19) was collected and authenticated by Dr. Montaser A. Alhammady, National Institute of Oceanography and Fisheries, Marine Biological Station, Hurghada, Egypt, from the Hurghada coast in May 2019. Four kilograms of the frozen soft coral were cut, extracted via 6 L of a mixture of 1:1 (MeOH–CH₂Cl₂), filtered, and dried under vacuum-afforded dark brown gum (167.8 g) using a rotary evaporator. The extract was further fractionated over silica gel column chromatography (6 × 120 cm) using n-hexane (100%), a step gradient of n-hexane–CH₂Cl₂, CH₂Cl₂ (100%), and CH₂Cl₂–MeOH until CH₂Cl₂–MeOH (1:1) afforded seven main fractions (NP-I to NP-VII). Fraction NP-4 (3.1 g) was subjected to silica gel column chromatography and generated three sub-fractions (NP-4A-C). The sub-fraction NP-4B (673.4 mg) was eluted by CHCl₃-MeOH (1:1) over a glass column packed with Sephadex LH-20 (3 × 120 cm) to afford compound ST-1 (181.7 mg). Compound ST-1 was analyzed via (i) NMR spectroscopic analysis (600 MHz Bruker NMR spectrometer, USA) in CD₃OD and (ii) mass spectroscopy (JEOL JMS-600 instrument (Tokyo, Japan) (Supplementary Materials Figures S1–S4). The chemical structure of ST-1 was constructed by comparison of its NMR with previously published data (Figures S1–S4) [22 (link)].

The degree of methacryloyl functionalization was quantified by using ¹H NMR according to a previously described method [36 (link)]. ¹H NMR spectra were collected by using a NMR spectrometer (JEOL Co. Ltd., Tokyo, Japan) with a single axis gradient inverse probe at a frequency of 300 MHz. Before the measurement, 20 mg of GelMA macromers was completely dissolved in 1 mL deuterium oxide containing 0.05 wt % 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt for calibration (Sigma-Aldrich, St. Louis, MO, USA). The gelatin without functionalization was also examined for calculating the degree of methacryloyl substitution using the following Equation (1):

H NMR spectra were analyzed using an NMR spectrometer (JEOL) with a single-axis gradient inverse probe at a frequency of 300 MHz. Before the measurement, 20 mg of GelMA macromers were completely dissolved in 1 ml of deuterium oxide containing 0.05% (w/v) 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt for calibration (Sigma-Aldrich). The gelatin without functionalization was also examined for calculating the degree of substitution (DoF) of MA using the following equation: $$\mathrm{DoF}=1-(\text{lysine methylene proton of GelMA})/(\text{lysine methylene proton of Gelatin})\times 100\mathrm{\%}$$

A 120 mg MWL was placed into a 5-mL vial and vacuum dried under P₂O₅ at ambient temperature for 24 h before sample preparation. The moisture-free MWL was dissolved at 0.75 mL DMSO-d₆ at 50 °C. The filtration was carried out in the same manner as for ¹H NMR samples mentioned above. The ¹³C and HSQC NMR analyses were conducted using an NMR spectrometer (600 MHz, JEOL, Tokyo, Japan) at the CURF, JBNU.