Cd3od

1D (¹H, ¹³C, and DEPT) and 2D (HSQC, HMBC, ¹H–¹H COSY, and NOESY) NMR spectra were obtained on a Bruker AM-500 spectrometer (Bruker, Germany) in CDCl₃ or CD₃OD (Qingdao Tenglong, China), and TMS was used as an internal reference. IR spectra were scanned on a Nicolet Magna-IR 550 spectrometer (Thermo Nicolet, United States) with KBr pellets. Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Japan). HR-ESI-MS spectra were obtained on an Agilent 6230 LC/TOF MS spectrometer (Agilent, United States). The prep-HPLC experiment was performed on an Agilent 1260 pump coupled with an analytical preparative ZORBAXSB column (21.2 × 500 m, 5 µm). Silica gel (300–400 mesh, Qingdao Haiyang, China) was used in column chromatography, and Dragendorff’s reagent was used in TLC analysis (GF₂₅₄ TLC plates, Qingdao Haiyang, China).

¹H NMR detected spectra of SPE-DOM were acquired with a Bruker Avance NMR spectrometer at 800.13 MHz (B₀ = 18.7 T) at 283 K. Around 200 μg (2 mg for forest soil extract) of solid, obtained by evaporation of SPE-DOM samples, was dissolved in ~50 μl CD₃OD (Merck. 99.95% ²H) solution and analyzed with a 5 mm z-gradient ¹H/¹³C/¹⁵N/³¹P QCI cryogenic probe (90° excitation pulses: ¹³C ~¹H ~10 μs) in sealed 1.7 mm Bruker MATCH tubes (forest soil: 150 μl CD₃OD, 3 mm MATCH tube). Acquisition conditions were identical to those described in Hertkorn et al. (2013) (link). The number of scans ranges from 272 (forest soil leachate) to 4,544 (groundwater inflow) in 1D ¹H NMR spectra (Bruker pulse sequence noesypr1d); further NMR acquisition conditions are given in Supplementary Table S2.

Cal-4 derivative 1 was synthesized according to the modified literature procedure [26 ]. Firstly, a mixture of tetrakis (chloromethyl) Cal-4 (216 mg, 0.22 mmol) and thiourea (200 mg, 2.6 mmol) was dissolved in DMF (15 mL). The mixture was then stirred at room temperature overnight under N₂. After the solvent was removed by vacuum distillation at 100 °C, the residue was dissolved in 20 mL of 1 M-aquoues Na₂CO₃. Finally, the product was extracted with CH₂Cl₂ (20 mL × 3), The combined organic layer was dried over MgSO₄, filtered, and concentrated to dryness to give glassy solid (200 mg, 94%). The product was characterized by ¹H and ¹³C NMR (400 and 100 MHz, CD₃OD, Bruker Co., Billerica, MA, USA). ¹H NMR (400 MHz, DMF-d6) δ 6.72 (s, 8 H), 4.75 (overlapped with NH₂), 4.67 (s, 8 H), 4.16 (s, 8 H), 4.12 (q, J = 6.6 Hz, 8 H), 3.20 (d, J = 11.5 Hz, 4 H), 1.20 (t, J = 6.6 Hz, 12 H); ¹³C NMR (100 MHz, CDCl₃) δ 185.9, 172.7, 171.7, 157.7, 136.9, 131.1, 129.8, 73.0, 62.3, 36.8, 32.7, 15.0. Cal-4 derivative 2 (carboxylic acid) was converted from Cal-4 derivative 1 (ethylester) by treatment of 0.1 M LiOH for 3 h. The molecular schemes of Cal-4 derivatives are shown in Figure 1.