*3.4. Purification*

The crude gum (240 g) was separated into ten fractions (Fr1–Fr10) on a silica gel VLC column using a stepwise gradient elution of petroleum ether (PE), PE-CH2Cl2 (1:1–0:1) followed by CH2Cl2-MeOH (1:0–1:1). Fr9 (26 g) was fractionated on Sephadex LH-20, eluted with CH2Cl2-MeOH (1:1), to obtain three subfractions (Fr9.1–Fr9.3). Fr9.2 (8 g) was further separated into five subfractions (Fr9.2.1–Fr9.2.5) by VLC on the RP-18 column using a stepwise gradient elution of MeOH-H2O (9:1–1:1), among which the elution of 40% MeOH–H2O gave compound **7** (9.2 mg). Compounds **1** (6.2 mg, tR 11.8 min) and **2** (32 mg, tR 18.7 min) were obtained from Fr9.2.2 (1.7 g) by semipreparative HPLC over an ODS column eluting with 15% MeCN-H2O containing 0.5 ‰ Et3N. Fr7 (12 g) was fractionated on Sephadex LH-20, eluted with MeOH-CH2Cl2 (1:1), to obtain four subfractions (Fr7.1–Fr7.4). Fr7.4 (3.3 g) was further purified by semipreparative HPLC over an ODS column eluting with 40% MeCN-H2O containing 0.5 ‰ TFA (trifluoroacetic acid) to yield compound **4** (7.6 mg, tR 16.5 min). Fr7.3 (1.3 g) was fractionated into four subfractions (Fr7.3.1–Fr7.3.5) on a RP-18 column using a stepwise gradient elution of MeOH-H2O (1:9–2:3). Fr7.3.2 (300 mg) was further separated by semipreparative HPLC on an ODS column eluted with 20% MeCN-H2O to yield compound **3** (3.1 mg, tR 7.8 min). Fr6 (17.6 g) was further fractionated on Sephadex LH-20 eluted with MeOH-CH2Cl2 (1:1) to afford four subfractions (Fr6.1–Fr6.4). Fr6.2 (1.1 g) was further separated by semipreparative HPLC on an ODS column eluted with 40% MeCN-H2O containing 0.5 ‰ TFA to yield compound **6** (16.3 mg, tR 15 min), while compound **5** (860 mg, tR 17.0 min) was purified from Fr6.4 (9 g) by semipreparative HPLC on an ODS column eluted with 65% MeCN-H2O. Pure compounds **5a** (8.8 mg, tR 39 min), **5b** (5.4 mg, tR 42 min), **5c** (7.3 mg, tR 44 min) and **5d** (6.8 mg, tR 46 min) were obtained from compound **5** by a careful separation on an ODS column eluted with 50% MeOH-H2O.

#### *3.5. The Preparation of Acetonide (***5e***) for Relative Configuration*

According to our procedure [16], compound **5a** (5 mg) in acetone (3 mL) was added to the mixture of 2,2-dimethoxypropane (1 mL), pyridinium *p*-toluenesulfonate (PPTS, 26 mg) and *<sup>N</sup>*,*<sup>N</sup>*-dimethylformamide (DMF, 1 mL). The resulting solution was stirred at room temperature (rt) for 12 h, and then 5 mL of H2O was added. The reaction solution was extracted with 15mL of CH2Cl2, and the organic phase was concentrated under reduced pressure. The residue was purified by semipreparative HPLC (95% MeOH-H2O) to yield the acetonide **5e** (3.4 mg, tR 5.7 min). Its structure was identified by ESIMS (Figure S65) and NMR data (Tables 3 and 4, Figures 4 and S66–S71).

#### *3.6. The Induced ECD Spectra of Compounds* **2***,* **5c***, and* **5d** *for Absolute Configuration*

According to a published procedure [16,17], analytical pure DMSO was dried with 4 Å molecular sieves and was used to prepare 0.6 mg/mL of Mo2(OAc)4 solution. To three pieces of this solution (each 1 mL, 1.40 μmol), compounds **2** (0.5 mg, 1.86 μmol), **5c** (0.8 mg, 1.90 μmol), and **5d** (0.8 mg, 1.90 μmol) were respectively added and the first ECD spectra of the mixtures were recorded immediately. Then, ECD spectra were continuously recorded every 10 min until stationary. The inherent ECD spectrum was subtracted. The observed signs of the diagnostic bands in the region of λmax 300–400 nm in the induced ECD spectra were correlated to the absolute configuration of the *ortho*-diol moiety.

(2*R*,3*R*,5*S*,9*R*,10*S*)-2,3,9,11-Tetrahydroxydrim-7-en-6-one (ustusol F, **1**): colorless oil; [α]23D −56.0 (*c* 0.11, MeOH); UV (MeOH) λmax (log *ε*) 232 (0.82) nm; ECD (1.76 mM, MeOH) λmax (Δ*ε*) 336 (+8.4), 271 (−3.2), 240 (−41.3), 215 (−12.8) nm; IR (KBr) *ν*max 3399, 2959, 1663, 1439, 1384, 1243, 1062, 1027 cm<sup>−</sup>1; 1H and 13C NMR see Table 1; HRESIMS *m*/*z* 285.1694 [M+H]+ (calcd for C15H24O5, 285.1697), or 283.1547 [M–H]− (calcd for C15H23O5, 283.1551).

(2*R*,3*R*,5*R*,9*S*,10*R*)-2,3,11-Trihydroxydrim-7-en-6-one (9-deoxyustusol F, **2**): yellow oil; [α]23D −56 (*c* 0.06, MeOH); UV (MeOH) λmax (log *ε*) 238 (1.65) nm; ECD (1.87 mM, MeOH) λmax (Δ*ε*) 334 (+6.8), 264 (−1.3), 240 (−18.3), 220 (−14.3) nm; IR (KBr) *ν*max 3398, 2942, 1659, 1440, 1382, 1237, 1152, 1060, 983 cm<sup>−</sup>1; 1H and 13C NMR see Table 1; HRESIMS *m*/*z* 269.1751 [M+H]+ (calcd for C15H24O4, 269.1747).

(3*S*,5*R*,9*R*,10*R*)-3,11,12-Trihydroxydrim-7-en-6-one (ustusol G, **3**): colorless oil; [α]23D −71 (*c* 0.04, MeOH); UV (MeOH) λmax (log *ε*) 240 (1.60) nm; ECD (1.87 mM, MeOH) λmax (Δ*ε*) 335 (+10.6), 265 (−2.6), 241 (−19.1), 205 (−71.7) nm; 1H and 13C NMR see Table 1; HRESIMS *m*/*z* 269.1750 [M+H]+ (calcd for C15H24O4, 269.1747).

(5*S*,6*R*,9*S*,10*S*,11*R*,2-*E*,4-*E*)-6-(11-Deoxy-11-hydroxystrobilactone A-6-yl)-5-carboxypenta-2,4-dienoate (ustusolate H, **4**): colorless solid; [α]25D −96 (*c* 0.2, MeOH); UV (MeOH) λmax (log *ε*) 264 (1.54) nm; ECD (0.64 mM, MeOH) λmax (Δ*ε*) 264 (−6.2), 232 (−3.3), 205 (−11.1) nm; IR (KBr) *ν*max 3434, 2953, 2926, 2856, 1684, 1640, 1460, 1398, 1310, 1260, 1208, 1136, 1028, 913 cm<sup>−</sup>1; 1H and 13C NMR see Table 2; HRESIMS *m*/*z* 391.1762 [M–H]– (calcd for C21H27O7, 391.1762).

((5*S*,6*R*,9*S*,10*S*)-Strobilactone A-6-yl) (2*E*,4*E*)-6,7-dihydroxyocta-2,4-dienoate (ustusolate I, **5**): light yellow oil; UV (MeOH) λmax (log *ε*) 265 (4.15) nm. 1H NMR (DMSO-*d*6, 500 MHz) *δ*H 1.83 (d, *J* = 13.6 Hz, 1H, H-1*α*), 1.95 (dd, *J* = 4.4, 13.6 Hz, 1H, H-1*β*); 1.59 (m, 1H, H-2*α*), 1.47 (m, 1H, H-2*β*); 1.20 (td, *J* = 3.2, 13.1 Hz, 1H, H-3*α*), 1.34 (d, *J* = 12.3 Hz, 1H, H-3*β*); 2.00 (d, *J* = 5.0 Hz, 1H, H-5); 5.59 (brs, 1H, H-6); 5.79 (brs, 1H, H-7); 4.88 (dt, *J* = 2.3, 12.6 Hz, 1H, H-12*α*), 4.78 (d, *J* = 12.6 Hz, 1H, H-12*β*); 1.06 (s, 3H, H-13); 0.92 (s, 3H, H-14); 1.07 (s, 3H, H-15); 5.94 (d, *J* = 15.3 Hz, 1H, H-2-); 7.20/7.23 (m, 1H, H-3-); 6.40/6.44 (m, 1H, H-4-); 6.30/6.34 (m, 1H, H-5-); 3.85/3.97 (m, 1H, H-6-); 3.49/3.56 (m, 1H, H-7-); 0.94/1.02 (d, *J* = 6.2 Hz, 3H, H-8-); 5.02 (brs, 1H, HO-6-); 4.61/4.66 (brs, 1H, HO-7-); 13C NMR (DMSO-*d*6,125 MHz) *δ*C 29.6 (CH2, C-1), 17.5 (CH2, C-2), 44.5 (CH2, C-3), 33.4 (C, C-4), 44.2 (CH, C-5), 65.8 (CH, C-6), 121.4 (CH, C-7), 136.6 (C, C-8), 73.2 (C, C-9), 37.3 (C, C-10), 174.4 (C, C-11), 68.3 (CH2, C-12), 18.3 (CH3, C-13), 32.2 (CH3, C-14), 24.4 (CH3, C-15), 165.51/165.50/165.49/165.47 (C, C-1-), 120.03/120.99/119.95/119.90 (CH, C-2-), 145.41/145.37/145.34/145.26 (CH, C-3-), 127.54/127.35/127.16/126.98 (CH, C-4-), 146.18/146.12/145.48/145.45 (CH, C-5-), 75.16/75.00/74.64/74.46 (CH, C-6-), 69.64/69.62/ 69.33/69.32 (CH, C-7-), and 19.34/19.26/18.26/18.24 (CH3, C-8-); ESIMS peak at *m*/*z* 419.1 for [M–H]– and *m*/*z* 464.9 for [M + HCO2]– (C23H32O7).

(2-*E*,4-*E*;6-,7--*erythro*)-Ustusolate I (**5a**): light yellow oil; [α]23D −35 (*c* 0.30, MeOH); UV (MeOH) λmax (log *ε*) 268 (4.15) nm; ECD (0.60 mM, MeOH) λmax (Δ*ε*) 255 (−8.3), 236 (−8.9), 208 (−21.2) nm; 1H and 13C NMR see Tables 3 and 4; ESIMS *m*/*z* 421.2 [M+H]+ (C23H32O7).

(2-*E*,4-*E*;*ent*-6-,7--*erythro*)-Ustusolate I (**5b**): light yellow oil; [α]23D −42 (*c* 0.30, MeOH); UV (MeOH) λmax (log *ε*) 261 (4.39) nm; ECD (0.60 mM, MeOH) λmax (Δ*ε*) 256 (−11.0), 234 (−8.9), 209 (−21.4) nm; 1H and 13C NMR see Tables 3 and 4; ESIMS *m*/*z* 421.2 [M+H]+ (C23H32O7).

(2-*E*,4-*E*,6-*R*,7-*R*)-Ustusolate I (**5c**): light yellow oil; [α]23D −105 (*c* 0.30, MeOH; UV (MeOH) λmax (log *ε*) 261 (4.42) nm; ECD (0.60 mM, MeOH) λmax (Δ*ε*) 256 (−8.6), 234 (−8.5), 208 (−22.9) nm; 1H and 13C NMR see Tables 3 and 4; ESIMS *m*/*z* 421.2 [M+H]+ (C23H32O7).

(2-*E*,4-*E*,6-*S*,7-*S*)-Ustusolate I (**5d**): light yellow oil; [α]23D −79 (*c* 0.29, MeO; UV (MeOH) λmax (log *ε*) 262 (4.36) nm; ECD (0.60 mM, MeOH) λmax (Δ*ε*) 259 (−10.4), 234 (−8.2), 208 (−18.9) nm; 1H and 13C NMR see Tables 3 and 4; ESIMS *m*/*z* 421.2 [M+H]+ (C23H32O7).

(2-*E*,4-*E*;6-,7--*erythro*)-Ustusolate I-6-,7--acetonide (**5e**): light yellow oil; [α]22D −102 (*c* 0.16, MeOH); UV (MeOH) λmax (log *ε*) 268 (4.15) nm; ECD (0.60 mM, MeOH) λmax (Δ*ε*) 256 (−15.1), 232 (−13.8), 210 (−39.4) nm; 1H and 13C NMR see Tables 3 and 4; ESIMS *m*/*z* 421.2 [M+H]+ (C26H36O7).

(5*S*,6*R*,9*S*,10*S*,2-*E*,4-*E*)-(Strobilactone A-6-yl)-5-carboxypenta-2,4-dienoate (ustusolate J, **6**): colorless solid; [α]20D −280 (*c* 0.65, MeOH); UV (MeOH) λmax (log *ε*) 265 (1.84) nm; ECD (0.64 mM, MeOH) λmax (Δ*ε*) 261 (−11.4), 232 (−8.9), 207 (−23.2) nm; IR (KBr) *ν*max 3400, 3320, 2950, 2928, 1658, 1615, 1460, 1385, 1290,1208, 1155, 1078, 970 cm<sup>−</sup>1; 1H and 13C NMR see Table 2; ESIMS *m*/*z* 459.5 [M–H]– (C21H26O7).

(2*S*,5*S*,9*R*,10*S*)-2,9,11-Trihydroxydrim-7-en-6-one (ustusol B, **7**): light yellow solid; [α]23D −140 (*c* 0.1, MeOH); UV (MeOH) λmax (log *ε*) 252 (1.33) nm; ECD (1.87 mM, MeOH) λmax (Δ*ε*) 335 (+11.9), 268 (−3.3), 241 (−66.9), 214 (−18.7) nm; IR (KBr) *ν*max 3400, 3320, 2950, 2928, 1658, 1615, 1460, 1385, 1290, 1208, 1155, 1078, 970 cm<sup>−</sup>1; 1H and 13C NMR see Table 1; ESIMS *m*/*z* 269.2 [M+H]+, 291.2 [M+Na]+ (C15H24O4).
