*3.3. Extraction and Isolation*

The freeze-dried and ground tunicate *C. stolonifera* (19 g) was extracted exhaustively with hexane (250 mL), DCM (250 mL) and MeOH (2 × 250 mL), respectively. The DCM and MeOH extracts were combined and the solvents were evaporated to yield a yellow residue (2.2 g). This extract was pre-adsorbed onto C18 (1 g) and packed dry into a small cartridge, which was connected to a C18 preparative HPLC column (5 μm, 21.2 × 150 mm). A linear gradient from 100% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) was performed over 60 min at a flow rate of 9 mL/min and 60 fractions (1.0 min each) were collected. Pure cnemidine A (**5**, 3.5 mg, 0.018% dry wt) and 11-hydroxyascididemin (**4**, 4.1 mg, 0.021% dry wt) were obtained in fractions 28, 29 and 32 respectively. Fraction 19 was further chromatographed on a Hypersil C18 HPLC column (5 μm, 10 × 250 mm) from 10% MeOH (0.1% TFA)/90% H2O (0.1% TFA) to 25% MeOH (0.1% TFA)/75% H2O (0.1% TFA) at a flow rate of 4 mL/min in 30 min to yield stolonine C (**3**, 0.1 mg, 0.0021% dry wt) in fraction 14 and stolonine A (**1**, 0.9 mg, 0.0047% dry wt) in fractions 17–18. Fractions 20 and 21 in the first chromatography were combined and purified using the Hypersil C18 HPLC column (5 μm, 10 × 250 mm) from 10% MeOH (0.1% TFA)/90% H2O (0.1% TFA) to 40% MeOH (0.1% TFA)/60% H2O (0.1% TFA) at a flow rate of 4 mL/min in 45 min to obtain stolonine C (**3**, 0.3 mg, 0.0021% dry wt in total) in fraction 33 and stolonine B (**2**, 1.2 mg, 0.0063% dry wt) in fractions 34–36.

Stolonine A (**1**): white, amorphous solid; UV (MeOH) λmax (log ε) 210 (3.8), 252 (3.5) and 325 (3.3) nm; IR (film) νmax 3307, 1681, 1205, 1049 and 802 cm−1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 1; (−)-HRESIMS *m*/*z* 295.0390 [M − H]<sup>−</sup> (calcd for [C12H11N2O5S]−, 295.0394, Δ −1.4 ppm).

Stolonine B (**2**): white, amorphous solid; UV (MeOH) λmax (log ε) 210 (3.8) and 240 (3.9) nm; IR (film) νmax 3386, 1684, 1204 and 1066 cm−1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 3; (−)-HRESIMS *m*/*z* 279.0441 [M − H]<sup>−</sup> (calcd for [C12H11N2O4S]−, 279.0445, Δ −1.4 ppm).

Stolonine C (**3**): white, amorphous solid; UV (MeOH) λmax (log ε) 210 (4.1) and 272 (3.8) nm; IR (film) νmax 3418, 1682, 1197 and 1034 cm−1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 4; (−)-HRESIMS *m*/*z* 318.0550 [M − H]<sup>−</sup> (calcd for [C14H12N3O4S]−, 318.0554, Δ −1.3 ppm).

Synthetic stolonine A (**1**): To a solution of taurine (12 mg, 0.096 mmol) in dry DMF (1 mL) was added HOBt (26 mg, 0.193 mmol) and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then cooled to 0 ◦C, EDCI (37 mg, 0.193 mmol) was added and continued stirring for 30 min at 0 ◦C. To this mixture was then added 3-indoleglyoxylic acid (**6**, 18 mg, 0.095 mmol) and the mixture was stirred for 21 h at rt. The crude product was concentrated *in vacuo* and separated by RP-HPLC (MeOH, H2O, 0.1% TFA) to obtain the synthetic stolonine A (18 mg, 64%); IR (film) νmax 3371, 1681, 1205, 1047, 802 and 749 cm<sup>−</sup>1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 5; (−)-HRESIMS *m*/*z* 295.0391 [M − H]<sup>−</sup> (calcd for [C12H11N2O5S]−, 295.0394, Δ −1.0 ppm).

Quinoline-2-carboxylic acid (**8**): To a solution of *o*-nitrobenzaldehyde (**7**, 302 mg, 2 mmol) in ethanol (5 mL) was added iron powder (504 mg, 9 mmol), followed by 0.1 N HCl (2 mL, 0.2 mmol) and the resulting mixture was vigorously stirred under reflux for 45 min. Methyl pyruvate (200 μL, 2 mmol) and powder KOH (135 mg, 2.4 mmol) were added slowly. The reaction mixture was stirred under reflux for 90 min and then cooled to rt. The crude product was purified by RP-HPLC (MeOH, H2O, 0.1% TFA) to yield **8** (190 mg, 55% in two steps); 1H NMR (500 MHz, DMSO-*d6*) δ 8.56 (1H, d, *J* = 8.5 Hz), 8.16 (1H, d, *J* = 8.0 Hz), 8.11 (1H, d, *J* = 8.5 Hz), 8.09 (1H, d, *J* = 8.0 Hz), 7.88 (1H, t, *J* = 8.0 and 7.5 Hz) and 7.74 (1H, t, *J* = 8.0 and 7.5 Hz); 13C NMR (125 MHz, DMSO-*d6*) δ 166.2 (COOH), 148.5 (C), 146.6 (C), 137.6 (CH), 130.5 (CH), 129.5 (CH), 128.7 (C), 128.5 (CH), 127.9 (CH) and 120.6 (CH). (+)-LRESIMS *m*/*z* 174.

Synthetic stolonine B (**2**): To a solution of taurine (13 mg, 0.104 mmol) in dry DMF (1 mL) was added HOBt (28 mg, 0.207 mmol) and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then cooled to 0 ◦C, EDCI (40 mg, 0.209 mmol) was added and continued stirring for 30 min at 0 ◦C. To this mixture was then added compound **8** (18 mg, 0.104 mmol) and the mixture was stirred for 48 h at rt. The pure synthetic stolonine B was purified by RP-HPLC (MeOH, H2O, 0.1% TFA) to obtain 21.8 mg (75% yield); IR (film) νmax 3383, 1677, 1201 and 1064 cm<sup>−</sup>1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 5; (−)-HRESIMS *m/z* 279.0446 [M − H]<sup>−</sup> (calcd for [C12H11N2O4S]−, 279.0445, Δ 0.4 ppm).

β-carboline-3-carboxylic acid (**10**): Formaldehyde (37%, 2 mL) was added to L-tryptophan methyl ester hydrochloride (**9**) (2.54 g, 10 mmol) in aqueous MeOH (10 mL, MeOH-H2O (10:1)). The resulting mixture was stirred at rt for 16 h. The reaction mixture was evaporated to dryness *in vacuo* and oxidized by activated MnO2 (2.5 g, 28.7 mmol) in benzene under reflux for 5 h. The hot solution was filtered through a bed of C18 to remove the MnO2 and the filter cake was washed with hot benzene. The crude product was suspended in a mixture of aq. NaOH 20% and MeOH (ratio 1:4) and heated to reflux for 45 min. The reaction was cooled to rt, evaporated to dryness *in vacuo* and then loaded onto RP-HPLC (MeOH, H2O, 0.1% TFA) yielding **10** (215 mg, 10% in three steps). 1H NMR (600 MHz, DMSO-*d6*) δ 12.07 (1H, s), 8.98 (1H, s), 8.92 (1H, s), 8.39 (1H, d, *J* = 8.4 Hz), 7.67 (1H, d, *J* = 8.4 Hz), 7.60 (1H, t, *J* = 7.2, 7.8 Hz), 7.31 (1H, t, *J* = 7.2, 7.8 Hz); 13C NMR (150 MHz, DMSO-*d6*) δ 166.8 (COOH), 141.1 (C), 137.4 (2xC), 133.1 (CH), 128.7 (CH), 127.8 (C), 122.2 (CH), 120.9 (C), 120.1 (CH), 117.2 (CH), 112.3 (CH). (+)-LRESIMS *m*/*z* 213.

Synthetic stolonine C (**3**): To a solution of taurine (5 mg, 0.040 mmol) in dry DMF (1 mL) was added HOBt (13.5 mg, 0.1 mmol) and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then cooled to 0 ◦C, EDCI (19 mg, 0.099 mmol) was added and continued stirring for 30 min at 0 ◦C. To this mixture was then added **10** (8 mg, 0.038 mmol) and the mixture was stirred for 16 h at rt. The crude product was concentrated *in vacuo* and separated by RP-HPLC (MeOH, H2O, 0.1% TFA) to obtain the synthetic stolonine C (4.8 mg, 40%); IR (film) νmax 3419, 1683, 1198 and 1034 cm−1; 1H (600 MHz, DMSO-*d6*) and 13C (150 MHz, DMSO-*d6*) NMR data are summarized in Table 5; (−)-HRESIMS *m*/*z* 318.0556 [M − H]<sup>−</sup> (calcd for [C14H12N3O4S]−, 318.0554, Δ 0.6 ppm).
