Pigments from the Puffball Calvatia rubro-flava
FULL PAPER
Jouan-Roussel at 20 °C. Ϫ IR: Pye Unicam SP 1100 and
PerkinϪElmer 1420. Ϫ NMR: Bruker AC 200 and WM 400, solv-
ent peak or TMS as internal standard. Ϫ MS: A.E.I. MS 30 and
(‘‘t’’, 3J ϭ 7.3 Hz, C-4), 148.9 (m), 154.0 (m), 166.4 (s, CONH2),
180.8 (s, C-1). Ϫ 13C NMR (100.6 MHz, [D6]DMSO): δ ϭ 14.7 (Q,
1J ϭ 140 Hz, CH3S), 43.8 (Q, 1J ϭ 141 Hz, CH3SO), 107.2 (Dd,
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MS 50 with data system DS 50. EI MS spectra were obtained at 1J ϭ 166, J ϭ 4.5 Hz, C-6), 112.7 (Dd, 1J ϭ 164, J ϭ 4.0 Hz, C-
70 eV with direct inlet. Ϫ TLC: Merck, silica gel 60 PF, solvent
systems (v/v): A: CHCl3/MeOH, 2:1; B: CHCl3/MeOH, 10:1; C:
nBuOH/AcOH/H2O, 4:1:1; D: iPrOH/HCO2H/H2O, 20:1:5. Ϫ Col-
2), 135.4 (t, 3J ϭ 7.0 Hz, C-4), 145.3 (m, C-5), 148.0 (m, C-3), 162.4
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(t, J ϭ 2.0 Hz, C-1), 162.6 (s, CONH2). Ϫ (ϩ)-FAB MS (glycerol
ϩ NaCl): m/z ϭ 296 [Mϩ ϩ Na], 318 [Mϩ Ϫ H ϩ 2Na]. Ϫ EI MS:
umn chromatography: Silica gel 60 (40Ϫ63 µm, Merck) and Lobar m/z (%) ϭ 217 (C8H11NO2S2, 1.3), 202.0125 (calcd. for C8H10O2S2:
RP-8 (size B, Merck). Ϫ Gel chromatography: Sephadex LH-20 202.0122, 25), 187 (C7H7O2S2, 24), 186 (C8H10OS2, 39), 156
(Pharmacia). Ϫ Analytical HPLC: Waters M 721, packing material (C7H8O2S, 8), 153 (C8H9OS, 42), 139 (C7H7OS, 8), 107 (C7H7O,
Lichrosorb RP-8, 7 µm, flow rate 1 mL/min. Eluent A: H2O/
9), 94 (C2H6S2, 21), 79 (CH3S2, 6), 63 (C2H7S, 8), 60 (CH4N2O,
MeOH, 9:1; eluent B: H2O/MeOH, 1:9. Linear gradient: 5 min: B 27), 47 (CH3S, 10), 44 (CONH2, 38), 43 (HNCO, 100), 42 (NCO,
50%, within 10 min to B 100%, then 15 min B 100%. UV/Vis detec- 19). Ϫ C9H11N3O3S2 ϫ H2O (273.3 ϩ 18): calcd. C 37.11, H 4.49,
tion at 245 and 435 nm. Waters 600 E Pump and System Controller N 14.42, O 22.0, S 22.0; found C 37.22, H 4.24, N 14.21, O 19.4,
with Photodiode Array Detector 990ϩ. All solvents were distilled
before use. The anhydrous solution of TBHP in toluene was pur-
chased from Fluka. Ϫ The elemental analyses were carried out by
Fa. Pascher, Bonn, and the Microanalytical Laboratory at the Uni-
versity of Bonn. Ϫ C. rubro-flava was collected in September 1983
in a meadow in the Marshall Forest, Great Smoky Mountains,
Georgia, USA (leg. et det. Dr. R. Baird).
S 19.7. Ϫ Sodium salt: C9H11N3O3S2Na (295.3): calcd. C 36.61, H
3.41, N 14.23; found C 36.58, H 3.89, N 13.95.
Oxyrubroflavin (2) (1:1 Mixture of Diastereomers): Amorphous, or-
ange-red solid, dec. at 200Ϫ210 °C. Ϫ [α]2D2 ϭ Ϫ860 (c ϭ 0.1,
MeOH). Ϫ Rf (TLC) ϭ 0.24 (system A), lemon-yellow spot. Ϫ UV
(MeOH): λmax (lg ε) ϭ 197 nm (4.19), 245 (3.98), 280 (sh, 3.88),
352 (4.01), 430 (3.18), 455 (sh, 3.12). Ϫ CD (MeOH): λmax (∆ε) ϭ
Isolation of Pigments 1, 2, 8, and Leucorubroflavin (4) from C. ru-
bro-flava: Air-dried fruit bodies of C. rubro-flava (13.2 g) were cut
and extracted exhaustively with MeOH. The extract was concen-
208 nm (ϩ8.27), 238 (Ϫ1.41), 269 (ϩ1.01), 316 (Ϫ0.63), 355
Ϫ1
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(ϩ0.46), 393 (ϩ0.43), 463 (Ϫ5.00). Ϫ IR (KBr): ν ϭ 3340 cm
(sh, m), 3040 (br., s), 2800 (m), 2695 (w), 1710 (sh, ss), 1690 (ss),
trated and the residue filtered through a short silica gel column 1587 (s), 1550 (s), 1475 (ss), 1420 (m), 1370 (ss), 1275 (s), 1240 (s),
(CHCl3/MeOH, 2:1). Column chromatography of the solution on 1215 (ss), 1175 (m), 1125 (w), 1060 (s), 1040 (ss), 1010 (s), 960 (s),
Sephadex LH 20 with MeOH yielded three colored zones. A bright 940 (m), 890 (m), 850 (m), 750(w), 720 (w), 675 (m), 645 (w). Ϫ 1H
yellow initial fraction contained oxyrubroflavin (2), leucorubrofla- NMR (400 MHz, CD3OD): δ ϭ 2.84 (s, 3 H), 2.89 (s, 3 H), 7.20
vin (4), and some rubroflavin (1), the orange-yellow main fraction (s, 1 H), 7.21 (s, 1 H). Ϫ 1H NMR (400 MHz, [D6]DMSO): δ ϭ
contained only 1. The last, orange-colored fraction consisted of a
mixture of 1 and deoxyrubroflavin (8). Preparative TLC was used
to separate 1, 2, and 4 on silica gel (system A). Compound 4 ap-
peared as narrow colorless zone (fluorescence quenching at
2.68 (s, 3 H), 2.76 (s, 3 H), 6.89 (s, 1 H), 6.92 (s, 1 H), 7.10Ϫ7.40
(br., NH and NH2). Ϫ 13C NMR (100.6 MHz, CD3OD): δ ϭ 44.7
(Q), 45.1 (Q), 118.4 (D), 118.5 (D) (signals of quat. C atoms not
visible). Ϫ 13C NMR (100.6 MHz, [D6]DMSO): δ ϭ 43.1 (Q, 1J ϭ
254 nm) below the main pigment, and was characterized without 141 Hz,), 43.8 (Q, 1J ϭ 140 Hz), 112.6 (Dd, 1J ϭ 166, 3J ϭ 5.0 Hz,
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further purification. The lemon-yellow minor pigment 2 showed
the lowest Rf value and was purified by repeated preparative TLC
on silica gel (system A) followed by column chromatography on
Sephadex LH-20 (eluent MeOH). Pigment 1 exhibited an interme-
2 C), 134.2 (t, J ϭ 6.5 Hz), 149.1 (m), 149.2 (m), 161.9 (s), 163.6
(t, 3J ϭ 2.0 Hz). Ϫ EI MS: m/z (%) ϭ 234 (C8H10O4S2, 7), 233
(C8H11NO3S2, 3), 218 (C8H10O3S2, 27), 203 (C7H7O3S2, 21), 202
(C8H10O2S2, 15), 201 (C8H9O2S2, 6), 187.99 (C7H8O2S2, 5), 187.96
diate Rf value and was obtained analytically pure by chromato- (C6H4O3S2, 23), 187 (C7H7O2S2, 18), 182 (C8H6OS2, 3), 173
graphy on Sephadex LH-20 (2 ϫ, eluent MeOH), followed by chro-
(C6H5O2S2, 18), 172 (C7H8O3S, 12), 156 (4), 155 (C7H7O2S, 7), 94
matography on a Lobar RP-8 column (eluent MeOH/H2O, 1:1).
(28), 85 (10), 63 (17), 45 (30), 44 (42), 43 (HNCO, 100). Ϫ
Yields: 90 mg of 1 (0.75% of dry-weight), 5 mg of 2 (0.04%), 5 mg C9H11N3O4S2 ϫ 0.5 H2O (298.3): calcd. C 36.20, H 4.02, N 14.08;
of 4 (0.04%), 2 mg of 8 (0.015%). found C 36.48, H 4.08, N 14.14.
Rubroflavin (1): Red needles, monohydrate. Ϫ M.p. 184Ϫ185 °C Deoxyrubroflavin (8): Red powder, m.p. 168Ϫ169 °C. Ϫ Rf
(dec.). Ϫ [α]2D2 ϭ Ϫ2180 (c ϭ 0.07, MeOH). Ϫ Rf (TLC) ϭ 0.57
(TLC) ϭ 0.77 (system A), 0.66 (system B), 0.38 (EtOAc), yellow
(system A); 0.33 (system B); 0.66 (system C); 0.73 (system D), yolk- spot. Ϫ UV (MeOH): λmax (lg ε) ϭ 212 nm (4.06), 232 (4.18), 253
˜
(4.16), 278 (4.05), 310 (sh, 3.58), 387 (3.97). Ϫ IR (KBr): ν ϭ 3460
yellow spot. Ϫ Analytical HPLC: tR ϭ 3.9 min. Ϫ UV (MeOH):
λmax (lg ε) ϭ 214 nm (4.01), 243 (3.92), 252 (3.89), 275 (3.65), 295 cmϪ1 (s), 3320 (m), 3080 (br., m), 2920 (w), 2790 (w), 1700 (ss),
(3.61), 435 (4.03), 460 (sh, 3.97). Ϫ CD (MeOH): λmax (∆ε) ϭ 221 1670 (sh, s), 1580 (s), 1545 (ss), 1450 (m), 1430 (s), 1350 (m), 1330
(Ϫ21.65), 247 (ϩ15.33), 265 (ϩ7.72), 297 (Ϫ0.86), 325 (ϩ4.61), 385 (s), 1230 (br., ss), 1205 (sh, ss), 1120 (m), 970 (m), 940 (m), 855
˜
(Ϫ5.47), 425 (ϩ1.07), 478 (Ϫ19.51) (Figure 1). Ϫ IR (KBr): ν ϭ
(m), 840 (s), 800 (m), 770, 720, 665, 635 (all w). Ϫ 1H NMR
3440 cmϪ1 (s), 3250 (br., s), 2920 (m), 2850 (w), 1700 (s), 1670 (s), (400 MHz, CDCl3): δ ϭ 2.32 (s, 3 H), 2.55 (s, 3 H), 4.80Ϫ5.20 (br.,
1570 (ss), 1465 (m), 1430 (m), 1360 (w), 1285 (sh, ss), 1230 (ss), 2 H),* 6.15 (d, 4J ϭ 1.8 Hz, 1 H), 6.22 (d, 4J ϭ 1.8 Hz, 1 H), 10.41
1185 (sh, ss), 1070 (s), 1055 (s), 1010 (m), 970 (m), 930 (sh, w), 845 (br. s, 1 H),* *exchangeable with D2O. Ϫ 1H NMR (400 MHz,
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(s), 775 (w), 675 (w). Ϫ H NMR (400 MHz, CD3OD): δ ϭ 2.41
CD3OD): δ ϭ 2.39 (s, 6 H), 6.55 (s, 2 H). Ϫ 13C NMR (100.6 MHz,
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(br. s, 3 H), 2.84 (s, 3 H), 6.44 (dq, J ϭ 2.3, J ϭ 0.25 Hz, 1 H), CD3OD): δ ϭ 15.6 (Q, J ϭ 138 Hz, 2 C), 109.9 (D, J ϭ 164 Hz,
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7.04 (d, J ϭ 2.3 Hz, 1 H). Ϫ H NMR (400 MHz, [D6]DMSO):
2 C), 137.1 (t, 3J ϭ 7.0 Hz, 1 C), 147.9 (m, 2 C), 166.1 (s, 1 C),
δ ϭ 2.25 (br. s, 3 H), 2.64 (s, 3 H), 5.97 (d, 4J ϭ 2.3 Hz, 1 H), 6.42 180.7 (t, 2J ϭ 3.0 Hz, 1 C). Ϫ 13C NMR (100.6 MHz, [D6]DMSO):
(br., 1 H),* 6.53 (br., 2 H),* 6.57 (d, 4J ϭ 2.3 Hz, 1 H), *exchange-
δ ϭ 14.9, 107.3, 136.1, 143.5, 161.1, 163.7. Ϫ EI MS: m/z (%) ϭ
able with D2O. Ϫ 13C NMR (100.6 MHz, CD3OD): δ ϭ 15.0 (Q, 257 (15) [Mϩ], 242 (C8H8N3O2S2, 7), 240 (C9H8N2O2S2, 5), 213
1J ϭ 138.5 Hz, CH3S), 44.7 (Q, J ϭ 140 Hz, CH3SO), 115.4 (Dd, (C8H9N2OS2, 8), 212 (5), 211 (11), 210 (C8H8N3O2S, 100), 201
1
1J ϭ 165, J ϭ 4.6 Hz), 116.9 (Dd, J ϭ 160, J ϭ 4.2 Hz), 132.2 (C8H11NOS2, 10), 200 (C7H8N2OS2, 4), 198 (C8H8NOS2, 11), 197
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Eur. J. Org. Chem. 2001, 3097Ϫ3104
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