1
Supplementary Methods
<Isolation and characterization>
Between May and October, the highly alkaline (pH 8.5–10.5) red secretion was
collected by wiping a hippopotamus's face and back with gauze once a day. The wet
gauzes were immediately refrigerated using dry ice. Within a day, the chemicals were
extracted using water. The coloured solution (about 10-5 M) was purified through gel
filtration on Sephadex G-15 eluted with H2O to give a red-brown solution and an orange
solution. The concentration of the solutions were estimated from their UV-VIS
spectrums. The resulting red-brown solution was chromatographed on Sephadex G-25
eluted with H2O, giving a brown coloured solution and a red coloured solution. To
avoid polymerization of the pigments, Sephadex G-15 and G-25 were added to the
orange and red coloured solutions, respectively, and the mixture was lyophilized. These
samples could be kept in a freezer for several months. Further purification of samples
was carried out using an ion exchange resin after elution from the gels.
To obtain the mass spectra of the pigments, the gel-supported sample was eluted
with 0.2 M phosphate buffer (pH 6.1) and the resulting eluate was applied to an anion
exchange resin, QAE Sephadex A-25, at pH 6.1. After elution with 1.7 M NaCl (for the
red pigment) or 2.3 M NaCl (for the orange pigment) / 0.2 M phosphate buffer (pH 6.1),
the eluate solution was dialyzed (MW 10,000) against H2O. The desalted solution was
applied to FAB-MS. To confirm the results of the FAB-MS, we also measured the LCMS (ESI).
For the LC-MS spectra measurement of the orange pigment, we used the sample
obtained after gel filtration. For the LC-MS spectra measurement of the red pigment, we
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prepared a sample in the following way: (1) elution of the gel-supported pigment with
0.2 M triethylamine-formic acid buffer (pH 6.1), (2) ion exchange chromatography on
QAE Sephadex A-25 (elute: 1.7 M NaCl), and (3) dialyzation (MW 10,000) against
H2O. Preparation of the samples for NMR analyses was performed in the same manner
as for samples for the FAB-MS, but D2O was used instead of H2O. The 1H NMR
spectra of the eluates from the anion exchange resin were measured without desalting.
Efforts to measure the
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C NMR spectra of the pigments failed because the pigments
were unstable under the necessary conditions and we were unable to use a concentrated
solution of the pigments. The IR spectra were not obtained because the solution was
highly diluted. The UV-VIS spectra were measured using the recovered solution of the
1
H NMR measurement; the sample amount was estimated from the 1H NMR spectra
using 3-trimethylsilyl-1-propanesulphonic acid sodium salt (DSS) as an internal
standard.
Data from the red pigment 2:
UV-VIS (0.1 M NaCl / 0.2 M phosphate buffer, pH 6.1) max (log ): 530 nm (3.95),
411 nm (4.08), 270 nm (4.31, shoulder), 240 nm (4.72) (Supplementary Figure 1);
1
H NMR (1.7 M NaCl / 0.2 M deuterated phosphate buffer, pH 6.1, DSS = 0.00 ppm) 
3.34 (2H, s), 6.44 (1H, s), 6.55 and 6.63 (each 1H, AB q, J = 11.0 Hz);
FAB-HRMS found m/z 329.0299 [M+H]+, calculated for C16H9O8 329.0297;
LC-MS (ESI) (50% CH3CN-H2O containing 1% acetic acid) positive m/z 329 [M+H]+,
negative m/z 327 [M-H]-.
Data from the orange pigment 3: UV-VIS (0.2 M NaCl / 0.2 M phosphate buffer, pH
6.1) max (log ): 511 nm (3.95), 418 nm (4.16), 271 nm (4.29, shoulder), 243 nm (4.73)
(Supplementary Figure 2);
3
1
H NMR (2.3 M NaCl / 0.2 M deuterated phosphate buffer, pH 6.1, DSS = 0.00 ppm) 
3.33 (2H, s), 6.46 (1H, s), 6.57 and 6.62 (each 1H, AB q, J = 10.4 Hz), 7.05 (1H, s);
FAB-HRMS found m/z 285.0419 [M+H]+, calculated for C15H9O6 285.0399;
LC-MS (ESI) (50% CH3CN-H2O containing 1% acetic acid) positive m/z 285 [M+H]+,
negative m/z 283 [M-H]-.
Antibacterial activity: Antibacterial activities were measured by microbroth dilution
method in nutrient broth medium with an inoculum of 106 cfu/ml at 37 °C for 18 h. In
the preliminary test, the red pigment 2 clearly shows growth inhibitory activities against
Pseudomonas aeruginosa A3 and Klebsiella pneumoniae PC1602 at 12.5 g/ml and
24.9 g/ml, respectively. Detailed biological study of the antibacterial activities of the
pigments is now in progress.
Chemical conversion of the red pigment 2 to the stable compound 1
After the ion-exchange chromatography, sodium dithionite was added to the red eluate
until the red colour disappeared. After acidification with 6 M HCl to pH 2, the mixture
was extracted with EtOAc. This was evaporated but the extracts were not allowed to
become dry, because the reduced compound was still unstable. A solution of
diazomethane in Et2O was added to the extracts. The resulting solution was evaporated
until completely dry and then dissolved in CH2Cl2. To the solution were added 2,6lutidine and t-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) at room
temperature and the mixture was stirred for 0.5 h. After the usual work up, the product
was purified using silica gel TLC and then recrystallized from MeOH to yield
colourless crystals of 1 suitable for X-ray crystallographic analysis. The asymmetric
centre at the C-9 of 1 was racemic.
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Data from 1: colorless prism; Rf = 0.67 (hexane : EtOAc = 2 : 1); mp 127-129 ºC;
UV (MeOH) max (log ): 337 nm (3.94), 327 nm (3.93), 312 nm (3.81), 276 nm (4.12),
267 nm (4.06), 215 nm (4.63), 207 nm (4.65);
IR maxKBr in cm-1: 3446, 2856, 1745, 1735, 1635, 1492, 1472, 1465, 1448, 1433, 1401,
1263, 1147, 1075, 1025, 853, 841;
1
H NMR (400 MHz, C6D6, solvent residual peak = 7.15 ppm)  0.22, 0.25, 0.27, 0.28
(each 3H, each s, four Me of two TBS), 1.11, 1.14 (each 9H, each s, two t-Bu of two
TBS), 2.99 (3H, s, 5-OMe), 3.27 (3H, s, 9-CO2Me), 3.38 (3H, s, 1’-CO2Me), 3.68 and
3.90 (each 1H, AB q, J = 15.7 Hz, 2 X H-1’), 5.03 (1H, s, H-9), 6.20 (1H, d, J = 8.8 Hz,
H-6), 6.56 (1H, d, J = 8.8 Hz, H-7), 6.90 (1H, s, H-2), 9.82 (1H, s, 4-OH);
C NMR (100 MHz, C6D6, C6D6 = 128.00 ppm)  -4.05, -3.96, -3.82, -3.77 (four Me of
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two TBS), 18.56 (2 X CMe3), 25.99 (CMe3), 26.09 (CMe3), 36.33 (C-1’), 51.40 (C-9),
51.65 (1’-CO2Me), 51.68 (9-CO2Me), 56.31 (5-OMe), 112.34 (C-6), 117.99 (C-7),
122.25 (C-2), 123.38 (C-3), 128.58 (C-4a), 131.78 (C-1a,5a), 133.93 (C-8a), 144.96 (C1), 145.49 (C-4), 146.58 (C-5), 147.73 (C-8),169.93 (9-CO2Me), 171.76 (1’-CO2Me);
FAB-HRMS found m/z 602.2739 [M]+, calculated for C31H46O8Si2 602.2731. X-ray
crystal data: C31H46O8Si2, M = 602.87, orthorhombic, space group Pna21, a = 11.486(1)
Å, b = 33.999(3) Å, c = 8.726(2) Å, V = 3407.8(9) Å3, Z = 4, Dcalc = 1.175 g cm-3. 
(MoK) = 0.148 mm-1, crystal size = 0.3 X 0.3 X 0.05 mm3, 4877 reflections measured,
4395 unique reflections. Refinement was based on F2 with Rw = [w(Fo2 Fc2)2/w(Fo2)2]1/2, w-1 = 2(Fo2) + (0.0385P)2 + 0.4258P, where P = (Fo2+2Fc2)/3 against
all the 4395 reflections. The R value, |Fo2-Fc2|/Fo2, was 0.040 for the 2367 reflections
with I > 2(I). The Rw value was 0.103. The absolute structure was confirmed by Flack
parameter, x = -0.04(16).
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<Preparation of 4>
The model compound 4 is also unstable in a concentrated solution. Therefore, it was
prepared from the corresponding tetrahydrodiquinone (preparation of which will be
described elsewhere) by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation
in CDCl3. The by-product, dihydroDDQ, was removed by filtration through silica gel.
Data from 4: 1H NMR (300 MHz, CDCl3, TMS=0.00 ppm)  6.67 (2H, d, J=10.2 Hz),
6.94 (2H, d, J=10.2 Hz), 7.38 (1H, s), 16.05 (1H, s);
UV-VIS (CHCl3) max nm (relative absorbance): 538 (1.00), 319 (2.84), 243 (7.22);
UV-VIS (CHCl3+Et3N) max nm (relative absorbance): 522 (1.00), 412 (1.08), 339
(1.85);
UV-VIS (MeOH) max nm (relative absorbance): 520 (1.00), 416 (1.23), 333 (2.43), 230
(8.59);
UV-VIS (50% MeOH-H2O) max nm (relative absorbance): 518 (1.00), 410 (1.60).