Supporting Information

© Wiley-VCH 2007

69451 Weinheim, Germany

S1

Chemical Defense of the Crust Fungus Aleurodiscus amorphus by a Tailor-Made Cyanogenic Cyanohydrin Ether

Bernhard L. J. Kindler and Peter Spiteller* Table of Contents

1) Experimental Section

1.1) General Experimental Procedures

1.2) Mushrooms

1.3) Mycelial Cultures and Cultivation Conditions

1.4) Detection and Derivatisation of Hydrocyanic Acid

1.5) HPLC Profiling

1.6) Isolation of Aleurodisconitrile (1) and Aleurodiscoester (2)

1.7) Synthesis of Aleurodiscoester (2)

1.8) Synthesis of the Model Compounds 3a, 3b, 3c and 3d

1.9) General Procedure for the Oxidation of the Model Compounds 3a, 3b, 3c or 3d with

Activated MnO2 in Aqueous MeOH

1.10) General Procedure for the Oxidation of the Model Compounds 3a, 3b, 3c or 3d with

Activated MnO2 in MeOH

1.11) Detection of the E and Z Isomer of the Quinonemethide 6d

1.12) Enzymatic Conversion of 1 to 2 with Mushroom Tyrosinase

2) Spectra

2.1) Detection of Hydrocyanic acid by GC-MS

2.2) UV, CD, NMR and Mass Spectra of Aleurodisconitrile (1)

2.3) UV, CD, NMR and Mass Spectra of Aleurodiscoester (2)

2.4) NMR and Mass Spectra of 9 and of Synthetic 2

2.5) NMR and Mass Spectra of 10a−10d and of the Model Compounds 3a−3d

2.6) Gas Chromatograms and Mass Spectra of the Oxidation Experiments with 3a−3d and

Activated MnO2 in Aqueous MeOH

2.7) Gas Chromatograms and Mass Spectra of the Oxidation Experiments with 3a−3d and

Activated MnO2 in MeOH

2.8) NMR Spectra of a mixture of 3d and the E and Z Isomer of the Quinonemethide 6d

2.9) HPLC Chromatogram of the Enzymatic Conversion of 1 in 2 with Mushroom

Tyrosinase

S2

1) Experimental Section

1.1) General Experimental Procedures: Evaporation of the solvents was performed

under reduced pressure using a rotary evaporator. Preparative HPLC separations were

performed using two Waters 590EF pumps equipped with an automated gradient controller

680 and a Kratos Spectroflow 783 UV-Vis detector. The samples were separated on a Luna

C-18 (2) column (5 µm, 15 × 250 mm, Phenomenex) using the following gradient program:

10 min at 99.9 % H2O/0.1 % AcOH, then within 40 min linear to 100 % MeOH; flow rate: 12

mL min-1; detection: UV at 250 nm. Analytical HPLC separations were performed using two

Waters 510 pumps equipped with a Waters 717 autosampler and a Waters 996 photodiode

array detector running under the software package Waters Millenium 2.10. The samples were

separated on a Luna C-18 (2) column (5 µm, 4.6 × 250 mm, Phenomenex) using the following

gradient program: 10 min at 99.9 % H2O/0.1 % AcOH, then within 40 min linear to 100 %

MeOH; flow rate: 1.0 mL min-1; detection: UV at 250 nm. UV spectra were recorded on a

Varian Cary 100 Bio UV-Vis spectrometer. Optical rotation values were measured with a

Jasco P-1030 polarimeter. CD spectra were obtained with a Jasco J-715 spectropolarimeter.

NMR spectra were recorded with a Bruker DMX 600 spectrometer equipped with a TXI cryo

probe (1H at 600.13, 13C at 150.9 MHz) and a Bruker DMX 500 spectrometer (1H at 500.11,

13C at 125.8 MHz). Chemical shifts were determined relative to the solvent CDCl3 (δΗ 7.26,

δC 77.00), CD3OD (δΗ

3.31, δC 49.00), and CD3CN (δΗ

1.93, δC 1.30) as internal standard.

EIMS spectra were obtained with a ThermoElectron DSQ instrument equipped with direct

insertion probe using EI at 70 eV. GC-EIMS spectra were recorded with a ThermoElectron

Trace DSQ coupled with a ThermoElectron Trace GC ultra equipped with a PTV injector. For

sample separation, a fused silica DB-5ms capillary column (15 m × 0.25 mm, coated with a

0.25 µm layer of liquid phase) and helium as carrier gas was used. Injection volumes were

0.2 − 0.5 µl of a 1 − 2 % (m/v) solution of pertrimethylsilylated samples. For

S3

pertrimethylsilylation N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was used.

Temperature program: 1 min isothermal at 50 °C, then 5 K/min up to 300 °C, finally 10 min

isothermal at 300 °C. Retention indices Ri according to Kováts were determined by injection

of a 0.2 µl sample of a standard mixture of saturated straight chain alkanes (C10 – C36).

MALDI-TOF spectra were recorded on a Bruker Daltonics Ultraflex TOF/TOF in the

reflector mode with (E)-2-cyano-3-(4-hydroxyphenyl)acrylic acid as matrix. HR-EIMS

spectra were obtained with a Finngian MAT 8200 instrument. HR-APCIMS and HR-

APCIMS/MS spectra were obtained with an LTQ-Orbitrap Spectrometer (Thermo Scientific).

The spectrometer was operated in positive mode (1 spectrum s-1; mass range: 50−1000) with

nominal mass resolving power of 60 000 at m/z = 400 with a scan rate of 1 Hz) with

automatic gain control to provide high-accuracy mass measurements within 2 ppm deviation

using polydimethylcyclosiloxane ([(CH3)2SiO]6, m/z = 445.120025) as internal lock mass.

The spectrometer was equipped with a Dionex HPLC system Ultimate 3000 consisting of

pump, UV detector � ���������, Flow Manager and autosampler (injection volume 0.5 µL).

Nitrogen was used as sheath gas (6 arbitrary units) and helium served as the collision gas. The

separations were performed with a Gemini C-18 column (Phenomenex, 3 µ, 0.3 × 150 mm)

using the following gradient program: 4 min at 99.9 % H2O/0.1 % HCO2H, then within 12

min linear to 49.45 % H2O/49.45 % CH3CN/0.1 % HCO2H, then linear over 2 min to 99.9 %

CH3CN/0.1 % HCO2H, flow rate: 4 µL min-1.

1.2) Mushrooms: Fruiting bodies of A. amorphus (leg. et det. B. L. J. Kindler and P.

Spiteller) were collected in April and May 2005, 2006 and 2007 on dead Abies alba trees at

Taubenberg near Oberwarngau (Bavaria) and at Gröbern near Schrobenhausen (Bavaria).

Voucher samples of A. amorphus are deposited at the Institut für Organische Chemie und

Biochemie II der Technischen Universität München, Germany. The mushrooms were frozen

and stored at −30 °C after collecting.

S4

1.3) Mycelial Cultures and Cultivation Conditions: For cultivation of A. amorphus

(CBS 109200, Centraalbureau voor Schimmelcultures, Amsterdam), 40 petri dishes (90 mm

diameter), each of them containing 25 g solid MEA medium, consisting of malt extract (30 g),

peptone (5.0 g), agar (15 g) and H2O (1000 mL), were inoculated with mycelia of A.

amorphus (CBS 109200) and incubated at 20 °C. Typically, the mycelia covered the whole

agar plate within four weeks.

1.4) Detection and Derivatisation of Hydrocyanic Acid: Fresh fruiting bodies of A.

amorphus (10 mg) were suspended in phosphate buffer (1 mL, 50 mM, pH 7.0),

homogenized, transferred into an Eppendorf tube and incubated at 25 °C for 5 min. Methylene

chloride (500 µL), pentafluorobenzyl bromide (3 µL) and polymer bound tributyl-

phosphonium bromide (5 mg) were added and the test tube was shaken vigorously for 30 min

at 25 °C. Then the organic and the water phase were separated by centrifugation, the organic

phase was removed and dried with anhydrous sodium sulfate. The organic phase was

concentrated to 10 µL under a nitrogen stream and 2 µL thereof were injected into the GC-

MS. In order to quantify the amount of HCN present in the fruiting bodies, a solution of HCN

(10 µg mL-1) in phosphate buffer (1 mL) was derivatized analogously and the areas of the ion

currents at m/z = 207 were compared to each other yielding 220 µg HCN per gram of the

fruiting bodies. The yields of HCN differ considerably from batch to batch depending on the

age and the condition of the fungi.

Pentafluorobenzyl cyanide: GC-MS: Ri = 1082, m/z (%) = 207 (100) [M]+, 206 (24), 188

(41), 181 (50), 180 (16), 179 (11), 161 (25), 157 (52).

1.5) HPLC Profiling: For comparison of the metabolite pattern of intact fruiting bodies

with that of mechanically injured fruiting bodies, methanol extracts both of intact and of

injured fruiting bodies were injected into the HPLC. According to the UV absorption at λ =

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250 nm in the HPLC, the extract of the intact fruiting bodies contained significantly more of

aleurodisconitrile (1) and only low amounts of aleurodiscoester (2), while the extract of the

injured fruiting bodies contained predominantly 2 and only low amounts of 1. If intact

fruiting bodies were suspended in methanol and then homogenized, the extract still contained

predominantly aleurodisconitrile (1) and only low amounts of aleurodiscoester (2), since

addition of methanol to the fruiting bodies denatures the enzyme(s) responsible for the

conversion of 1 to 2 after injury of the fruiting bodies. In order to compare the metabolite

pattern of intact fruiting bodies with that of mycelial cultures, methanol extracts both of intact

fruiting bodies and of mycelial cultures were injected into the HPLC. At λ = 250 nm, only the

extract of the fruiting bodies showed significant absorptions indicating the absence of 1 and 2

in mycelial cultures of A. amorphus (1: Rt = 28.2 min, 2: Rt = 30.4 min).

1.6) Isolation of Aleurodisconitrile (1) and Aleurodiscoester (2): Fresh fruiting bodies

(737 mg) were extracted with MeOH (1 × 100 mL) at 25 °C under stirring for 3 h until

complete decolorization of the fruiting bodies was observed. The slightly yellow extract was

then concentrated in vacuum at 40 °C. The resulting residue was dissolved in 1 mL of an 1:1

mixture of H2O and MeOH, prepurified with an RP-18 cartridge and separated by preparative

HPLC (for separation conditions see “General Experimental Procedures”). 737 mg of the

frozen fruiting bodies yielded 2.9 mg (0.39 %) of aleurodisconitrile (1) and 1.2 mg (0.16 %)

of aleurodiscoester (2). Yields of 1 and 2 can differ considerably from batch to batch

depending on the age and the condition of the fungi.

Aleurodisconitrile (1): Colorless solid; HPLCprep:

Rt = 19.4 min; 25][ Dα = +11 cm3 g-1 dm-1 (c = 0.00137 g

cm-3 in MeOH); CD (MeOH): λ (∆ε) = 210 nm (+1.8);

1H NMR (600 MHz, CD3OD, 330 K): = 2.07−2.13

1

HO

H3CO

OH

O

CN

CO2H

NH2

141’

6’’’

4’’’

1’’

S6

(m, 1H, H-3), 2.24−2.29 (m, 1H, H-3), 3.66 (dd, 1H, J = 7.0, 4.9 Hz, H-2), 3.74−3.78 (m, 1H,

H-4), 3.85−3.89 (m, 1H, H-4), 3.88 (s, 3H, 5’’-OCH3), 5.34 (s, 1H, H-1´), 6.67 (d, 1H, J = 1.8

Hz, H-6´´´), 6.68 ppm (d, 1H, J = 1.8 Hz, H-2´´´); 1H NMR (600 MHz, CD3OD, 298 K): =

2.07−2.12 (m, 1H, H-3), 2.23−2.28 (m, 1H, H-3), 3.66 (dd, 1H, J = 7.0, 4.9 Hz, H-2),

3.71−3.76 (m, 1H, H-4), 3.83−3.89 (m, 1H, H-4), 3.87 (s, 3H, 5’’-OCH3), 5.35 (s, 1H, H-1´),

6.67 ppm (m, 2H, H-2´´´/H-6´´´); 13C NMR (151 MHz, CD3OD, 298 K): = 31.85 (C-3),

54.44 (C-2), 56.77 (OCH3), 67.74 (C-4), 72.02 (C-1´), 104.06 (C-6´´´), 109.54 (C-2´´´),

118.72 (C-1´´), 125.53 (C-1´´´), 136.79 (C-4´´´), 147.02 (C-3´´´), 149.93 (C-5´´´), 173.56 ppm

(C-1); UV (MeOH): λmax (lgε) 212 (4.07), 246 (3.31), 274 nm (2.79); LC-HR-APCIMS: Rt =

15.4 min, m/z: 297.10788 [M+H]+, calcd for C13H17N2O6 297.10866; LC-HR-APCIMS/MS

(parent ion m/z 297, 25eV): m/z (%): 280.08098 (100) [C13H14NO6]+, 279.09694 (58)

[C13H15N2O5]+, 252.08605 (41) [C12H14NO5]

+, 251.10205 (49) [C12H15N2O4]+, 221.09146 (9)

[C11H13N2O3]+, 194.06826 (77) [C9H10N2O3]

+, 168.06512 (3) [C8H10NO3]+.

Aleurodiscoester (2): Colorless solid; HPLCprep:

Rt = 20.1 min; 25][ Dα = +70 cm3 g-1 dm-1 (c = 0.00135 g

cm-1 in MeOH); 1H NMR (600 MHz, CD3OD, 300 K):

= 2.17−2.23 (m, 1H, H-3), 2.34−2.40 (m, 1H, H-3),

3.67 (dd, 1H, J = 7.3, 5.5 Hz, H-2), 3.88 (s, 3H, 5’’-OCH3), 4.38−4.46 (m, 2H, H-4), 7.20 (d, 1H,

J = 1.9 Hz, H-6´´), 7.24 ppm (d, 1H, J = 1.9 Hz, H-2´´); 13C NMR (151 MHz, CD3OD, 300 K):

= 32.07 (C-3), 53.90 (C-2), 56.74 (5’’-OCH3), 62.28 (C-4), 106.21 (C-6´´), 112.07 (C-2´´), 121.08

(C-1´´), 141.07 (C-4´´), 146.44 (C-3´´), 149.22 (C-5´´), 168.23 (C-1´), 174.83 ppm (C-1); UV/Vis

(MeOH): λmax (lgε) 217 (3.57), 277 nm (3.18); LC-HR-APCIMS: Rt = 15.2 min, m/z: 286.09161

[M+H]+, calcd for C12H16NO7 286.09268. LC-HR-APCIMS/MS (parent ion m/z 286, 25eV): m/z

(%): 268.08118 (56) [C12H14NO6]+, 240.08653 (0.3) [C11H14NO5]

+, 167.03365 (100) [C8H7O4]+,

139.03914 (0.2) [C7H7O3]+, 120.06535 (0.5) [C4H10NO3]

+, 102.05482 (1) [C4H8NO2]+.

2

HO

H3CO

OH

O CO2H

O NH2

141’

2’’4’’

S7

Pertrimetylsilyl derivative of 2: GC-MS: Ri = 2620, m/z (%): 573 (1) [M]+, 558 (0.3) [M–

OCH3]+, 456 (1) [M–CO2Si(CH3)3]

+, 385 (0.5), 328 (0.5), 312 (3), 311 (11) [M–

OCH2CH2CH(NHSi(CH3)3)CO2Si(CH3)3]+, 253 (1), 246 (2), 223 (5), 202 (3), 147 (3), 129

(13), 128 (100), 73 (20) [Si(CH3)3]+, 56 (7), 45 (1).

1.7) Synthesis of Aleurodiscoester (2)

2-[(Benzyloxy)carbonyl]-4-[3,4-bis(benzyloxy)-5-methoxybenzoyloxy]butanoic acid

benzylester (9): 3,4-Bis(benzyloxy)-5-meth-

oxybenzoic acid (200 mg, 0.549 mmol) was

dissolved in anhydrous CH2Cl2 (4 mL),

molecular sieve 4 Å (40 mg) was added and the

mixture was treated with thionyl chloride (0.8 mL, 11.0 mmol) and stirred for 30 min under

argon. The solvent was evaporated under reduced pressure and the residue was treated two

times with anhydrous toluene (4 mL) which was removed immediately each time in vacuum

to remove excess thionyl chloride. Anhydrous CH2Cl2 (5 mL) was added under argon and the

mixture was treated with collidine (0.1 mL, 0.549 mmol) and 2-benzyloxycarbonylamino-4-

hydroxybutyric acid benzyl ester (37.7 mg, 0.110 mmol). After stirring for 12 h under argon,

CH2Cl2 (100 mL) and H2O (50 mL) were added. The aqueous phase was extracted for 2 times

with CH2Cl2 (25 mL). The combined organic phases were extracted with brine (50 mL) and

then dried over anhydrous Na2SO4. The solvent was removed under reduced pressure and the

crude product was immediately subjected to flash chromatography on silica gel (hexane/ethyl

acetate 5:1).

Yield: 60.0 mg (79 %). Yellow oil; TLC: Rf = 0.10 (silica gel, hexane/EtOAc 5:1); 1H

NMR (500 MHz, CDCl3, 300 K): = 2.24−2.29 (m, 1H, H-3), 2.34−2.38 (m, 1H, H-3), 3.86

(s, 3H, 5’’-OCH3), 4.41 (dd, 2H, J = 5.7, 5.7 Hz, H-4), 4.63−4.66 (m, 1H, H-2), 5.01−5.14 (m,

8H, CH2Ph), 5.58 (d, 1 H, J = 7.7 Hz, NH), 7.24−7.44 (m, 20H, 4 × CH2Ph), 7.27 (d, 1H, J ≈

9

PhCH2O

H3CO

OCH2Ph

O CO2CH2Ph

O NHCO2CH2Ph

141’

2’’4’’

S8

3.5 Hz, H-6´´), 7.36 ppm (d, 1H, J ≈ 3.5 Hz, H-2´´); 13C NMR (126 MHz, CDCl3, 300 K): =

31.38 (C-3), 51.55 (C-2), 56.20 (5’’-OCH3), 60.72 (C-4), 67.20 (CH2Ph), 67.43

(NHCO2CH2Ph), 71.12 (CH2Ph), 74.94 (CH2Ph), 107.17 (C-6´´), 108.76 (C-2´´), 124.83 (C-

1´´), 127.54 (CH2Ph), 127.91 (CH2Ph), 127.93 (CH2Ph), 128.05 (CH2Ph), 128.12 (CH2Ph),

128.20 (CH2Ph), 128.41 (CH2Ph), 128.46 (CH2Ph), 128.51 (CH2Ph), 128.57 (CH2Ph), 134.88

(CH2Ph), 136.04 (CH2Ph), 136.63 (CH2Ph), 137.35 (CH2Ph), 141.79 (C-4´´), 152.32 (C-3´´),

153.45 (C-5´´), 155.77 (NHCO2CH2Ph), 165.79 (C-1´), 171.66 ppm (C-1); UV/Vis (MeOH):

λmax (lgε) 209 (4.74), 267 nm (4.07); EIMS (70 eV): m/z (%): 689 (0.003) [M]+, 581 (0.1),

554 (0.01), 526 (0.01), 490 (2), 255 (3), 181 (14), 108 (11) [C7H8O]+, 91 (100) [CH2Ph]+, 79

(10), 77 (5); MALDIMS: m/z (%): 712 (100) [M+Na]+, 728 (16) [M+K]+.

Aleurodiscoester (2): 2-[(Benzyloxy)carbonyl]-4-

[3,4-bis(benzyloxy)-5-methoxybenzoyloxy]butanoic

acid benzylester (9, 20 mg, 0.0290 mmol) was

dissolved in MeOH (5 mL), palladium on charcoal (10

mg, 10 % Pd) was added. Then, a stream of hydrogen was slowly bubbled through the

suspension for 30 min. The Pd/C was removed with an RP-18 cartridge and the solvent was

removed under reduced pressure. The crude product was separated by preparative HPLC (for

separation conditions see “General Experimental Procedures”).

Yield: 2.85 mg (35 %). Colorless solid; HPLCprep: Rt = 20.1 min; 25][ Dα = +58 cm3 g-1 dm-1

(c = 0.00019 g-1 dm-1 in MeOH); 1H NMR (500 MHz, CD3OD, 300 K): = 2.20−2.27 (m, 1H,

H-3), 2.36−2.43 (m, 1H, H-3), 3.71 (dd, 1H, J = 7.2, 5.4 Hz, H-2), 3.89 (s, 3H, 5’’-OCH3),

4.38−4.47 (m, 2H, H-4), 7.20 (d, 1H, J = 1.8 Hz, H-6´´), 7.24 ppm (d, 1H, J = 1.8 Hz, H-2´´);

13C NMR (126 MHz, CD3OD, 300 K): = 31.59 (C-3), 53.74 (C-2), 56.79 (5’’-OCH3), 62.14

(C-4), 106.27 (C-6´´), 112.16 (C-2´´), 121.25 (C-1´´), 140.82 (C-4´´), 146.36 (C-3´´), 149.20

(C-5´´), 168.18 (C-1´), 173.66 ppm (C-1); UV/Vis (MeOH): λmax (lgε) 217 (3.57), 277 nm

2

HO

H3CO

OH

O CO2H

O NH2

141’

2’’4’’

S9

(3.18); LC-HR-APCIMS: Rt = 15.2 min, m/z: 286.09186 [M+H]+, calcd for C12H16NO7

286.09268; LC-HR-APCIMS/MS (parent ion m/z 286, 25eV): m/z (%): 268.08120 (51)

[C12H14NO6]+, 240.08628 (0.2) [C11H14NO5]

+, 167.03366 (100) [C8H7O4]+, 139.03889 (0.1)

[C7H7O3]+, 120.06537 (1) [C4H10NO3]

+, 102.05484 (2) [C4H8NO2]+.

1.8) Synthesis of the Model Compounds 3a, 3b, 3c and 3d

General Procedure for the Preparation of the Cyanhydrines 10a, 10b, 10c and 10d: The

corresponding aromatic aldehyde (1.19 mmol) was dissolved in trimethyl orthoformate (5 mL,

45.6 mmol) and methanol (0.2 mL) under argon. p-Toluenesulfonic acid (10 mg, 0.0581

mmol) and molecular sieve 4 �������� � ���� �������� ������� ������ �� ��������������

2 h. The solvent was evaporated under vacuum and then trimethylsilyl cyanide (2 mL, 15.9

mmol) and SnCl2 (45 mg, 0.237 mmol) were added. The mixture was stirred for 12 h in an

argon atmosphere at room temperature. The excess of trimethylsilyl cyanide and trimethyl

orthoformate was removed at 40 °C in vacuum and the crude product was immediately

subjected to flash chromatography on silica gel (Column dimensions: 10 cm × 1 cm).

2-Methoxy-2-(4-trimethylsilyloxyphenyl)-acetonitrile (10a): Yield:

193 mg (69 %). Colorless oil; TLC: Rf = 0.02 (silica gel, hexane/EtOAc

10:1); 1H NMR (500 MHz, CDCl3, 300 K): = 0.27 (s, 9H, Si(CH3)3, 3.52

(s, 3H, 2-OCH3), 5.12 (s, 1H, H-2), 6.88 (d, 2H, J = 8.6 Hz, H-3´/H-5´), 7.36

ppm (d, 2H, J = 8.6 Hz, H-2´/6´); 13C NMR (126 MHz, CDCl3, 300 K): =

0.17 (Si(CH3)3), 57.05 (2-OCH3), 72.03 (C-2), 117.11 (C-1), 120.54 (C-3´/C-5´), 126.23 (C-

1´), 128.89 (C-2´/6´), 156.63 ppm (C-4´); UV/Vis (MeOH): λmax (lgε) 202 (3.61), 227 (3.19),

274 nm (2.63); GC-MS: Ri = 1583, m/z (%): 235 (33) [M]+, 220 (42) [M−CH3]+, 204 (100)

[M−OCH3]+, 179 (7), 151 (19), 149 (9), 135 (10), 121 (7), 95 (8), 73 (57) [Si(CH3)3]

+.

10a

OSi(CH3)3

NC OCH3

1

2

2’

4’

S10

2-(3-Methoxy-4-trimetylsilyloxyphenyl)-2-methoxyacetonitrile

(10b): Yield: 208 mg (66 %). Colorless oil; TLC: Rf = 0.11 (silica gel,

hexane/EtOAc 15:1); 1H NMR (500 MHz, CDCl3, 300 K): = 0.25 (s, 9H,

Si(CH3)3), 3.53 (s, 3H, 2-OCH3), 3.85 (s, 3H, 3’-OCH3), 5.11 (s, 1H, H-2),

6.87 (d, 1H, J = 8.1 Hz, H-5´), 6.94 (dd, 1H, J = 8.1, 2.0 Hz, H-6´), 6.98

ppm (d, 1H, J = 2.0 Hz, H-2´); 13C NMR (126 MHz, CDCl3, 300 K): = 0.30 (Si(CH3)3),

55.59 (3’-OCH3), 57.11 (2-OCH3), 72.23 (C-2), 110.88 (C-2´), 117.13 (C-1), 120.24 (C-6´),

120.99 (C-5´), 126.68 (C-1´), 146.07 (C-4´), 151.39 ppm (C-3´); UV/Vis (MeOH): λmax (lgε)

202 (3.33), 247 (sh, 2.76), 289 nm (sh, 2.50); GC-MS: Ri = 1718, m/z (%): 265 (36) [M]+,

250 (58) [M−CH3]+, 235 (73) [M−CH2O]+, 234 (47) [M−OCH3]

+, 220 (6), 204 (100)

[M−(CH2O+OCH3)]+, 174 (7), 151 (5), 73 (36) [Si(CH3)3]

+, 59 (5), 45 (9); HR-EIMS: m/z:

265.1137 [M]+, calcd for C13H19NO3Si 265.1134.

2-[3,4-Bistrimethylsilyloxyphenyl)-2-methoxyacetonitrile (10c):

Yield: 259 mg (67 %). Colorless oil; TLC: Rf = 0.16 (silica gel,

hexane/EtOAc 15:1); 1H NMR (500 MHz, CDCl3, 300 K): = 0.26 (s,

18H, 2 × Si(CH3)3), 3.50 (s, 3H, 2-OCH3), 5.09 (s, 1H, H-2), 6.86 (d,

1H, J = 7.5 Hz, H-5´), 6.96−6.98 ppm (m, 2H, H-2´/H-6´); 13C NMR

(126 MHz, CDCl3, 300 K): = 0.29 (2 × Si(CH3)3), 56.89 (2-OCH3), 71.86 (C-2), 117.07 (C-

1), 120.27 (C-2´), 121.07 (C-6´), 121.18 (C-5´), 126.63 (C-1´), 147.01 (C-3´), 148.08 ppm (C-

4´); UV/Vis (MeOH): λmax (lgε) 205 (5.75), 237 (4.96), 283 nm (4.68); GCMS: Ri = 1774, m/z

(%): 323 (37) [M]+, 292 (26), [M−OCH3]+, 251 (5), 220 (4), 209 (100), 204 (70), 193 (11),

174 (7), 166 (5), 135 (3), 73 (43) [Si(CH3)3]+.

2-(3-Methoxy-4,5-bistrimethylsilyloxyphenyl)-2-methoxy-

acetonitrile (10d): Yield: 254 mg (61 %). Colorless oil; TLC: Rf

= 0.15 (silica gel, hexane/EtOAc 15:1); 1H NMR (500 MHz,

10b

4’

2’

2

1

OSi(CH3)3

NC OCH3

OCH3

OSi(CH3)3

NC OCH3

OSi(CH3)3

1

2

2’

4’

10c

10d

4’

2’

2

1

OSi(CH3)3

NC OCH3

OCH3(H3C)3SiO

S11

4’

2’

2

1

OH

NC OCH3

3a

CDCl3, 300 K): = 0.22 (s, 9H, Si(CH3)3), 0.25 (s, 9H, Si(CH3)3), 3.50 (s, 3H, 2-OCH3), 3.82

(s, 3H, 3’-OCH3), 5.07 (s, 1H, H-2), 6.61 (d, 1H, J = 2.1 Hz, H-6´), 6.65 ppm (d, 1H, J = 2.1

Hz, H-2´); 13C NMR (126 MHz, CDCl3, 300 K): = 0.27 (Si(CH3)3), 0.51 (Si(CH3)3), 55.66

(3’-OCH3), 56.90 (2-OCH3), 72.19 (C-2), 104.24 (C-2´), 113.09 (C-6´), 117.03 (C-1), 125.43

(C-1´), 137.69 (C-4´), 147.37 (C-5`), 152.48 ppm (C-3´); UV/Vis (MeOH): λmax (lgε) 211

(6.11), 243 (5.25), 270 nm (4.12); GC-MS: Ri = 1899, m/z (%): 353 (23) [M]+, 322 (14)

[M−OCH3]+, 239 (100), 234 (49), 224 (10), 223 (7), 191 (3), 73 (41) [Si(CH3)3]

+; HR-EIMS:

m/z: 353.1477 [M]+, calcd for C16H27NO4Si2 353.1479.

General Deprotection Procedure for the Preparation of the Cyanohydrins 3a�3d: The

corresponding cyanohydrin 10a−10d (0.6 mmol) was dissolved in a degassed 1:1 mixture (10

mL) of water and methanol and stirred at room temperature until the reaction had been

finished. The course of each reaction was monitored by TLC. The solvent and the

trimethylsilanol was then evaporated in vacuum. The corresponding residues consisting of the

model compounds 3a−3d were pure according to their 1H NMR spectra.

2-(4-Hydroxyphenyl)-2-methoxyacetonitrile (3a): Yield: 98 mg (99 %).

Colorless solid; TLC: Rf = 0.09 (silica gel, hexane/EtOAc 2:1); 1H NMR (500

MHz, CD3OD, 300 K): = 3.45 (s, 3 H, 2-OCH3), 5.27 (s, 1 H, H-2), 6.83 (d,

2H, J = 8.6 Hz, H-3´/H-5´), 7.30 ppm (d, 2H, J = 8.6 Hz, H-2´/H-6´); 13C

NMR (126 MHz, CD3OD, 300 K): = 57.15 (2-OCH3), 72.81 (C-2), 116.60 (C-3´/C-5´),

118.86 (C-1), 126.12 (C-1´), 130.10 (C-2´/C-6´), 160.01 ppm (C-4´); UV/Vis (MeOH): λmax

(lgε) 201 (5.96), 231 (5.77), 275 nm (4.82); EIMS (70 eV): m/z (%): 163 (30) [M]+, 148 (4),

137 (6), 132 (100) [M−OCH3]+, 121 (13), 104 (4), 94 (3), 77 (11), 65 (3), 51 (5). HR-EIMS:

m/z: 163.0632 [M]+, calcd for C9H9NO2 163.0633.

S12

2-(4-Hydroxy-3-methoxyphenyl)-2-methoxyacetonitrile (3b): Yield:

113 mg (97 %). Colorless solid; TLC: Rf = 0.21 (silica gel, hexane/EtOAc

2:1); 1H NMR (500 MHz, CD3OD, 300 K): = 3.45 (s, 3H, 2-OCH3), 3.86

(s, 3H, 3’-OCH3), 5.26 (s, 1H, H-2), 6.84 (d, 1H, J = 8.1 Hz, H-5´), 6.94

(d, 1H, J = 8.1 Hz, H-6´), 7.01 ppm (s, 1 H, H-2´); 13C NMR (126 MHz,

CD3OD, 300 K): = 56.44 (3’-OCH3), 57.21 (2-OCH3), 72.93 (C-2), 111.90 (C-2´), 116.27

(C-5´), 118.84 (C-1), 121.67 (C-6´), 126.55 (C-1´), 149.11 (C-4´), 149.30 ppm (C-3´);

UV/Vis (MeOH): λmax (lgε) 205 (6.14), 237 (5.55), 264 (5.22), 280 nm (5.21); EIMS (70 eV):

m/z (%): 193 (35) [M]+, 162 (100) [M−OCH3]+, 147 (11), 119 (10), 43 (12); HR-EIMS: m/z =

193.0740 [M]+, calcd for C10H11NO3 193.0739.

2-(3,4-Dihydroxyphenyl)-2-methoxyacetonitrile (3c): Yield: 105 mg

(98 %). Colorless solid; TLC: Rf = 0.16 (silica gel, hexane/EtOAc 2:1); 1H

NMR (500 MHz, CD3OD, 300 K): = 3.42 (s, 3H, 2-OCH3), 5.19 (s, 1H,

H-2), 6.80 (m, 2H, H-5´/H-6´), 6.91 ppm (s, 1H, H-2´); 13C NMR (126

MHz, CD3OD, 300 K): = 57.13 (2-OCH3), 72.83 (C-2), 115.49 (C-2´),

116.31 (C-5´), 118.84 (C-1), 120.43 (C-6´), 126.53 (C-1´), 146.74 (C-3´), 147.85 ppm (C-4´);

UV/Vis (MeOH): λmax (lgε) 205 (5.26), 236 (4.47), 283 nm (4.20); EIMS (70 eV): m/z (%):

179 (44) [M]+, 161 (3), 148 (100) [M−OCH3]+, 137 (11), 130 (5), 119 (5), 110 (5), 102 (9), 81

(3), 75 (5), 63 (3), 51 (4); HR-EIMS: m/z: 179.0581 [M]+, calcd for C9H9NO3 179.0582.

2-(3,4-Dihydroxy-5-methoxyphenyl)-2-methoxyacetonitrile (3d):

Yield: 123 mg (98 %). Colorless solid; TLC: Rf = 0.13 (silica gel,

hexane/EtOAc 2:1); 1H NMR (500 MHz, CD3OD, 300 K): = 3.44 (s,

3H, 5’-OCH3), 3.85 (s, 3H, 5’-OCH3), 5.23 (s, 1H, H-2), 6.61 (d, 1 H,

J = 1.9 Hz, H-6´), 6.62 ppm (d, 1H, J = 1.9 Hz, H-2´); 13C NMR (126

MHz, CD3OD, 300 K): = 56.71 (5’-OCH3), 57.11 (2-OCH3), 73.08 (C-2), 103.97 (C-6´),

109.42 (C-2´), 118.84 (C-1), 125.77 (C-1´), 136.62 (C-4´), 146.92 (C-3´), 149.84 ppm (C-5´);

OH

NC OCH3

OCH3

1

2

2’

4’

3b

OH

NC OCH3

OH

1

2

2’

4’

3c

OH

NC OCH3

OHH3CO

1

2

2’

4’

3d

S13

UV/Vis (MeOH): λmax (lgε) 211 (6.07), 245 (5.33), 271 nm (4.82); EIMS (70 eV): m/z (%):

209 (40) [M]+, 194 (5) [M − CH3]+, 178 (100) [M−OCH3]

+, 167 (6), 163 (11), 148 (8), 135

(14); HR-EIMS: m/z: 209.0688 [M]+, calcd for C10H11NO4 209.0688.

1.9) General Procedure for the Oxidation of the Model Compounds 3a, 3b, 3c or 3d

with Activated MnO2 in Aqueous MeOH: Activated MnO2 (1.0 mg, 0.0115 mmol) was

suspended in an 1:1 solution (500 µL) of MeOH and H2O containing 1 % AcOH and added

to the corresponding model compound 3a, 3b, 3c or 3d (0.00575 mmol) dissolved in a stirred

1:1 solution (500 µL) of MeOH and H2O containing 1 % AcOH. After certain periods of time,

samples were withdrawn (50 µL). In order to remove the MnO2 and to stop the oxidation

reaction each sample was filtrated with MeOH through an RP-18 cartridge. The solvents were

evaporated at 50 °C in vacuum and the residue was pertrimethylsilylated with MSTFA and

analyzed by gas chromatography.

R1 = H, R2 = H: 5a

R1 = OMe, R2 = H: 5b

R1 = OH, R2 = H: 5c

R1 = OMe, R2 = OH: 5d

HO

R2

OMeR1

OMeNC

HO

R2

OMeR1

O

R1 = H, R2 = H: 4a

R1 = OMe, R2 = H: 4b

R1 = OH, R2 = H: 4c

R1 = OMe, R2 = OH: 4d

HO

R2

OMeR1

CN

R1 = H, R2 = H: 3a

R1 = OMe, R2 = H: 3b

R1 = OH, R2 = H: 3c

R1 = OMe, R2 = OH: 3d

Table S1. Results of the oxidation of 3a with activated MnO2 in MeOH/H2O/AcOH[a]

t in h relative amount of 3a relative amount of 4a relative amount of 5a

0 100 % 0 % 0 %

3 99.9 % 0.1 % 0 %

24 99.0 % 1.0 % 0 %

[a] The relative amounts of the products were determined by GC-MS.

S14

Pertrimethylsilylated 3a = 10a: See section 1.8.

Pertrimethylsilylated 4a: GC-MS: Ri = 1485, m/z (%): 224 (65) [M]+, 209 (100) [M–

CH3]+, 193 (28) [M–OCH3]

+, 177 (21), 149 (13), 135 (21), 97 (4), 91 (11), 89 (15), 73 (12)

[Si(CH3)3]+, 59 (5).

Table S2. Results of the oxidation of 3b with activated MnO2 in MeOH/H2O/AcOH[a]

t in h relative amount of 3b relative amount of 4b relative amount of 5b

0 100 % 0 % 0 %

3 98.8 % 1.2 % 0 %

24 87.8 % 12.2 % 0 %

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 3b = 10b: See section 1.8.

Pertrimethylsilylated 4b: GC-MS: Ri = 1645, m/z (%): 254 (31) [M]+, 239 (53) [M–

CH3]+, 224 (100) [M–CH2O]+, 193 (48), 179 (4), 165 (7), 163 (5), 149 (4), 137 (8), 135 (4),

119 (3), 104 (5), 89 (5), 73 (13) [Si(CH3)3]+, 59 (5).

Table S3. Results of the oxidation of 3c with activated MnO2 in MeOH/H2O/AcOH[a]

t in min relative amount of 3c relative amount of 4c relative amount of 5c

0 100 % 0 % 0 %

1 71.2 % 28.1 % 0.7 %

5 46.4 % 50.3 % 3.3 %

10 19.6 % 76.5 % 3.9 %

30 2.8 % 96.8 % 0.4 %

40 0 % 99.5 % 0.5 %

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 3c = 10c: See section 1.8.

S15

Pertrimethylsilylated 4c: GC-MS: Ri = 1716, m/z (%): 312 (16) [M]+, 281 (4) , 235 (1),

224 (2), 207 (1), 193 (100), 165 (7), 137 (5), 133 (1), 73 (25) [Si(CH3)3]+.

Pertrimethylsilylated 5c: GC-MS, Ri = 1814, m/z (%): 353 (15) [M]+, 322 (100) [M–

OCH3]+, 312 (7), 281 (2), 239 (16), 234 (23), 219 (6), 193 (59), 180 (2), 165 (7), 163 (2), 137

(4), 100 (2), 73 (30) [Si(CH3)3]+, 59 (1), 45 (7).

Table S4. Results of the oxidation of 3d with activated MnO2 in MeOH/H2O/AcOH[a]

t in min relative amount of 3d relative amount of 4d relative amount of 5d

0 96.0 % 3.0 % 1.0 %

1 0 % 92.3 % 7.7 %

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 3d = 10d: See section 1.8.

Pertrimethylsilylated 4d: GC-MS, Ri = 1856, m/z (%): 342 (12) [M]+, 327 (2), 311 (2),

254 (2), 223 (100), 195 (5), 137 (3), 133 (3), 89 (2), 73 (26), 59 (1), 45 (5).

Pertrimethylsilylated 5d: GC-MS, Ri = 1921, m/z (%): 383 (6) [M]+, 352 (30) [M–

OCH3]+, 342 (14), 311 (3), 269 (8), 264 (11), 254 (4), 223 (100), 195 (6), 137 (4), 133 (3), 73

(32) [Si(CH3)3]+.

1.10) General Procedure for the Oxidation of the Model Compounds 3a, 3b, 3c or 3d

with activated MnO2 in MeOH: Activated MnO2 (1.0 mg, 0.0115 mmol) was suspended in

MeOH (500 µL, HPLC grade, contains approx. 0.03 % H2O) and

added to a stirred methanolic solution (500 µL) of the corresponding

model compound 3a, 3b, 3c or 3d (0.00575 mmol). After certain

periods of time, samples were withdrawn (50 µL). In order to remove

the MnO2 and to stop the oxidation reaction each sample was filtrated

HO

R2

OMeR1

OMeMeO

R1 = H, R2 = H: 11a

R1 = OMe, R2 = H: 11b

R1 = OH, R2 = H: 11c

R1 = OMe, R2 = OH: 11d

S16

with MeOH through an RP-18 cartridge. The solvent was removed by a stream of nitrogen

and the crude mixture was pertrimethylsilylated with MSTFA and analyzed by gas

chromatography.

Table S5. Results of the oxidation of 3a with activated MnO2 in MeOH[a]

t in h rel. amount of 3a rel. amount of 4a rel. amount of 5a rel. amount of 11a

0 100 % 0 % 0 % 0 %

24 100 % 0 % 0 % 0 %

[a] The relative amounts of the products were determined by GC-MS.

Table S6. Results of the oxidation of 3b with activated MnO2 in MeOH[a]

t in h rel. amount of 3b rel. amount of 4b rel. amount of 5b rel. amount of 11b

0 95.3 % 3.9 % 0.8 % 0 %

24 92.5 % 6.6 % 0.9 % 0 %

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 5b: GC-MS: Ri = 1759, m/z (%): 295 (9) [M]+, 280 (6) [M–CH3]+,

264 (100) [M–OCH3]+, 234 (30), 224 (13), 219 (9), 204 (7), 193 (12), 165 (8), 149 (7), 73

(18) [Si(CH3)3]+.

Table S7. Results of the oxidation of 3c with activated MnO2 in MeOH[a]

t in min rel. amount of 3c rel. amount of 4c rel. amount of 5c rel. amount of 11c

0 100 % 0 % 0 % 0 %

5 83.5 % 0.8 % 15.7 % 0 %

10 66.9 % 1.3 % 31.0 % 0.8 %

20 54.1 % 1.2 % 43.5 % 1.2 %

60 23.0 % 1.6 % 72.1 % 3.3 %

120 2.8 % 2.0 % 89.1 % 6.1 %

S17

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 11c: GC-MS: Ri = 1753, m/z (%): 358 (3) [M]+, 327 (69) [M–

OCH3]+, 312 (16), 281 (3), 239 (12), 224 (4), 207 (2), 193 (100), 179 (3), 165 (8), 137 (5),

105 (4), 73 (35) [Si(CH3)3]+.

.

Table S8. Results of the oxidation of 3d with activated MnO2 in MeOH[a]

t in min rel. amount of 3d rel. amount of 4d rel. amount of 5d rel. amount of 10d

0 97.4 % 1.1 % 1.5 % 0 %

5 49.1 % 4.8 % 44.9 % 1.2 %

10 29.8 % 9.4 % 59.8 % 1.0 %

20 0 % 31.4 % 68.6 % 0 %

[a] The relative amounts of the products were determined by GC-MS.

Pertrimethylsilylated 11d: GC-MS, Ri = 1862, m/z (%): 388 (2) [M]+, 357 (17) [M–

OMe]+, 342 (9), 269 (6), 254 (5), 223 (100), 195 (5), 133 (3), 105 (3), 89 (3), 73 (36)

[Si(CH3)3]+, 59 (2), 45 (6).

1.11) Detection of the E and Z Isomer of the Quinonemethide 6d

2-(3,4-Dihydroxy-5-methoxyphenyl)-2-methoxy-

acetonitrile (3d, 20 mg, 0.096 mmol) was

dissolved in CD3CN (2 mL). After addition of

molecular sieve (4 Å, 20 mg) activated MnO2 (17

mg, 0.19 mmol) the mixture was stirred for 20

min at 25 °C. Then MnO2 was removed by centrifugation and the yellow solution was dried

with molecular sieve (4 Å, 20 mg). The solution was concentrated to 0.5 mL in a stream of

nitrogen and immediately analyzed by NMR. In the course of the reaction, the two isomers

E-6d and Z-6d were generated. Yield: 14 % E-6d, 14 % Z-6d, 72 % 3d).

Z-6d

4’

2’

2

1

O

H3CO CN

OCH3HO

O

NC OCH3

OCH3HO

2

2’

4’

E-6d

1

S18

E-6d: 1H NMR (600 MHz, CD3CN, 300 K): = 3.78 (s, 3 H, 5´-OCH3), 4.04 (s, 3 H, 2-

OCH3), 6.67 (d, 1 H, J = 2.2 Hz, H-2´), 6.80 (d, 1 H, J = 2.2 Hz, H-6´); 13C NMR (151 MHz,

CD3CN, 300 K): = 56.64 (5´-OCH3), 61.24 (2-OCH3), 103.06 (C-6´), 107.06 (C-2´), 113.17

(C-1´), 134.40 (C-2), 150.79 (C-3´), 153.06 (C-5´), 176.62 (C-4´), C-1 is probably obscured

by the carbon resonance at = 118.29 of the CN group of CD3CN.

Z-6d: 1H NMR (600 MHz, CD3CN, 300 K): = 3.80 (s, 3 H, 5´-OCH3), 4.03 (s, 3 H, 2-

OCH3), 6.54 (d, 1 H, J = 1.9 Hz, H-6´), 6.86 (d, 1 H, J = 1.9 Hz, H-2´); 13C-NMR (151 MHz,

CD3CN, 300 K): = 56.51 (5´-OCH3), 61.24 (2-OCH3), 103.30 (C-2´), 107.17 (C-6´), 113.14

(C-1´), 134.65 (C-2), 150.60 (C-3´), 153.26 (C-5´), 176.60 (C-4´), C-1 is probably obscured

by the carbon resonance at = 118.29 of the CN group of CD3CN.

1.12) Enzymatic Conversion of 1 to 2 with Mushroom Tyrosinase

Aleurodisconitrile (1, 0.20 mg, 0.68 µmol) was

dissolved in phosphate buffer (500 µL, 50 mM, pH =

7.0) and mushroom tyrosinase (0.1 mg, EC 1.14.18.1,

CAS number: 9002-10-2) was added. After 0 min, 30

min and 120 min samples (50 µL) were withdrawn and injected into the analytical HPLC (1:

Rt = 28.2 min, 2: Rt = 30.4 min, 12: Rt = 29.4 min).

Table S9. Results of the oxidation of 1 with mushroom tyrosinase.

t in min relative amount of 1 relative amount of 2 relative amount of 12

0 100 % 0 % 0 %

30 35 % 25 % 40 %

120 0 % 65 % 35 %

12O

MeO

O

O

CN

CO2H

NH2

S19

2) Spectra 2.1) Detection of Hydrocyanic acid by GC-MS 2.1.1) GC Chromatogram of Pentafluorobenzylcyanide

5 6 7 8 9 10 11 12 13 14 15Time [min]

0

20

40

60

80

100

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

RT: 5.90

Area: 523848

RT: 5.91

Area: 118285

NL:1.65E5m/z = 206.50-207.50Standard

NL:4.16E4m/z = 206.50-207.50

FruitingBodies

S20

2.1.2) GC-MS of Pentafluorobenzylcyanide from Fruiting Bodies

40 60 80 100 120 140 160 180 200 220 240m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

207

157188

181

161

11768 71 112

67

2.1.3) GC-MS of Pentafluorobenzylcyanide (Standard)

40 60 80 100 120 140 160 180 200 220 240m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

207

157

181

18840

161

11793 13044 99

S21

2.2) UV, CD, NMR and Mass Spectra of Aleurodisconitrile (1) 2.2.1) UV-Vis Spectrum of 1 in MeOH

200 250 300 350 400

0.0

0.2

0.4

0.6

0.8 212

246

274

Abs

orpt

ion

nm

2.2.2) CD Spectrum of 1 in MeOH

200 250 300 350 400-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0 210

∆ε

[Lm

ol-1cm

-1]

nm

CD Spectrum of (R)-Amygdaline in MeOH

200 250 300 350 400-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

201213

∆ε [L

mol

-1cm

-1]

nm

S22

2.2.3) 1H NMR (600 MHz, CD3OD, 330 K) of 1

2.53.03.54.04.55.05.56.06.5 ppm

2.0833

2.0935

2.0979

2.1041

2.1085

2.1183

2.2455

2.2495

2.2539

2.2579

2.2616

2.2667

2.2703

2.2743

2.2827

3.3100

3.6519

3.6600

3.6636

3.6717

3.7356

3.7444

3.7484

3.7513

3.7571

3.7601

3.7641

3.7725

3.8477

3.8572

3.8634

3.8671

3.8766

3.8828

3.8872

4.5630

5.3415

6.6738

6.6767

6.6829

6.6859

1.0

1.0

1.0

1.0

4.0

1.0

2.0

2.2.3) 1H NMR (600 MHz, CD3OD, 330 K) of 1

6.6656.6706.6756.6806.6856.6906.695 ppm

6.6738

6.6767

6.6829

6.6859

2.0

S23

2.2.3) 1H NMR (600 MHz, CD3OD, 330 K) of 1

1.82.02.22.42.62.83.03.23.43.63.84.04.2 ppm

2.0730

2.0833

2.0935

2.0979

2.1041

2.1085

2.1183

2.1286

2.2367

2.2455

2.2495

2.2539

2.2579

2.2616

2.2667

2.2703

2.2743

2.2791

2.2827

2.2915

3.3100

3.6519

3.6600

3.6636

3.6717

3.7356

3.7444

3.7484

3.7513

3.7571

3.7601

3.7641

3.7725

3.7769

3.8477

3.8572

3.8634

3.8671

3.8766

1.0

1.0

1.0

1.0

4.0

2.2.3) 1H NMR (600 MHz, CD3OD, 298 K) of 1

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

2.0877

2.1008

2.1121

2.1220

2.2294

2.2382

2.2426

2.2469

2.2506

2.2597

2.2626

2.2674

2.2718

2.2751

2.2842

3.3100

3.6541

3.6622

3.6662

3.6739

3.7067

3.7155

3.7199

3.7287

3.7308

3.7349

3.7436

3.7509

3.7641

3.8324

3.8419

3.8485

3.8518

3.8576

3.8740

3.8868

4.8662

5.3503

6.6687

1.0

1.0

1.0

1.0

4.0

1.0

2.0

S24

2.2.3) 1H NMR (600 MHz, CD3OD, 298 K) of 1

6.656.666.676.686.69 ppm

6.6687

2.0

2.2.3) 1H NMR (600 MHz, CD3OD, 298 K) of 1

1.82.02.22.42.62.83.03.23.43.63.84.04.2 ppm

2.0665

2.0760

2.0877

2.1008

2.1121

2.1220

2.2294

2.2382

2.2426

2.2469

2.2506

2.2597

2.2626

2.2674

2.2718

2.2751

2.2842

3.3100

3.6541

3.6622

3.6662

3.6739

3.7067

3.7155

3.7199

3.7287

3.7308

3.7349

3.7436

3.7509

3.7641

3.8324

3.8419

3.8485

3.8518

3.8576

1.0

1.0

1.0

1.0

4.0

S25

2.2.4) 13C NMR (151 MHz, CD3OD, 298 K) of 1

405060708090100110120130140150160170 ppm

31.8472

49.0000

54.4401

56.7657

67.7369

72.0175

104.0581

109.5386

118.7200

125.5285

136.7862

147.0192

149.9348

173.5590

S26

2.2.5) COSY (600 MHz, CD3OD, 330 K) of 1

ppm

2.22.42.62.83.03.23.43.63.84.0 ppm

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

S27

2.2.6) HSQC (600 MHz, CD3OD, 330 K) of 1

ppm

2.53.03.54.04.55.05.56.06.57.0 ppm

30

40

50

60

70

80

90

100

110

S28

2.2.7) HMBC (600 MHz, CD3OD, 330 K) of 1

ppm

2.02.53.03.54.04.55.05.56.06.57.0 ppm

20

40

60

80

100

120

140

160

180

200

S29

2.2.8) NOESY (600 MHz, CD3OD, 320 K) of 1

ppm

2.53.03.54.04.55.05.56.06.5 ppm

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

S30

2.2.9) HR-APCIMS

290 300 310 320 330 340 350m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

297.10788

286.09189

C13H17O6N2

C12H16O7N1

2.2.10) HR-APCIMS/MS (Parent Ion m/z 297, 25 eV)

100 120 140 160 180 200 220 240 260 280 300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

280.08098C 13H14O 6 N1

194.06826C 9 H10O 3 N2

251.10205C 12H15O 4 N2

221.09146C 11H13O 3 N2168.06512

C 8 H10O 3 N1

279.09694C 13H15O 5 N2

252.08605C 12H14O 5 N1

S31

2.3) UV, CD, NMR, and Mass Spectra of Aleurodiscoester (2) 2.3.1) UV-Vis Spectrum of 2 in MeOH

200 300 400

0.0

0.1

0.2

0.3

0.4

0.5

277

217

Abs

orpt

ion

λ [nm]

2.3.2) CD Spectrum of 2 There are no cotton effects in the CD. 2.3.3) 1H NMR (600 MHz, CD3OD, 300 K) of 2

1.01.52.02.53.03.54.04.55.05.56.06.57.0 ppm

2.1744

2.1852

2.1962

2.1973

2.2084

2.2095

2.2205

2.2314

2.3425

2.3523

2.3542

2.3638

2.3737

2.3766

2.3885

2.3980

3.3100

3.6600

3.6691

3.6722

3.6813

3.8841

4.3763

4.3864

4.3957

4.4058

4.4167

4.4187

4.4288

4.4310

4.4405

4.4478

4.4500

4.4596

4.8695

7.1989

7.2019

7.2338

7.2369

1.0

1.0

1.0

3.0

2.0

1.0

1.0

S32

2.3.3) 1H NMR (600 MHz, CD3OD, 300 K) of 2

7.197.207.217.227.237.247.25 ppm

7.1989

7.2019

7.2338

7.2369

1.0

1.0

2.3.3) 1H NMR (600 MHz, CD3OD, 300 K) of 2

2.02.53.03.54.04.5 ppm

2.1678

2.1686

2.1744

2.1852

2.1962

2.1973

2.2084

2.2095

2.2205

2.2314

2.3425

2.3523

2.3542

2.3638

2.3737

2.3766

2.3885

2.3980

3.3100

3.6600

3.6691

3.6722

3.6813

3.8841

4.3763

4.3864

4.3957

4.4058

4.4167

4.4187

4.4288

4.4310

4.4405

4.4478

4.4500

4.4596

1.0

1.0

1.0

3.0

2.0

S33

2.3.4) 13C NMR (151 MHz, CD3OD, 300 K) of 2

405060708090100110120130140150160170 ppm

32.0730

49.0000

53.9042

56.7354

62.2766

106.2085

112.0733

121.0760

141.0668

146.4361

149.2236

168.2302

174.8331

2.3.5) COSY (600 MHz, CD3OD, 300 K) of 2

ppm

7.167.187.207.227.247.267.287.30 ppm

7.16

7.17

7.18

7.19

7.20

7.21

7.22

7.23

7.24

7.25

7.26

7.27

7.28

7.29

7.30

S34

2.3.5) COSY (600 MHz, CD3OD, 300 K) of 2

ppm

2.22.42.62.83.03.23.43.63.84.04.24.44.6 ppm

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

S35

2.3.6) HSQC (600 MHz, CD3OD, 300 K) of 2

ppm

2.22.42.62.83.03.23.43.63.84.04.24.44.6 ppm

35

40

45

50

55

60

S36

2.3.6) HSQC (600 MHz, CD3OD, 300 K) of 2

ppm

7.167.187.207.227.247.267.287.30 ppm

106

107

108

109

110

111

112

113

114

115

S37

2.3.7) HMBC (600 MHz, CD3OD, 300 K) of 2

ppm

2.53.03.54.04.55.05.56.06.57.07.5 ppm

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

S38

2.3.8) NOESY (600 MHz, CD3OD, 300 K) of 2

ppm

2.02.53.03.54.04.55.05.56.06.57.0 ppm

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

S39

2.3.9) HR-APCIMS of 2

290 300 310 320 330 340 350m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

286.09161C12H16O7N1

2.3.10) HR-APCIMS (Parent Ion m/z 286, 25 eV) of 2

100 120 140 160 180 200 220 240 260 280 300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

167.03365C 8 H7 O 4

268.08118C 12H14O 6 N1

120.06535C 4 H10O 3 N1

240.08653

C 11H14O 5 N1

139.03914

C7H7O3

102.05482

C 4 H8 O 2 N1

S40

2.3.11) GC-MS of 2 Pertrimetylsilylated with MSTFA, Ri = 2620

50 100 150 200 250 300 350 400 450 500 550 600m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

128

73

129311

56 223147 202 31224645 573253 456385328 558

S41

2.4 NMR and Mass Spectra of 9 and of Synthetic 2

2.4.1.1) 1H NMR (500 MHz, CDCl3, 300 K) of 9

2.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm

2.2380

2.2516

2.2666

2.2794

2.2912

2.3414

2.3528

2.3689

2.3814

3.8555

4.4027

4.4144

4.4258

4.6256

4.6373

4.6502

4.6619

5.0055

5.0297

5.0844

5.0954

5.1115

5.1192

5.1419

5.5673

5.5827

7.2442

7.2600

7.2754

7.2776

7.2893

7.3117

7.3143

7.3227

7.3396

7.3539

7.3685

7.3828

7.4243

7.4390

2.0

3.0

2.0

1.0

8.0

1.0

22.0

2.4.1.2) 13C NMR (126 MHz, CDCl3, 300 K) of 9

405060708090100110120130140150160170 ppm

31.3781

51.5466

56.1978

60.7209

67.1044

67.4347

71.1152

74.9440

77.0000

107.1651

108.7627

124.8328

127.5359

127.9066

127.9336

128.0482

128.1156

128.1965

128.4055

128.4594

128.5066

128.5740

134.8834

136.0361

136.6293

137.3505

141.7860

152.3151

153.4476

155.7664

165.7900

171.6613

S42

2.4.1.3) EIMS of 9

50 100 150 200 250 300 350 400 450 500 550 600 650 700m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

91.1

181.2108.179.177.1

255.2 490.3346.3271.2 363.3 581.4436.3 526.4 689.5627.4

500 520 540 560 580 600 620 640 660 680 700m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

581.4

582.5

526.4 554.4 583.5524.4 527.5 580.6

506.5 546.6 689.5627.4

S43

2.4.2.1) 1H NMR (500 MHz, CD3OD, 300 K) of Synthetic 2

1.52.02.53.03.54.04.55.05.56.06.57.0 ppm

2.2029

2.2161

2.2286

2.2312

2.2458

2.2583

2.2719

2.3613

2.3731

2.3866

2.3984

2.4024

2.4163

2.4281

3.3100

3.6991

3.7097

3.7134

3.7244

3.8864

4.3778

4.3903

4.4013

4.4138

4.4251

4.4361

4.4383

4.4497

4.4589

4.4614

4.4732

4.8413

7.2014

7.2051

7.2370

7.2407

1.0

1.0

1.0

3.0

2.0

1.0

1.0

2.4.2.2) 13C NMR (126 MHz, CD3OD, 300 K) of Synthetic 2

405060708090100110120130140150160170 ppm

31.5884

49.0000

53.7388

56.7856

62.1446

106.2700

112.1615

121.2549

140.8235

146.3578

149.2024

168.1778

173.6581

S44

2.4.2.3) HR-APCIMS of Synthetic 2

100 150 200 250 300 350m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

286.09186C 12H16O 7 N1

102.05491C4 H8 O 2 N1

300.10730C13H18O 7 N1

268.08142C12H14O 6 N1

167.03375C

8H

7O

4120.06543

C 4 H10O 3 N1

2.4.2.4) HR-APCIMS (Parent Ion m/z 286, 25 eV) of Synthetic 2

100 150 200 250 300 350m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

167.03366C 8 H7 O 4

268.08120C 12H14O 6 N1

102.05484C4 H8 O 2 N1

240.08628C11H14O 5 N1

139.03889C 7 H7 O 3

120.06537C4 H10O 3 N1

S45

2.5) NMR and Mass Spectra of 10a�10d and of the Model Compounds 3a�3d 2.5.1.1) 1H NMR (500 MHz, CDCl3, 300 K) of 10a

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.2747

3.5174

5.1236

6.8728

6.8900

7.2600

7.3520

7.3693

9.0

3.0

1.0

2.0

2.0

2.5.1.2) 13C NMR (126 MHz, CDCl3, 300 K) of 10a

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

0.1681

57.0471

72.0320

77.0000

117.1146

120.5389

126.2282

128.8908

156.6293

S46

2.5.1.3) EIMS of 10a

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

204

73

220

235

151 205

1351499545 221121 179 23620643

2.5.2.1) 1H NMR (500 MHz, CDCl3, 300 K) of 10b

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.2472

1.5372

3.5277

3.8471

5.1133

6.8570

6.8731

6.9325

6.9366

6.9487

6.9531

6.9740

6.9780

7.2600

9.0

3.0

3.0

1.0

1.0

1.0

1.0

S47

2.5.2.2) 13C NMR (126 MHz, CDCl3, 300 K) of 10b

140 120 100 80 60 40 20 0 ppm

0.3030

55.5912

57.1146

72.2275

77.0000

110.8794

117.1281

120.2424

120.9906

126.6799

146.0665

151.3917

2.5.2.3) EIMS of 10b

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

204

235

73250

234

265

174 205236

2514559 266220151

S48

2.5.3.1) 1H NMR (500 MHz, CDCl3, 300 K) of 10c

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.2578

3.4991

5.0855

6.8482

6.8632

6.9556

6.9710

6.9754

7.2600

18.0

3.0

1.0

1.0

2.0

2.5.3.2) 13C NMR (126 MHz, CDCl3, 300 K) of 10c

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

0.2895

56.8854

71.8635

77.0000

117.0674

120.2693

121.0715

121.1793

126.6259

147.0102

148.0752

S49

2.5.3.3) EIMS of 10c

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

209

73204

323

292210

45 193324211174 293

166

2.5.4.1) 1H NMR (500 MHz, CDCl3, 300 K) of 10d

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.2160

0.2523

3.5028

3.8203

5.0748

6.6080

6.6124

6.6494

6.6535

7.2600

9.0

9.0

3.0

3.1

1.0

1.0

1.0

S50

2.5.4.2) 13C NMR (126 MHz, CDCl3, 300 K) of 10d

140 120 100 80 60 40 20 0 ppm

0.2692

0.5119

55.6586

56.8989

72.1938

77.0000

104.2396

113.0903

117.0337

125.4260

137.6876

147.3741

152.4769

2.5.4.3) EIMS of 10d

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

239

234

73

353240

322224

45 223 354241 32319135559

S51

2.5.5.1) 1H NMR (500 MHz, CD3OD, 300 K) of 3a

3.54.04.55.05.56.06.57.0 ppm

3.3100

3.4471

4.8369

5.2711

6.8212

6.8384

7.2957

7.3129

3.0

1.0

2.0

2.0

2.5.5.2) 13C NMR (126 MHz, CD3OD, 300 K) of 3a

5060708090100110120130140150160 ppm

49.0000

57.1497

72.8086

116.6038

118.8620

126.1151

130.0989

160.0147

S52

2.5.5.3) EIMS of 3a

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

132

163

12177 13316213751 76 104 1649465 7850 105

2.5.6.1) 1H NMR (500 MHz, CD3OD, 300 K) of 3b

3.54.04.55.05.56.06.57.0 ppm

3.3100

3.4479

3.8564

4.8113

5.2612

6.8281

6.8443

6.9279

6.9440

7.0137

3.0

3.0

1.0

1.0

1.0

1.0

S53

2.5.6.2) 13C NMR (126 MHz, CD3OD, 300 K) of 3b

5060708090100110120130140150 ppm

49.0000

56.4418

57.2103

72.9299

111.8986

116.2667

118.8350

121.6728

126.5532

149.1080

149.3035

2.5.6.3) EIMS of 3b

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

162

193

43 147 163119 161

19416712470 19264 91 133109

S54

2.5.7.1) 1H NMR (500 MHz, CD3OD, 300 K) of 3c

3.54.04.55.05.56.06.5 ppm

3.3100

3.4156

4.8212

5.1864

6.7999

6.8017

6.9132

3.0

1.0

2.0

1.0

2.5.7.2) 13C NMR (126 MHz, CD3OD, 300 K) of 3c

5060708090100110120130140150 ppm

49.0000

57.1294

72.8288

115.4915

116.3139

118.8417

120.4258

126.5263

146.7420

147.8542

S55

2.5.7.3) EIMS of 3c

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

148

179

149137102

16175 180119130110 17851 63 81

2.5.8.1) 1H NMR (500 MHz, CD3OD, 300 K) of 3d

3.54.04.55.05.56.06.5 ppm

3.3100

3.4424

3.8531

4.8318

5.2260

6.6056

6.6092

6.6213

6.6254

3.0

3.0

1.0

2.0

S56

2.5.8.2) 13C NMR (126 MHz, CD3OD, 300 K) of 3d

60708090100110120130140150 ppm

49.0000

56.7115

57.1093

73.0783

103.9715

109.4181

118.8418

125.7713

136.6241

146.9241

149.8361

2.5.8.3) EIMS of 3d

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

178

209

179135

163148

167 194 210129119

S57

2.6) Gas Chromatograms and Mass Spectra of the Oxidation Experiments with 3a�3d

and Activated MnO2 in Aqueous MeOH

2.6.1.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products of 3a

with Activated MnO2 in H2O/MeOH 1:1 with 1 % AcOH

15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0Time (min)

020406080

100

020406080

100

Rel

ativ

e A

bund

ance

020406080

100RT: 18.18Area: 505275698

RT: 18.12Area: 156786358

RT: 15.97Area: 171908

RT: 18.22Area: 681386992

RT: 15.95Area: 7025295

180 min

24 h

See section 2.5.1.3 for the mass spectrum of pertrimethylsilylated 3a = 10a

2.6.1.2) GC-MS of Pertrimethylsilylated 4a

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

209

224

193

135 177210

8940 149 22573 915944 97

0 h

3a 4a

S58

2.6.2.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products

of 3b with Activated MnO2 in H2O/MeOH 1:1 with 1 % AcOH

15 16 17 18 19 20 21 22 23 24 25Time (min)

020406080

100

020406080

100

Rel

ativ

e A

bund

ance

020406080

100RT: 21.28Area: 1997918851

RT: 21.08Area: 121093421

RT: 19.55Area: 1521248

RT: 21.03Area: 8336894

RT: 19.53Area: 1161551

180 min

24 h

See section 2.5.2.3 for the mass spectrum of pertrimethylsilylated 3b = 10b

2.6.2.2) GC-MS of Pertrimethylsilylated 4b

40 60 80 100 120 140 160 180 200 220 240 260 280 300m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

224

239193

254

40 223 22524073 194

137 226165 25545 59 89 104 163 195 241135 149 17975 119

0 min

3b 4b

S59

2.6.3.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products

of 3c with Activated MnO2 in H2O/MeOH 1:1 with 1 % AcOH

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0Time (min)

050

100

050

100

050

100

050

100

Rel

ativ

e A

bund

ance

050

100

050

100RT: 22.36Area: 24726187

RT: 22.40Area: 72040487RT: 21.11

Area: 28399685 RT: 23.10Area: 802278

RT: 21.16Area: 66364091 RT: 22.40

Area: 61226786RT: 23.11Area: 4351844

RT: 21.16Area: 54402586 RT: 22.34

Area: 13960012 RT: 23.11Area: 2756745

RT: 21.18Area: 92018482

RT: 22.31Area: 2624749

RT: 23.10Area: 397815

RT: 21.13Area: 31664638

RT: 23.10Area: 148229

1 min

5 min

10 min

30 min

40 min

See section 2.5.3.3 for the mass spectrum of pertrimethylsilylated 3c = 10c

2.6.3.2) GC-MS of Pertrimethylsilylated 4c

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

193

73

312194

16545 195137 313281224163 207133 235

3c 4c 5c 0 min

S60

2.6.3.3) GC-MS of Pertrimethylsilylated 5c

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

322

193

73323

234

239 353324194

31216545 219 354137 195 24074 100 180 327163 28159

S61

2.6.4.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products of 3d

with Activated MnO2 in H2O/MeOH 1:1 with 1 % AcOH

22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

RT: 25.02Area: 709390339

RT: 24.00Area: 21993971 RT: 25.28

Area: 7356272

RT: 24.00Area: 47462746

RT: 25.25Area: 3978967

1 min

See section 2.5.4.3 for the mass spectrum of pertrimethylsilylated 3d = 10d

2.6.4.2) GC-MS of Pertrimethylsilylated 4d

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

223

73

224342

19545 225 34313775 133 254 32719389 31159

3d 4d 5d 0 min

S62

2.6.4.3) GC-MS of Pertrimethylsilylated 5d

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

223

73352

224342

264269 353

195 38345 225254137 35413374 31159 193

S63

2.7) Gas Chromatograms and Mass Spectra of the Oxidation Experiments with 3a�3d

and Activated MnO2 in MeOH

2.7.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products of 3a

with Activated MnO2 in MeOH

15 16 17 18 19 20 21 22 23 24 25Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

RT: 18.23Area: 1152516773

RT: 18.08Area: 162546834 24 h

See section 2.5.1.3 for the mass spectrum of pertrimethylsilylated 3a = 10a

0 h

3a

S64

2.7.2.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products of 3b

with Activated MnO2 in MeOH

15 16 17 18 19 20 21 22 23 24 25Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

RT: 21.16Area: 293722528

RT: 19.53Area: 11901014 RT: 22.00

Area: 2560051

RT: 21.06AA: 38084958

RT: 19.53Area: 2727384 RT: 22.01

Area: 380037

24 h

See section 2.5.2.3 for the mass spectrum of pertrimethylsilylated 3b = 10b

See section 2.6.2.2 for the mass spectrum of pertrimethylsilylated 4b

2.7.2.2) GC-MS of Pertrimethylsilylated 5b

50 100 150 200 250 300 350m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

264

265

234

73224193 264

219 295165 204 280149 235182916952 107 207 297

0

0 h

3b

4b 5b

S65

2.7.3.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation

Products of 3c with Activated MnO2 in MeOH

20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0Time (min)

0

100

0

100

0

100

0

100

Rel

ativ

e A

bund

ance

0

100

0

100

0

100

RT: 22.35Area: 345531819

RT: 22.40Area: 525709803 RT: 23.09

Area: 98721281RT: 21.06Area: 4911016

RT: 22.40Area: 358747709 RT: 23.13

Area: 165811594RT: 21.06Area: 6988303

RT: 21.86Area: 4449290

RT: 22.36Area: 231698530

RT: 23.13Area: 186152509

RT: 21.86Area: 5501966

RT: 21.06Area: 4958193

RT: 23.15Area: 256411553

RT: 22.35Area: 157310943RT: 21.86

Area: 8659152RT: 21.06Area: 5998697

RT: 23.15Area: 213697429

RT: 22.31Area: 68265552RT: 21.86

Area: 9844044RT: 21.06Area: 4677733

RT: 23.16Area: 334742301

RT: 21.86Area: 22678444

RT: 22.28Area: 10606476RT: 21.06

Area: 7576231

5 min

10 min

20 min

40 min

60 min

120 min

See section 2.5.3.3 for the mass spectrum of pertrimethylsilylated 3c = 10c

See section 2.6.3.2 for the mass spectrum of pertrimethylsilylated 4c

See section 2.6.3.3 for the mass spectrum of pertrimethylsilylated 5c

2.7.3.2) GC-MS of Pertrimethylsilylated 11c

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

193

327

73

328312194

23945 165 329195

137 313105 224 358281179 20759 163 240 359

3c 4c 5c 11c 0 min

S66

2.7.4.1) Gas Chromatograms of Pertrimethylsilylated Samples of the Oxidation Products of 3d

with Activated MnO2 in MeOH

23.8 24.0 24.2 24.4 24.6 24.8 25.0 25.2 25.4Time (min)

0

50

100

0

50

100

0

50

100

Rel

ativ

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bund

ance

0

50

100

RT: 24.86Area: 93394951

RT: 25.24Area: 1429288RT: 23.99

Area: 991406

RT: 24.84Area: 50950055

RT: 25.28Area: 46500166

RT: 23.99Area: 5002973

RT: 25.26Area: 21878293

RT: 24.79Area: 10891854RT: 23.99

Area: 3454348

RT: 25.24Area: 1065066

RT: 23.98Area: 425388

5 min

10 min

20 min

RT: 24.09Area: 1272961

RT: 24.09Area: 383685

See section 2.5.4.3 for the mass spectrum of pertrimethylsilylated 3d = 10d

See section 2.6.4.2 for the mass spectrum of pertrimethylsilylated 4d

See section 2.6.4.3 for the mass spectrum of pertrimethylsilylated 5d

2.7.4.2) GC-MS of Pertrimethylsilylated 11d

50 100 150 200 250 300 350 400m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

223

73

40 357224

34245 358269195 25475 2258959 133105 193

0388

3d 5d 4d

0 min

11d

S67

2.8) NMR Spectra of a mixture of 3d and the E and Z Isomer of the Quinonemethide 6d

1H NMR (600 MHz, 300 K, CD3CN) of a mixture of E-6d, Z-6d and 3d

2.53.03.54.04.55.05.56.06.57.07.5 ppm

1.93

00

2.14

68

3.42

65

3.78

343.

8002

3.83

574.

0271

4.03

77

5.14

24

6.53

736.

5406

6.60

896.

6122

6.62

856.

6316

6.67

306.

6765

6.79

416.

7976

6.86

286.

8663

3.00

0.60

0.60

3.00

0.60

0.60

1.00

0.20

1.00

1.00

0.20

0.20

0.20

13C NMR (126 MHz, 300 K, CD3CN) of a mixture of E-6d, Z-6d and 3d

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

1.30

00

56.5

064

56.6

413

56.9

682

57.5

412

61.2

387

72.6

649

103.

0606

103.

0842

103.

3033

104.

0212

107.

0614

107.

1693

109.

0770

113.

1386

118.

2921

118.

7371

126.

0849

135.

5629

145.

7824

149.

1260

150.

7911

176.

6028

176.

6197

S68

COSY (500 MHz, 300 K, CD3CN) of a mixture of E-6d, Z-6d and 3d

ppm

6.56.66.76.86.97.0 ppm

6.45

6.50

6.55

6.60

6.65

6.70

6.75

6.80

6.85

6.90

6.95

7.00

S69

HSQC (600 MHz, 300 K, CD3CN, aliphatic region) of a mixture of E-6d, Z-6d and 3d

ppm

3.43.63.84.04.24.44.64.85.05.2 ppm

56

58

60

62

64

66

68

70

72

74

S70

HSQC (600 MHz, 300 K, CD3CN, aromatic region) of a mixture of E-6d, Z-6d and 3d

ppm

6.456.506.556.606.656.706.756.806.856.906.957.00 ppm

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

S71

HMBC (600 MHz, 300 K, CD3CN) of a mixture of E-6d, Z-6d and 3d

ppm

3.54.04.55.05.56.06.57.0 ppm

60

70

80

90

100

110

120

130

140

150

160

170

S72

HMBC (600 MHz, 300 K, CD3CN, aromatic region) of a mixture of E-6d, Z-6d and 3d

ppm

6.46.56.66.76.86.97.0 ppm

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

S73

NOESY (600 MHz, 300 K, CD3CN) of a mixture of E-6d, Z-6d and 3d

ppm

4.04.55.05.56.06.57.07.5 ppm

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

S74

2.9) HPLC Chromatogram of the Enzymatic Conversion of 1 to 2 with Mushroom

Tyrosinase

Incubation time: 30 min.

1 2 12