SUPPLEMENTARY MATERIAL

Alternaria toxins in South African sunflower seeds: Cooperative study

Sebastian Hickert†, ‡, Lena Hermes†, Lucas Maciel Mauriz Marques§, Christine Focke†, Benedikt

Cramer†, Norberto Peporine Lopes§, Bradley Flett∥, ⊥ and Hans-Ulrich Humpf*†, ‡

†Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45,

48149 Münster, Germany.

‡NRW Graduate School of Chemistry, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany.

§Departamento de Física-Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,

Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.

∥ARC-Grain Crops Institute, Private Bag X1251, Potchefstroom 2520, South Africa.

⊥Unit of Environmental Sciences and Management, North-West University, Private Bag

X6001, Potchefstroom 2520, South Africa.

*Corresponding author (Tel: +49 251 83 33391; Fax: +49 251 83 33396; E-mail:

humpf@wwu.de)

1

Production of Alternariol mono methyl ether-3-O-ß-D-glucoside (AME-3G)

A solution of 0.41 mg AME in 150 µL MeCN was added to 40 mL potato dextrose medium

which had been incubated with Rhizopus oryzae (DSM 907, German Collection of

Microorganisms and Cell Cultures, Braunschweig, Germany) for five days at 30 °C on a

shaking incubator at 170 rpm. The mixture was incubated for three further days under the

same conditions. Afterwards the mycelium was separated from the aqueous phase by

filtration and the mycelium extracted with MeCN. The aqueous phase and the MeCN phase

were combined and the MeCN removed using a rotary evaporator at 40 °C water bath

temperature. Prior to preparative HPLC the raw extract (approximately 30 mL) was

fractionated on a solid phase extraction (SPE) column (Strata C18-E with 10 g sorbens, 60 mL,

Phenomenex, Aschaffenburg, Germany) with attached water pump jet. The SPE column was

activated with 50 mL MeCN followed by 50 mL H2O. Afterwards the raw extract was loaded

on the SPE column and fractionated into eight fractions (50 mL each) with increasing MeCN

percentage (0% MeCN, 10%, 20%, 25%, 30%, 35%, 40% and 100%) which were checked for

the presence of AME-3G and AME by HPLC-DAD (Jasco X-LC, Jasco Labor- und Datentechnik,

Groß-Umstadt, Germany with X-LC 3159AS autosampler, DG-2080-53 degasser, PU-2085

Plus pump, LG-2080-02S gradient mixer, and MD-2010 Plus detector). An Eclipse XDB-C18

column (150 x 4.6 mm, 5 µm, Agilent Technologies, Waldbronn, Germany) was operated at a

flow rate of 1.0 mL/min with MeCN (1% FA, mobile phase A) and H2O (1% FA, mobile phase

B) as eluents. The injection volume was 10 µL. The gradient started at 20% A for 2.0 min, was

increased to 100% A at 11.0 min, held for 2 min and the column was equilibrated for 2 min

prior to the next injection. AME and AME-3G were monitored at 256 nm. AME-3G eluted

after 6.5 min and AME after 8.4 min. The UV maxima of AME-3G recorded simultaneously

2

with the DAD detector were 256, 254 and 330 nm which match those of AME (256, 254,

330 nm).

The SPE fraction containing AME-3G (35% MeCN) was reduced to approx. 3 mL using a rotary

evaporator. 500 µL aliquots were subjected to preparative HPLC (Degasys Populaire DP4010

degasser (VDS Optilab, Montabaur, Germany), two Jasco PU 2086/2087 pumps with 2 mL

injection loop and UV-2070/2075 detector (256 nm), Eclipse XDB-C18, 250 mm x 9.4 mm,

5 µm column at 4 mL/min isocratically with MeCN/H2O (2:3, v/v)). AME-3G eluted after

5.2 min and the peaks for AME-3G of all HPLC runs were collected, the MeCN evaporated

with a rotary evaporator and the aqueous residue freeze dried. The complete amount of

AME-3G was subjected to 1H-NMR (DD2 600 MHz, Agilent, Santa Clara, CA, USA) in

perdeuterated dimethyl sulfoxide (see Figure S2 for the structure of AME-3G with numbered

atoms).

(DMSO-d6): δ [ppm]: 11.79 (1H, 7-OH), 7.31 (d, J = 1.9 Hz, 1H, 10-CHaromat), 6.99 (d, J = 4.0 Hz,

1H, 4-CHaromat), 6.98 (d, J = 2.4 Hz, 1H, 2-CHaromat), 6.69 (d, J = 1.9 Hz, 1H, 8-CHaromat), 5.03 (d,

J = 7.4 Hz, 1H, 1´-CH), 3.93 (3H, 12-CH3), 3.71 (1H, 6´-CH), 3.45 (1H, 6´-CH), 3.44 (1H, 5´CH),

3.16 - 3.28 (2´-CH (1H), 3´-CH (1H), 4´-CH (1H)), 2.81 (3H, 11-CH3).

The spectroscopic data are consistent with data for synthetic AME-3G (Mikula et al. 2013).

DMSO was removed at 40 °C in a stream of nitrogen. As the amounts of isolated AME-3G

were too low to be weighed the concentration was estimated after enzymatic cleavage to

AME. To that end all AME-3G recovered from the NMR tube was dissolved in MeOH (4 mL).

All incubations were as well carried out with AME as well to ensure that no degradation of

the parent toxin occurs. 50 µL of the solution of AME-3G were incubated with 20 mU/mL

ß-glucosidase (from almond, ≥ 2 U/mg, Sigma Aldrich, Steinheim, Germany) in 50 mM

3

sodium phosphate buffer (pH 7.4, total volume 100 µL) in a 2 mL polypropylene reaction

tube at 37 °C for four hours. The reaction mixture was checked for remaining AME-3G and

the amount of AME quantified by external calibration (0.5 - 10 µg/mL AME, six calibration

points) by HPLC-DAD (method as described above). After the incubation AME-3G was not

detectable and the control incubation with AME showed no decrease. The same incubations

were performed with α-glucosidase (from Saccharomyces cerevisiae, 19.3 U/mg, Sigma

Aldrich, Steinheim, Germany) as well to confirm the ß-configuration of AME-3G. As to be

expected the amount of AME-3G was constant in these experiments. The concentration

calculated of AME-3G thus was 44.7 µg/mL.

High Resolution Mass Spectrometry (HRMS, instrument: LTQ-Orbitrap XL (Thermo Scientific,

Dreieich, Germany) with an Ion Max Heated Electrospray Ionization (HESI) source coupled

with a Nexera HPLC system (Shimadzu, Duisburg, Germany) with a SIL-20AXR autosampler, a

DGU-20A5R degasser, a CBM-20A communications bus module and a CT0-10ASVP column

oven) was used to confirm the exact mass and sum formula of AME-3G. Ten µL of the toxin

solution diluted with H2O (1+1, v/v) were injected on a Nucleodur C18 HTech column

(100 x 2 mm, 3 µm, Macherey Nagel) equipped with a Universal RP guard column (2 x 4 mm,

Macherey Nagel) which was operated at 40 °C. The applied gradient consisted of MeCN (1%

FA, eluent A) and H2O (1% FA, eluent B) and started at 250 µL/min and 10% A. The

proportion of A was increased to 100% at 20 min, held for 5 min and the column was

equilibrated with the starting conditions for 5 min. The HRMS parameters were 3.0 kV spray

voltage, -35 kV capillary voltage, 220 °C capillary temperature, -110 V tube lens voltage,

350 °C vaporizer temperature, 40 arbitrary units (arb) sheath gas flow rate, 20 arb aux gas

flow rate, 5 arb sweep gas flow rate and a resolution of 60000. A full spectrum was recorded

in the range m/z = 90-1000. The deprotonated molecular ion (Rt = 15.41 min) found [M-

4

H]- = m/z 433.1138 and the even more intense formiate adduct [M+CH2O2-H]- = m/z 479.1195

match the calculated exact masses (433.1135, ∆ 0.91 ppm / 479.1190 ∆ 1.04 ppm) and the

sum formula of C21H22O10.

The purity of AME-3G was calculated by HPLC coupled to evaporative light scattering

detection (ELSD) and found to be ≥ 99.5%. 10 µL of the toxin solution were injected into the

HPLC-system (Shimadzu, Tokyo, Japan with SIL-20A autosampler, DGU-20A3 pump and ELSD-

LT detector). The column used was an Eclipse XDB-C18 column (150 x 4.6 mm, 5 µm, Agilent

Technologies, Waldbronn, Germany) with an Universal RP guard column (4 x 3 mm,

Macherey Nagel) operated at 1 mL/min with MeCN (A) and H2O (B) as mobile phases. The

gradient started at 5% A for 5 min, was changed to 100% A at 20 min, kept constant for

5 min and the column was equilibrated 5 min prior to the next injection. The ELSD

parameters were a temperature of 40 °C and 2.5 bar compressed air.

5

Isolation of valine-tenuazonic acid

Valine-tenuazonic acid (Val-TeA) was isolated from crude extracts of Alternaria alternata

grown on agar plates (DSM 12633, German Collection of Microorganisms and Cell Culture,

Braunschweig, Germany). For culture conditions see our recent publication (Hickert et al.

2016). The ethyl acetate extract was evaporated to dryness using a rotary evaporator at 40°C

water bath temperature and dissolved in 20 mL MeOH/H2O (1+4, v/v). Solid remainders

were removed by centrifugation and the solution was checked for the presence of

tenuazonic acid analogues by HPLC-MS/MS in negative ionization mode (1200 series HPLC

(Agilent, Waldbronn, Germany) coupled to an API 3200 mass spectrometer (Sciex,

Darmstadt, Germany)). The SRM transitions for all possible amino-acid analogues were

calculated based on the proposed fragmentation pathway (Asam et al. 2013) for TeA. All

other settings were based on the parameters of TeA which were optimized after syringe

pump infusion (1 µg/mL, MeCN/H2O, 1+1, v/v, 7 µL/min). The parent masses used can be

found in Table S3. The fragments used were m/z = 138.7, 112.0 and 82.9. A declustering

potential of -50 V, collision energies of -30 V and collision cell entry and exit potentials of -

10 V were used with a dwell time of 10 ms per SRM transition. The source temperature was

400 °C, a curtain gas flow of 2.06 x 105 Pa zero air and nebulizer gas pressures of 2.41 x 105

and 3.10 x 105 Pa were used. The collision gas pressure was 1.33 x 10-2 Pa. 10 µL of the

extract were injected on a Synergi Hydro-RP column (50 x 2 mm, 2.5 µm, Phenomenex) with

a KrudCatcher filter (Phenomenex) operated at 50 °C. The gradient (MeCN 1% FA (A) and

H2O 1% FA (B)) started at 5% A at 450 µL/min for 1 min, increased to 35% A and 500 µL/min

at 4 min, was kept constant for 1.5 min, increased further to 100% A and 600 µL/min at

7 min for 1.5 min. The column was equilibrated for 2 min prior to the next injection. The first

3 min of each run were diverted to waste to avoid unnecessary contamination of the ion

6

source. The only detectable analyte besides TeA (tR = 5.5 min) was Val-TeA. (tR = 4.7 min).

The raw extract was fractionated on a SPE column (see isolation of AME-3G) using different

proportions of MeCN and H2O (both 1% FA). The SPE column was activated with 40 mL

MeCN followed by 40 mL H2O. After the extract had been loaded on the column, fractions

with an increasing proportion of MeCN (5, 10, 30, 40, 50, 60, 70, 80, 90, 100% MeCN, 40 mL

each) were collected and analyzed by HPLC-MS/MS as described above. The fraction

containing both TeA and Val-TeA (30% MeCN, note: the 40% MeCN fraction contained more

TeA than the 30% fraction but only small amounts of Val-TeA) was evaporated to dryness

using a rotary evaporator and dissolved in 4 mL MeOH/H2O (1+1, v/v). Aliquots of 500 µL

were subjected to preparative HPLC (for apparatus see isolation of AME-3G). A Supelco

Ascentis RP-Amide column (250 x 10 mm, 5 µm, Sigma Aldrich, Steinheim, Germany) was

operated at a flow rate of 3.5 mL/min. A linear binary gradient with MeOH (eluent A) and

H2O (eluent B, both 1% FA) starting at 30% A for 3 min was applied. The proportion of A was

increased to 100% A at 13 min and the column was equilibrated for 3.5 min prior to the next

injection. The UV-detector was set to 277 nm. Peaks of Val-TeA (tR = 8.7 min) and TeA

(tR = 10.0 min) were collected separately, neutralized with 10% aqueous NH3 and the organic

solvent removed using a rotary evaporator. After freeze drying the remainder was extracted

twice with 4 mL CHCl3 to separate TeA and Val-TeA from ammonium formate salts. The CHCl3

extracts were evaporated to dryness in a stream of N2 at 40 °C. TeA was obtained as a yellow

oil while Val-TeA was an orange oil. The complete amount of Val-TeA and 20 mg of TeA (total

amount about 240 mg) were dissolved in 600 µL perdeuterated chloroform (CDCl3) and used

for 1H-NMR measurements (DPX-400, Bruker, Rheinstetten, Germany). 1H-NMR spectra for

Val-TeA as well as for TeA show signals for both enol- as well as keto-forms of both

7

substances (see Figure S1). The major form of TeA is reported to be the keto-tautomer with

about 80% (Steyn and Wessels 1978).

1H-NMR Val-TeA, keto-form (400 MHz, CDCl3): δ [ppm]: 7.02 (1H, 1-NH), 3.74 (1H, d,

J = 3.5 Hz, 5-CH), 2.47 (3H, 7-CH3), 2.29 - 2.16 (1H, m, 8-CH), 1.04 (3H, d, J = 7.0 Hz, 9- or 10-

CH3), 0.84 (3H, d, J = 6.8 Hz, 9- or 10-CH3).

1H-NMR Val-TeA, enol-form (400 MHz, CDCl3): δ [ppm]: 6.78 (1H, 1-NH), 3.90 (1H, d,

J = 3.9 Hz, 5-CH), 2.50 (3H, 7-CH3), 2.29 - 2.16 (1H, m, 8-CH), 0.88 (3H, d, J = 6.7 Hz, 9- or 10-

CH3). The duplet expected around 1 ppm is only visible as a highfield shifted shoulder in the

signal at 1.04 ppm of the keto-form.

Comparison of the 5-CH signals (3.74/3.90 ppm) and 7-CH3 signals (2.47/2.50 ppm) allows to

estimate the distribution of both tautomers. Depending on the proton chosen, proportions

of 24.5 ± 1.1% of the enol are present which is consistent with other tetramic acids (Steyn

and Wessels 1978).

For comparative reasons, an aliquot of the isolated TeA alongside to Val-TeA was analyzed as

well:

1H-NMR TeA, keto-form (400 MHz, CDCl3): δ [ppm]: 7.58 (1H, 1-NH), 3.76 (1H, d, J = 3.3 Hz, 5-

CH), 2.42 (3H, 7-CH3), 1.98 – 1.90 (1H, m, 8-CH), 1.39 – 1.33 (1H, m, 9-CH), 1.25 – 1.14 (1H,

m, 9-CH´), 0.99 (3H, d, J = 6.9 Hz, 11-CH3), 0.86 (3H, t, J = 7.2 Hz, 10-CH3).

1H-NMR TeA enol-form (400 MHz, CDCl3): δ [ppm]: 7.52 (1H, 1-NH), 3.94 (1H, d, J = 3.4 Hz, 5-

CH), 2.47 (3H, 7-CH3), 0.94 (3H, t, J = 7.5 Hz, 10-CH3), 0.75 (3H, d, J = 6.8 Hz, 11-CH3). The

multiplet signals for 8-CH, 9-CH and 9-CH´ protons were overlaid by those of the dominant

keto-form.

8

Described comparison of keto- and enol-form protons leads to 22.2 ± 1.0% of the enol form.

Besides signals for the tautomeric keto- and enol-forms of TeA further signals for the epimer

of TeA, allo-tenuazonic acid (allo-TeA) are detectable for both tautomers of allo-TeA. Most

signals are overlaid by signals for the predominant epimer TeA but the signal for 7-CH3 is

clearly visible at 3.85 ppm (keto-form, d, J = 2.8 Hz) and 4.02 ppm (enol-form, d, J = 2.2 Hz).

Comparison of the signals for both tautomers of allo-TeA indicates that 21.2% of the enol

form are present. As only the protons of 7-CH3 can be used, no standard deviation can be

calculated. Additionally, the 7-CH3 signals for both keto-forms of TeA and allo-TeA can be

compared to estimate the proportion of allo-TeA present in the sample. This leads to 18.9%

of allo-TeA of the sum of both epimers. Note: As the amino acid residue of Val-TeA contains

no stereocenter, there is no allo-valine-tenuazonic acid (see Figure S1). The remaining

stereocenter at C5 theoretically allows two enantiomers ((5S)-3-acetyl-5-isopropyl-tetramic

acid and (5R)-3-acetyl-5-isopropyl-tetramic acid). As enantiomers are neither distinguishable

by conventional NMR spectroscopy nor by achiral HPLC, the configuration of the proton at

C5 cannot be determined. The NMR solvent was evaporated to dryness and the amount of

Val-TeA determined gravimetrically to be 32.4 mg. The molecular formula of Val-TeA was

confirmed by HPLC-HRMS in positive ionization mode (see isolation of AME-3G for

apparatus). 15 µL (30 µg/mL Val-TeA, MeOH/H2O, 1:1, v/v) were injected on a Synergi Hydro-

RP column (50 x 2 mm, 2.5 µm, Phenomenex) with a KrudCatcher filter (Phenomenex). A

linear binary gradient with MeOH (1% FA, eluent A) and H2O (1% FA, eluent B) at 450 µL/min

and a column oven temperature of 50 °C was used. The gradient started at 2.5 % A for 3 min,

was increased to 97.5% A at 10 min which were held constant for 2 min. The column was

equilibrated for 3 min prior to the next injection. The HRMS parameters were 3.5 kV spray

voltage, 20 kV capillary voltage, 380 °C capillary temperature, 150 V tube lens voltage, 400 °C

9

vaporizer temperature, 40 arbitrary units (arb) sheath gas flow rate, 20 arb aux gas flow rate,

10 arb sweep gas flow rate and a resolution of 30000. A full spectrum was recorded in the

range m/z = 75-750. HPLC-HRMS analysis of Val-TeA (Rt = 6.6 min) shows predominantly

complex ions of two molecules Val-TeA and one metal ion. The most prominent ions are

m/z = 420.0968 and 405.1324 which correspond to the ions [2M+Fe III-2H]+ (calculated

m/z = 420.0984, ∆ = 1.0 ppm) and [2M+CaII-H]+ (calculated m/z = 405.1339, ∆ = 0.9 ppm)

while only small amounts of the protonated molecule [M+H]+ are visible with m/z = 184.0964

(calculated m/z = 184.0974, ∆ = 0.7 ppm) which all fit to the sum formula of C9H13NO3 for Val-

TeA. These unusual complex ions are the most prominent ions in HPLC-HRMS of TeA

(tR =7.4 min) as well as the most intense ions found were m/z = 433.1641 and 448.1285

which as well correspond to the ions [2M+FeIII-2H]+ (calculated m/z = 448.1297, ∆ = 0.6 ppm)

and [2M+CaII-H]+ (calculated m/z = 433.1651, ∆ = 0.5 ppm) while only small amounts of the

protonated molecule [M+H]+ are visible with m/z = 198.0534 (calculated m/z = 198.1130,

∆ = 0.2 ppm). All three ions suggest the molecular formula of C10H15NO3.

The purity of Val-TeA was calculated after HPLC-DAD analysis (for apparatus see the HPLC-

DAD analysis of AME-3G). 10 µL of a 50 µg/mL solution in MeOH/H2O (1:1, v/v) were injected

on a Nucleodur Phenyl-Hexyl column (150 x 2 mm, 3 µm, guard column of the same material

(2 x 4 mm), Macherey Nagel, Düren, Germany) with a linear binary gradient using MeCN

(1% FA, eluent A) and H2O (1% FA, eluent B) at 1 mL/min. The gradient started at 5% for one

minute, was linearly increased to 100% A at 15 min, held constant for 5 min and afterwards

the column was equilibrated 7 min. Val-TeA eluted after 13.8 min and the purity was > 95%

on all wavelengths. The UV-absorption maximum for Val-TeA was 277 nm. The molar

absorptivity value in MeCN was determined in quintuplicate as described (Hickert et al.

2015) and found to be (1.250 ± 0.013) x 104 L/mol/cm at 277 nm.

10

11

Isolation of altenuisol and altenusin

Altenuisol (ALTSOH) and altenusin (ALTS) were isolated from culture extracts of Alternaria

alternata from a previous study as described above for Val-TeA. For ALTSOH and ALTS,

cultures of another strain (MRI 1277, Max Rubner Institute, Karlsruhe, Germany) on rice

were used. See the recent publication for culture conditions (Hickert et al. 2016). The

MeOH/acetone extract (1:1, v/v) was evaporated to dryness and treated as described for

Val-TeA. The described HPLC-MS/MS method was extended with further SRMs for the not

yet isolated compounds altenusin (Vishwanath et al. 2009), altertoxin II, stemphylotoxin II,

altertoxin III and altenuisol (Liu. and Rychlik 2015) from literature using mass spectrometers

comparable to the one used in this study. The additional MS/MS parameters in negative

ionization mode are displayed in Table . Besides a small signal for altertoxin II, intense signals

for ALTS (Rt = 5.8 min) and ALTSOH (Rt = 6.4 min) were detected. These peaks were further

investigated using product ion spectra in a range of m/z = 50-300 with the same

instrumental setup as described and a collision energy of -30 V. Fragment ions for ALTS were

m/z = 271.1, 255.6, 244.7, 229.9, 212.1, 202.0, 173.8, 160.0, 145.5 and 69.9 and for ALTSOH

m/z = 257.8, 229.9, 200.8, 186.0, 173.7, 161.2 and 158.2. After SPE fractionation, the

fractions containing ALTS (30 and 40% MeCN) and ALTSOH (50% MeCN) were evaporated,

dissolved in 4 mL MeOH/H2O (1:1, v/v) and subjected to preparative HPLC (for apparatus see

isolation of AME-3G). A Reprosil-Pur 120 C18-AQ column (250 x 10 mm, 5 µm, 5 x 2 mm

guard column of the same material, Dr. Maisch, Ammerbuch-Entringen, Germany) was

operated at a flow rate of 2.5 mL/min and 256 nm for the purification of ALTSOH and

296 nm for ALTS. The gradient (MeCN (A)/H2O (B)) for ALTSOH started at 50% A for 1 min,

was increased to 100% A at 20 min, held for 2 min and the column was equilibrated for

4 min. For ALTS, the starting conditions were 62.5% A for 3 min which were increased to 70%

12

A at 15 min and increased further to 100% A at 16 min for 3 min. Afterwards the column was

equilibrated for 5 min. The peaks for ALTS (tR = 16.5 min) and ALTSOH (tR = 13.6 min) were

collected, the organic solvent removed using a rotary evaporator and the residue freeze

dried. The complete amount of ALTSOH was subjected to 1H-NMR (DPX-400, Bruker,

Rheinstetten, Germany) in perdeuterated acetone (acetone-d6, 400 MHz): δ [ppm]: 8.73 (1H,

OH), 7.58 (1H, 1-CH), 7.06 (1H, d, J = 2.2 Hz, 10-CH), 6.85 (1H, 4-CH), 6.52 (1H, d, J = 2.3 Hz, 8-

CH), 3.99 (3H, O-CH3). The data are consistent with literature data (Nemecek et al. 2012).

The NMR solvent was removed in a stream of N2 at 30 °C and the remainder dissolved in

4 mL MeOH. HPLC-HRMS analysis of ALTSOH (Rt = 8.0 min) using the same parameters as

described for Val-TeA gives the protonated molecule [M+H]+ with m/z = 275.0545 as the

predominant ion formed which matches the sum formula C14H10O6 (calculated

m/z = 275.0556, ∆ = 0.5 ppm). The purity of ALTSOH was calculated after HPLC-DAD analysis

(for apparatus see the HPLC-DAD analysis of Val-TeA). 10 µL of the diluted toxin solution

(1:1, v/v with H2O) were injected on a Synergi Hydro-RP column (50 x 2 mm, 2.5 µm, with

KrudCatcher filter, Phenomenex) with a linear binary gradient with MeCN (1% FA, eluent A)

and H2O (1% FA, eluent B) at 300 µL/min. The gradient started at 5% A for 3 min, was linearly

increased to 100% A at 20 min, held constant for 2 min and afterwards the column was

equilibrated 3 min. ALTSOH eluted after 13.8 min and the purity was > 95% on all

wavelengths. The UV-absorption maxima for ALTSOH were 217, 256 and 346 nm. As the

amount of ALTSOH was too low to be determined gravimetrically, it was calculated using the

molar absorptivity values of 4.677 x 104 L/mol/cm at 256 nm and 1.585 x 104 L/mol/cm at

345 nm from literature (Zwickel et al. 2016). Depending on the wavelength used, a

concentration of 137.7 ± 1.8 µg/mL was calculated. An aliquot was diluted to 100 µg/mL.

13

The amount of isolated ALTS was determined gravimetrically and found to be 17.6 mg.

5.0 mg of the isolated product ALTS were dissolved in 600 µL acetone-d6 and used for 1H-

NMR analysis (DPX-400, Bruker, Rheinstetten, Germany).

1H-NMR of ALTS (acetone-d6, 400 MHz): δ [ppm]: 11.65 (1H, OH), 8.73 (1H, OH), 7.58 (1H, 3´-

CH), 7.06 (1H, d, J = 2.1 Hz, 4-CH), 6.85 (1H, 6´-CH), 6.52 (1H, d, J = 2.2 Hz 6-CH), 3.99 (3H, O-

CH3). The signal for the C-2´ methyl group to be expected around 2 ppm is overlaid by the

solvent signal. The data are consistent with literature (Nakanishi et al. 2014). The purity of

ALTS was calculated after HPLC-DAD analysis (for apparatus see the HPLC-DAD analysis of

AME-3G). 10 µL (50 µg/mL, MeOH/H2O, 1:1, v/v) were injected on a ReproSil-Pur C18 AQ

column (150 x 4 mm, 3 µm, 4 x 2 mm guard column of the same material, Dr. Maisch,

Ammerbuch-Entringen, Germany) with a linear binary gradient with MeCN (1%FA, eluent A)

and H2O (eluent B) at 1 mL/min. The gradient started at 20% A at 1 mL/min for 1 min, was

linearly increased to 100% A at 23 min, held constant for 4 min and afterwards the column

was equilibrated for 3 min. ALTS eluted after 8.1 min and the purity was > 95% on all

wavelengths. The UV-absorption maxima for ALTS were 199, 215 and 296 nm. The molecular

formula for ALTS was confirmed by HPLC-HRMS using the same parameters described for

Val-TeA. A different column (Nucleodur C18 Gravity, 100 x 2 mm, 3 µm, 4 x 2 mm guard

column of the same material, Macherey Nagel) with MeCN and H2O (both 0.1% FA) was

used. The most intense ion (Rt = 10.3 min) found was [M-H]- with m/z = 289.0715 which

matches the molecular formula C15H13O6 (calculated m/z = 289.0712, ∆ = 0.8 ppm). The molar

absorptivity values in MeCN were determined in quintuplicate as described (Hickert et al.

2015) and found to be (3.039 ± 0.090) x 104 L/mol/cm at 199 nm,

(2.676 ± 0.033) x 104 L/mol/cm at 215 nm and (0.578 ± 0.003) x 104 L/mol/cm at 296 nm.

14

Figure S1: Tautomerism of TeA, allo-TeA and Val-TeA. The predominant form in solution is the keto-form (left).

15

Figure S2: Structures of ALTS, ALTSOH and AME-3G with numbered atoms.

16

Figure S3: HPLC-MS/MS chromatograms of tenuazonic acid (TeA) and allo-tenuazonic acid (allo-TeA). A: mixture of standard with TeA and allo-TeA, B: Sunflower sample containing TeA without any detectable allo-TeA and C: Sunflower seed sample containing 5.8 ± 1.7% allo-TeA besides 94.2% TeA.

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Figure S4: Cell viability of HT-29 cells after incubation with tenuazonic acid (TeA) and valine-tenuazonic-acid (Val-TeA) for 48 h determined by CCK-8 assay. The incubations were carried out in triplicate with cells from three different passages (n = 9). * Statistically significant (p ≤ 0.05) compared to solvent control, ** statistically highly significant (p ≤ 0.01) compared to solvent control, ## statistically highly significant (p ≤ 0.01) compared to 100 µM Val-TeA.

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Figure S5: EPI mass spectra recorded in positive ionization mode of A: tenuazonic acid (TeA) and B: valine-tenuazonic acid (Val-TeA). The spectra were recorded with HPLC and MS parameters analogously to the screening method for analogues of TeA. Collision energy 21 V, 750 µg/kg each spiked on a blank sample. The fragments used for the screening for further analogues of TeA are underlined.

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Table S1: Calibration levels given in µg/kg.

Analyte 1st level 2nd level 3rd level 4th level 5th level 6th level 7th levelAAL TA1 + TA2 3.8 7.5 23 70 140 210 280ALT + iso-ALT 12 24 72 230 450 680 900ALTSOH 3.5 6.9 21 65 130 190 260AME 0.9 1.9 5.7 18 35 53 71AME-3G 2.8 5.6 17 53 110 160 210AOH 6.6 13 40 120 250 370 500ATX-I 18 36 110 340 680 1000 1400ATX-II 18 35 110 340 680 1000 1400TeA + allo-TeA 62 120 370 1200 2300 3500 4700Val-TeA 93 190 560 1800 3500 5200 7000TEN 0.9 1.7 5.2 16 33 49 65

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Table S2: SRM parameters of all analytes. The quantifier transition is highlighted.

Analyte Parent ion [m/z]

tR[min] Fragment ions [m/z]

Fragment ion intensity ratios [cps/cps ± SD]

DP [V]

CE [V]

AAL TA1 + TA2 [M+H]+ = 522.4 4.8 328.4/310.4/292.4 1.00/0.70±0.02/0.70±0.06 115 35/39/43ALT [M+H]+ = 293.1 5.0 257.1/239.2/229.0 4.61±0.35/1.00/0.95±0.06 75 22/31/31iso-ALT [M+H]+ = 293.1 5.0 257.1/239.2/229.0 47.3±13.9/1.00/9.20±2.92 75 22/31/31ALTS [M-H]- = 289.0 3.4 270.8/244.9/229.8 0.06±0.01/1.00/0.46±0.01 -85 -22/-25/-32ALTSOH [M-H]- = 273.0 3.8 257.6/186.1/174.1 1.00/0.16±0.01/0.15±0.01 -120 -44/-46/-22AME [M-H]- = 271.0 4.4 255.8/227.8/212.8 1.00/0.34±0.03/0.15±0.01 -105 -33/-41/-51AME-3G [M-H]- = 433.1 3.2 269.9/226.8/170.8 1.00/0.47±0.21/0.11±0.01 -135 -44/-80/-88AOH [M-H]- = 257.0 4.0 214.8/212.9/147.0 0.46±0.03/1.00/0.32±0.03 -125 -32/-33/-39ATX-I [M-H]- = 351.0 3.3 332.9/315.2/262.9 1.00/0.84±0.06/0.45±0.13 -90 -16/-23/-41ATX-II [M-H]- = 349.0 3.7 330.7/312.9/303.0 1.00/0.30±0.02/0.56±0.06 -95 -21/-33/-35TeA [M+H]+ = 198.2 5.1 153.1/139.2/124.9 1.16±0.26/0.51±0.05/1.00 80 21/22/24allo-TeA [M+H]+ = 198.2 5.1 153.1/139.2/124.9 1.13±0.10/0.44±0.06/1.00 80 21/22/24Val-TeA [M+H]+ = 184.2 4.5 167.0/138.9/125.2 0.45±0.06/1.18±0.17/1.00 65 20/20/23TEN [M+H]+ = 415.2 5.4 312.2/256.2/132.0 1.00/0.53±0.02/0.43±0.03 115 31/44/62

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Table S3: Molecular weight and m/z of putative analogues of TeA.

Amino acid Molecular weight amino acid [Da]

Molecular weight TeA-analogue [Da]

[M-H]- of TeA-analogue [m/z]

[M+H]+ of TeA-analogue [m/z]

Glycine 75 141 140 142Alanine 89 155 154 156Serine 105 171 170 172Proline 115 181 180 182Valine 117 183 182 184Threonine 119 185 184 186Cysteine 121 187 186 188Isoleucine 131 197 196 198Leucine 131 197 196 198Asparagine 132 198 197 199Aspartic acid 133 199 198 200Glutamine 146 212 211 213Lysine 146 212 211 213Glutamic acid 147 213 212 214Methionine 149 215 214 216Histidine 155 221 220 222Phenylalanine 165 231 230 232Arginine 174 240 239 241Tyrosine 181 247 246 248Tryptophane 204 270 269 271

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Table S4: SRM parameters for screening of not yet isolated metabolites.

Analyte Parent ion [m/z] Fragment ions [m/z] DP [V] CE [V]Altenuisol [M-H]- = 273.0 258.0/186.0 -50 -30/-40Altenusin [M-H]- = 289.0 245.0/230.0 -50 -30/-30Altertoxin III [M-H]- = 347.0 330.0/285.0/319.0 -60 -50/-20/-20Altertoxin II [M-H]- = 349.0 331.0/313.0/261.0 -60 -50/-20/-20Stemphylotoxin II [M-H]- = 347.0 330.0/285.0/319.0 -60 -50/-20/-20

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