Electronic Supplementary Material Corrected A. Preiss 04042012

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Electronic Supplementary Material corrected A. Preiss 04042012

Sampling procedure

Samplingwas carried out accordingto DIN 38402 -A 13 andDIN 38402 -A 21witha GrundfosMP1pump (maximum flow rateof approximately25 L/min) fitted with PVC tubing and an outlet tap at the end of the pump rod to restrict contamination. The depth atwhich the pumpwasmounted, depended on theconstructionofthe groundwater monitoring wellandthe sampling level and was 15 m at well 5001, 18 m at well 5002 and 22 m at well 5003. Prior to sampling, the wells were purged until the in-situ parameters (pH, temperature, electrical conductivity, redox potential and oxygen content) reached constant values and the well volume was exchanged at least 3 times. All samples were collected in washed and heated glass bottles of 1 L (well 5001) and 2L (wells 5002 and 5003) and were treatedwithsodium azide to prevent microbial activity. During transport, the bottles were cooled and kept in the laboratory in a cooling room in the dark at 7°C until further preparations were carried out.

Solid Phase Extraction

The remaining aqueous phase of each sample was treated for 20 min on a rotaryevaporatorat 60° C and10.000 kPa to remove traces of dichloromethane, which would otherwise have interfered with the solid phase extraction. The aqueous phase was then adjustted to pH 2 with phosphoric acid and sucked over a LiChrolut EN cartridge (200 mg) conditioned with 3 mL methanol, 3 mL acetonitrile and 10mL water. The cartridge was left dripping wet and the analytes were eluted with 2 x 3 mL acetonitrile/water (80/20 % v/v).

GC-MS conditions

GC
Injector
Injector purge-off time
Carrier gas
Column
Initial temperature
Temperature program
Injection volume
Detector
Transfer line temperature
Acquisition mode
Mass spectral data base / Agilent 6890 series GC-system
splitless, 250°C
0.5 min
helium
30 m x 0.25 mm id, HP-5MS , 0.25 µm
60°C
1 min at 60°C, with a rate of 10°C/min to 170°C, then with 4°C/min to 280°C which was kept for another 5 min
1 µL
MSD 5973
280°C
Scan
NIST 98

HPLC-UV

HPLC fractionation was performed at 30° C with an HPLC system consisting of two pumps (Waters 510) an autosampler (Waters 717 plus) and a Waters 996 PDA detector (Waters, Milford, MA, USA). For the separation a Purospher RP 18e column (sorbent: high-purity silica particles C18 with special modification and deactivation of the surface; pore size: 9 nm; dimensions: 4.0 x 250 mm, 5 µm) (Merck, Darmstadt, Germany) was used. The mobile phase consisted of acetonitrile (A) and 0.2% aqueous formic acid (B). The chromatographic runs were started with 20% acetonitrile which was held for 2 minutes and then raised within 12 min to 70%. This composition was kept for a further 7 min. Between 21 and 27 min acetonitrile was again reduced to its initial value and then the system rinsed for 10 min. The flow rate was 1.0 mL/min.

HPLC-NMR

The HPLC system consisted of a Bruker Saxonia LC-22 pump (Bruker, Leipzig, Germany), a Rheodyne 7725i injection valve with a 50 µL loop (Cotati, USA), a Bischoff Lambda 1010 UV detector (Bischoff, Leonberg, Germany) and a Bruker BPSU-36 peak sampling unit. The NMR spectrometer was a Bruker Avance DRX 600 (Bruker BioSpin, Rheinstetten, Germany) equipped with a 4-mm z-gradient flow probe (1H/13C inverse, active detection volume 120 µL).

The HPLC-1H NMR runs were carried out on a Merck Purospher RP 18e column (4.0 x 250 mm, 5 µm). Two on-flow runs with different chromatographic conditions were performed.

On-flow 1: solvent A was acetonitrile, solvent B was a mixture of acetonitrile (30 % v/v) and 10 mmol trifluoroacetic acid-D1 in D2O (70 % v/v). The runs were started with 1% A, and the eluent composition was held for 10 min. The percentage of A was then increased within 150 min to 35% and kept at this composition for further 50 min. The flow rate was 0.1 mL/min, 50 µL of the sample were injected.

On-flow 2: solvent A was a mixture of acetonitrile (30 % v/v) and 10 mmol trifluoroacetic acid-D1 in D2O (70 % v/v), solvent B was 10 mmol trifluoroacetic acid-D1 in D2O. The runs were started with 50% A, and the eluent composition was held for 10 min. The percentage of A was then increased within 180 min to 99% and kept at this composition for further 50 min. The flow rate was 0.1 mL/min, 50 µL of the sample were injected.

The 1H NMR data were acquired using the Bruker lc2wetdcus sequence for multiple solvent suppression. Free induction decays (FIDs) were collected into 16 K data points (sweep width 9615.4 Hz) with a relaxation delay of 0.8 s and a flip angle of 90° (9.7µs). The NMR chromatogram was recorded within 162 rows, each row consisting of 48 FIDs.

As far as 1H chemical shifts were determined from the NMR chromatograms, they were referenced to acetonitrile (1H,  = 2.00 ppm).

HPLC-MS and flow injection MS

For the HPLC-MS investigations, an HPLC system (Waters, Milford, MA, USA) consisting of two pumps 510, an autosampler WISP 712, and a UV detector 486 coupled to an Esquire-LC mass spectrometer with an ESI source(Bruker Daltonics, Bremen, Germany) was used.

HPLC separation was carried out on a Merck Purospher RP 18e column (4.0 x 250 mm, 5µm) using the following gradient: solvent A was acetonitrile, solvent B a solution of 0.2% formic acid in H2O. The gradient was identical with that of the HPLC-UV method. The flow rate was 1.0 mL/min, 10 µL of the sample were injected.

MS ionization parameters were as follows: ESI negative/positive ionization; skim voltage was varied between 15.2 and 36.2 V; dry gas: 8L/min nitrogen at 300 °C; nebulizing gas: nitrogen 103.421 kPa. Scan range 40 to 500 m/z; 16 averages per spectrum.

MSn experiments (ESI negative/positive ionization) of the samples obtained from HPLC fractionation were carried out in the flow injection mode. Typical conditions were as follows: flow: 400 µL/h; skim voltage was varied between 7.9 and 30.8 V; dry gas: 5L/min nitrogen at 300°C; nebulizing gas: nitrogen 75.842 kPa. Scan range: 40 to 800m/z; target mass: 200 m/z; fragmentation amplitude: 0.4-1.0 V; isolation width: 3 m/z; 16 averages per spectrum; rolling average: 3.

Off-line NMR

For the off-line NMR measurements also the Bruker Avance DRX 600 spectrometer was used, equipped with a 2.5-mm 1H/13C inverse dual probe head with z-gradient.

Later, further measurements were carried out on an Avance 2 equipped a with the 5 mm TCI cryo probe (z-gradient, 1H/13C inverse) at 600 MHz.Spectra were acquired in CD3CN/D2O (50:50 v/v %) with standard Bruker pulse sequences. 1H-NMR spectra (600 MHz) : zg 30, sweep width 6602 or 7788 Hz, 32K data points, 2.4 sec relaxation delay, 64-1024 scans; 2D COSYgs, 2D COSYlr spectra: cosygs, cosygpqf, cosylr, cosygpl, size 2 K x 256 data points, 8-64 scans; 2D NOESY spectra: noesyphpr,spectra size 1 K x 512 data points

13C NMR data were determined by 2DHSQC and 2D HMBC experiments.

2D HSQC spectra: hsqcetgpsi2 , spectra size 1 or 2 K x 512 K data points, 16-64 scans; 2D HMBC spectra: inv4gslplrnd, hmbcgplpndqf, spectra size 1 or 2 K x 512 data points, 16-64 scans. The average CH long-range coupling was set to 7.7 Hz. The 2D spectra were processed as 2 K x 1 K data matrices. Shifted squared sine window functions were applied in both dimensions. Chemical shifts were referenced to the residual solvent peak of CD3CN (1H, δ = 1.97 ppm; 13C, δ = 1.32 ppm).

Semiquantitave estimation of components was performed from the 1H NMR spectrum acquired with the pulse sequence zg 30 and a relaxation delay of 30 sec.The residual signal of the CD3CN which had beenpreviously calibratedwas used as internal standard.

Reagents and reference compounds

Acetonitrile (NMR grade) was purchased from Honeywell Specialty Chemicals (Seelze, Germany), deuterium oxide (99.9 atom %), deuterium oxide (100 atom %) and acetonitrile-D3 (100 atom%) from Deutero GmbH (Kastellaun, Germany). Water for HPLC separation was prepared using a Milli-Q purification system from Millipore (Milford, CT, USA). Acetonitrile (HPLC-analyzed) was purchased from Baker (Deventer, The Netherlands), solid phase cartridges LiChrolut EN (200 mg) and formic acid (98-100 %)from Merck (Darmstadt, Germany), 2-amino-5-chloro-4-methylbenzenesulfonic acid ( 97%) from ABCR (Karlsruhe, Germany), 4-methylcyclohexane-1,2-dicarboxylic acid anhydride (97 %) from Sigma-Aldrich (Taufkirchen, Germany) and naphthalene-1,6-disulfonic acid was provided by Dr. Anke Putschew, (Technical University of Berlin, Germany).

1H NMR, 13C NMR and MS data of identified or tentatively identified compounds

NMR and MS data of U2, U4, U5, U8, U9 and U11 have been extracted from the HPLC-NMR and HPLC-MS runs, NMR and MS data of U1, U3, U6, U7, U10, U12, U13 and U14 have been generated from the isolated unknown components.

Aromaticsulfonic acids

U1: naphthalene-1,6-disulfonic acid

1H NMR, δ (ppm): 8.71 (d, 3J8,7 = 9.0 Hz, 1H, H-8), 8.34 (d, 4J5,7 = 1.9 Hz, 1H, H-5), 8.14 (dd, 3J2,3 = 7.3 Hz, 4J2,4 = 1.2 Hz, 1H, H-2), 8.12 (d, 3J4,3 = 8.2 Hz, 1H, H-4), 7.90 (dd, 3J7,8 = 9.0 Hz, 4J7,5 = 1.9 Hz, 1H, H-7), 7.60 (dd, 3J3,2 = 7.3 Hz, 3J3,4 = 8.2 Hz, 1H, H-3).

U3: 2-amino-5-chloro-4-methylbenzenesulfonate (CTL acid)

1H NMR, δ (ppm): 7.50 (s, 1H, H-6), 6.70 (s, 1H, H-3), 2.23 (s, 3H, H3-7),

13C NMR, δ (ppm): 144.1 (C-2), 140.4 (C-4), 128.2 (C-6), 127.3 (C-1), 122.0 (C-5), 120.1 (C-3), 20.1 (C-7),

neg. ESI-MS, m/z: 220 [M-H], MS², m/z: 156 [M-H-SO2], 120 [M-H-SO2-HCl],

pos. ESI-MS, m/z: 222 [M+H]+, MS², m/z: 204 [M+H-H2O]+, 156 [M+H-H2SO2]+,

144 [M+H-H2SO3]+.

U6: naphthalene-1-sulfonic acid

1H NMR, δ (ppm): 8.65 (dd, 3J8,7 = 8.8 Hz, 4J8,6 = 1.2 Hz, 1H, H-8), 8.05 (dd, 3J4,3 = 7.2 Hz, 4J4,2 = 1.2 Hz, 1H, H-4), 8.00 (d, 3J2,3 = 7.8 Hz, 1H, H-2),7.95 (dd, 3J5,6 = 8.4 Hz, 4J5,7 = 1.2 Hz, 1H, H-5), 7.63 (ddd, 3J7,8 = 8.8 Hz, 3J7,6 = 7.2 Hz, 4J7,5 = 1.2 Hz, 1H, H-7 ), 7.57 (ddd, 3J6,7 = 7.2 Hz, 3J6,5 = 8.4 Hz, 4J6,8 = 1.2 Hz, 1H, H-6), 7.51 (dd,3J3,4 = 7.2 Hz, 3J3,2 = 7.8 Hz, 1H, H-3),

neg. ESI-MS, m/z: 207 [M-H], MS², m/z: 143 [M-H-SO2].

U7: naphthalene-2-sulfonic acid

1H NMR, δ (ppm): 8.28 (s, broad, 1H, H-1), 8.00-7.93 (m, 3H, H-8, H-4, H-5), 7.79 (dd , 3J3,4 = 8.7 Hz, 4J3,1 = 1.2 Hz, 1H, H-3), 7.566-7.611 (m, 2H, H-6, H-7),

neg. ESI-MS, m/z: 207 [M-H], MS², m/z: 143 [M-H-SO2],

pos. ESI-MS, m/z: 209 [M+H]+, MS², m/z: 191 [M+H-H2O]+, 163 [M+H-CH2O2]+,

144 [M+H-HSO2]+, 128 [M+H-HSO3]+.

U11: 4-isopropylbenzenesulfonic acid (cumenesulfonic acid),

1H NMR, δ (ppm): 7.63 (pd, 2H, H-2, H-6), H-3,H-5: 7.34 (pd, 2H, H-3, H-5), (H-7,not detectable), 1.18 (d, 6H, H3-8, H3-9),

neg. ESI-MS, m/z: 199 [M-H].

Dicarboxylatepolyethyleneglycols (DCPEGn).

U2: 3,6,9,12,15-Pentaoxaheptadecane-1,17-dioic acid (DCPEG4)

1H NMR, δ (ppm): 4.129 (s, 4H, α-CH2-), 3.650 (m, 4H, β-CH2-), 3.614 (m, 4H, γ-CH2), 3.594 (m, 8H, chain-CH2-),

neg. ESI-MS, m/z: 309 [M-H].

U4: 3,6,9,12,15,18-Hexaoxaicosane-1,20-dioic acid (DCPEG5)

1H NMR, δ (ppm): 4.126 (s, 4H, α-CH2-), 3.648 (m, 4H, β-CH2-), 3.612 (m, 4H, γ-CH2), 3.591(m, 12H, chain-CH2-),

neg. ESI-MS, m/z: 353 [M-H], pos. ESI-MS, m/z: 355 [M+H]+.

U5: 3,6,9,12,15,18,21-Heptaoxatricosane-1,23-dioic acid (DCPEG6)

1H NMR, δ (ppm): 4.123 (s, 4H, α-CH2-), 3.646 (m, 4H, β-CH2-), 3.611 (m, 4H, γ-CH2), 3.588 (m, 16H, chain-CH2-),

neg. ESI-MS, m/z: 397 [M-H], pos. ESI-MS, m/z: 399 [M+H]+.

U8: 3,6,9,12,15,18,21,24-Octaoxahexacosane-1,26-dioic acid (DCPEG7)

1H NMR, δ (ppm): 4.121(s, 4H, α-CH2-), 3.644 (s, 4H, β -CH2-), 3.610 (m, 4H, γ-CH2), 3.585 (m, 20 H, chain-CH2-),

neg. ESI-MS, m/z: 341 [M-H], pos. ESI-MS, m/z: 343 [M+H]+.

U9: 3,6,9,12,15,18,21,24,27-Nonaoxanonacosane-1,29-dioic acid (DCPEG8)

1H NMR, δ (ppm): 4.119 (s, 4H, α-CH2-), 3.642 (s, 4H, β -CH2-), 3.609 (m, 4H, γ-CH2), 3.583 (m, 24 H, chain-CH2-),

neg. ESI-MS, m/z: 385 [M-H], pos. ESI-MS, m/z: 387 [M+H]+.

Heterocycles

U10: (1-oxo-4-methyl-pyrrolo[1,2-a]pyrazin-2(1H)yl)acetic acid

1H NMR, δ (ppm): 7.32 (dd, 3J5,6=2.6 Hz, 4J5,7=1.5 Hz, 1H, H-5), 7.09 (dd, 3J6,7=4.0 Hz, 4J5,7=1.5 Hz, 1H, H-7), 6.69 (dd, 3J6,7=4.0 Hz, 3J5,6=2.6 Hz, 1H, H-6), 6.44 (q, unresolved, 1H, H-3), 4.42 (s, 2H, H2-10), 2.30 (d, 4J3,12=1.3 Hz, 3H, H3-12).

neg. ESI-MS, m/z: 205 [M-H], MS², m/z: 161 [M-H-CO2], 147 [M-H-C2H2O2],

pos. ESI-MS,: m/z: 207 [M+H]+, MS², m/z: 189 [M+H-H2O]+,161 [M+H-CH2O2]+,

MS3, m/z: 133 [161-C2HNO]+.

U12: (1-oxo-3,4-dimethyl-pyrrolo[1,2-a]pyrazin-2(1H)yl)acetic acid

1H NMR, δ (ppm): 7.34 (dd, 3J5,6=2.6 Hz, 4J5,7=1.6 Hz, 1H, H-5), 7.05 (dd, 3J6,7=4.0 Hz, 4J5,7=1.6 Hz, 1H, H-7), 6.66 (dd, 3J6,7=4.0 Hz, 3J5,6=2.6 Hz, 1H , H-6), 4.64 (s, 2H, H2-10) 2.36 (s, 3H, H3-13), 2.20 (s, 3H, H3-12).

13C NMR, δ (ppm): 174.0 (C-11), 157.8 (C-1), 121.3 (C-3), 118.5 (C-5), 114.6 (C-4), 113.5 (C-6), 111.2 (C-7), 46.4 (C-10), 14.9 (C-12), 14.2 (C-13), C-8, not detectable because of low substance concentration.

neg. ESI-MS, m/z: 219 [M-H], MS², m/z: 201 [M-H-H2O], 175 [M-H- CO2],MS3,

m/z: 120 [175- C2HNO], pos. ESI-MS, m/z: 221 [M+H]+, MS², m/z: 203 [M+H-H2O]+,175 [M+H-CH2O2]+.

U14: 2-(N-hydroxymethyl)-6-methyl-1H-indole-1,3-dione.

1H NMR, δ (ppm): 7.76, (d, 3J4,5=8.7 Hz, 1H, H-4), 7.63, (dq, unresolved, 1H, H-7) 7.30, (dd, 3J4,5=8.7 Hz, 4J5,7=1.2 Hz, 1H, H-5), 5.21 (s, 2H, H2-10), 2.45 (d, unresolved, 3H, H3-11).

13C NMR, δ (ppm): 173.0 (C-3), 172.7 (C-1), 145.6 (C-8), 143.6 (C-9), 138.5 (C-6) 130.8 (C-5), 117.9 (C-4), 116.7 (C-7), 59.9 (C-10), 22.0 (C-11).

pos. ESI-MS, m/z: 192 [M+H]+, MS², m/z: 146 (M+H-CH2O2]+, 91 (M+H-C3H3NO3]+,

MS³, m/z: 91 [146- C2HNO]+, 65 [91- C2H2]+.

Alicycles

U13A: 4-methylcyclohexane-1,2-dicarboxylic acid (isomer A)

1H NMR, δ (ppm): 3.16 (1H, H-1), 2.43 (1H, H-2), 2.05 (1H, H-6a) 1.88 (1H, H-3a), 1.72 (1H, H-3b), 1.71 (1H, H5a), 1.38 (1H, H-4), 1.25 (1H, H-6b), 0.92 (1H, H-5b), 0.84 (3H, H3-9).

13C NMR, δ (ppm): 177.7 (C-7), 42.5 (C-1), 43.4 (C-2), 36,8 (C-6), 34.1 (C-5), 28.8 (C-4), 24.7 (C-3), 22.0 (C-9), C-8, not detectable because of low substance concentration.

U13B: 4-methylcyclohexane-1,2-dicarboxylic acid (isomer B)

1H NMR, δ (ppm): 3.13 (1H, H-1*), 2.50, (1H, H-2*), 2.11(1H, H-6a*),1.87 (1H, H-3a*), 1.58 (1H, H-6b*),1.52 (1H, H-5a*), 1.390 (1H, H-4*),1.35 (1H, H-3b*), 0.83 (1H, H-5b*), 0.88 (3H, H3-9*).

13C NMR, δ (ppm): 177.6 (C-7), 43.8 (C-2), 41.7 (C-1), 33.1 (C-3), 32.3 (C-4), 31.1 (C-5), 28.6 (C-6), 22.5 (C-9), C-8, not detectable because of low substance concentration.

neg. ESI-MS, m/z: 185 [M-H], MS², m/z: 167 [M-H-H2O], 141 [M-H-CO2], MS³, m/z:

139 (167-CO), 123 (167-CO2),

pos. ESI-MS, m/z: 187 [M+H]+, MS², m/z: 169 [M+H-H2O]+, MS², m/z: 141 [169-CO]+, 123 [169-CH2O2]+, MS4, m/z: 95 [123-CO]+, 95 [141- CH2O2]+.