Supporting Information. Transformation Pathways of the Recalcitrant Pharmaceutical Compound

Transcription

1 Supporting Information Transformation Pathways of the Recalcitrant Pharmaceutical Compound Carbamazepine by the White-Rot Fungus Pleurotus ostreatus: Effects of Growth Conditions Naama Golan-Rozen 1 ; Bettina Seiwert 2 ; Christina Riemenschneider 2 ; Thorsten Reemtsma 2 ; Benny Chefetz 1 and Yitzhak Hadar 1 Content Table S1 HPLC conditions for separation of CBZ, EP-CBZ and dioh-cbz. S2 Table S2 MRM parameters for quantification of CBZ, EP-CBZ and dioh-cbz. S3 Table S3 Identification of transformation products formed during solid-state fermentation. S4 Figure S1 14 C-CBZ labeling position S6 Figure S2 Figure S3 Figure S4 Accumulation of 14 C-CO 2 formed during degradation of 14 C-CBZ by P. ostreatus in solid-state fermentation on cotton stalks. Peak area trends of transformation products identified during dissipation of acridine as primary substrate. Dissipation of carbamazepine at an environmentally relevant concentration by P. ostreatus during solid-state fermentation on cotton stalks S6 S7 S8 S1

2 Table S1. HPLC conditions for separation of CBZ, EP-CBZ and dioh-cbz. Time Solvent A (%) Solvent B (%) Flow (min) Water Acetonitrile (ml (with.1% (with.5% min -1 ) AcOH) AcOH) Column temperature was 4 C, injection volume was 5 µl. S2

3 Table S2. MRM parameters for quantification of CBZ, EP-CBZ and dioh-cbz. Compound Fragmentati on voltage Collision energy (ev) MRM transitions Carbamazepine 14 Carbamazepine D 2 13 C ,11-Epoxy carbamazepine ,11-Epoxy carbamazepine, D trans-1,11-dihydro-1,11- dihydroxy carbamazepine S3

4 Table S3. Identification of transformation products formed during solid-state fermentation. Transformation product Retention time (min) measured m/z M+H Error (ppm) Fragments (m/z) Predicted formula (fragments) Verified by a 9-OH acridine C 13 H 1 NO proposed 42 OH-9-acridine carboxylic acid (a d) 4.64, 4.81, 5.47, 5.71 Reference C 14 H 1 NO proposed 27 (1 isomer) C 14 H 8 NO 2 (1.4) TP C 16 H 12 N 2 O proposed This study 1-OH-CBZ C 15 H 15 N 2 O TP (neg: ) C 14 H 9 NO (C 14 H 11 NO 2 ) , C 15 H 13 N 2 O (4.6); C 14 H 12 N (3.6) proposed C 13 H 1 N (.6) proposed This study TP C 15 H 12 NO C 14 H 8 NO 2 proposed This study * The structure of TP 254 is proposed by the sum formula and the loss of CH3OH by fragmentation to yield the m/z S4

5 a Identification of 9-OH acridine, OH-9-acridine carboxylic acid (a d), 1-OH-CBZ, TP 297 TP 254 and TP 28: 9-OH acridine exhibited the same exact mass ( Da) as acridone, but it differed from it in its retention time. For that reason, an aromatic structure is proposed. According to De Voogt et al. [1], the carbon atom at position 9 is most likely to be hydroxylated due to its lower electron density than the other carbons. OH-9-acridine carboxylic acid eluted at four different retention times, fitting the four possible isomers (listed as OH-9- acridine carboxylic acid a d in Table 1). They eluted later than the 9-acridine carboxylic acid reported by Salame et al. [2]. The structure of 1-OH-CBZ is proposed according to water loss leading to m/z , and to the characteristic fragment m/z (C 14 H 12 N), which ensured the structure of CBZ. This fragmentation pattern was compared to a standard compound. TP 297 (C 16 H 12 N 2 O 4 ) is proposed to be the methoxylated BQD. Formation of the fragment m/z (C 14 H 8 NO 2 ) supports this assumption as it is formed by loss of methanol and CNO. The proposed structure of TP 254 was determined by the sum formula and the loss of CH 3 OH leading to the m/z TP 28 (C 14 H 9 NO) was detected in positive-ion mode, but the same compound was also detected in negative-ion mode, where m/z (C 14 H 1 NO 2 ) was observed. For the proposed structure, deprotonation is possible and water loss in positive-ion mode as observed for dioh-cbz is likely to occur. However, this fragment (m/z ) is quite nonspecific. S5

6 Figure S1. 14 C-CBZ labeling position % Radiactivity (accumulative) Live Control Incubation time (days) Figure S2. Accumulation of 14 C-CO 2 formed during degradation of 14 C-CBZ by P. ostreatus in solid-state fermentation on cotton stalks. Plotted values are means ± SD of three replicates S6

7 Peak area Acridine Acridone acridine 9-acridine carboxaldehyde carboxaldehyde 9-acridine carboxylic acid OH acridine a b c d 9-OH acridine trans OH-9-acridine Hydroxyl 9-acridine carboxylic acid acid 3 TP m/z Incubation time (days) Figure S3. Peak area trends of transformation products identified during dissipation of acridine as primary substrate by P. ostreatus in solidstate fermentation on cotton stalks. Plotted values are means ± SD of three replicates. S7

8 Figure S4. Dissipation of carbamazepine at an environmentally relevant concentration (.25 µg g -1 ) and formation of 1,11-carbamazepine epoxide and 1,11-carbamazepine trans diol by P. ostreatus in solid-state fermentation on cotton stalks. Plotted values are means ± SD of three replicates. S8