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ESR 4: Marta Saccomanno: October 31 - November 21 2016

Title: Biotransformation of SF5 substituded aminophenols

Host University: Department of Computational Biology and Biomolecular NMR Spectroscopy, MFPL, Vienna University, Austria

Accommodation: Rented accommodation, Klimsh gasse 8, Vienna

Science: Several strains belonging to Pseudomonas and Comamonas spp. had shown to be able to use SF5 substituded aminophenol (1) as a sole growth substrate but the biodegradation process of this compound is yet unknown. GC-MS analysis on resting cell experiments and growing cultures supernatants allowed the detection of 3-pentafluorosulfanyl-catechol (2) and N-acetylated derivatives (3) of the starting substrate but as previous experimental work demonstrated none of these intermediates takes part to the growth process.

Aim of the academic secondment at MFPL is to further investigate the biotransformation of 1 availing of 19F NMR aiming to detect other fluorine containing intermediates occurring as consequence of bacterial metabolism.

Analysis of metabolites occurring in resting cell experiments and growing cultures

(Experiments previously conducted at University College Dublin). This section involved NMR on chloroform extracts and aqueous supernatants from growing cultures and different setups of resting cell experiments.

Results

From the analysis of supernatants resulted evident that different bacterial strains show different biodegradation patterns of compound 1.

No intermediate was detected in the organic fraction of growth experiments suggesting mineralisation of 1.

Resting cell experiments in the presence or absence of yeast extract 1% were analysed and the results compared. When yeast extract was present the biotransformation of 1 occurred at a higher extent.

Compound 3 was detected in Comamonas testosteroni, Pseudomonas pseudoalcaligenes KF707, Pseudomonas knackmussii B13, consistently to what previously detected by GC-MS. 19F NMR signals of 3 were compared to a standard (4) providing supporting evidence of the chemical structure of this intermediate.

New SF5 intermediates were detected in the organic fraction and in the aqueous fraction of resting cell supernatants along with fluoride ion. The identity of the new SF5 intermediates, particularly the water soluble SF5 metabolites, needs to be further investigated.

In situ biotransformation of SF5 aminophenols.

Resting cell experiments were conducted in phosphate buffer supplemented with yeast extract 1% and 1 (0.2 mM) for Pseudomonas spp. SH12, Comamonas testosteroni, Pseudomonas putida KT2440 and Pseudomonas pseudoalcaligenes KF707. The in situ experiment was set in a sterile NMR tube at room temperature and controls with the same composition were made in 50 mL conical flasks providing optimal shaking and temperature. The progress of the biodegradation of 1 was monitored by 19F NMR from time zero until 6 days of incubation.

Results

Biodegradation of 1 was recorded overtime either in the in situ experiment and in the shaking flasks. The reaction in the NMR tube occurred at a slower rate as consequence of the limited oxygen availability while the biodegradation occurred faster in the control flasks when optimal conditions were provided to the cells. The control experiments in the conical flasks showed almost full degradation of 1 within 96 hours (quantitative experiment), a signal for fluoride ion occurred for all strains tested within the first 24 hours of incubation.

New Skills acquired

  • Understanding of the 19FNMR and its applications;
  • To do a 90° pulse calibration while shifting compounds, buffers and solvents;
  • Ability to set customised experiments;
  • Deeper understanding of NMR technique advantages and limitations;
  • Topspin software;
  • Quantitative NMR;
  • Effective management of time;
  • Planning and performing experiments in a different scientific context;
  • Interdisciplinary communication within different scientific fields;
  • Understanding of interactions and working dynamics in a larger research group;
  • Quick and effective adaptation to a new working environment;
  • Improvement of interpersonal skills and networking;
  • Team-working.