Rapid identification of atypical tetracyclines using tandem mass spectrometric fragmentation patterns

Martin Šala; Drago Kočar; Tadeja Lukežič; Gregor Kosec; Milan Hodošček; Hrvoje Petković

doi:10.1002/rcm.7252

Rapid identification of atypical tetracyclines using tandem mass spectrometric fragmentation patterns

Rapid Commun Mass Spectrom. 2015 Sep 15;29(17):1556-1562. doi: 10.1002/rcm.7252.

Authors

Martin Šala¹, Drago Kočar², Tadeja Lukežič³, Gregor Kosec^{3

4}, Milan Hodošček¹, Hrvoje Petković^{3

5

6}

Affiliations

¹ National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia.
² Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva cesta 5, SI-1000, Ljubljana, Slovenia.
³ Acies Bio, d.o.o., Tehnološki park 21, SI-1000, Ljubljana, Slovenia.
⁴ Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, (CIPKeBiP), Jamova cesta 39, SI-1000, Ljubljana, Slovenia.
⁵ Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria, CSIC, C/Albert Einstein, 22, 39011, Santander, Spain.
⁶ Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia.

PMID: 28339151
DOI: 10.1002/rcm.7252

Abstract

Rationale: When applying biosynthetic engineering approaches at the early stages of drug discovery, e.g. aiming to develop novel tetracycline analogues, target compounds are generally produced by engineered microorganisms in low yields. Rapid and reliable identification of metabolites with desired structural modification directly from bacterial cultures is therefore of great importance.

Methods: Structural elucidation of atypical tetracyclines was carried out by fragmentation applying electrospray ionisation tandem mass spectrometry (ESI-MS/MS) (triple quadrupole - linear ion trap; Applied Biosystems 4000 QTRAP) and a high-resolution mass spectrometer (Agilent Technologies 6224 TOF). Fragmentation patterns were obtained either with direct injection or by applying separation of target compounds with high-performance liquid chromatography (HPLC) prior to mass spectrometry. In-source and CID fragmentation were compared. Theoretical calculations of target structures using the Gaussian programme suite were carried out with the aim of strengthening experimental structural elucidation.

Results: Recombinant strains of Amycolatopsis sulphurea producing atypical tetracyclines chelocardin, modified chelocardin analogues (9-demethylchelocardin and 2-carboxyamido-2-deacetyl-chelocardin (CDCHD), and anhydrotetracycline (ATC) were analysed by collision-induced dissociation (CID) fragmentation with higher collision energies to yield structurally important fragments which were identified. We have demonstrated that ATC is more prone to fragmentation compared to its epimer, which was further supported by comparison of both structures calculated with ab initio calculations.

Conclusions: We have demonstrated that fragmentation patterns of atypical tetracyclines in CID-MS spectra enable rapid structural elucidation of target metabolites produced by cultures of genetically engineered bacteria. This method is of significant importance for early stages of drug development considering that isolation of target metabolites produced at low concentration is challenging. Copyright © 2015 John Wiley & Sons, Ltd.