Discovery of tanshinone derivatives with anti-MRSA activity via targeted bio-transformation

Abstract

Two potent anti-MRSA tanshinone glycosides (1 and 2) were discovered by targeted microbial biotransformation, along with rapid identification via MS/MS networking. Serial reactions including dehydrogenation, demethylations, reduction, glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447. In addition, tanshinosides B (2) showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 μg/mL. This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.

Keywords: Biotransformation; Glycosylation; Mucor rouxianus; Tanshinone IIA.

Fig. 1

Structure of isolated tanshinosides A–D (1–4), tanshinone IIA (5) and tanshinone I (6).

Fig. 2

Process of targeted micro-titer microbial biotransformation for anti-infective compounds. I. Rapid micro-titer microbial biotransformation screening: a) 203 microbes were selected for this study; b) Master plates with a spore solution were stored in a −80 °C freezer; c) Assay plates, containing 2 mL medium and 100 μL spores solution transferred from master plates; d) EtOAc extraction of the fermentation broth; e) HPLC-DAD-UV analysis of EtOAc extract. The samples containing potential new products were highlighted for further flash chromatographic fractionation; f) antibacterial evaluation of the fractions. A heat map was generated to indicate the most promising samples; g) The MS/MS data were collected and MS/MS networking was applied to identify the bioconverted products rapidly. II. Starting substrate: Tan IIA, isolated from TCM S. miltiorrhiza. III. Large-scale fermentation and isolation of biotransformed compounds and their structures were determined by means of spectroscopic techniques.

Fig. 3

Molecular network of tanshinone IIA biotransformation products of strain M. rouxianus AS 3.3447. (a) Tanshinones molecular family (boxed and enlarged) from the tanshinone IIA and fungus AS 3.3447 biotransformed products MS/MS clusters. (b) MS2 spectra of biotransformed products on tanshinone IIA (m/z 441.15 and 459.20) revealed glycosylation via biotransformation.

Fig. 4

Observed ¹H—¹H COSY, key HMBC, and ROESY correlations of 1–4.

Fig. 5

The bactericidal activities of tanshinosides A (1) and B (2) against MRSA and S. aureus (SA). Compounds were added to cultures at time zero, and samples were processed as described in the Materials and Methods. A: in vitro time-kill curve of tanshinoside A (1) against MRSA; B: in vitro time-kill curve of tanshinoside B (2) against MRSA; C: in vitro time-kill curve of vancomycin against MRSA; D: in vitro time-kill curve of tanshinoside A (1) against SA; E: in vitro time-kill curve of tanshinoside B (2) against SA; F: in vitro time-kill curve of vancomycin against SA.