Gut microbiota of human fecal samples exposed to entacapone and bioorthogonal drug derivatives

To investigate the impact of entacapone on the gut microbiota, we used synthesized entacapone and two alkyne-tagged derivatives, which were designed by replacing the terminal methyl groups with alkyne moieties. These compounds were evaluated as molecular probes through ex vivo incubations with human fecal samples from three healthy participants and selected E. coli strains. Fecal samples were processed under anaerobic conditions, resuspended in supplemented M9 medium, and incubated with native or modified entacapone. Samples were collected at various time points and stored for downstream analyses. Following incubation, alkyne-tagged compounds were conjugated to fluorescent azides via click chemistry, and labeled bacterial cells were analyzed via fluorescence-activated cell sorting (FACS) and identified by 16S rRNA gene amplicon sequencing. DNA was extracted using a commercial kit with mechanical lysis, and the V4 region of the 16S rRNA gene was amplified using primers 515F/806R. Sequencing libraries were prepared using a PCR-free protocol and sequenced on an Illumina NovaSeq platform (2 x 250 bp). Paired-end reads were demultiplexed and processed using standard bioinformatics tools.