Ultraflex instrument

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for species identification; the procedure was performed as described by Bizzini and Greub, 2010 [45 (link)].
Briefly, all suspected isolates were subcultured on Mueller–Hinton agar (Oxoid Ltd., Basingstoke, UK) supplemented with 5% sheep blood at 37 °C for 24 h. Pure colonies were put in 300 μL of water, vortexed and then precipitated with 900 µL ethanol (96% vol/vol) (Carl Roth GmbH, Karlsruhe, Germany). After centrifugation for 5 min at 10,000× g, the pellet was resuspended with a volume of 50 μL of 70% (vol/vol) formic acid (Sigma Aldrich Chemie GmbH, Steinheim, Germany) and 50 μL of acetonitrile (Carl Roth GmbH, Karlsruhe, Germany). After centrifugation for 5 min at 10,000× g, 1.5 μL of the supernatant was spotted onto a MTP 384 Target Plate Polished Steel TF (Bruker Daltonik GmbH, Bremen, Germany) and allowed to air dry at room temperature. A droplet of 2 μL of matrix solution (α-cyano-4-hydroxycinnamic acid) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was added on top of each sample spot and again the spots were completely dried. Spectra recording was performed using an Ultraflex instrument (Bruker Daltonik GmbH, Bremen, Germany) and data were automatically analyzed using the Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany).

The identification of bacterial species was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) according to the procedure described previously [29 (link)]. Briefly, a water suspension of cultured bacteria (300 μl) was precipitated with 900 µL of ethanol (96% vol/vol; Carl Roth GmbH, Karlsruhe, Germany) and centrifuged for 5 min at 10,000 × g. The pellet was resuspended in 50 μl of 70% (vol/vol) formic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), followed by adding 50 μl of acetonitrile (Carl Roth GmbH), mixing, and centrifugation for 5 min at 10,000 × g. Then, 1.5 μl of the supernatant were spotted onto a MTP 384 Target Plate Polished Steel TF (Bruker Daltonik GmbH, Bremen, Germany) and air-dried. The dried material was overlaid with 2 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid (in a solution of 50% acetonitrile, 2.5% trifluoroacetic acid) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Air-drying at room temperature followed. Spectra recording and data analysis were done with an Ultraflex instrument and Biotyper 3.1 software (Bruker Daltonik GmbH) [30 (link)].

All starting materials and reagents were obtained commercially and used without further purification unless otherwise specified. 4 Å molecular sieves were flame-dried under vacuum and cooled to rt under a N₂ atmosphere immediately before use. The reactions were monitored by thin-layer chromatography (TLC) on glass-packed precoated silica gel plates and visualized with a UV detector or charring with 10% H₂SO₄ in EtOH (v/v). The purification of products was accomplished by flash column chromatography on silica gel (200–300 mesh). NMR spectra were recorded on a Bruker Avance III 400 or Avance II 600 spectrometer (¹H at 400 or 600 MHz, ¹³C at 100 or 150 MHz) with chemical shifts reported in ppm using TMS as the internal standard. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m), with coupling constants (J) in hertz. MALDI-TOF mass spectrometry was performed with a Bruker Ultraflex instrument by applying the matrix of 2,5-dihydroxybenzoic acid (DHB). The high resolution electron spray ionization mass spectra (HR-ESI-MS) were obtained using a Waters Micromass-LCT Premier-XE mass spectrometer.