Unity 500

The active fraction D was purified by HPLC (Waters: controller 600, pump 600, dual λ absorption detector 2487, Linear Recorder); column C18 (250 × 7.8 mm² UP ODS); mobile phase: linear gradient of methanol–H₂O from 0 to 100% for 52 min; flow rate: 1 mL/min, λ detection at 220 nm and at 420 nm. The column temperature was 30 °C. The injection volume was 200 µL. Under these conditions, the retention time was recorded at 17.6 min. This fraction was shown to contain two main peaks, D₁ and D₂, but only D₂ showed both phosphate-solubilizing and anti-microbial activities.
After purification of the active compound present in D₂ by HPLC, the latter was subjected to spectroscopic studies. ¹H NMR spectroscopy: Varian Unity 500 (500 MHz), Bruker AMX 500 (500 MHz), Varian Inova 500 (500.33 MHz). ¹³C NMR spectroscopy: Varian Unity 500 (125.8 MHz), Varian Inova 500 (125.8 MHz). Chemical shifts were measured relative to tetramethylsilane as an internal standard. The homonuclear and heteronuclear 1D and 2D NMR spectra were recorded on a Varian Inova 500 instrument. Mass spectrometry was performed with an LCC ion-trap mass spectrometer (INSERM, Purpan, Toulouse). Samples were analyzed by electrospray ionization in both negative and positive ion mode, and the full-scan mass range (m/z) was 100–2000.

Dendrimer G₃Si(SC₂H₄NHFITC)(SC₂H₄CO₂Na)₃₁ was synthesized following the protocol described in the bibliography with the appropriate modifications described [50 (link)]. The procedure is described below. Solvents and the chemicals were purchased from commercial sources and used without prior treatment. Thiol-ene reactions were carried out using a UV lamp that radiates vertically (high efficiency with VL-115.L Viber Lourmat filter, 365 nm emission, 30 W, 1100 µW/cm²). NMR experiments were performed on Varian spectrometers, Mercury-300 or Unity-500 spectrometers or Bruker AV400, at ambient temperature. The chemical shifts (ppm) were measured relative to the residual signal of ¹H of the deuterated solvents. Infrared spectra were measured in the IR-FT Perkin-Elmer Spectrum 2000, and ultraviolet spectra were taken from the UV-Vis spectrophotometer Perkin-Elmer Lambda 35.