Prominence chromatograph

All solvents were used as received from commercial suppliers. All of the other reagents used in the synthesis were purchased from Sigma‒Aldrich except the reagent for the phosphoramidation reaction ((S)-isopropyl 2-(((S)-(perfluorophenoxy) (phenoxy) phosphoryl) amino) propanoate), purchased from ALCHIMIA LIMITED. All reactions were carried out under an argon atmosphere. Reactions were monitored with analytical TLC on silica gel 60-F254 precoated aluminum plates and visualized under UV (254 nm). Purification was performed by column chromatography on silica gel (35–70 μM) or by crystallization. NMR data were recorded on a Bruker Fourier 80, or Bruker 500 MHz spectrometer in the deuterated solvents indicated, and the spectra were calibrated on TMS. Chemical shifts (δ) are quoted in ppm, and J values are quoted in Hz. In reporting spectral data, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dddd (doublet of double doublets) and m (multiplet). Mass spectrometry analysis was performed on a Shimadzu LCMS-8045. HPLC was carried out on a Shimadzu Prominence chromatograph equipped with a DAD detector (Xterra RP18, 250 × 4.6 mm 5 mm – part n°:186000496 column). Solvents were used as HPLC grade. Structural details are highlighted in the Supplementary Chemical information.

Fructose, glucose and sucrose content were determined by means of High Performance Liquid Chromatography (HPLC) [35 ]. Samples for chromatographic analyses were 3-fold diluted with ultrapure deionized water and filtered through a 0.2 μm nylon membrane filter. Analyses were performed using a Shimadzu Prominence chromatograph (Shimadzu Corp., Tokyo, Japan) equipped with a Luna NH₂ column (250 mm × 4.6 mm) (Phenomenex, Torrance, CA, USA). Samples were loaded onto a 10 µL injection loop and eluted at 40 °C with 80:20 acetonitrile:water (ACN:H₂O) mobile phase at a flow rate of 1.2 mL/min. The detection of compounds was performed using Shimadzu RID-20A refractive index detection, at a temperature of 30 °C. Integration and quantification were performed using the LabSolutions software (Shimadzu Corp., Kyoto, Japan).

The obtained propolis extracts were filtered with Iso-Disc™ Filters PTFE-25-2, diameter 25 mm, pore size 0.20 μm (Supelco Analytical™, Bellefonte, PA, USA) and subjected to HPLC. A Shimadzu Prominence chromatograph equipped with an auto sampler SIL-20AC HT, photodiode array detector SPD-M20A, and LC solution 1.21 SP1 chromatography software (Shimadzu, Kyoto, Japan) were used. Separations were achieved using a 100 × 4.60 mm, C18 reversed-phase column, 2.6-μm particles with solid core and porous outer layer (Kinetex™, Phenomenex^®, Torrance, CA, USA). Binary gradient of deionized water (WCA R03 DP ECO, COBRABiD Aqua, Warsaw, Poland) adjusted to pH 2 with phosphoric acid (Merck) and filtered with 47-mm nylon membrane filter 0.20 μm (Phenex™, Phenomenex^®, Torrance, CA, USA) as mobile phase A and MeCN (acetonitrile for HPLC ≥ 99.9%, Merck) as phase B was used as follows: 0 min–12.5% B; 25.0 min–40% B; 34.0 min–60% B; 37.0 min–95% B; 37.1 min–12.5% B; 40 min–stop. The flow rate was set to 2.0 mL/min, oven temperature 45 °C, injection volume 1 μL. Peak identification was carried out by comparison of retention time as well UV spectra with standards. The content of the determined compounds was calculated in mg per 100 mL of propolis extract.

Concentration of carbohydrates (sucrose, glucose and fructose) in ciders, apple and pear juices and ethanol content of ciders was assessed by the means of high performance liquid chromatography (HPLC). Samples of cider and juice were 3-fold diluted with de-ionized water and filtered through 0.2 µm nylon membrane filter. Analyses were performed using Shimadzu Prominence chromatograph (Shimadzu Corp., Tokyo, Japan) equipped with Rezex ROA-Organic Acid H+ column (300 × 7.8 mm) (Phenomenex, Torrance, USA). The samples were loaded into 20 µL injection loop and eluted at 50 °C with 0.005 M H₂SO₄ as a mobile phase at a flow rate of 0.4 mL min⁻¹. The compounds were detected using Shimadzu RID-10A refractive index detector; detection temperature was maintained at 50 °C. Integration and quantification of chromatograms were performed using Chromax 10 software (Pol-Lab, Warsaw, Poland). Degree of attenuation was calculated using the formula below: