Stearates

Pollen samples were obtained through fieldwork in the publicly funded institutions in the Republic of Croatia: the Ruđer Bošković Institute and the University of Zagreb. The study is a part of government-funded research, and therefore has been conducted with the full cooperation of the administrations of the involved institutions. The field studies did not involve endangered or protected species.
Samples of pollen were collected at two facilities of the University of Zagreb; the Botanical Garden of the Faculty of Science and the Botanical Garden “Fran Kušan” of the Faculty of Pharmacy and Biochemistry. Additional samples of common wind-pollinated species were collected at the facilities of the Rudjer Boskovic Institute and the Faculty of Science of the University of Zagreb. All the locations are in the City of Zagreb. 813 samples were collected altogether, belonging to 300 plant species (Table S1 in File S1). Pollen samples were collected during five pollination seasons (2009–2013). The pollen samples were collected directly from plants at flowering time, either by shaking flowers (anemophilous species) or collecting mature anthers (entomophilous species). The samples were kept in paper bags at r.t. for 24 hours (together with anthers for entomophilous species), and afterwards transferred to vials and stored at −15°C; in general their IR spectra were recorded within 48 h after sampling.
For identification of basic biochemicals in pollen a set of model compounds that included lipids, carbohydrates and proteins was measured to correlate with high positive or negative values in the principal component analyses loadings plots. Spectra of crystal lipids and carbohydrates were recorded above their melting temperature, and again at r.t. after cooling to obtain spectrum of amorphous phase (liquid and/or glass phase). Tristearin (1,3-di(octadecanoyloxy)propan-2-yl octadecanoate), triolein (2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl (Z)-octadec-9-enoate), phosphatidistearoylcholine (1,2-distearoyl-rac-glycero-3-phosphocholine), phosphatidioleylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine), stearic acid (octadecanoic acid), oleic acid ((9Z)-octadec-9-enoic acid), cellulose, amylose, amylopectin, sucrose, trehalose, fructose, glucose, gluten, α-casein, β-casein, κ-casein were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, United States), and used without further purification.

The reference standards of 18 analytes examined in this study (Table 1) were obtained from Sigma (St. Louis, MO). The deuterium-labeled internal standards (ISs) used were: 2,2,3,4,4,6-d₆-cholesterol and 2,2,3,4,4,6-d₆-cholesteryl stearate (C/D/N Isotopes, Pointe-Claire, Quebec, Canada) for cholesterol and CEs, respectively; and 25,26,26,26,27,27,27-d₇-4β-OHC and 25,26,26,26,27,27-d₆-27-OHC (Avanti Polar Lipids, Alabaster, AL) for three cholesterol precursors and eight OHCs. The trimethylsilylating (TMS) agents, N-methyl-N-trifluorotrimethylsilyl acetamide (MSTFA), ammonium iodide (NH₄I), and dithioerythritol (DTE), were purchased from Sigma. The hybrid SPE-precipitation cartridge (H-PPT) (1 ml, 30 mg) was supplied by Supelco (Bellefonte, PA). All organic solvents were of analytical and HPLC grade and were purchased from Burdick and Jackson (Muskegon, MI).

LB liquid medium was used to explore the optimal growth, lipase production temperature and pH of the WCO-9 strain, and ρ-nitrophenyl decanoate (C10), ρ-nitrophenyl laurate (C12), ρ-nitrophenyl myristate (C14), ρ-nitrophenyl palmitate (C16) and ρ-nitrophenyl stearate (C18) was chosen as the substrate for the lipase activity assay [34 (link)]. Oil degradation assays were performed on rhodamine B oil plates [35 (link)] using corn oil, soybean oil, peanut oil, canola oil and olive oil mixed with a solution of 4% polyvinyl alcohol (PVA) in a 1:3 volume ratio and emulsified by sonication for 10 min. Then, 25 mL of the emulsion was added to 200 mL of medium and stained with 10% rhodamine B solution. The WCO-9 and A. junii ATCC 17908 strains were activated by plate streaking, and single colonies were incubated in a liquid medium at 30 °C and shaken at 200 r/min for 12 h to prepare a seed solution, which was then inoculated in fresh liquid medium starting with an OD₆₀₀ of 0.1. When both strains reached an OD₆₀₀ of 0.7 ± 0.05, the bacterial concentrations of the two strains were adjusted to the same OD₆₀₀ value. The oil plate was punched, and 10 μL of the two bacterial solutions with the same OD₆₀₀ value was injected into the well and incubated at 30 °C for 4 days. The plate was observed under UV light, and the size of the transparent circle was measured as the parameter of oil degradation capabilities. The oil degradation activity was tested by GB/T_23535-2009 method with the five oils mentioned above.

SG-17, an MMP-sensitive lipopeptide (SKK(stearate)SGPLGIAGQSK(stearate)KS), was prepared by microwave (MW)-assisted Fmoc solid-phase peptide synthesis (Fmoc-SPPS) on Wang resin (loading between 0.2–0.4 mmol/g), using a CEM Liberty Blue synthesizer. Coupling reactions were performed with amino acids (5 eqs., 0.2 M in DMF), using diisopropyl carbodiimide (DIC, 0.5 M in DMF) and Oxyma Pure (1 M in DMF) as coupling reagents. The MW synthesis cycle entailed 15 s at 75 °C–170 W, followed by 110 s at 90 °C–40 W. N-Fmoc deprotection was performed using 20% piperidine in DMF with a MW cycle entailing 15 s at 75 °C–155 W, followed by 60 s at 90 °C–50 W. Final cleavage in solution entailed shaking the resin for 3 h in a 90:2.5:2.5:5 TFA/TIPS/H₂O/phenol mixture. The lipopeptide was then purified in reverse-phase chromatography with a Biotage Isolera instrument equipped with a C18 column. 0.1% TFA in water was used as phase A and 1% TFA in ACN as phase B, going from 20 to 100% phase B in 18 min (Rt = 16.6 min for MSLP-1). Electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR TOF-MS) on a Q-TOF Synapt G2-Si yielded m/z 1503.7206 [M+2H]²⁺, 702.8170 [M+3H]³⁺ values for the pure lipopeptide.