Sodium Methoxide

The fatty acid (FA) compositions of the insect and plant oils and baked products (cookies) made from the respective oils (20 mg each), were analyzed as fatty acid methyl esters (FAMEs) following previous methods [25 ]. A solution of sodium methoxide (15 mg/mL) was prepared in dry methanol and was added (500 µL) to the samples. The samples were vortexed for 1 min, sonicated for 5 min, and incubated at 60 °C for 1 h, thereafter quenched by adding 100 μL deionized water followed by vortexing for another 1 min. The resulting methyl esters were extracted using gas chromatography (GC)-grade hexane (1000 µL; Sigma–Aldrich, St. Louis, MO, USA) and centrifuged at 14,000 rpm for 5 min. The supernatant was dried over anhydrous Na₂SO₄ and analyzed (1.0 µL) by GC-MS on a 7890A gas chromatograph linked to a 5975 C mass selective detector (Agilent Technologies Inc., Santa Clara, CA, USA). The GC was fitted with a (5%-phenyl)-methylpolysiloxane (HP5 MS) low bleed capillary column (30 m × 0.25 mm i.d., 0.25 µm; J&W, Folsom, CA, USA). Helium at a flow rate of 1.25 mL min⁻¹ served as the carrier gas. The oven temperature was programmed from 35 °C to 285 °C, with the initial temperature maintained for 5 min, with a rise at 10 °C min⁻¹ to 280 °C, and then held at this temperature for 20.4 min. The mass selective detector was maintained at ion source temperature of 230 °C and a quadrupole temperature of 180 °C. Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. Fragment ions were analyzed over 40–550 m/z mass range in the full scan mode. The filament delay time was set at 3.3 min. Serial dilutions of the authentic standard methyl octadecenoate (0.2–125 ng/μL) and hexanal (1–280 ng/μL) were analyzed by GC-MS in full scan mode to generate a linear calibration curves (peak area vs. concentration) which gave coefficient of determinations R² = 0.9997) and 0.9997 for methyl octadecenoate and hexanal. These regression equations were used for the external quantification of the different fatty acids and volatile organic compounds (VOCs) respectively.
A Hewlett-Packard (HP Z220 SFF intel xeon) workstation equipped with ChemStation B.02.02. acquisition software was used. The mass spectrum was generated for each peak using Chemstation integrator set as follows: initial threshold = 5, initial peak width = 0.1, initial area reject = 1, and shoulder detection = on. The compounds were identified by comparison of mass spectral data and retention times with those of authentic standards and reference spectra published by library–MS databases: National Institute of Standards and Technology (NIST) 05, 08, and 11. The insect and plant oils as well as the insect-based oil cookies were analyzed for FAMEs in triplicate, with each replicate collected from a different batch of respective samples.

LL-III and g-LL-III peptides were synthesized using an ABI 433A peptide synthesizer (Applied Biosystem, Foster City, CA, USA) with standard Fmoc chemistry on a 0.1 mmol scale. The acid labile H-PAL ChemMatrix resin, with a substitution of 0.20 mmol/g, was used as solid support. Amino acids were activated in situ with 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as coupling reagent. Peptide cleavage from the resin and sidechains deprotection was accomplished using a mixture of 94% trifluoroacetic acid (TFA), 2.5% H₂O, 2.5% triisopropylsilane (TIS), and 1% ethanedithiol (EDT), yielding the peptides with amidated C-terminal. The crude peptides were precipitated in cold diethyl ether and dried under reduced pressure. The synthesis yields were 58% and 63% for LL-III and g-LL-III, respectively, based on HPLC analysis of the crude peptides (Figure. S1 and S2 panel A). Removal of the acetyl groups from protected hydroxyls of N-acetylglucosamine (GlcNAc) was achieved by treating the crude g-LL-III peptide (1.5 mM) with a solution of sodium methoxide (~ 10 mM) in methanol at pH 9. Milder conditions with respect to literature procedures were used to prevent amino acids racemization. The reaction was carried out overnight at room temperature, under magnetic stirring. Complete deprotection was assessed by RP-HPLC–MS analysis (Figure. S2 panel B). A shift of the retention time (RT) from 26.74 to 26.32 min was observed upon sugar deprotection, due to the higher hydrophilic character of the fully deprotected product with respect to its precursor.