Setting up N-acyl amino acid synthesis reaction under wet-dry cycles using GMO and an amino acid

Most of the protocol used in this study for NAA synthesis and analysis was adapted from our previously standardized protocol for NAA synthesis from phospholipids and amino acids³. In a typical reaction, adequate volumes of 10 mg/ml methanol stocks of GMO and OA were taken together in a glass vial, and methanol was evaporated under vacuum to form a dry lipid film. This film was then hydrated with 200 mM glycine (Gly) solution of pH 9.8, to get a final 200 µl reaction mixture containing 3 mM each of GMO and OA, and 200 mM Gly. OA was used in the reaction mixture to increase the overall solubility of GMO, which is otherwise poorly soluble in water. Given the buffering capacity of Gly between pH 8.6 to 10.6 (pKa of α-amino group of Gly is 9.6), a slightly higher concentration of Gly (200 mM) was used so that Gly can also maintain the reaction pH in addition to acting as one of the reactants. The reaction mixture was then subjected to three wet-dry cycles at 90 °C on a heating block (RCT basic, IKA). The duration of each wet-dry cycle was 24 hours, where water was allowed to evaporate to dryness at 90 °C, and this dried mixture was again rehydrated after 24 h with 200 µl of ultrapure water (18.2 MΩ-cm) to complete one wet-dry cycle. After rehydration, the solution was briefly vortexed to remix the reaction components. The same procedure was followed for subsequent wet-dry cycles. The final rehydration was done with 200 µl of ultrapure water two times with vortexing to recover the entire reaction content. This reaction solution was then stored at 4 °C until further analysis. Similar reaction conditions and experimental procedure was followed for NAA synthesis from GMO and other amino acids (alanine, valine, lysine, and serine), and also for the other control reactions, excepting for a few variations as mentioned. For the GMO + Lys reaction, pH was set to 9.1 to account for the lower pKa of the α-amino group of Lys (8.9). For the control reaction that did not contain Gly, a solution of GMO + OA (3 mM each) was prepared in 200 mM CHES buffer pH 9.8, which was subjected to three wet-dry cycles at 90 °C. For the OA + Gly control reaction, 3 mM OA solution was prepared in 200 mM Gly pH 9.8 and subjected to three wet-dry cycles at 90 °C.