Enzyme-free synthesis of natural phospholipids in water

Abstract

All living organisms synthesize phospholipids as the primary constituent of their cell membranes. Enzymatic synthesis of diacylphospholipids requires preexisting membrane-embedded enzymes. This limitation has led to models of early life in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate an enzyme-free synthesis of natural diacylphospholipids by transacylation in water, which is enabled by a combination of ion pairing and self-assembly between lysophospholipids and acyl donors. A variety of membrane-forming cellular phospholipids have been obtained in high yields. Membrane formation takes place in water from natural alkaline sources such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained. This high-yielding non-enzymatic synthesis of natural phospholipids in water opens up new routes for lipid synthesis in artificial cells and sheds light on the origin and evolution of cellular membranes.

Fig. 1 |. Enzyme-free synthesis of natural phospholipids.

a, De novo synthesis of diacylphospholipids in water leading to an in situ self-assembled membrane. LPC, lysophosphatidylcholine; PC, phosphatidylcholine. The leaving group is shown in red; the structures of r¹ are shown in panel c. b, The proposed reaction intermediate. c, reactive fatty acyl derivatives: acyl donors 2 (0.75 mM), with which reactions were carried out with 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine 1a (0.5 mM) in the presence of Na₂CO₃/NaHCO₃ buffer (pH = 10.6) at 37 °C for 5 h.

Fig. 2 |. Predicted effects of thioester charge on phospholipid synthesis.

a, Energetics from B3LYP-D3 density functional theory calculations on transacylation reactions. Tetrahedral intermediates iNt1 and iNt2 resemble the transition states of the corresponding addition steps. iNt1 is destabilized by over 7 kcal mol⁻¹ compared with iNt2. b, Optimized structures of reaction intermediates. The negatively charged sulfonate group of iNt1 is distal from the phosphate group to avoid a disfavoured charge repulsion interaction, whereas the positively charged side chain of iNt2 moves into a geometry that will increase the favorable interaction with the phosphate group to stabilize the tetrahedral intermediate. Distances are shown in ångströms.

Fig. 3 |. Enzyme-free formation of phospholipid membranes.

The reaction was carried out by mixing 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine 1a (0.5 mM) and oleoylation reagent 2b (0.75 mM) in different solvents at 37 °C. a, Fluorescence micrographs are shown: Na₂CO₃/NaHCO₃ buffer (pH = 8.8) was used as a solvent; samples were taken from the reaction mixture at different time points and were stained by using 0.1 mol% Nile red dye. Scale bars, 10 μm. b, A fluorescence micrograph is shown: Lost City vent fluid was used as a solvent; the sample was taken from the reaction mixture after 48 h and was stained by using 0.1 mol% Nile red dye. Scale bar, 5 μm. c, A negative staining transmission electron micrograph is shown: mono Lake water was used as a solvent; the sample was taken from the reaction mixture after 5 h. Scale bar, 200 nm. d, A fluorescence micrograph of vesicles formed in mono Lake water (pH = 10) containing the pH indicator dye HPTS, two hours after the external media was exchanged to citrate buffer (pH = 4.6). Scale bar, 10 μm.