Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments

It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.


I.a Giant vesicle (GV) preparation by the natural swelling method
Lipids (mixture of commercial lipids or crude extracts from reaction mixtures, with or without the addition of 0.01-0.2 mol% DOPE-Rh) were dissolved in dichloromethane (typically, 2 mL) and poured in a 10 mL cylindrical thick-wall round-bottom glass tube. The solvent was completely evaporated under reduced pressure using a rotatory evaporator. The resulting thin lipidic film was further dried for 30 minutes at 1 mbar/25 °C, and next hydrated for 16 hourswithout shakingwith the aqueous buffer, termed "Isolution" (composed of 200 mM sucrose in 5 mM or 200 mM of Na-bicine, pH 8.5 or 200 mM sucrose in 25 mM Tris-HCl, pH 7.5), in order to obtain an overall 1-2 mM lipid concentration. The hydration temperature was 25 °C. When needed, 1-10 M calcein was included in the I-solution. Three volumes of the thus obtained GVs were diluted with one volume of an aqueous isotonic buffer solution termed "O-solution" (composed of 200 mM glucose in 5 mM or 200 mM of Na-bicine, pH 8.5 or 200 mM glucose in 25 mM Tris-HCl, pH 7.5), and centrifuged at 5,000 rpm for 10 minutes in a bench-top Eppendorf mini-centrifuge. GVs were pelletted down in the Eppendorf tube due to the density difference between the I-solution and the O-solution. The supernatant was carefully removed and the pelletwhich appears pink-red when DOPE-Rh is presentwas re-suspended in 100 L of fresh O-solution.
Note that Mix A and Mix B were hydrated by using one of the above mentioned I-solutions (pH 7.5 or pH 8.5 ) depending on the lipid composition. Exploratory experiments showed that a lower pH value (7.5) is advantageous for the hydration of PA-containing lipid mixtures. Consequently, reconstituted lipid mixtures (main text, Table 1) were treated with the I-solution based on 25 mM Tris-HCl, 200 mM sucrose (pH 7.5). These observations fit with previously published reports on conventional PA-based vesicles (Hauser and Gains, 1982;Hauser et al., 1983).

I.c Flow cytometry
GV samples were analysed using a BD LSRFortessa X-20 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) cell analyser. Forward scatter, side scatter and green fluorescence data were collected using a 488 nm laser with a power of 50 mW as excitation light source. For the forward scatter and the side scatter a photodiode detector with a 488/10 bandpass filter and a photomultiplier tube with a 488/10 bandpass filter have been used, respectively. The quartz cuvette flow cell is gel-coupled by refractive index-matching optical gel to the fluorescence objective lens (1.2 NA) for optimal collection efficiency. Emitted light from the gel-coupled cuvette was delivered by fiber optics to the detector arrays. The flow rate was 12 µL/min. The morphologic plot was side scatter vs. forward scatter and green emission was recorded in the wavelength range 500-550 nm. The number of GVs analysed in each run was about 10,000.

II.a General procedure for the simulated prebiotic formation of amphiphiles
In a typical experiment an Eppendorf tube (volume 2 ml), was filled with 0.025 mmol of rac-DOG (1, 15.

II.b General procedure for control experiments
In a typical experiment rac-DOG (1) was mixed with cyanamide (2a) or urea (2b) without phosphate sources 3a or 3b. Similar reactions were performed in the absence of 2a or 2b but in the presence of 3a or 3b. Molar ratios, reaction times and conditions were the same for all control experiments. All control reactions were made in triplicate and in both reaction conditions used in the general procedures.

II.c General procedure for the extraction of amphiphiles from crude prebiotic mixtures
The crude mixtures were carefully dissolved in a minimum amount of water (13 mL) and transferred in a small separation funnel (10 mL) where the mixture was extracted three times with CHCl3 or CH2Cl2 (3x5 mL). The combined organic layers were collected and evaporated in a small round bottom flask. Aliquots of those residues were submitted to NMR spectroscopic and vesiculation studies. TLC (eluent systems A or B) of the water phases showed no presence of amphiphiles and or lipophilic compounds.  Table S1. Composition of crude prebiotic mixtures containing racemic DOPA (4) and DOPE (5) together with starting material rac-DOG (1), byproducts rac-MOPA (4b), rac-MOG (7a), sec-MOG 7b) and oleic acid (8)

V Synthesis of racDOG (1) and racMOG (7a)
Racemic dioleoyl glycerol (racDOG, 1) was obtained in three steps from commercial glycerol. Selective protection was performed with triphenylmethyl chloride (TrtCl) in the presence of a catalytic amount of 4-dimethylaminopyridine (DMAP). Rac3triphenylmethylglycerol (9) was crystallized from dichloromethane/pentane and used without further purification. Subsequently, 9 was acylated with oleoyl chloride in the presence of DMAP. Compound 10 was obtained as a viscous oil and the deprotection of 10 was carried out with a catalytic amount of HCl in MeOH/CHCl3 1:1 v/v. Scheme S1. Synthetic pathway of rac-1,2-dioleoyl-sn-glycerol (1, rac-DOG ) Racemic mono-oleoyl glycerol (racMOG, 7a -isopropylidene-DL-glycerol. Acylation was performed as described for the synthesis of compound 10. The compound 11 was deprotected by using a small amount of acidic resin (Amberlyst ® 15) and the product was obtained after filtration and evaporation. This kind of synthesis represents a novelty with respect to other syntheses reported in the literature for obtaining these type of compounds, including an enzymatic synthesis.

V.b 1,2-O,O-Dioleoyl-DL-glyceryl-3-O-triphenylmethyl ether (10)
To a stirred solution of 9 (2.23 g, 6.7 mmol) in 50 mL of CHCl3 was added oleoyl chloride (5.03 g, 5.52 ml, 16.7 mmol) and DMAP (2.06 g, 16.7 mmol). The resulting solution was stirred overnight at room temperature. The excess of oleoyl chloride was decomposed by addition of 50 ml solution of NaHCO3 (0.4 M) and the resulting bisphasic solution was left stirring for 15 min. The biphasic solution was extracted with CHCl3 (2 × 50 mL), and the combined organic phases were washed with 2 × 10 mL of brine and dried over Na2SO4. Evaporation of the solvent followed by chromatography over freshly activated SiO2 with CHCl3 gave 3.40 g (3.39 mmol, 58.2%) 10 as a white solid. Rf (hexane/

V.c 1,2-O,O-Dioleoyl-DL-glycerol (1)
A solution of CHCl3-MeOH containing 0.22 ml of concentrated HCl (37%) was cooled to 0°C and to this a solution, prepared by dissolving 2g         Figure S39. An additional image of GVs from Mix A hydration (in 5 mM bicine, pH 8.5; 200 mM sucrose inside/glucose outside), stained with DOPE-Rh (0.1 mol%). The red staining allows the detection of lipid-rich regions in the pictured particles. Calcein filled vesicles appear as a green circle surrounded by a thin red layer. In contrast, particles appearing red are lipid-rich. They might include quite large lipid particles (a), calcein-containing in the presence of captured small lipid particles or small vesicles (b), GVs with foam-like internal structure (c), or lipid clumps (d). In some cases GVs inside GVs (multivesicular vesicles or vesosomes) can be observed, as in the inset (note the different calcein-filling pattern).  Table 1 in the article). M1 and M3, which contain 30 and 60 mol% of DOPA, have been hydrated in the I-solution made of 25 mM Tris-HCl, 200 mM sucrose (pH 7.5). In contrast, M2 and M4, which contain 30 and 60 mol% of DOPE, have been hydrated with 200 mM Nabicine, 200 mM sucrose (pH 8.5). Calcein-containing GVs are found in good amounts in samples M1 and M3, whereas the mixture remained essentially not hydrated for samples M2 and M4. Arrow 1 indicates a GVs which is probably multilamellar, as evident by its highly red fluorescence due to high amount of the lipid marker DOPE-Rh; arrow 2 indicates a lipid-filled spherical particle, probably surrounded by a lipid bilayer; arrow 3 indicates poorly hydrated lipid clumps; arrow 4 indicates a GVs in a sample which contain also lipid-rich particles, which appear red due to abundance of DOPE-Rh (arrow 5). Figure S41. Pictures of GVs prepared from mixture M5 in 200 mM bicine pH 8.5; and 200 mM sucrose inside/200 mM glucose outside. Note that DOPC is 60 mol% (Table S3). GVs lumen appears green due to encapsulated calcein fluorescence, lipid membranes appear red due to the co-hydration of DOPE-Rh (0.02 % w/v).