N-Carboxyanhydride-Mediated Fatty Acylation of Amino Acids and Peptides for Functionalization of Protocell Membranes

Abstract

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, N^α-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

Figure 1

Val-NCA-mediated acylation of amino acids and peptides. (a) Schematic of Val-NCA-mediated peptide acylation in the presence of either oleic acid or octanoic acid. (b–e) Overlay of extracted high-resolution mass spectrometry (HRMS) spectra ([M – H]⁻¹) for peptide or acyl peptide products observed in 24 h incubated reaction mixtures starting from Val-NCA (20 mM), tryptophan (W) (20 mM), EPPS (300 mM), and either oleic acid (20 mM) (b and c) or octanoic acid (20 mM) (d and e). For similar HRMS analyses of the oligo-valine products (V_n) [R² = CH(CH₃)₂], see Figure S1, SI). HRMS experiments were carried out in negative mode. Panels (b) and (d) highlight the W-containing native peptides observed in the presence of oleic acid and octanoic acid, respectively. Panels (c) and (e) highlight the W-containing oleoylated (Ol-) and octanoylated (Oc-) products, respectively. See the SI for tabulation of mass errors and an HPLC-extracted ion chromatogram showing the product distribution observed in the case of oleic acid (b, c).

Figure 2

Possible pathways of a reaction between Val-NCA (1) and a carboxylate nucleophile. Product 3 also represents acyl-Val since free Val (from hydrolysis of 1) reacts with 4 via path iii.

Figure 3

Mechanistic investigation of the formation of a mixed anhydride from Val-NCA 1 and octanoic acid. (a) (a) Schematic of the reaction between 1 and either ¹⁶O-enriched or ¹⁸O-enriched octanoic acid with observable [M – H]^–1 ions. (b) MS/MS product ion spectra highlighting the isolated ¹⁶O-enriched species and its fragmentation product. (c) MS/MS product ion spectra highlighting the isolated ¹⁸O-enriched species and their fragmentation products.

Figure 4

Oleic acid membrane growth (a) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane growth (b) upon oleate (OA) micelle and Ol-arginine (Ol-Arg) addition. The change in membrane surface area is reported upon the addition of various additives to 7.5 mM oleate or POPC vesicles in 200 mM bicine buffer, pH 8.5. OA micelles or Ol-Arg were dissolved by addition of either 4 mM NaOH or 4 mM HCl, respectively, and control additions of either the base or acid alone are indicated. n = 3; error bars represent standard deviation.

Figure 5

Microscopy of RNA-membrane localization with Ol-Arg. Confocal images of a 5′ Alexa Fluor-647 labeled 15-mer RNA oligonucleotide (green) association with giant vesicles composed of (a) 100% oleic acid (OA) (b) 100% POPC vesicles. An NBD-PE dye (red, panel a) and Lissamine Rhodamine PE dye (red, panel b) was used to label OA and POPC vesicle membranes, respectively. (Top rows, panels a and b) In the absence of Ol-Arg (control), RNA does not associate with either OA or POPC vesicles. (Bottom rows, panels a and b) Upon incubation with RNA, OA containing 25 mol % Ol-Arg and POPC vesicles containing 50 mol % Ol-Arg internalize or localize RNA. Increased regions of RNA fluorescence indicate aggregates of RNA that have bound the membrane. Differential interference contrast (DIC) images (gray) and fluorescence images (NBD or Rhodamine (red), AlexaFluor-647 (green), and a merge of the Rhodamine and Alexa Fluor-647 channels) are shown for each field of view to show the location of RNA with respect to the vesicle membranes. Images are contrast adjusted. Scale bars represent 10 μm.