Hydroxy acid conjugation to lipids increases structural and hydrolytic stability

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Nature Communications volume 17 , Article number: 6973 ( 2026 ) Cite this article

Contemporary life requires functional polymers and phospholipid-derived compartmentalization. Mutualistic relationships between compartmentalization and polymerization have never been demonstrated in an abiotic scenario. As both the polymerization of hydroxy acids and the primitive cell-like aggregation of short-chain fatty acids are well known, we study cooperative interactions between compartmentalization and polymerization using these two classes of molecules as a model system. To that end, we explore the formation of various hydroxy acid-fatty acid conjugates. All reactions produce two types of condensation products: hydroxy acid oligoesters and lipid-conjugated oligoesters. We find that conjugation of hydroxy acids to fatty acids leads to the reduction of fatty acid critical aggregation concentration by an order of magnitude. Furthermore, hydroxy acid oligomers are protected against hydrolysis only upon conjugation to fatty acids. Our work offers meaningful insights into the role of self-assembly and cooperative chemistry as selective driving forces in lipid-polymer co-evolution.

One of the puzzling questions in origins of life research is which pathways might allow a direct evolutionary linkage between compartmentalization and polymerization 1 . The existence of compartments is essential in promoting Darwinian evolution, forming microenvironments in which cellular chemistry can occur 2 , 3 . In the prebiotic context, compartmentalization offers physical mechanisms to promote polymerization by localizing different building blocks at high concentration and in close proximity. Compartmentalization can also hinder polymer hydrolysis, which is thermodynamically favorable in aqueous environments. This effect can be achieved, for example, when molecules partition to, or are shielded by, the membrane interface, or when the compartment lumen differs from the bulk solution in pH or ionic strength 4 , 5 , 6 , 7 , 8 , 9 , 10 . In modern biology, the role of compartmentalization is dominantly played by phospholipids 11 , which are the key building blocks of cell membranes, although other mechanisms of sub-cellular compartmentalization exist, such as membraneless liquid-liquid phase separation condensates 12 . Membranes formed by the self-aggregation of phospholipids and other contemporary lipids possess low permeability and physical durability, allowing the membranes to act as selective barriers 13 . Such properties, while desirable from a biological perspective, may raise difficulties in the context of prebiotic environments, wherein greater diffusion of small molecules through the membrane or other transport mechanisms were likely necessary in the absence of highly evolved specific membrane transporters 14 .

Phospholipids are complex amphiphiles and likely emerged at later stages of chemical evolution, although several prebiotic routes for their synthesis have been suggested 15 , 16 , 17 , 18 , 19 . The core building blocks of phospholipids, fatty acids, are found in model prebiotic reactions via Fischer, Tropsch type synthesis 20 , 21 , 22 , 23 and are abundant in meteorites 23 , 24 , 25 , 26 , although they are considerably shorter than the fatty acids found in contemporary phospholipids. While most abundant prebiotic fatty acids are commonly short (up to C12-C15) 26 , 27 , contemporary phospholipids contain long-chain acids (typically C16-C26) 28 . Fatty acids are perhaps the simplest model of protocell building blocks 29 , and their self-assembly into vesicles has been thoroughly studied 7 , 30 , 31 , 32 . By themselves, fatty acids suffer from poor physical stability, which may affect their robustness as protocells. However, this fragility can be attenuated under certain conditions, such as in the gel phase 33 or through cooperative aggregation in the presence of other building blocks that help to stabilize fatty acid vesicles 20 , 34 . For example, the presence of even small amounts of fatty alcohols, mono-acyl glycerides, and alkylamines reduces the critical vesicle concentration (CVC) and stabilizes decanoic acid vesicles at a wider range of pH conditions and over a greater range of salt concentrations 31 , 35 , 36 . Amino acids, peptides, nucleobases, and sugars also stabilize decanoic acid vesicles via noncovalent interactions 37 , 38 , 39 , 40 , 41 . Other non-covalent and covalent associations of lipids have been reported 42 , 43 , 44 , 45 , 46 , providing evolutionary advantages and underlining the importance of cooperation between different classes of chemical species during molecular evolution 33 , 34 , 47 .

The molecular diversity on Early Earth was immense, and included various organic molecules that were either delivered exogenously by meteorites or produced from fundamental building blocks in situ 25 , 48 , 49 , 50 , 51 , 52 . In the context of lipids, such diversity can assist in stabilizing lipid vesicles non-covalently, but also holds the potential path towards the formation of more complex and robust amphiphiles. Hydroxy acids (HAs) are highly attractive prebiotic molecules in the context of origins of life; they serve as metabolites in extant biology, exhibit high reactivity, and play a catalytic role in prebiotic peptide synthesis 53 . HAs such as glycolic acid and lactic acid have been found to facilitate peptide bond formation via ester-amide exchange under wet-dry cycling and drying reactions 54 , 55 . HAs have been suggested as key non-biomolecular players in prebiotic chemistry, and the polymerization and aggregation properties of their resulting oligoesters have been thoroughly studied 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 .

All biopolymers result from condensation-dehydration reactions and can hydrolyze in water 47 , 64 . Hence, in a prebiotic context, formation of polymers by itself is insufficient, and strategies to impart hydrolytic stability are perhaps of equivalent importance. Therefore, it is important to unravel plausible molecular mechanisms that enable simultaneous polymerization, hindered hydrolysis, and compartmentalization.

Here, we investigate whether simple reactions between prebiotically plausible fatty acids and hydroxy acids can generate molecular systems in which oligomerization, protection from hydrolysis, and compartmentalization become directly coupled. To that end, we investigated the formation of prebiotic amphiphiles obtained by reacting decanoic acid (DA) and additional fatty acids with four different HAs. Under simple drying conditions, we were able to produce a wide variety of lipid products that were characterized via various analytical and structural techniques. Our results indicate that DA is mostly reactive towards lactic acid, although lipid-conjugated oligoesters comprised of DA and HAs were obtained for all tested HAs. We then asked whether these conjugates alter properties that could matter functionally in prebiotic settings. Specifically, we examined how conjugation affects amphiphile assembly, permeability-related behavior, and resistance of hydroxy acid esters to hydrolysis. Our results indicate synergistic interactions and pure molecular mutualism, where, on one hand, conjugation of hydroxy acids to lipids promoted greater self-assembly propensity of the lipids, and on the other hand, this conjugation protected hydroxy acid esters from hydrolysis. Moreover, the resulting lipid-conjugated oligoesters exhibited greater permeability compared to unreacted DA. Together, these results identify lipid-conjugated oligoesters as a model system in which covalent coupling between two molecular classes gives rise to emergent physicochemical consequences and highlight the importance of cooperative interactions between different classes of molecules in shaping early chemical evolution.

We investigated esterification reactions between decanoic acid (10-carbon long fatty acid, DA) and four different HAs: glycolic acid (GA), L-lactic acid (LA), L-malic acid (MA), and L-phenyllactic acid (PLA) (Fig. 1 ), to produce the O -acylhydroxy acids. Decanoic acid and single HAs (either GA, LA, MA, or PLA) were allowed to react under dry conditions in the absence of water, other than water originating in the raw materials for 7 days at 85 °C at a 1:1, 1:2, and 1:4 molar ratio (in favor of the HAs). Following the reaction, we analyzed the resulting products via LC-UV-MS, FTIR, and NMR. We found that all four HAs reacted to produce either hydroxy acid oligoesters (oligomers of pure HAs) or lipid-conjugated oligoesters containing oligoesters covalently bound to DA (Fig. 2A, B ). For all HAs tested, the DA-HAs conjugates contained one DA and n-HAs, as expected. Typically, the number of products and their concentration (both hydroxy acid oligoesters and lipid-conjugated oligoesters) increased as the amount of the HA increased, with the exception of PLA, for which no significant differences were observed as the molar ratio increased (Figs. S1, S21 ). When examining the effect of DA:LA molar ratio on DA conversion across different batch sizes, we observed similar trends for 200 and 400 μmol DA, with conversion increasing as the LA fraction increases (Figs. S22, 23 ). However, for 800 μmol DA, conversion reaches a plateau at a 1:2 molar ratio (DA:LA).

Mixtures of decanoic acid with each of the four alpha hydroxy acids were allowed to react at 85 °C for 7 days under dry conditions to produce hydroxy acid oligoesters or lipid-conjugated oligoesters.

HPLC chromatograms were obtained for DA:LA reaction products at 1:1, 1:2, and 1:4 molar ratios. Two types of products, including LA oligoesters and DA-LA conjugated oligoesters were formed ( A ). HPLC chromatograms were obtained for DA:HA reaction products at a 1:4 molar ratio (DA:HA) for the four tested HAs ( B ). HPLC chromatogram obtained for DA:LA reaction product at a 1:4 molar ratio. The labeled signals are lipid-conjugated oligoesters of DA-LA n with up to 12LA-mer conjugated to DA ( C ). MS spectrum (negative mode) was obtained for the identification of the 1LA1DA reaction product. The obtained m/z values correspond to [M-H] - and [2M-H] - ions ( D ). Source data are provided as a Source Data file.

The identification of oligomer length and composition was achieved using LC-MS (Fig. 2C, D , Figs. S24, S157 , Tables S1, S9 ). Out of the four HAs tested, we found that LA produced the longest lipid-conjugated oligoesters (12LA1DA), while GA, PLA, and MA produced similar length oligomers of up to 5, 6 HA (Table 1 ). As for hydroxy acid oligoesters, MA produced the longest oligomers (16MA) and LA, GA, and PLA produced up to 13-,12-, and 7-mer, respectively. Interestingly, in the absence of DA, LA oligomerized to form products of up to 18-mer and possibly longer species (Tables S4, S6 , Figs. S74, S126 ), suggesting that the formation of DA-LA conjugated oligoesters came at the expense of LA oligoesters. The oligomerization degree of the HAs formed in the absence of DA is in good agreement with previous studies, although we observed deviations in the maximum oligomer length detected, likely due to differences in analytical methodology 61 , 65 . The formation of the esterification products was further confirmed by FTIR (Figs. S158, S166 ) and NMR measurements (Figs. S167, S187 ). A shift in the carbonyl stretching in FTIR and downfield shifts of the alpha protons in NMR indicated that ester bonds were formed in all tested mixtures upon drying.

We measured the conversion of DA into products in the various reactions via HPLC and found that the highest consumption was observ