2023 年 20 巻 1 号 論文ID: e200012
While it is often believed that the origins of life required participation of early biomolecules, it has been recently proposed that “non-biomolecules”, which would have been just as, if not more, abundant on early Earth, could have played a part. In particular, recent research has highlighted the various ways by which polyesters, which do not participate in modern biology, could have played a major role during the origins of life. Polyesters could have been synthesized readily on early Earth through simple dehydration reactions at mild temperatures involving abundant “non-biological” alpha hydroxy acid (AHA) monomers. This dehydration synthesis process results in a polyester gel, which upon further rehydration, can assemble into membraneless droplets proposed to be protocell models. These proposed protocells can provide functions to a primitive chemical system, such as analyte segregation or protection, which could have further led to chemical evolution from prebiotic chemistry to nascent biochemistry. Here, to further shed light into the importance of “non-biomolecular” polyesters at the origins of life and to highlight future directions of study, we review recent studies which focus on primitive synthesis of polyesters from AHAs and assembly of these polyesters into membraneless droplets. Specifically, most of the recent progress in this field in the last five years has been led by laboratories in Japan, and these will be especially highlighted. This article is based on an invited presentation at the 60th Annual Meeting of the Biophysical Society of Japan held in September, 2022 as an 18th Early Career Awardee.
Polyesters could have been synthesized readily on early Earth through simple dehydration reactions at mild temperatures involving abundant alpha hydroxy acid (AHA) monomers on early Earth. This dehydration synthesis process results in a polyester gel, which upon further rehydration, can assemble into membraneless protocell which could potentially provide functions to a primitive chemical system leading to chemical evolution from prebiotic chemistry to nascent biochemistry. Investigating primitive polyesters and polyester-based protocells is of utmost importance to understand how “non-biomolecules” could have contributed to the origins of life.
The origins of life took place over billions of years starting from the available prebiotic chemicals and chemistries on early Earth and arriving at modern biology. How such processes took place is a focus of the origins of life field, which has recently blossomed into a vibrant, diverse research community encompassing planetary scientists, geologists, physicists, chemists, biologists, and more [1–3]. In particular, one of the most fascinating unanswered questions (among many!) in prebiotic chemistry is what the first molecules of life looked like, i.e., what their chemical makeup was, how they polymerized into functional polymers, and how these polymers reacted with each other to lead to assembly of the first cells on Earth, i.e., protocells. An essential part of prebiotic chemistry seeks to answer this question [4–6].
There are generally two main candidates for the first biopolymers on Earth, both derived from modern biology: peptides and RNA. Peptides are simplistic versions of proteins, and can perform structural and/or catalytic functions. Similarly, RNA are also present in modern biology, and can perform both catalytic functions and act as a genetic vessel. Thus, given the various functions of peptides and/or RNA, the “peptide world” and “RNA world” theories were born (and later also merged to become the “peptide-RNA world” or “pre-RNA” theories) [7–12]. However, it is also plausible that the first driver of prebiotic chemistry need not have been functional biopolymers, but rather nonenzymatic metabolic cycles on early Earth (also known as the “metabolism first” theory) [13–15]. While there are various merits and demerits to considering each of these theories, we do not focus on this here as it is out of our scope, and a number of articles (referenced above) have already focused on each of these topics.
Nearly all of these theories have focused on how biomolecules or biological-like processes could have emerged on early Earth. And while this is certainly reasonable, given that we have only one example of what “life” should look like, we are likely being subjected to survival bias [16], i.e., a type of cognitive bias humans are susceptible to when we gather information and make new knowledge only on “successful” entities that are persistent or apparent to us, while overlooking those that are not. Hence, as an alternative to the biocentric origins of life narrative and to minimize the potential of such survivor bias, we conjecture that: there may be other “non-biological” chemistries which could have also contributed to the origins of life that did not survive to lead to modern biology. Indeed, such “non-biological” prebiotic chemicals were highly abundant on early Earth, and far more abundant than prebiotic biological molecules [17]. For example, while biomolecules such as amino acids can be produced as a result of atmospheric discharge (which could have been plausible on early Earth), the products spectra are quite broad and a significant number of “non-biomolecules” is also simultaneously synthesized [18]. Similarly, while nucleotides and amino acids can be detected in carbonaceous chondrites (one possible method for prebiotic chemical delivery to Earth), non-canonical nucleotides and amino acids are also detected in the same samples [19,20]. Thus, it is indeed relevant to at least consider the relevance of “non-biomolecules” during the origins of life.
One of the more abundant “non-biomolecules” that may have been abundant on early Earth are alpha hydroxy acids (AHAs). AHAs are monomers that are congeners to amino acids, but with the amino group replaced by a hydroxyl group, and can be found environmentally in volcanic or other hydrothermal settings [21,22], in extraterrestrial bodies such as meteorites [23–25] or UV-irradiated comets [26], or as products of atmospheric discharge [18] or photochemical [27] reactions. Solutions of AHAs can then undergo dehydration synthesis through drying, at mild geological locales such as at the banks of bodies of water or through seasonal/diurnal cycles, resulting in accumulation of polyester products [17]. This synthetic process is thermodynamically favorable, and can produce a large amount of polyester products in a number of different fresh or saltwater environments on early Earth, which could then go on to assemble into membraneless droplets with potential function upon rehydration (such as through precipitation or the tidal cycle). Here, we review the recent advances in understanding aspects of the synthesis of primitive polyesters and subsequent membraneless microdroplet assembly, and allude to the future work required to further understand the role of polyesters at the origins of life.
AHAs, such as lactic acid (LA), have been used to synthesize polylactate (PLA) polyesters for a number of years by ring-opening polymerization (ROP) of lactides (the cyclic dimer of LA), mostly for industrial or medical purposes [28,29]. A number of these processes have relied on various catalysts [30], and although the chemistry itself is of interest in a prebiotic context, the existence of specific catalysts on early Earth is unlikely, leading to the necessity to discover a non-enzymatic method of AHA polymerization. Luckily, the thermodynamics of AHA dehydration synthesis are quite favorable, as the ester bond formation Gibbs Free Energy is around 0 kJ/mol (at physiological conditions) [31], and thus perhaps there are alternative methods to generate polyesters from AHAs. In fact, a number of studies have shown the potential for various polyester systems to form through heating of dry AHA powders [32,33]. Given that it appeared that heat itself could provide sufficient energy to drive the polymerization reaction, finding a prebiotically plausible mechanism to remove water from AHAs (which would lead to chemical equilibrium shifting to favor polyester formation) could be one mechanism to produce polyesters prebiotically.
In regards to the origins of life, Alexander Rich was perhaps the first to suggest the participation of AHAs in early biological systems in 1971 [34]. His idea was based on a paper published a year earlier showing ribosomes’ ability to catalyze ester-bond formation [35], However, to our knowledge, his suggestion did not gain any momentum, perhaps limited by the more highly explored biomolecular-centric origins of life view or a lack of a mild geological avenue capable of hosting prebiotic chemistry at the time.
Since then, the seminal study performed by Mamajanov, et al. in 2014 was perhaps the first to investigate the prebiotic possibility of AHA polymerization through evaporation of malic acid (a prebiotically plausible AHA) solutions via heating, a plausible mechanism of condensation of water [36]. The researchers decided to first heat dried malic acid at 50°C, over 18 hours. After this, the system was rehydrated, and allowed to sit for another 12 hours at different temperatures between 70°C and 95°C, temperatures that could have been achieved on early Earth, such as through hot springs or volcanic environments. The researchers discovered that higher temperature reactions resulted in greater polymerization propensity, with a smaller amount of unreacted malic acid remaining at the higher temperatures. Mass spectrometric analysis revealed the existence of polyesters of length up to 10 residues. Mamajanov, et al. then designed a system utilizing repeated “wet-dry cycles” with alternating dehydration (of constant temperature) and rehydration (of variable temperature between 60–85°C) phases of malic acid, with a combined period of about 24 hours. While a majority of polymers, which formed during the dehydration phase, hydrolyzed back to the monomer state during the rehydration phase, a small amount of intermediate length polymers (up to 8-mers) could be maintained during the wet phase at lower temperatures (i.e., 60°C). While the authors then go on to optimize cycle duration, temperature, etc., this was an important early demonstration in the feasibility of polyester polymerization from simple AHA under prebiotically plausible early Earth conditions.
Further work building upon this seminal study then focused on increasing the complexity of polyester-based primitive systems. For example, in 2015, Forsythe, et al. mixed different combinations of AHA (lactic acid; LA) and amino acid (glycine; G) monomers, and then subjected mixtures of these monomers to wet-dry cycles [37]. Drying of pure LA resulted in production of linear polymers, whereas drying of pure G resulted in no polymers. However, mixed LA/G systems resulted in depsipeptide products containing both LA and G residues. LA/alanine (A) systems also revealed that with increasing numbers of cycles, more A residues became incorporated into the depsipeptide products. Over time, it appeared that amino acid residues “outcompeted” AHA residues due to the thermodynamic stability of the peptide bond compared to the ester bond. However, at earlier points in the wet-dry cycles, ester bonds may be preferred as their production is more thermodynamically favorable. The authors suggest that dehydrating mixtures of both AHAs and amino acids on early Earth to form depsipeptides could have been an intermediate product that led to formation of primitive peptide products at larger scale. This work has then been expanded upon to additionally study more aspects of primitive depsipeptide polymerization, including transition metal catalysis [38], depsipeptide-RNA stabilization [39], and selection of biological amino acids [40], among others [41–45].
However, increasing chemical complexity of primitive polyesters can also be accomplished by using purely AHA-containing systems, which is the focus of this review. In 2018, Chandru, et al. increased the chemical diversity of the starting materials to react up to five different AHAs with different functional groups (LA, glycolic acid (GA), leucic acid (MA), 2-hydroxy-4-(methylsulfanyl)butanoic acid (SA), and phenyllactic acid (PA); all form exclusively linear polymers) simultaneously through dynamic combinatorial chemistry combined with dehydration synthesis between 60–120°C [46]. Using high-resolution Fourier transform ion cyclotron resonance MS (FT-ICR-MS), the researchers were able to find that all five AHA monomers successfully incorporate into polymer products after dehydration, suggesting the plausibility that even higher chemical complexity could be afforded within this system (Fig. 1). Similarly, Chandru, et al. showed that chemical diversity could be increased not only through increasing the library size, but also significantly increasing the library diversity in chemical functionality, and performed a study which showed that cyclic starting materials (such as glycolide or lactide, which are cyclic dimers of GA and LA, respectively), could also form polyester products after dehydration synthesis [47]. As early Earth likely contained a large diversity of chemicals, different starting materials similar or derived from AHAs could still result in formation of polyester products. However, while it is possible to physically increase the chemical diversity infinitely by mixing an unlimited number of AHA monomers in the initial library or by increasing the chemical functionality of the starting monomers (which actually may indeed by the only mechanism to approach simulating the true chemical diversity of early Earth), it is difficult from a practical point of view for tractability reasons, as complex chemical mixtures are practically difficult to analyze in the laboratory (such as the advanced FT-ICR-MS technique used in the previous study).
A zoomed-in region of an FT-ICR-MS spectra of polyester products derived from dehydration synthesis of a mixture of five different AHAs (LA, GA, MA, SA, and PA), showing incorporation of each residue within the product chain. Figure reprinted with permission from [46] under a Creative Commons license.
Another strategy is to increase the chemical diversity of the system step-wise to observe whether single changes in components result in changes in polyester polymerization. For example, in 2021, Jia, et al. were able to successfully incorporate a basic AHA, 4-amino-2-hydroxybutyric acid (4a2h), into polyester products by mixing 4a2h with another neutral AHA (such as LA or PA) [48]. These basic polymers could be synthesized through formulations containing different ratios of 4a2h to other AHAs, suggesting that fine-control of polyester product composition is possible. Similarly, Afrin, et al. decided to study the effect of monomer chirality on primitive polyester polymerization reactions, and discovered that for LA and PA, starting monomer chirality (D-, L-, or DL-) did not matter and all of the polymer product lengths were similar [49]. However, for other AHAs, this may not be the case and further exploration of a greater AHA sequence space is necessary. However, in the same study, it was found that polymer length could be tuned by changes in temperature, with higher temperatures (up to 120°C) resulting in longer polyester products (Fig. 2). This suggests that changing the environmental conditions of the synthesis reaction could result in different reaction products, and should be further probed in the future. Previous studies suggested that the polymerization reaction equilibrium constant favors hydrolysis of the polymers into monomers at room temperature, while favoring further polymerization at higher temperatures [28]. However, density functional theory (DFT) estimates of the Gibbs Free Energy of the reaction suggested that thermodynamic factors alone could not explain this observation, and that there may be kinetic factors involved [49].
Increasing synthesis temperature results in increasing product chains of LA and PA, up until 120°C. Modified (contrast was modified slightly to improve distinguishability between the gray and the black bars) and reprinted with permission from [49] under a Creative Commons license.
Finally, while the previously highlighted studies all focused on increasing chemical complexity of primitive polyester-based systems, it is also possible to increase their structural complexity as well. Up until now, nearly all of the mentioned systems resulted in linear or mostly linear polymer products. However, if there is more than one carboxyl group and/or hydroxyl group on the starting materials (even if they are not hydroxyacids), then the resulting product may be branched. For example, Mamajanov, et al. were able to produce so-called hyperbranched polyesters through complete drying over 72 hours at 85°C of mixtures of glycerol (containing three hydroxyl groups) and citric acid (containing three carboxyl groups and one hydroxyl group), both of which are also prebiotically available [50]. Mamajanov and Cody then follow to increase the chemical diversity of the starting materials in an attempt to effect an increase in the structural and functional diversity of hyperbranched polyester products [51]. Increasing the starting library of glycerol and citric acid to also include triethanolamine, followed by heating to 110°C without dehydration, resulted in hyperbranched polyester products containing a tertiary amine. Other studies probing synthesis of primitive hyperbranched polyesters through wet-dry cycles have also been performed [52], which suggest the prebiotic plausibility of such complex structures on early Earth. Modern enzymes are inherently globular, and this globular structure may be a key to their catalytic function. In particular, hyperbranched polymers form similar globular structures, and thus it was proposed by Mamajanov and Cody that the globular structures afforded by primitive hyperbranched polymers (in particular, polyesters), could lead to primitive catalytic structures [51].
Once polyesters are formed on early Earth, what can they contribute to the origins of life? For example, replacement of citric acid in the ternary starting mixture with methylsuccinic acid or adipic acid also resulted in similar hyperbranched polyester products, which even had the ability to act as catalysts to the Kemp elimination reaction by increasing the reaction rate by two- to three-fold, thus suggesting that polyesters with sufficient chemical, structural, and functional complexity could have been early catalysts (i.e., “protoenzymes”) on early Earth before the emergence of peptide or protein-based catalysts [51]. Further development of this system revealed that hyperbranched polyesters could even scaffold photocatalytic zinc sulfide nanoparticles [53]. However, other than the literature described here, there are not many studies which focus on prebiotic hyperbranched polyesters, and we suggest that this should be a priority for future study.
Nevertheless, another possibility could be through macromolecular self-assembly, which was absolutely essential to the origins of life [54–56]. However, macromolecular self-assemblies could take many forms, for example droplets [57,58], crystals [59,60], fibers [61,62], or even more complex architectures [63,64]. However, one type of macromolecular self-assembly likely to have been relevant for origins of life were primitive compartments, also known as protocells, which themselves may have eventually evolved into modern cells (although it is not a strict requirement) [65–67]. These protocells may have imbued important functions into primitive evolving chemical systems, such as homeostasis [68], movement/diffusion [69,70], biomolecular compartmentalization [71,72], scaffolding of more complex structures [73,74], division [75,76], or more. Nevertheless, it is still unclear what structure the first protocells on Earth took. Some have proposed lipid membrane-based vesicles [72,75], while other researchers prefer to study alternative systems, such as membraneless droplets generated from phase separation [77–79]. For example, Oparin’s initial proposal of coacervates as protocells has led to a significant discussion on the importance of membranes at the origins of life, and whether a membraneless compartment may be equally as important [67,78,80,81]. Whether one is more “correct” than the other is still up to debate. Nevertheless, given the fact that phase-separated membraneless droplets, such as coacervates, can scaffold lipid membranes around them [74,82,83], suggests that perhaps such membraneless protocells may have preceded membrane-bound protocells and are worth more investigation.
In particular, Jia, et al. discovered in 2019 that homopolyesters synthesized through a single dehydration cycle of pure mixtures of AHAs (LA, PA, MA, and SA) at 80°C over one week resulted in a gel-like product, and upon rehydration in 4:1 water:acetonitrile solution, formed a turbid suspension [84]. This mixture was composed of a large population of membraneless droplets visualizable through light microscopy (Fig. 3), and are thought to assemble due to the fact that the long-chain polyester products (some of which reached more than 40 residues in length) were more thermodynamically stable in the condensed phase, resulting in membraneless droplet formation. These droplets eventually coalesced over longer time periods (on the order of days), while also exhibiting stability to transient high temperatures and also showed the ability to segregate biomolecules (e.g., a protein and RNA) and small molecules (e.g., chemical dyes), and Jia, et al. thus proposed that membraneless polyester droplets could have been a relevant protocell model. In fact, starting from the same five AHAs that Chandru, et al. studied in 2018, other than pure GA, the researchers found that polyesters synthesized through dehydration of all other equimolar combinations of these five AHAs (for example, all singular, binary, ternary, quaternary, and quinary mixtures) all resulted in formation of membraneless droplets, suggesting that this assembly property may be general to certain long-chain polyesters.
Membraneless polyester droplets assembled after dehydration-rehydration of different AHA monomer solutions (500 mM, ~1 week, 80°C), including homopolyesters composed of lactic acid (LA), leucic acid (MA), 2-hydroxy-4-(methylsulfanyl)butanoic acid (SA), and phenyllactic acid (PA). Homopolyesters of glycolic acid (GA) did not form droplets, while heteropolyesters including LA, GA, SA, MA, and PA still resulted in droplet formation. Reprinted with permission from [84]. Copyright Jia, T.Z., Chandru, K., et al., with an exclusive License to Publish to NAS. Scale bars of main images are 100 μm; scale bars of insets are 10 μm.
It is indeed a requirement for polyesters to be of a certain length in order to assemble into droplets, as Afrin, et al. found in 2022 [49]. In this study, increasing lengths of polyesters correlated to increasing droplet assembly propensity, and it is hypothesized that depending on the composition of the polyesters, there is a minimum length requirement before droplet assembly becomes physically possible. Jia, et al. also found in 2021 that the primary sequence of the polyester chains also had a significant effect on droplet assembly propensity [48].
In this study, increasing ratios of incorporated 4a2h (a basic AHA which may imbue positive charge) on a polyester chain containing another neutral AHA resulted in lower assembly propensity (Fig. 4). This is likely due to the increased charge of the polyester leading to greater solubility within the aqueous media, resulting in fewer assembled droplets. Additionally, incorporating 20% charged 4a2h into a polyester resulted in acquisition of RNA segregation ability to some polyester droplets, suggesting that even small changes in chemical composition could lead to changes in or emergence of physical properties. Additional studies with step-wise increases in chemical complexity in AHA and polyester systems are thus needed to further probe the functional space of primitive polyester droplets.
Results showing the relationship between polyester composition (increasing ratios of 4a2h) and droplet assembly propensity. Reprinted with permission from [48] under a Creative Commons license.
Here, we reviewed the current state-of-the-art of primitive polyester synthesis and membraneless droplet assembly. While there has been significant progress made in the last 5–8 years, there still remain many unanswered questions related to the relevance of polyesters to the origins of life. We propose four specific areas of primitive polyester research in which further studies are necessary:
1) In particular, the polyester chemical space studied so far is quite small, with the number of different AHAs probed in the low double-digits. We suggest future studies to increase the area of primitive polyester chemical space that can be analyzed, and in particular to use more computational or combinatorial chemistry methods to further correlate polyester chemical space to polymerization and assembly propensity.
2) Considering their possible ubiquity within the prebiotic chemical milieu, polyesters may catalytically contribute to driving primitive reactions (as shown in [51]). Indeed, while some seminal studies have explored the plausibility of primitive catalysis by polyester systems [53], the prebiotic functionality of polyesters as “protoenzymes” must be further explored. However, which reactions were the most important/abundant reactions on early Earth requiring catalysis is under debate, resulting in difficulty in determining which reactions to focus on when assigning “protoenzymes” to reactions (however, this is an issue with other primitive catalytic molecules as well). Similarly, polyesters can not form hydrogen bonds to fold into 3D structures (like proteins), thus resulting in the lack of a clear active site for catalysis, necessitating a different view of how such catalysis can occur.
3) The environmental conditions of early Earth were very diverse, containing a variety of geologies such as minerals including clays, micas, and serpentines, while also supplying a wide range of temperatures, pressures, salinities, and other variables; thus, the physicochemical properties of the various potential environmental microenvironments equally broad [85]. While researchers have probed polyester polymerization and assembly under different temperature conditions [49], there are an infinite number of other plausible environmental conditions (such as salinities, pH, etc.) that must also be probed.
4) One of the more important aspects of protocells is their ability to interact with different primitive biomolecules, such as peptides, lipids, and/or nucleic acids. Further studies to incorporate different biomolecular systems into the polyester microdroplets are required. These studies may include the potential of different molecular self-assemblies (which themselves have been shown to assemble within membrane coacervate droplets) to assemble within polyester droplets [86–88].
5) Previous studies have considered condensed phase gels as relevant primitive structures that could segregate or protect prebiotic molecules [89–91]. Thus, one recent proposal is for polyester gels, which can form following dehydration of AHAs (but before the rehydration process which assembles droplets) or other primitive compounds [52], to act as vessels to protect molecules from degradation. In particular, these prebiotic gels are proposed to have the potential to have been utilized as panspermia seeds (Fig. 5). Panspermia seeds are structures that would have been able to transport chemicals from one planet (donor) to another planet (recipient); these encapsulated chemicals then would be released from the seed on the recipient planet, eventually leading to the origin of life. However, such panspermia seeds must survive the harsh conditions of space (including radiation, temperature fluctuation, microgravity, etc.) and also simultaneously protect the encapsulated chemicals from degradation [92]. Further studies characterizing the stability of polyester gels in extraterrestrial conditions, such as cosmic radiation or microgravity, are required.
Proposal for polyester gels to act as material-based Pansmermia seeds (M-BPS), and protect and transport compartmentalized analytes during extraterrestrial travel to a recipient planet. Reprinted with permission from [92], copyright John Wiley and Sons.
No conflicts of interest.
T.Z.J. and K.C. wrote the manuscript.
No new data was generated.
K.C. is supported by the Research Encouragement Grant (GGPM-2021-057) by the National University of Malaysia and Visitor Grant (ZF-2022-008). T.Z.J. is supported by JSPS Grant-in-aid and 21K14746. T.Z.J. is a member of the Earth-Life Science institute (ELSI) at the Tokyo Institute of Technology, which is sponsored by a grant from the Japan Ministry of Education, Culture, Sports, Science and Technology as part of the World Premier International Research Center Initiative. T.Z.J. would like to thank the Biophysical Society of Japan (BSJ) for being awarded the Earth Career Presentation Award at the 60th annual BSJ meeting in Hakodate, Japan in 2022, which led to the opportunity to submit this review article. We would like to thank the following researchers who have assisted us throughout the years with fruitful discussions and collaborations regarding some of the ideas written in this paper: Nicholas Guttenberg, Chaitanya Giri, Yayoi Hongo, Christopher Butch, Irena Mamajanov, Henderson James Cleaves II, Rehana Afrin, Tomohiro Usui, Kunihiro Myojo, Niraja Bapat, Ajay Verma, Mahendran Sithamparam, Nirmell Satthiyasilan, Chen Chen, Davide Sarpa, Ruiqin Yi, Sudha Rajamani, Anna Wang, Po-Hsiang Wang, and many others.
