Amino acid analogues provide multiple plausible pathways to prebiotic peptides

Abstract

Prebiotic peptide synthesis has consistently been a prominent topic within the field of the origin of life. While research predominantly centres on the 20 classical amino acids, the synthesis process encounters significant thermodynamic barriers. Consequently, amino acid analogues are being explored as potential building blocks for prebiotic peptide synthesis. This review delves into the pathway of polypeptide formation, identifying specific amino acid analogues that might have existed on early Earth, potentially participating in peptide synthesis and chemical evolution. Moreover, considering the complexity and variability of the environment on early Earth, we propose the plausibility of coevolution between amino acids and their analogues.

1. Introduction

The emergence of the constituent molecules of life on early Earth, and their subsequent polymerization to form functional structures, remains a pivotal inquiry in the field of the origin of life. Peptides are essential for the sustenance of existing organisms, playing a central role in life's functions and information mechanisms [1]. These molecules facilitate diverse biological functions through intricate replication mechanisms within organisms [2]. Notably, investigations indicate that factors like thermal conditions and pH levels in the primordial ocean minimally impacted the stability of monomer molecules, specifically amino acids, constituting peptides. Amino acids demonstrated stability as small organic molecules under conditions of pressure, high temperature and salt concentration [3,4]. In the context of the origin of life, peptides resulting from amino acid polymerization exhibited structural stability in extreme environments and during salt-induced peptide formation (SIPF) reactions [2]. In comparison, nucleic acid molecules do not possess strong stability even under mild chemical and thermal conditions. Therefore, it is highly likely that functional short peptides could have spontaneously formed under the conditions on primitive Earth. Additionally, peptides can serve as replicable information molecules, acting as substitutes for nucleic acids before the emergence of the RNA world. Furthermore, research by Carny et al. has demonstrated that peptides exhibit catalytic abilities in chemical synthesis [5–7]. Consequently, peptides played a crucial role in the origin and development of life billions of years ago during the emergence of life.

The synthesis of peptides under prebiotic conditions has consistently been a focal point of interest. Abiotic synthesis of peptides involves two key steps: first, the synthesis of amino acids or amino acid analogues, followed by the polymerization of these building blocks to form amide bonds [8]. In 1953, Stanley Miller successfully achieved synthesis of organic small molecules from inorganic counterparts in the laboratory using reducing gases (H₂, H₂O, CH₄ and NH₃) under spark discharge conditions [9]. Current research indicates that the early atmosphere contained only a small amount of reducing substances. Bada suggested that a neutral environment (N₂, CO₂) can still generate a significant amount of non-biological organic compounds, including the formation of amino acids [10]. Amino acids present in the experiments of Miller's & Urey [11,12], extraterrestrial meteorites [13,14] and extant organisms have been consistently studied as potential building blocks for prebiotic peptide synthesis. However, the spontaneity of amino acid condensation into polypeptides in aqueous solutions is relatively low due to obstacles such as high activation energy, unfavourable thermodynamic conditions, and side reactions [15,16].

The challenge of forming peptides from amino acids in model prebiotic reactions has led to the exploration of amino acid analogues. These analogues play a crucial role in prebiotic chemistry, serving as fundamental components in supporting the origin of life [17]. Analysing residues from Miller's discharge experiment [12,18], Bada found that hydroxyl compounds were preferentially synthesized, while subsequent HCN and aldehydes/ketones dissolved into the aqueous phase [10]. These compounds then undergo amino acid synthesis through the Strecker reaction pathway. Additionally, Peltzer & Bada detected hydroxyl acids in carbonaceous meteorites [13], suggesting that in the study of origin of life molecules such as peptides, whether through geochemical or extraterrestrial meteorite pathways, consideration should extend beyond individual amino acids to include corresponding amino acid structural units.

The environment on early Earth was intricate and diverse [10], featuring an unimaginable array of compounds in the aqueous phase. It is likely that these substances interacted with amino acids, facilitating the synthesis of prebiotic peptides. This paper reviews potential amino acid analogues (α-hydroxy acid, mercaptan, amino acid amide (AA-NH₂), α-N-carbamoyl amino acid (CAA), α-aminonitrile (α-AA-CN)) in prebiotic sources and prospects the co-evolution of amino acids and their analogues in peptide prebiotic synthesis.

2. Amino acid analogues

2.1. α-Hydroxy acid

From the perspective of the origin of life, α-hydroxy acids emerge as potential small organic molecules present on early Earth. Evidence supporting the concomitant presence of α-hydroxy acids and α-amino acids (α-AA) is derived from multiple sources, including meteorites that reached early Earth and laboratory-produced inorganic small molecules [10,13]. There is even a proposition that polyesters formed through the polymerization of α-hydroxy acids might serve as ancestral precursors to peptides, eventually replaced by peptides during chemical and biological evolution [19].

Weber conducted experiments involving the heat treatment of glyceric acid at 80°C, resulting in the production of oligomers [20]. Subsequent analysis revealed that polymers containing up to 25 monomers could be synthesized through thermal contraction and reaction. The studies conducted by Ohtani et al. demonstrated the successful thermal shrinkage and reaction of l-malic acid at temperatures exceeding 100°C [21,22]. These findings suggest that α-hydroxyl acids can effectively undergo polymerization, forming polyester structures when subjected to drying and moderate heating (figure 1a). In-depth investigations by Mamajanov into the relationship between ester bond formation rates and the hydrolysis rate of polyester in the temperature range of 60–95°C showed that the polyester formed by l-malic monomers reached a stable concentration state and average length after several dry and wet cycles. Mamajanov postulates that the stable coexistence of oligomers with monomers is a vital mechanism for the emergence of the first functional biopolymers [23].

Figure 1.

(a) Polyesters are formed through the reaction of hydroxyl acid. (b) Depsipeptides are formed by reacting polyesters with amino acids.

Interestingly, Frenkel-Pinter et al. conducted experiments on the polymerization of positively charged protein amino acids (lysine (Lys), arginine (Arg), histidine (His)) and non-protein amino acids (ornithine (Orn), 2,4-diaminobutyric acid (Dab) and 2,3-diaminopropionic acid (Dpr)) with hydroxy acids to form depseptides without enzymes and activators. The results revealed that compared to the non-protein amino acids Orn, Dab and Dpr, protein amino acids Lys, Arg and His readily and preferentially polymerize into linear depsipeptides through α-amino [24]. Furthermore, in 2020, Frenkel-Pinter et al. found that oligomers containing Arg, His and Lys in depsipeptides generally exhibit higher enhancement of RNA duplex thermal stability compared to those containing Orn, Dab or Dpr [25].

The experimental system mentioned above illustrates the process of α-hydroxy acid reactions leading to the formation of polyesters. In the study of life's origin, it is imperative to avoid narrowly focusing solely on the polymerization of a single molecule and instead consider the diverse array of other building blocks present in the prebiotic environment [26,27].

In a model prebiotic reaction driven by the wet-cold/dry-heat cycle, Forsythe et al. discovered that the polymerization of α-hydroxy acids and α-AA results in the formation of depsipeptides containing both ester and amide linkages. Forsythe et al. used l-lactic acid (LA) and glycine in the wet phase at 65°C and the dry phase at 85°C for continuous dry–wet cycles. Mass spectrometry analysis revealed changes in the proportion of LA and glycine in depsipeptides with the number of cycles. The hot dry phase witnessed the evaporation of solvent water, leading to the formation and exchange of ester–amide bonds. In the cold and wet phase, the stability of the amide bond surpassed that of the ester bond, resulting in the preference for ester bond hydrolysis [28,29]. Consequently, as the number of cycles increases, depsipeptides gradually enriched peptide oligomers containing amide bonds, with lengths reaching up to 10 residues (figure 1b).

In the mixed system of α-hydroxy acid and α-AA, continuous dehydration and hydrolysis reaction cycles generate oligomers with higher molecular weight and enhanced functionality [30,31]. Importantly, this peptide polymer holds greater biological significance on prebiotic Earth, as depsipeptides formed through the polymerization of ester and amide bonds may serve as precursors to classical peptides. Therefore, investigating the existence of prebiotic polymers under plausible conditions is of paramount importance for elucidating the origins of life [32].

2.2. Mercaptan (mercaptoacids and α-amino thioacids)

Vogel proposed that a sulfous vortex of superheated water and seeping magma provides an environment conducive to the birthplace of life [33]. Huber speculated that the high temperatures of the primitive Earth and violent volcanic eruptions may have catalysed the formation of initial prebiotic molecules from iron sulfide and nickel sulfide minerals near volcanic vents [21]. Experiments conducted by Huber & Wächtershäuser demonstrated that the combination of CO with metal sulfide at high temperatures resulted in the formation of acetic acid, serving as a starting material for amino acid synthesis [34,35]. This process expands the reaction pathways facilitated by prebiotic peptides, underscoring the essential role of sulfur in the origin of life [36].

Heinen & Lauwers discovered that FeS, H₂S and CO₂ can generate various organic sulfides in an anaerobic water environment, with mercaptan identified as a significant organic product [37]. Frenkel-Pinter et al. suggested that the abundance of mercaptan on prebiotic Earth [16], while De Duve speculated that thioesters play a key role in the origin of life based on their importance in the anabolism and catabolism of living organisms (figure 2a) [38].

Figure 2.

(a) Thioesters are formed through the reaction of mercaptoacids. (b) Thiodepsipeptides are formed through the reaction of thioesters with amino acids. (c) The oxidation reaction of α-amino thioacids for the synthesis of α-peptides.

The relationship between thioesters and amino acids is a hot topic in the origin of life field. In 1953, Wieland et al. discovered that peptide bonds can form in an aqueous environment through thioester intermediates [39]. Weber found the formation of glycine-thiodepsipeptides and N-acetyl-S-lactoylcysteine, considered as energy-rich prebiotic molecules [40,41]. In 1998, Weber investigated the role of mercaptan in the synthesis of alanine and homoserine catalysed by formaldehyde and glycolaldehyde, revealing mercaptan as a catalyst for the formation of alanine and homoserine [42]. The study also showed the formation of amino acid thioester, a precursor to thiodepsipeptides. In 2005, Weber successfully synthesized peptides consisting of 20 residues in the presence of mercaptan, emphasizing their relevance to the process of life's origin [43].

Frenkel-Pinter et al. discovered that the dry-down reaction between mercaptoacids and amino acids produced co-oligomers (thiodepsipeptides) with up to hexameric, with various compositions of mercaptoacids and amino acids [16]. Thioester bonds in the thiodepsipeptides can be converted to amide bonds by thioester–amide exchange (figure 2b), a relationship used to study intermediate compound formation and template-assisted replication [44]. Nanda et al. investigated the formation of dominant functional polymers through β-sheet polymerization, demonstrating that β-sheet-driven peptide replication can be used for significant enrichment of naturally occurring condensation products [45], and Fisher et al. explored the conversion efficiency of thioester bonds in thiodepsipeptides to study the structural stability of peptide folding during thioester–amide exchange [46–51]. Dadon et al. found that substituting thioester bonds for amide bonds did not significantly affect the folding pattern of thiodepsipeptides [52]. As we all know, short peptide molecules have the ability to form ordered structures and catalyse chemical reactions [5]. Studies on thiodepsipeptides suggest that they may have existed on early Earth, complementing classical peptides and participating together in the process of chemical evolution of life, both in terms of structural stability and functional catalytic effects.

Okamoto et al. utilized α-amino thioacids [53] as amino acid analogues to synthesize amide bonds in a region-selective peptide bond formation experiment. Fe (III) was employed as an oxidant, and the highest tetramer of α-thioacid form oligomers was detected within 5 min using liquid chromatography-mass spectrometry (LCMS). The unique oxidation potential of sulfuric acid led to the formation of a diaminoacyl–disulfide structure [54–58]. Because the carbonyl carbon in the diacyl–disulfide moiety is highly electrophilic, Okamoto et al. speculated that diaminoacyl–disulfide would undergo an intramolecular S-to-N-acyl transfer, leading to the predominant formation of an α-peptide bond (figure 2c).

The co-condensation of mercaptoacids with amino acids to form thiodepsipeptides, and the simple oxidation of α-amino thioacids to form amino acid polymers, all underscore the irreplaceable importance of sulfides in the formation of prebiotic peptide bonds.

2.3. Amino acid amide

Amino acids and their analogues play an indispensable role in the living organisms, potentially serving as the fundamental constituents in the prebiotic inventory that facilitated the emergence of life [17]. Among these analogues, AA-NH₂ is considered to be a significant candidate that could have potentially existed during the prebiotic stage [59], playing a crucial role in the formation of prebiotic peptides in primitive oceans [60].

In 1959, Oró et al. discovered the formation of amino acids and other compounds by heating an aqueous mixture of formaldehyde and hydroxylamine hydrochloride [61]. An enhanced marine medium facilitated the reaction between formaldehyde and hydroxylamine, leading to the formation of hydrogen cyanide, which underwent the Strecker reaction to form nitrile. Subsequent hydrolysis of the nitrile resulted in the formation of amide, further hydrolysing into acid and ultimately synthesizing glycine [62]. In 1960, Oró et al. proposed a novel method of polypeptide synthesis, which placed Gly-NH₂ in an aqueous solution at 100°C and heated it for 20 h [63]. Analysis of the resulting product revealed that polyglycine exhibited a polymerization degree of up to 40. This experiment suggests the plausible hypothesis that the majority of α-AA-NH₂ can undergo polymerization under analogous liquid or gas phase conditions.

In the study of Nishizawa et al. on the polymerization of Gly-NH₂, the generation of Gly₂ was observed. Furthermore, the co-reaction of Gly-NH₂ and Gly₂ resulted in the formation of Gly₃, along with a minor quantities of tetramers and pentamers [60]. This indicates that peptide formation using Gly-NH₂ can occur through a stepwise reaction. Alkaline catalysts, such as nucleobases, were found to promote peptide formation in this reaction. Yanagawa et al. investigated the morphology of the polymer formed by Gly-NH₂ in different fluctuating systems (neutral aqueous solutions). The results showed that the glycine polymers were widely aggregated under various conditions to form stable organized structures, such as leaf-like structure, stacked disc structure and sheet structure [64].

Parker et al. mentioned in an article that the pKa value of the NH₂ group of Gly-NH₂ is lower than that of the NH₂ group of glycine, resulting in a lower protonation degree of the NH₂ group of Gly-NH₂ and an increased protonation degree of the NH₂ group of glycine [59]. Consequently, Gly-NH₂ acts as a better nucleophile than glycine and is more efficient at forming amide bonds in peptide prebiotic synthesis [65] (figure 3).

Figure 3.

Peptides are formed through the reaction of amino acid amides.

AA-NH₂, acting as both reactants and logical intermediates, can play a dual role in the initial synthesis of peptides. Additionally, their superior nucleophilic reactivity compared to amino acids positions them as potential substrates for prebiotic peptide synthesis.

2.4. α-N-carbamoyl amino acid

Taillades conducted an investigation into the thermodynamics and kinetics of the HCHO/HCN/NH₃ reaction system in aqueous solution with a pH of 8, leading to the formation of α-hydroxyacetonitrile and α-aminoacetonitrile [66]. Notably, formaldehyde was found to catalyse the α-hydroxyacetonitrile reaction, producing α-aminoamides that serve as precursors α-AA. Additionally, CO₂ catalysed the α-aminoacetonitrile reaction, yielding hydantoins, precursors of α-N-carbamoyl-amino acids (CAAs). Collet et al. subjected CAAs to a nitrosation reaction with NO and O₂ compounds, resulting in the production of α-AA-N-carboxyanhydrides (NCA) [67,68], which have the same configuration and quantitative yield as precursors of peptides [17,69]. Vayaboury et al. synthesized CAA using potassium cyanate in water, and continued with a nitrosation reaction to produce NCA [70]. Commeyras et al. proposed that a mixture containing hydrogen cyanide [71], carbonyl compound [72], ammonia, alkyl amine, carbonic anhydrides, etc., led to the formation of α-AA and CAA. Commeyras et al. believed that on primitive Earth, the reaction of CAA with high-energy molecules NO_x might have been a key driving force for the molecular evolution of life [73].

Flores & Leckie, through heating α-AA in a phosphate-buffered aqueous solution, revealed cyanate-mediated amide bond formation [74]. α-AA were converted to CAA by cyanate, and CAA was activated to NCAs by nitrification reaction, which promotes the formation of amide bonds (figure 4a). Taillades treated NCA with aqueous carbonate at pH 9 to produce polymers ranging from trimer to nonamer [75]. In 2002 [76], the synthesis process of prebiotic peptides by CAA driven by nitrogen oxides was envisioned. The reaction pathway consisted of eight sequential steps, including initial CAA synthesis, NCA synthesis [17,77] and hydrolysis, and subsequent polycondensation into a peptide [78]. Consecutive cycles facilitated the expansion of peptide synthesis through the NCA reaction, leading to the gradual accumulation and increased complexity of peptides [79–84].

Figure 4.

(a) Peptide generation by CAA. (b) Urea reacts with amino acids/peptides to produce CAA/carbamoyl peptides.

Danger et al. speculated that CAAs could partially spontaneously converted to NCA through an elimination pathway, contributing to the origin of life and explaining the breakdown of urea at moderate pH [85–90]. We found that under reasonable prebiotic reaction conditions, amino acids reacted with urea to produce CAAs, and dipeptides and tripeptides react with urea to form carbamoyl peptides (figure 4b) [91]. These findings suggest that CAAs could potentially exert a significant influence on the evolution of peptides.

2.5. α-Aminonitrile

Patel et al. and Islam et al. demonstrated that α-AA-CN can be synthesized through organic reactions [92,93]. Despite being a straightforward method for peptide prebiotic synthesis, the efficiency of AA-CN synthesis is low [94,95], and its transformation into amino acids typically requires strong acidic or alkaline conditions, which are not in line with the prevailing conditions for the chemical origin of prebiotic life. AA-SH [53,96] is considered by Maurel & Orgel as a potential alternative to bio-thioesters due to its exceptional water stability and high oxidation properties [58,97,98]. However, its efficiency in peptide linkage formation is limited, and it presents challenges in peptide formation.

The study conducted by Paventi & Edward revealed that aldehydes were initially transformed into AA-CN [99], which subsequently underwent conversion to imidazolidin-4-thiones. Subsequent hydrolysis of the imidazolidin-4-thiones yielded thioamides. Canalvelli et al. [100] proposed a more reasonable and simple approach to achieve continuous peptides synthesis using sulfide-mediated AA-CN [101–103].

The N-acylation of AA-CN not only reduces the degradation of peptides induced by diketopiperazine, but also activates the nitrile group for thiolysis. Ac-AA-SNH₂ was generated through sulfidation of Ac-AA-CN, and subsequently converted to Ac-AA-SH with high hydrolysis efficiency. Canavelli et al. found that Ac-AA-SH formed after Ac-AA-CN thiolation was seven times more efficient than AA-SH analogizes formed after AA activation. Ac-AA-SH was structurally stable and resistant to destruction by related activators present in the reaction system [96,100]. Incubating Ac-AA-SH with AA-CN and ferricyanide produced Ac-AA₂-CN rich in amide bonds, and this connection has been shown to be achieved with high yields under various activator conditions [104], even at low concentrations of AA-CN, leading to high polymer yields. Cysteine not only serves as a raw material for coenzymes but also plays an important role in oxidation–reduction and free radical reactions, leading to it being considered a product of evolution. However, cysteine nitrile (Cys-CN) is unstable due to the incompatibility between the aminothiol of cysteine and the nitrile group. Foden et al. discovered that dehydroalanine activated by nitrile can convert serine into cysteine, and N-acyl cysteines can catalyse peptide bond formation. This suggests that cysteine acts as both a precursor for prebiotic peptides and a catalyst for peptide synthesis [105].

The experimental results demonstrate the feasibility of reacting Ac-AA-SH, produced through Ac-AA-CN thiolysis, with AA-CN to form amide bonds, enabling continuous peptide synthesis (figure 5) [106]. Consequently, AA-CN can be employed as a means for peptide prebiotic synthesis.

Figure 5.

Aminonitrile reaction to form peptide.

The synthesis of peptides from amino acids has consistently been a prominent area of research in prebiotic conditions. The data collected in this review indicate that amino acid analogues (α-hydroxyl acid, mercaptoacid and α-amino thioacid, AA-NH₂, α-N-CAA, α-AA-CN) not only coexisted with amino acids on early Earth but also underwent interconversion with amino acids. Furthermore, the thermodynamic barriers they need to overcome during polymerization are lower than those of amino acid systems. In the intricate and diverse environment of early Earth, analogues of amino acids facilitate peptide synthesis through distinct pathways, expanding the repertoire of peptide formation routes. Consequently, these analogues may hold significant biological implications in prebiotic peptide synthesis.

3. Conclusion and perspectives

The diverse array of small organic molecules generated on primitive Earth managed to endure the challenging conditions of the constantly fluctuating chemical and physical environment. The diversity and structural stability of prebiotic biomolecules and their constituents prove to be of paramount importance in the process of chemical evolution and their widespread availability [2]. The chemical properties and functional structures of individual biomolecules alone are insufficient to navigate the complexity of the prebiotic environment. Thus, a dynamic combination of biomolecules appears more plausible in the evolutionary trajectory of the origin of life.

The synthesis of prebiotic peptides extends beyond specific amino acid species. On one hand, the low reactivity of amino acids poses challenges for the polymerization of amide bonds. On the other hand, the diversity and complexity of the external environment dictate that the evolution of peptides is influenced by other molecules. The emergence of life is an evolutionary process within a dynamic chemical network. Restricting the study to amino acid polymerization alone limits the potential functionality of diverse biopolymers. Amino acids and their analogues are believed to have played a crucial role from the outset, emphasizing the importance of considering the involvement of these analogues in peptide formation from a dynamic perspective [107].

Early Earth's relatively cool temperatures, near-neutral water environment, and the presence of small volcanic islands and oases are considered crucial factors in understanding the origins of life. While earlier discussions have highlighted the potential formation of peptides from amino acid analogues, it is imperative to recognize the differing conditions required for peptide formation. Peptide formation from hydroxy acids necessitates acidic conditions, typically occurring at temperatures ranging from 65 to 85°C, which could have manifested during the local dry–wet cycles prevalent on early Earth. Similarly, the polymerization reactions required for peptide formation from AA-NH₂ also demand elevated temperatures, a condition plausibly met in the hydrothermal environments of early Earth. Furthermore, the activation of amino acids into their N-carboxy anhydrides (NCAs) through NOx-driven nitrosation is noteworthy. This activation process, followed by dissolution into the ocean, may have plausibly occurred in tidal beach environments. Additionally, the synthesis of peptides from AA-CNs necessitates the presence of continuous H₂S and activators, likely abundant in lakes proximal to volcanic activity. The reaction of mercaptoacids' with amino acids exhibits a broad tolerance for pH and temperature ranges, facilitating peptide formation through simple dehydration reactions, yielding products up to hexamers. These diverse pathways offer viable routes for peptide synthesis, underscoring the complexity and adaptability of the chemistry on early Earth in facilitating the emergence of life.

In the context of the prebiotic Earth, the functional cooperation and coevolution of amino acids and their analogues are reasonable. The incorporation of amino acid analogues into the chemical synthesis of research peptides enables the formation of more advanced structures along the biological evolution ladder [8]. The utilization of amino acid analogues has resulted in a diverse and intricate synthesis of polypeptides through multiple pathways, posing an ongoing challenge in selecting functional prebiotic polymers from the vast mixture and achieving subsequent amplification [45]. Nevertheless, we believe that finding a solution to this challenge may illuminate the evolution of life molecules.

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Declaration of AI use

We have used AI-assisted technologies in creating this article.

Authors' contributions

L.Z.: writing—original draft, writing—review and editing; J.Y.: conceptualization, funding acquisition, validation, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This work was supported by the Fundamental Research Funds for the Provincial Universities of Zhejiang (grant no. SJLY2023007), the National Natural Science Foundation of China (grants nos. 42388101, 92256203, 42003062).