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. 2024 Apr 29;4(5):1752–1757. doi: 10.1021/jacsau.4c00243

Spontaneous Peptide Ligation Mediated by Cysteamine

Abid Barat †, Matthew W Powner †,*
PMCID: PMC11134366  PMID: 38818061

Abstract

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The fundamental and universal nature of life’s exploitation of peptides suggests they must have played a vital role during the onset of life, but their spontaneous chemoselective synthesis in water remains unknown. Aminonitriles (1) are widely accepted as prebiotic precursors of both amino acids and peptides, but they do not spontaneously polymerize in water to yield peptides. Here, we demonstrate that the simple prebiotically plausible aminothiol, cysteamine (5), participates in Strecker chemistry to furnish β-mercaptoethyl-α-aminonitriles (8) and β-mercaptoethyl-amino acids (16), which are predisposed to spontaneously form peptides in water. Intramolecular thiol catalyzed ligation is faster, higher-yielding, and more α-selective than previously reported prebiotic peptide ligation chemistries, enabling, for example, the highly regioselective α-ligation of Asp- and Glu-dinitriles in quantitative yields. Our findings suggest that cysteamine (5), the thiol bearing moiety of the universal thiol cofactor coenzyme A, may have played an important role in the selective chemical synthesis of prebiotic α-peptides.

Keywords: cysteamine, nitrile, peptide, prebiotic, Strecker

The universal genetic code establishes that peptide biosynthesis predated life’s last universal common ancestor;1−5 however, peptide biosynthesis is highly evolved1−7 and could not spontaneously appear in its current form at the onset of life. Therefore, peptide biosynthesis must have been preceded by nonenzymatic peptide syntheses during the early stages of evolution;8 however, the nature of prebiotic peptide synthesis remains elusive despite decades of research. Particularly, the spontaneous, chemoselective formation of prebiotic α-peptides in neutral water has not been demonstrated.

Chemical strategies for peptide synthesis rely upon monomer activation and coupling to the N-terminal amine of a growing peptide.9−11 However, interestingly, this stands in stark contrast to biosynthesis; both ribosomal and nonribosomal peptide syntheses proceed in the opposite direction, from the N- to C-terminus, and both rely upon activation of the growing peptide chain toward nucleophilic addition of the monomer amine.6,12−14 This observation suggests that there may be important clues in the direction and strategy of peptide biosynthesis that would facilitate prebiotic peptide synthesis.

Seeking to explore the advantages of the biomimetic strategy, we have previously recognized that aminonitriles 1 and peptide nitriles 2 are thermodynamically activated but kinetically stable substrates for prebiotic peptide synthesis (Figure 1).15−19 Coupling these nitriles, through transformation into thioacids 3 or thioimidates 4 (Figure 1B), allows efficient protecting-group-free peptide synthesis in water. However, despite these advances, the spontaneous synthesis of peptides remains an elusive goal with direct ligation of α-aminonitriles yielding amino-imidazoles that block peptide synthesis (Figure 1A).20

Figure 1.

Figure 1

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(A) Direct aminonitrile ligation would yield aminoimidazoles rather than peptides. (B) Previous work: Prebiotic nitrile mediated peptide ligations. (C) Previous work: Prebiotic synthesis of pantetheine. (D) This work: Intramolecular thiol-catalyzed ligation of peptide-nitriles and reductive fragmentation yielded peptide 11. R = amino acid side chain; R′ = alkyl.

We recently demonstrated a chemoselective prebiotic synthesis of pantetheine (Figure 1C),20 the functional fragment of the universal cofactor, coenzyme A, which is essential throughout all domains of life as an acyl-carrier.13,14 The deep-seated role of pantetheine in biochemistry, for example in the Krebs cycle, fatty acid, and polyketide syntheses, has suggested that thiols may have played a key role in prebiotic chemistry, the “Thioester World” model for the origins of life.21−23 Importantly, in the context of peptides, pantetheine is required in nonribosomal protein modules to generate active thioesters for peptide elongation, and it has been previously proposed that this mode of peptide synthesis may predate ribosomal peptide synthesis during the evolution of life.21 Therefore, our prebiotic pantetheine synthesis opens exciting questions about the role of pantetheine and its precursor, cysteamine (5),20,24−27 in prebiotic peptide synthesis. Specifically, whether 5 could play a role in developing and directing a mechanism for spontaneous peptide synthesis in water that would prevent amino-imidazole formation and foreshadow its incorporation into nonribosomal peptide synthesis as a component of pantetheine.

We reasoned that if 5 (or it is oxidized disulfide, cystamine 6) were to participate in Strecker synthesis, with hydrogen cyanide (HCN) and an aldehyde 7, the reaction would afford β-mercaptoethyl-aminonitrile 8 (Figure 2), where a thiol catalyst, a nucleophilic amine,28 and an electrophilic nitrile are built into one molecule. We anticipated that tethering a thiol-catalyst, required for catalytic peptide ligation (CPL; Figure 1B),17,18 to the amine coupling partner (9) would greatly enhance the rate of peptide ligation and importantly would lead to spontaneous peptide ligation in near-neutral water (Figure 1D).15−19 We also recognize that the thiol-tethered ligation products would be β-mercaptoethyl-peptoids 10, which undergo reductive fragmentation to yield proteinogenic peptides 11 (Figure 1D).29−31

Figure 2.

Figure 2

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Strecker synthesis of mercaptoalkyl-aminonitriles and mercaptoalkyl-amino acids in water. The reaction of aldehyde 7 and aminothiol (5 or 13) yields thiazolidine 12 or thiazinane 14, which react with HCN to yield β-mercaptoethyl-aminonitrile 8 or γ-mercaptopropyl-aminonitrile 25. Hydrolysis of β-mercaptoethyl-aminonitrile 8 or γ-mercaptopropyl-aminonitrile 25 is observed to yield amino acid 16 or 17, respectively, under alkaline conditions. R = amino acid side chain; R′ = H, alkyl, CH2CH2OH, or CH2CH2NH2.

To begin our investigation, we first incubated cysteamine (5) and formaldehyde (7a) in water. We observed quantitative formation of thiazolidine 12a (R = H) across a broad pH range (pH 5–9; Figure 2; Supplementary Figures 1–4). Incubating aldehyde 7a with a mixture of aminothiols (5 and 13) yielded a mixture of thiazolidine 12a and thiazinane 14a (R = H; Supplementary Figure 5). However, selective formation of thiazolidine 12a was observed in equimolar competition with other amines, including ammonia 15a (R′ = H), ethylamine 15b (R′ = Et), ethanolamine 15c (R′ = CH2CH2OH), and ethylene diamine 15d (R′ = CH2CH2NH2) (Figure 2; Supplementary Figures 6–9), demonstrating the selective association of aminothiols with aldehydes that is required for Strecker chemistry to furnish mercaptoalkyl-amino acids.

The addition of HCN to thiazolidine 12a, at pH 8–10, led to the formation of a heterogeneous precipitate, which underwent acid catalyzed hydrolysis to yield mercaptoethyl-amino acid 16G (R = H; 55%; Supplementary Figure 11). Hydrolysis of the precipitate to 16G implicated the in situ Strecker synthesis of aminonitrile 8G, and its reaction onward by CPL. CPL is curtailed at highly alkaline pH,18 therefore we next investigated the Strecker reaction of thiazolidine 12 at pH 13, where we expected precipitate formation would be avoided if the reaction proceeded through CPL. As anticipated at pH 13, the addition of HCN to 12a directly yielded glycine 16G (65%) (Figure 2; Supplementary Figure 12). Under the same conditions, thiazolidine 12b (R = Me) yielded alanine 16A (64%) and thiazinane 14a yielded glycine 17G (62%; Figure 2; Supplementary Figures 13–20). While unlikely to be prebiotic, these high pH reactions demonstrate that the reaction of thiazolidine 12 with HCN proceeded via a Strecker reaction.

The Strecker reaction is widely considered to be the foremost pathway for prebiotic amino acid synthesis;15,32 however, α-ketoacids 18 play a role in amino acid biosynthesis.33−35 Therefore, we next incubated 18 with cysteamine (5) (Figure 3). Quantitative conversion of 18 to thiazolidine 19 was observed across a broad pH range (pH 5–9). Thiazolidine 19 formation was highly selective in competition with other amines 15 (Supplementary Figures 26–29).

Figure 3.

Figure 3

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Synthesis of mercaptoalkyl-amino acid from α-keto acids in water. The reaction of α-ketoacid 18 and cysteamine 5 yields thiazolidine 19 that undergoes hydride reduction to yield amino acid 16. R = amino acid side chain.

Reduction of thiazolidine 19 with NaBH3CN was observed across a range of acidic-to-neutral conditions (pH 3–6; Figure 3), for example furnishing glycine 16G (90%), alanine 16A (77%), and valine 16V (69%) in good-to-excellent yield. In the presence of ammonia (15a; 2 equiv), the reduction of 19a (R = H) led to the selective formation of 16G (85%)—no glycine from reductive amination with ammonia (15a) was observed (Supplementary Figure 32), demonstrating the ambident nucleophilicity of 5 can drive the selective synthesis of β-mercaptoethyl-amino acids (16). However, these amino acids are not activated, and a prebiotic borohydride equivalent remains to be discovered; therefore, we returned our attention to nitrile chemistry.

Having observed the synthesis and spontaneous CPL reaction of aminonitrile 8, we next investigated the Strecker reaction of cystamine (6). We suspected thiol-oxidation would render disulfide-aminonitrile 20 stable and block CPL-type nitrile reactions (Figure 4); however, we also recognized that disulfide 6 could be reduced by HCN,36 and if sufficiently fast, this reduction would block the synthesis of disulfide 20. However, we suspected disulfide reduction would be inhibited by the formation of cyanohydrin 21.37−39 Therefore, we were pleased to observe that incubating cyanohydrin 21a (R = H) and cystamine (6) at pH 9.5 yielded disulfide 20G (50–60%; Supplementary Table 3). Demonstrating thiol oxidation (5 → 6) stabilized β-mercaptoethyl-aminonitriles (8) by curtailing the spontaneous CPL reaction that they are predisposed to undergo.

Figure 4.

Figure 4

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Reductive synthesis of mercaptoalkyl-aminonitrile in water. The reaction of 7 and HCN yields cyanohydrin 21, which reacts with cystamine 6 to yield β,β′-disulfide-α,α′-dinitrile 20 under Strecker conditions, that undergoes disulfide reduction with tris(2-carboxyethyl)-phosphine (TCEP) to yield aminonitrile 8. Aminonitrile 8 undergoes spontaneous ligation at pH 7 to yield peptoid 22 and spontaneous hydrolysis at pH 5 to yield amino acid 16. (B) R = amino acid side chain.

Reduction of disulfide-dinitrile 20 was observed to yield aminonitrile 8 (>95%) across a broad pH range (Figure 4; Supplementary Figures 50–57); under all conditions, except extremely acidic conditions, 8 was observed to react spontaneously to yield secondary products. At acidic pH, the ligation of 8 was not observed; nitrile 8 was observed to spontaneously hydrolyze to amino acid 16 (80% after 7 days; Figure 4) at pH 5, likely via intramolecular thiol-catalyzed hydrolysis, and upon further acidification (pH 1), 8·HCl was observed to be stable, for example yielding glycine 8G·HCl (66%) or alanine 8A·HCl (85%; Figure 4; Supplementary Figures 50, 51, 62, and 63). At neutral-to-alkaline pH (pH 7.0–9.5), aminonitrile 8 underwent spontaneous ligation; at neutral pH, ligation of nitrile 8G was observed to yield cyclic dipeptoid 22GG (65%) in good yield (Figure 4; Supplementary Figures 53–56). This demonstrates the ligation of 8 with a second equivalent of 8 to yield a dimeric peptoid, which remains activated at the C-terminus, resulting in cyclization to yield 22GG. Prior studies of α-aminonitrile–nitrile ligation have yielded imidazoles, blocking amide bond synthesis (Figure 1A);20 however, the incorporation of cysteamine (5) into the aminonitrile, through Strecker reactions, leads to efficient peptide synthesis by aminonitrile coupling.

To further examine the efficacy of cysteamine-mediated nitrile ligation, we next investigated the reaction of peptide nitrile 2G with amino acid 16G. After 24 h, at pH 7, this ligation furnished peptoid 10a (>80%) as a mixture of two rotamers (Table 1; Supplementary Figures 64–66). The ambident nature of 16 resulted in rapid ligation with 2, via the intramolecular reaction of thioimidate (4) with the tethered amine. The secondary amine moiety of 9 prevents tautomerization to a stable thiazoline,40 and so progresses through a transient thiazolinium 23 to furnish amide 10 (Figure 1D). The ligation was observed to be highly pH dependent (Table 1). At pH 5, only partial conversion to 10a was observed in 24 h, whereas at pH 9 near quantitative formation of 10a was observed within 3 h. The ligation of β-mercaptoethyl-amino acids (16) with a wide range of peptide-nitriles 2 resulted in high yields of peptoid 10 (Figure 5). Sterically demanding β-branched nitriles ligated slowly but afforded good-to-excellent yields of 10.

Table 1. Ligation of Glycyl Nitrile with β-Mercaptoethyl Glycine.

graphic file with name au4c00243_0007.jpg

entry 2G/mM 16G/mM T/°C pH buffera time/h yield/%
1 115 100 r.t. 5 ABS 24 7
2 130 100 r.t. 7b   24 85
3 100 100 r.t. 7 PBS 24 84
4 100 100 25 7 PBS 15 79
5 100 100 40 7 PBS 15 90
6 100 100 60 7 PBS 15 91
7 110 100 20 9 PBS 3 >95
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a

Ligation of nitrile 2G and glycine 16G to form peptoid 10a. 500 mM buffer solution; PBS = phosphate; ABS = acetate; BBS = borate. r.t. = room temperature.

b

Final pH = 7.8.

Figure 5.

Figure 5

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Ligation of peptide–nitriles with mercaptoalkyl-amino acids. Ligation of nitriles 2 (37.5–50 mM) with β-mercaptoethyl-amino acid 16 or γ-mercaptopropyl-amino acid 17 (1 equiv) in PBS (pH 7, 500 mM) or BBS (pH 9, 500 mM) at room temperature. *60 °C. R = acetyl or peptide. R′ and R″ = rac-amino acid side chain as indicated by subscript single letter code.

The ligation was strongly promoted by the five-atom intramolecular disposition41 of the amine and thioimidate moieties formed upon reaction of amino acid 16 with nitrile 2 but also achieved through a six-atom disposition (Figure 5; 2G + 17a → 24a). However, incubating equimolar 16G and 17G with nitrile 2G selectively yielded 10a as the major product (10a/24a 77:23; Supplementary Figure 88), demonstrating preferential ligation of amino acid 16G derived from cysteamine (5).

Next, to explore the selectivity of coupling amino acid 16 with proteinogenic α-amidonitriles, we incubated 16G with nonproteinogenic-nitriles. β-Alanine nitrile (2βA) and α-aminoisobutyric acid nitrile (2Aib) were both incubated with 16G and nitrile 2G. Exclusive ligation of 16G with 2G, forming 10a, was observed (Figure 6; Supplementary Figures 131–133), demonstrating that the α-amide selectivity observed in nitrile-activated CPL,17 is retained with intramolecular thiol-catalysis. We next investigated the reaction of the dinitriles. Incubating α,β-dinitrile 2EX or α,γ-dinitrile 2DX with 16G led to exclusive ligation through the α-nitrile (Figure 6C; Supplementary Figures 134, 135, 139, and 140). Moreover, upon further incubation of β-nitrile 10q (up to 2 days) with excess 16G, we did not observe β-nitrile ligation. This remarkable selectivity differentiates α-nitriles from longer homologues; it is also of note that α-ligation, in 2DX, is significantly accelerated by the electron-withdrawing effect of the β-nitrile (Supplementary Figures 137 and 138).

Figure 6.

Figure 6

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Selective ligation of α-amidonitriles with β-mercaptoethyl-glycine. (A) The reaction of 2G (100 mM), 16G (100 mM), and 2Aib (100 mM) in PBS (pH 7, 500 mM) yields 10a (74%) after 24 h. (B). The reaction of 2G (100 mM), 16G (100 mM), and 2βA (100 mM) in PBS (pH 7, 500 mM) yields 10a (87%) after 24 h. (C) The reaction 2DX (50 mM) and 2EX (50 mM) with 16G (50 mM) in PBS (pH 7, 500 mM) yields 10q (>95%) and 10r (>95%) after 1.5 and 10 h, respectively.

Finally, having recognized that fragmentation of peptoid 10 would furnish α-peptide 11 (Figure 1D),29,30 we incubated 10a with TCEP at pH 8.5 and observed the formation of peptide 11GG (70%) in good yield. It is expected that other phosphines (PR3), and potentially phosphine (PH3), can achieve this fragmentation. While PH3 is the primary volatile form of phosphorus and a trace constituent of the Earth’s atmosphere, it is likely produced biologically (or industrially) on the Earth.42−46 It remains unclear whether phosphines would be prebiotically available, and other prebiotic reagents may be required to induce this fragementation.44−46 Exploration of the prebiotic conditions that could achieve this fragmentation on the early Earth remains to be tested; however, the effective and spontaneous ligation of cysteamine-peptoids warrants further study of this fragmentation in a prebiotic context.

Our observations support a scenario in which nitriles may have served as activated substrates for peptide synthesis on the primordial Earth, with cysteamine (5) playing a role in their ligation. It is of note that 5 is a component of pantetheine,20,47−49 which is essential throughout all domains of life as an acyl-carrier, suggesting that 5 could have been intimately associated with peptide synthesis since the origins of life.

Acknowledgments

The authors thank the Engineering and Physical Sciences Research Council (grants EP/X011755/1, EP/P020410/1 to M.W.P.) and the Simons Foundation (grant 1154101 to M.W.P.), Powner group members for helpful discussions, and K. Karu (UCL Mass Spectrometry) and Dr A. Aliev (UCL NMR).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.4c00243.

  • Additional experimental details, materials, and methods, including NMR spectra of ligation reactions and isolated compound, mass spectrometry, and IR spectroscopic data (PDF)

Author Present Address

Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, U.K

Author Contributions

M.W.P. conceived the research. A.B. and M.W.P. designed and analyzed the experiments and wrote the manuscripts. A.B. conducted the experiments. CRediT: Abid Barat data curation, formal analysis, investigation, methodology, writing-original draft, writing-review & editing; Matthew William Powner conceptualization, formal analysis, funding acquisition, project administration, resources, supervision, writing-original draft, writing-review & editing.

The authors declare no competing financial interest.

This paper was published ASAP on April 24, 2024 with incorrect versions of Figures 2 and 4, and reposted on April 30, 2024 with incorrect versions of Figures 1 and 2 (production errors). The figures were corrected and the revised paper was reposted on May 1, 2024.

Supplementary Material

au4c00243_si_001.pdf (9.8MB, pdf)

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