Ghinwa Ajram

Jean-Christophe Rossi

Laurent Boiteau

Robert Pascal
email: robert.pascal@umontpellier.fr

A reassessment of prebiotically relevant

Keywords: peptides, activating agents, self-organization, origin of life

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Life's complexity arose from a limited number of building blocks, including α -amino acids, nucleotides, or fatty acids. The abiotic polymerization or self assembly of these monomers or their derivatives gave birth to the three subsystems (peptides, nucleic acids, and lipid membranes) that are responsible for the main functions observed in living cells. Complex systems could therefore be formed from their interactions, which resulted eventually into the self-organization of the first living organisms [START_REF] Ruiz-Mirazo | Prebiotic systems chemistry: new perspectives for the origins of life[END_REF]. In addition to the requirement for building blocks, polymers and supramolecular assemblies as material components for building the living entities, the emergence and evolution of life correspond to a state in which change is driven by a continuous research for increased Dynamic Kinetic Stability (DKS) [START_REF] Pross | What is life? How chemistry becomes biology[END_REF]. This behaviour is expressed by entities able to reproduce themselves and thus to grow exponentially when resources are not limited. Maintaining a drive towards higher DKS requires an environment able to provide energy and thus to keep the system in a far from equilibrium situation. An analysis of the kinetic requirements for the occurrence of DKS as a driving force in simple chemical systems (i.e. replicating entities, autocatalysts) led to the conclusion that the entities must be reproduced through a cycle that proceeds irreversibly [START_REF] Pascal | Suitable energetic conditions for dynamic chemical complexity and the living state[END_REF][START_REF] Pascal | Life, metabolism and energy[END_REF][START_REF] Pross | How and why kinetics, thermodynamics, and chemistry induce the logic of biological evolution[END_REF]. As a result, chemical systems capable of evolving toward the living state would have to spend a cost of irreversibility by dissipating energy and making the reverse process slow enough compared to the time scale of the reproduction cycle (meaning that the flux of reagents crossing the higher activation barrier in the reverse direction becomes negligible) [START_REF] Pascal | Suitable energetic conditions for dynamic chemical complexity and the living state[END_REF][START_REF] Pascal | Life, metabolism and energy[END_REF][START_REF] Pross | How and why kinetics, thermodynamics, and chemistry induce the logic of biological evolution[END_REF][START_REF] Pascal | Energy flows, metabolism and translation[END_REF]. This approach suggests that energy must not only be provided to the system to allow for the synthesis of biomolecules or the polymerization of building blocks but must, for the most part, be dissipated without serving any other purpose than satisfying this requirement for kinetic irreversibility and that strong activation processes should be involved in the process of self-organization. For that reason, sources of free energy are required to constrain self-organization as far as the scope of the chemical perception of the origin of life is concerned. This analysis is supported by considering peptide biosynthesis, shared with minute differences by all extant living organisms, which reveals one of the paradoxes of life's emergence and early evolution. Among the intermediates of the process, aa-AMPs, known as aminoacyladenylates (Scheme 1), are produced by a reaction of ATP with amino acids in the presence of a set of aminoacyl-tRNA-synthetases (aaRSs) the enzymes responsible for the reaction. aaAMPs possess free energies of hydrolysis values ( Δ G°' = ca. -70 kJ mol -1 ) among the highest of the metabolites present in the cells [START_REF] Wells | Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases[END_REF]. The aaRS enzymes not only increase the rate of formation of adenylates from ATP and free amino acids owing to their kinetic activity but also stabilize the intermediate that remains sequestrated in the local environment provided by the enzyme, which therefore thermodynamically contributes to the reaction as well. Without that stabilizing contribution from the enzyme, the equilibrium values of the concentration of free aa-AMP formed from ATP and amino acids would be negligible [START_REF] Wells | Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases[END_REF], which raises the chicken-and-egg question of how this process could have emerged in the absence of enzymes. This observation strongly suggests that translation evolved in an environment providing strong activation processes in which adenylates or other mixed anhydrides could be available from alternative pathways [START_REF] Pascal | From the prebiotic synthesis of α-amino acids towards a primitive translation apparatus for the synthesis of peptides[END_REF][START_REF] Pascal | Energetic constraints on prebiotic pathways: application to the emergence of translation[END_REF]. Several groups, including ours, have provided evidence that α -amino acids and peptides, strongly activated in the forms of N-carboxyanhydrides (NCAs) [START_REF] Biron | Amino acid N-carboxyanhydrides: activated peptide monomers behaving as phosphate-activating agents in aqueous solution[END_REF][START_REF] Biron | Expeditious, potentially primordial, aminoacylation of nucleotides[END_REF][START_REF] Leman | Amino acid dependent formation of phosphate anhydrides in water mediated by carbonyl sulfide[END_REF] or 5(4H)-oxazolones [START_REF] Liu | Evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides[END_REF][START_REF] Liu | Mixed anhydride intermediates in the reaction of 5(4H)oxazolones with phosphate esters and nucleotides[END_REF][START_REF] Liu | 5(4H)-Oxazolones as effective aminoacylation reagents for the 3-terminus of RNA[END_REF], respectively, react spontaneously with inorganic phosphate and phosphate esters to give mixed anhydrides including aaAMPs at equilibrium in diluted neutral aqueous solution (Scheme 2). One of the most logical consequences of these observations is that the possible formation of peptides, involving for instance mild wetting/drying cycles [START_REF] Forsythe | Ester-mediated amide bond formation driven by wet-dry cycles: a possible path to polypeptides on the prebiotic Earth[END_REF][START_REF] Yu | Elongation of model prebiotic proto-peptides by continuous monomer feeding[END_REF][START_REF] Rodriguez-Garcia | Formation of oligopeptides in high yield under simple programmable conditions[END_REF] at early stages of life, does not prevent the need of processes involving strong reagents for life to emerge. As far as strong activating agents are involved, Ncarboxyanhydrides (NCAs) or 5(4H)-oxazolones are essential intermediates during the activation of αamino acids or peptides, respectively [START_REF] Danger | Pathways for the formation and evolution of peptides in prebiotic environments[END_REF]. Some examples of prebiotically plausible processes for the formation of these activated intermediates have been reported in the literature. N-Carbamoyl amino acids yield NCAs by nitrosation in the dry state [START_REF] Taillades | N-Carbamoyl amino acid solidgas nitrosation by NO/NOx: A new route to oligopeptides via alpha-amino acid N-carboxyanhydride. Prebiotic implications[END_REF] or, more directly, by heating neutral aqueous solution [START_REF] Danger | The peptide formation mediated by cyanate revisited. N-Carboxyanhydrides as accessible intermediates in the decomposition of Ncarbamoylamino acids[END_REF]. Carbonyl sulfide (COS) is also efficient in producing NCAs from free amino acids in the presence of oxidizing or alkylating agents and transition metal ions. [START_REF] Leman | Carbonyl sulfide-mediated prebiotic formation of peptides[END_REF]. 5(4H)-Oxazolones are formed when acyl-amino acids or C-terminal residues in peptides are activated by strong activating agents like carbodiimides [START_REF] Danger | 5(4H)-oxazolones as intermediates in the carbodiimide-and cyanamide-promoted peptide activations in aqueous solution[END_REF]. The latter reaction significantly increases the rate of polymerization of amino acids promoted by carbodiimides [START_REF] Danger | 5(4H)-oxazolones as intermediates in the carbodiimide-and cyanamide-promoted peptide activations in aqueous solution[END_REF]. Though being considered as a drawback leading to a loss of chirality in the synthesis of peptides, 5(4H)-oxazolone formation has a very different outcome in prebiotic chemistry starting from racemic reagents since it may also be considered as a tool to induce a stereomer imbalance during polymerization [START_REF] Danger | 5(4H)-oxazolones as intermediates in the carbodiimide-and cyanamide-promoted peptide activations in aqueous solution[END_REF]. However, carbodiimides are not likely activating agents in an origin of life perspective. Cyanamide constitutes an alternative but its reactions turned out to be tedious [START_REF] Danger | 5(4H)-oxazolones as intermediates in the carbodiimide-and cyanamide-promoted peptide activations in aqueous solution[END_REF] even at 80°C. We therefore considered the published work on amino acid condensation agents [START_REF] Brack | From amino acids to prebiotic active peptides: A chemical reconstitution[END_REF][START_REF] Brack | From interstellar amino acids to prebiotic catalytic peptides: A Review[END_REF]. Many other reagents including cyanamide, dicyanamide, dicyandiamide (DCDA), diaminomaleonitrile (DAMN, HCN tetramer), and cyanogen leading to peptide bond formation can be found in the literature, although the yields were usually limited [START_REF] Hulshof | Prebiotic condensation reactions in an aqueous medium: a review of condensing agents[END_REF]. However, general conclusions are not easily drawn from these studies involving different conditions, different analytical methods, different starting materials, and in some cases using elevated temperatures or extreme values of pH. We realized that the formation of oxazolones from dipeptides constitutes a simple and practical way to characterize strong carboxyl activating agents (Scheme 3). The chiral instability of oxazolones rapidly results in a partial change of the configuration of the α -carbon of the amino acid residue, which can easily be monitored by analytical methods (e.g. HPLC) through the formation of diastereomers thus revealing the effect of powerful activating agents. The aim of the present work is to use this tool as a mean of investigating the reactivity of a series of prebiotically plausible condensing agents (Scheme 4) under conditions compatible with the environment of the primitive earth.

Experimental

Reagents and solvents were purchased from Sigma-Aldrich or Bachem, and were used without further purification.

Dicyclohexyl ammonium methyl 3 phosphate was prepared as described earlier [START_REF] Dueymes | Facile synthesis of simple mono-alkyl phosphates from phosphoric acid and alcohols[END_REF]. All aqueous solutions were prepared using pure water (

Results

The activating agents shown in Scheme 4 were checked for strong activation using the dipeptide models 1-LL (R 1 = H) and 2-LL (R 1 = Ac) as starting materials (HPLC retentions times 9.6 min and 16.2 min respectively, method A, Scheme 3). Any formation of 5(4H)oxazolones from these substrates should result in the epimerization of the C-terminal alanine residue yielding the diastereomers 1-LD (R 1 = H) and 2-LD (R 1 = Ac) [START_REF] Beaufils | Diastereoselectivity in prebiotically relevant 5(4H)-oxazolonemediated peptide couplings[END_REF] (HPLC retentions times 16.5 min and 19.9 min respectively, cf. Figure 1). Monitoring products is facilitated by the presence of the aromatic side-chain chromophore of tyrosine ( λ max = 273 nm). Activation was carried out in a neutral to moderately acidic pH range considered as representative of aqueous environments of the primitive Earth with an atmosphere enriched in CO2 under conditions chosen to remain close to those reported in the literature for such experiments. The efficiency of the method was checked using the EDC activation reagent (Table 1 entries 1 and 2) known to induce 5(4H)-oxazolone formation [START_REF] Beaufils | Diastereoselectivity in prebiotically relevant 5(4H)-oxazolonemediated peptide couplings[END_REF] even in the case of unprotected dipeptide 1-LL, which does not yield diketopiperazine owing to specific stereochemical properties [START_REF] Beaufils | The activation of free dipeptides promoted by strong activating agents in water does not yield diketopiperazines[END_REF] though in this case hydrophobic products having 23.3 to 24.9 min retention times were observed and correspond very probably to longer oligomers formed by reaction of the oxazolone with the free dipeptide. The advantage of this procedure is that the detection of epimerization gives information on the activation step independently of subsequent processes that are dependent on the concentration of nucleophile and other parameters that should influence the fate of the system provided that activation works. The results of the activation experiments using different reagents and conditions are listed in Table 1. None of the reagents checked during our investigations proved to reproduce an activity reaching that of EDC and, in most cases, no activity could be detected. Significant epimerization of the starting material was observed using cyanamide (Figure 1, Table 1, entry 7), confirming earlier preliminary results [START_REF] Danger | 5(4H)-oxazolones as intermediates in the carbodiimide-and cyanamide-promoted peptide activations in aqueous solution[END_REF], and the related dicyanamide (Table 1, entries 17 and 18). However, the process required heating of the mixture to 80-90°C for long periods.

Other reagents observed earlier to induce free amino acid polymerization in solution turned out to be inert with the acetylated dipeptide. This was indeed the case for cyanogen ( [START_REF] Brotherton | The synthesis and chemistry of cyanogen[END_REF] and then to the carbamoylation of free amines [START_REF] Taillades | A pH-dependent cyanate reactivity model: application to preparative N-carbamoylation of amino acids[END_REF]. The activation observed in the literature should therefore result from the formation of carbamoyl-amino acids and their further conversion into NCAs as demonstrated by a reexamination of the activation of amino acids by cyanate [START_REF] Danger | The peptide formation mediated by cyanate revisited. N-Carboxyanhydrides as accessible intermediates in the decomposition of Ncarbamoylamino acids[END_REF]. The result obtained from DAMN may be accounted for in a similar way since it was suggested in the literature that it can disproportionate into cyanogen and amino acetonitrile [START_REF] Mamajanova | HCN polymers characterized by solid state NMR: Chains and sheets formed in the neat liquid[END_REF]. No other combination of reagents proved to be effective. Interestingly, even the procedure reported recently [START_REF] Tsanakopoulou | Cyanamide as a prebiotic phosphate activating agent -catalysis by simple 2-oxoacid salts[END_REF] to improve the rate of formation of cyclic phosphodiesters by a catalytic cycle based on the reactivity of glyoxylate or pyruvate with cyanamide at moderate temperature proved to be ineffective (Table 1, entry 11) demonstrating that, in this case, the increase in rate is brought about at the expense of the strength of the activating agent. In the same way, the use of a nucleophilic methyl phosphate buffer expected to react with cyanamide to give a reactive adduct proved to be ineffective (Table 1, entry 9). Further attempts to add metal ions to the reaction mixture, with the aim of binding both the reagent and the activating agent and bringing reactive centres in close proximity in the coordination sphere of the metal failed.

A theoretical analysis of the conditions required for a steady state based on DKS led to the conclusion that energy has to be provided to the system with a potential equivalent or exceeding than that of visible light [START_REF] Pascal | Suitable energetic conditions for dynamic chemical complexity and the living state[END_REF][START_REF] Pascal | Life, metabolism and energy[END_REF]. It would have been logical to investigate potential activation processes based on visible or UV irradiation of solutions containing our reagents and a series of potential activating agent precursors. However, preliminary experiments demonstrated that the dipeptide models used in the present study were not stable under irradiation at 254 nm. These investigations will require the development of a more sophisticated methodology avoiding a light-sensitive substrate.

Conclusions.

The presence of strong activating agents or of energyrich carriers is a crucial parameter to induce selforganization as a consequence of a selection process among variants of entities capable of reproducing themselves [START_REF] Pross | What is life? How chemistry becomes biology[END_REF][START_REF] Eigen | Selforganisation of matter and the evolution of biological macromolecules[END_REF][START_REF] Lifson | On the crucial stages in the origin animate matter[END_REF][START_REF] Szathmáry | Sub-exponential growth and coexistence of non-enzymatically replicating templates[END_REF]. In addition to the need of unlimited possibilities of variations that can be brought about by mutations in the sequence of a polymer, approaches based on the requirements for establishing DKS states emphasize the need for sources of energy capable of feeding these systems and holding them in a state that is sufficiently far from equilibrium for a reproduction loop to work in an irreversible way [START_REF] Pascal | Towards an evolutionary theory of the origin of life based on kinetics and thermodynamics[END_REF].

The results of the present study indicate that a process able to activate the C-terminal end in a peptide in a way consistent with this approach is not available yet. Indeed, carbodiimides are not likely prebiotic reagents and cyanamide does not constitute an appropriate alternative because of its sluggish reaction even at high temperature. The only established processes leading to strong activation in prebiotically plausible environments are at present those based on the formation of N-carboxyanhydrides of amino acids [START_REF] Taillades | N-Carbamoyl amino acid solidgas nitrosation by NO/NOx: A new route to oligopeptides via alpha-amino acid N-carboxyanhydride. Prebiotic implications[END_REF][START_REF] Danger | The peptide formation mediated by cyanate revisited. N-Carboxyanhydrides as accessible intermediates in the decomposition of Ncarbamoylamino acids[END_REF][START_REF] Leman | Carbonyl sulfide-mediated prebiotic formation of peptides[END_REF]. The occurrence of a presently unknown catalytic process similar to that already established with oxoacids [START_REF] Tsanakopoulou | Cyanamide as a prebiotic phosphate activating agent -catalysis by simple 2-oxoacid salts[END_REF] but that do not reduce the potential of cyanamide reagent could be a possibility. In the absence of a catalytic pathway, the lack of reactivity of cyanamide and other nitriles is a drawback but it must be acknowledged that limited spontaneous breakdown rates are also necessary for the emergence of a metabolism in which reactants must be protected by kinetic barriers [START_REF] Eschenmoser | Chemistry of potentially prebiological natural products[END_REF][START_REF] Eschenmoser | Question 1: Commentary referring to the statement "The origin of life can be traced back to the origin of kinetic control" and the question "Do you agree with this statement; and how would you envisage the prebiotic evolutionary bridge between thermodynamic and kinetic control?" stated in Section 1.1[END_REF]. Compounds containing triple bonds including nitriles and cyanamides are very likely candidate for this role [START_REF] Pascal | Energy flows, metabolism and translation[END_REF]. The kinetic barriers protecting them from spontaneous hydrolysis actually represent a property allowing them to migrate from the location of their formation to that of a self-organizing system potentially requiring a different context. This hypothesis is however also dependent on the occurrence of efficient catalytic systems that could trigger the transfer of their energy potential into the metabolism of a self-reproducing entity. In a systems chemistry approach to the origin of life, a very attractive possibility for the conversion of the free energy of nitriles could involve an autocatalytic process able to contribute to a steady state behaving in conformity with a DKS drive and associated with possibilities of variations allowing the initiation of an open-ended evolutionary process. Finally, this observation means that the difficulty in detecting any possibility of strong activation of C-terminus in peptide must not be considered as a definitive conclusion since 5 the height of the kinetic barriers associated with nitrile reactions may turn out to become an advantage and should encourage further investigations in this direction [START_REF] Mariani | A Light-Releasable Potentially Prebiotic Nucleotide Activating Agent[END_REF], provided that catalysts are found that conserve the strong activation character. This potential of nitriles or other simple species bearing triple bonds in activation might be on the contrary considered as in line with the role of hydrogen cyanide that has been recently uncovered in the formation of a wide range of biochemical building blocks through photocatalytic redox cycles [START_REF] Ritson | Prebiotic synthesis of simple sugars by photoredox systems chemistry[END_REF][START_REF] Patel | Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism[END_REF][START_REF] Xu | 01499j Scheme 1 The biochemical pathway of activation of amino acids. tRNA aminoacylation by the two step reaction of α-amino acids via aminoacyl adenylate (aa-AMP) intermediates. Scheme 2 Formation of mixed anhydrides from NCA and 5(4H)-oxazolones[END_REF].

Additional files

Additional file 1 -Supporting Information (pdf format) including the description of the preparation of starting materials and reagents and the detailed description of the experiments corresponding to the data provided in Table 1. (HCN tetramer)

Table 1 Screening of a variety of activating agents and reaction conditions to detect the epimerization of the two dipeptides H-Tyr(Me)-L-Ala-OH 1-LL and Ac-Tyr(Me)-L-Ala-OH 2-LL in buffered aqueous solution (100 mM MES unless otherwise mentioned). The solutions resulting from the activation experiments were analyzed by HPLC (method A, detection 273 nm unless otherwise mentioned) and potential products were characterized by mass spectrometry or NMR. Abbreviations: s.m. and n.d. stand for starting material and not detected, respectively.

Entr y

Substrate

7 Scheme 3 Figure 1

 731 Scheme 3 The epimerization of free (1-LL) or acetylated (2-LL) Tyr(Me)-Ala dipeptides as a tool to monitor strong activation.

Table 1

 1

	, entries 3 and 21) formed in situ
	by the reaction of CuSO4 and KCN [30] and
	diaminomaleonitrile (DAMN, entries 5, 6 and 22). The
	acetylated dipeptide 2-LL remained unchanged in these
	experiments revealing that the activity observed
	previously should result from a pathway different from
	direct carboxyl group activation. The replacement of

Acknowledgements

This work was supported by a grant from the Agence Nationale de la Recherche (ANR-14-CE33-0020 to the PeptiSystems project). The authors thank the COST action CM1304 Emergence and Evolution of Complex Chemical Systems for facilitating interactions with other European groups.

Author details 1 DSBC, CC17006, IBMM, Place E. Bataillon, F-34095 Montpellier Cedex, France Author's contributions GA and JCR designed and performed the experiments. RP supervised the project. GA, JCR, LB and RP analyzed results and wrote the paper.

Competing interests

The authors declare that they have no competing interests.