# Sulfur Amino Acids: From Prebiotic Chemistry to Biology and Vice Versa

Sparta Youssef-Saliba, Yannick Vallée

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Sparta Youssef-Saliba, Yannick Vallée. Sulfur Amino Acids: From Prebiotic Chemistry to Biology and Vice Versa. Synthesis: Journal of Synthetic Organic Chemistry, 2021, 53 (16), pp.2798 - 2808. [hal-04910546](https://hal.science/hal-04910546v1)

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# Sulfur amino acids: from prebiotic chemistry to biology and vice versa

Sparta Youssef-Saliba, Yannick Vallée

Université Grenoble Alpes, DCM, CNRS, Grenoble, France

## Abstract

Two sulfur containing amino acids are included in the list of the 20 classical protein amino acids. A methionine residue is introduced at the start of the synthesis of all current proteins. Cysteine, thanks to its thiol function, plays an essential role in a very large number of catalytic sites. Here we present what is known about the prebiotic synthesis of these two amino acids and homocysteine, and we discuss their introduction into primitive peptides and more elaborate proteins.

1 Introduction

2 Sulfur sources

3 Prebiotic synthesis of cysteine

4 Prebiotic synthesis of methionine

5 Homocysteine and its thiolactone

6 Methionine and cystine et proteins

7 Prebiotic scenarios using sulfur amino acids

8 Introduction of Cys and Met in the genetic code

9 Conclusion

## 1 Introduction

While many questions arise as to how life came to be on Earth (or on any other planet), one thing is certain: it is primarily a matter of organic chemistry, probably facilitated by some metals.¹ The objective of this mini-review is to briefly summarize what is known (or what we think we know) about the prebiotic synthesis of a particular type of "molecular breaks of life", sulfur amino acids, and how they may have entered prebiotic peptides. From the point of view of an organic chemist, this amounts to questioning the possible role of the thiol and thioether functions, and precisely because the reactivity of these functions is very specific (in particular for thiols), one can imagine that they were difficult to circumvent.

In the proteome pl data base,² containing 5029 proteomes, cysteine accounts for 1.38% of all residues (significantly less in Archaea, 0.98%, and Bacteria, 0.94%), and methionine for 2.32%. They are both underrepresented compared to the expected average of 5% calculated on the basis of the 20 usual protein amino acids. Indeed, in this representative data base, cysteine is the rarest residue in Archaea and Bacteria, and it is second only to tryptophan in the global set. Despite this, if it does not seem unthinkable to replace all Met residues with other hydrophobic residues, Leu for example, in proteins, and after that to keep a proteome globally compatible with life, such substitution seems quite impossible for Cys residues.

Cysteine, due to its thiol function, exhibits specific properties such that it is not clear which other protein amino acid could replace it (except in some cases serine and threonine). This is reflected in the overrepresentation of cysteine in catalytic sites, which sharply contrasts with their weak overall presence. The "catalytic propensity" of all amino acid residues, which characterizes the tendency of a residue, when it is present in a protein, to be so in a catalytic site, has been estimated on the basis of a set of 178 enzymes.³ It was found to be *ca.* 4.7 for Cys, second only to His (*ca.* 6.7), when it was less than 1 for Ser and Thr, and only *ca.* 0.3 for Met (meaning than when Met is present in an enzyme, it is generally not part of a catalytic site). Somehow, Cys residues are pretty rare, but when present most of them take part in a catalytic action.

The irreplaceable role of cysteine in catalytic sites argues for its early introduction in the history of life on Earth. Other arguments, including its relative rarity in proteins, point in the opposite direction to its later introduction. Both types of arguments will be discussed in this
review, where we will first introduce what is known about the possible prebiotic synthesis of sulfur amino acids, starting with the plausible sources of sulfur on the early Earth.

## 2 Sulfur sources

SO₂ and H₂S are frequently abundant in gaseous emissions of volcanoes,⁴⁻⁸ in which tiny amounts of OCS have also been detected. In our O₂ containing atmosphere, SO₂ can then be oxidized to SO₃, hence forming sulfates. Elementary sulfur deposits, such as those observed near fumaroles, result from the condensation of gaseous S₂ or from H₂S oxidation. It is thus probable that SO₂ (and sulfites), H₂S, and in lesser amounts S₈ and OCS, were all present on the primitive Earth. Because of the lack of O₂, the presence of SO₃ and sulfates could be regarded as less assured. However, they might have been present, may be formed from disproportionation processes from sulfites. Formation of methanethiol is observed in hydrothermal systems at midocean ridges (where however it might be of biological origin)⁹ and in terrestrial hydrothermal pools.¹⁰ Pyrite, FeS₂, as well as other mineral sulfides (chalcopyrite, FeCuS₂, sphalerite, ZnS...),¹¹ are reasonable sulfur-delivering candidates in "on the rocks"¹² heterogeneous prebiotic chemistry scenarios.

Today, sulfates are the most available source of sulfur in water. Sulfate-reducing archaea and bacteria are able to reduce them to sulfites, then to H₂S, which is used in the synthesis of sulfur amino acids.¹³ This chemistry is highly dependent on ATP (Scheme 1).¹⁴,¹⁵ Sulfates first form adenosine 5'-phosphosulfate 1, which is then phosphorylated to phosphoadenosine 5'-phosphosulfate 2. Reduction of 2 delivers sulfite anions which are reduced to H₂S thanks to an NADPH-dependent enzyme.

### Scheme 1 Reduction of sulfate anions to sulfide via phosphosulfates 1 and 2

The complexity of this process, with the necessity to reduce sulfates into sulfides, makes it probable that H₂S was the most common primitive source of the sulfur atoms introduced in organic molecules (even though thiosulfate anions could also intervene,¹⁶,¹⁷ if they were present).¹⁸,¹⁹ The sulfur cycle then evolved over time to what it is today.²⁰,²¹

## 3 Prebiotic synthesis of cysteine

The first claim of cysteine production under possible prebiotic conditions has been published in 1971.²² The authors observed that some cysteine, as well as its disulfide, cystine, was formed during the UV irradiation of a gaseous mixture of methane, ethane, ammonia and hydrogen sulfide at high temperature (Scheme 2). In certain experiments, serine, cystamine, cystic acid, taurine and methionine were also detected. The very poor yields obtained were said to be due at least in part to the possible transformation of cysteine into pyruvic acid and alanine thiosulfuric (or thiosulfonic) acid, so that, without these transformations, acceptable yields of cysteine and cystine might have been reached this way on the primitive Earth. Cysteine was only detected as "trace" (or said to be "present") in experiments mimicking hydrothermal conditions, published in 1992.²³ Mixtures of KCN, HCl, NaSH and formaldehyde in water were heated under a CO₂/H₂ atmosphere in the presence of various mineral phase (containing *e.g.* pyrite or clay). Depending on the used mineral phase and the temperature, different amounts of some amino acids were obtained, including not only cysteine, but also methionine.
**Scheme 2** Some products obtained from a gaseous mixtures containing $H_2S$ at high temperature

Cysteine itself has not been detected in the mixture of products resulting from a Miller type experiments run in the presence of $H_2S$,²⁴ in which however, other sulfur compounds were observed (Scheme 3). As this mixture was analyzed nearly half a century after the experiment was carried out, a period during which it was not protected against $O_2$, it might be that some of these compounds were formed by oxidation of some cysteine initially formed during the experiment. It should however be noticed that the most obvious oxidized form of cysteine, cystine, was not detected.

Thus, if its direct "geologic generation" cannot be ruled out, such processes would likely have delivered only tiny quantities of cysteine and it might well be that it was formed in significant amount only at a time when more classical organic chemistry was running in the oceans.

**Scheme 3** Some products obtained in a Miller-type experiment using $H_2S$ as a sulfur source

It has been shown that the Strecker reaction of mercaptoacetaldehyde 3 (which exist mostly as a dimer in water) delivers the corresponding aminonitrile (Scheme 4), which upon hydrolysis forms first cysteine amide, then cysteine.²⁵ However, it is not clear how 3 could be formed in the primitive ocean. Theoretical calculations showed that its formation from the reaction of hydroxyacetaldehyde 4 with $H_2S$ should be a difficult process.²⁶ It can be obtained from chloroacetaldehyde 5. However, the early existence of this halogen derivative on Earth is disputable.²⁷

**Scheme 4** Synthesis of cysteine from mercaptoacetaldehyde 3

Some N-acetyl cysteine derivatives have been synthesized from serine nitrile 6 (Scheme 5).²⁸ This nitrile, which is the Strecker product of hydroxyacetaldehyde, was bis-acetylated to compound 7 with thioacetic acid. This reaction was promoted by ferricyanide anion, an oxidant. 7 was then transformed into dehydroalanine derivative 8, via acetic acid elimination. Treatment of 8 by $H_2S$ led to the N-acetylated thioamide 9. This interesting process is very close to one of today's biological ways to cysteine. However, it makes use of an oxidative acetylating process, whose prebiotic character may be questioned, and the synthesis of cysteine itself was not mentioned.

**Scheme 5** Synthesis of acylated derivatives of cysteine from hydroxyacetaldehyde cyanohydrine 6

Thus, the way cysteine was synthesized in the primitive ocean, if it was, is still uncertain. In today's biology, it is produced from serine (representative ways presented in Scheme 6), the sulfur source being $H_2S$,²⁹ thiosulfate anions³⁰ or homocysteine 10.³¹,³² It could well be that this was already the case in prebiotic chemistry.²⁸ By the way, thinking about the serine + homocysteine pathway (via cystathionine 11) may lead an intriguing question: did homocysteine predate cysteine?

**Scheme 6** Representative biological ways to cysteine
## 4 Prebiotic synthesis of methionine

Methionine was present in the aforementioned mixture of products resulting from a Miller experiment run in the presence of H₂S.²⁴ Its production from methane, nitrogen, ammonia, water, and hydrogen sulfide or methanethiol, activated by spark discharges, was already mentioned in 1972.³³,³⁴ As acrolein was also observed in the obtained mixtures, the authors proposed that this aldehyde was the precursor of methionine, *via* a way similar to its industrial synthesis (Scheme 7): [1,4]-addition of MeSH, followed by a Strecker reaction, leading to the aminonitrile **12**, which upon hydrolysis yields methionine. From an organic chemistry point of view, this seems to be the most logical synthesis of methionine, assuming that HCN was abundant on the early Earth (or at least regularly formed from methane and ammonia or dinitrogen for instance).³⁵

**Scheme 7** Synthesis of methionine from acrolein

Methionine was also detected (in less than 1% yield), when ammonium thiocyanate was irradiated with UV light in water,³⁶ and in mixtures resulting from heating formaldehyde and hydroxylamine at 105°C in water, in the presence of various metal cations (Fe³⁺, Cu²⁺, Zn²⁺...).³⁷

Overall, partly because of the high reactivity of the thiol function, it could well be that the prebiotic synthesis of methionine was easier than that of cysteine. Today the biological syntheses of these two amino acids are interlinked. In the classical transsulfuration pathway (Scheme 8) succinyl-homoserine **13** reacts with cysteine to form cystathionine **11**, which is cleaved to pyruvate and homocysteine **10**. Methylation of **10**, then leads to methionine.³⁸ In some organisms H₂S (or even MeSH) is used in place of cysteine, but adding that cysteine is often obtained from homocysteine, which can be seen as both a product and a precursor of methionine, the triangle connecting cysteine, homocysteine and methionine is obvious.
Furthermore, except when the sulfur source is MeSH, methionine is derived from homocysteine, which gives this non-protein amino acid a central position in the metabolism between cysteine and methionine, and could lead to imagine making it a prebiotic predecessor of these two amino acids.

**Scheme 8** Transsulfuration way to methionine

## 5 Homocysteine and its thiolactone

Homocysteine is not a protein constituent but its chemistry is so closely related to that of methionine and cysteine that it is difficult to ignore in a prebiotic scenario.
It has been detected in Miller type experiments, in which it is supposed to derive from acrolein (Scheme 9).³³ If this is the case, the aminonitrile **14** should be an intermediate. **14** has been synthesized (in a non-prebiotic manner), and it has been shown that its first evolution product in water is not homocysteine itself but its thiolactone **15**.³⁹ It is therefore possible that this thiolactone existed on a primitive Earth rich in cyanides. It is pretty stable in water, although it eventually hydrolyzes to homocysteine (faster in basic water of course), and could have participate in a de Duve's thioester world.⁴⁰

**Scheme 9** Proposed synthesis of homocysteine thiolactone **15** from acrolein
Interestingly, it has been shown that homocysteine is able to interact with Methionyl-tRNA synthetase (in place of methionine) during the translation process, forming Hcy-AMP. However, thanks to an editing mechanism, Hcy is not introduced in the protein under synthesis, but Hcy-AMP is broken into AMP and thiolactone **15** (Scheme 10).<sup>41,42</sup> This may indicate that with a less efficient editing process (or no editing at all), Hcy might have been introduced in primitive peptide chains.

**Scheme 10** Editing process of homocysteine on MetRS

Why cysteine eventually became a protein amino acid and not homocysteine? There is no obvious answer to this question. The use of both of them would have made them almost duplicates, but no more than aspartic and glutamic acids, or asparagine and glutamine. Furthermore, wondering about the absence of homocysteine in proteins should also lead to thinking about the serine / homoserine alternative. The only certain fact is that today, when present in excess in an organism, homocysteine exhibits toxic properties related in particular (but not only) to its ability to form its thiolactone.<sup>43,44</sup>

**6 Methionine and cysteine in proteins**

The most striking role of methionine in today's proteins is to start their synthesis. A Met residue is introduced first during protein synthesis in Archaea and Eukaryotes. In Bacteria, the first residue is *N*-formyl-Met. In our "genetic world", this is a consequence of the fact that AUG,<sup>45</sup> the codon for Met, is also the start codon. Curiously, however, even when protein synthesis is not triggered by the AUG codon, but by an alternative codon (e.g. GUG, UUG, AUU, CUG), which happens in 17% of cases in *Escherichia coli*, it is still fMet which is introduced (and not for instance Leu, or *N*-formyl-Leu, as it could be expected).<sup>46-49</sup> A pre-genetic (pre-RNA world) explanation to this could be that Met, as the "start residue", was selected prior to the start codon.<sup>50</sup> However, as the chemistry of methionine makes it difficult to explain this,<sup>51,52</sup> it could in fact be that the first starting amino acid was rather homocysteine (or one of its derivatives) which via its thiolactone could have aminoacylated any preexisting peptide chain in the N-terminal position.<sup>39</sup>

Otherwise, Met residues are sometimes used to complex metal cations (but much less often than Cys).<sup>53,54</sup> They are involved in redox processes (forming the corresponding sulfoxide)<sup>55</sup> and frequently participate in the stabilization of protein structure thanks to the interaction of their sulfur atom with aromatic groups.<sup>56</sup> Despite this, as already indicated, it does not seem insurmountable to replace them with other hydrophobic residues in many cases. It was thus possible to replace the 13 Met residues of the heme domain of a human cytochrome, consisting in total of 463 residues, by norleucine residues without destroying its activity (which on the contrary was reinforced).<sup>57</sup>

With no Met residues in proteins, living organisms would have been different, but this absence would most probably not have been completely crippling for the development of some efficient form of life. It is therefore possible that methionine was only used in proteins because it was otherwise necessary in metabolism (and therefore present in all proto-cells). Conversely to what was done in the experiment presented above, it might be that Met took the place of other hydrophobic residues during molecular evolution.<sup>58</sup> If this is true, before Met, the most common first residues in early proteins may well have been Ile (current codons AUU, AUC, AUA, with only the last base differentiating them from AUG), Val (inter alia GUG) and/or Leu (inter alia UUG and CUG, first base differentiating them from AUG).<sup>59</sup>
The absence of cysteine in today's proteomes would be much more detrimental. First, the formation of disulfide bridges between Cys residues play an essential role in establishing and maintaining the secondary structure of proteins.⁶⁰⁻⁶² These bridges are common (they are present in one third of human proteins),⁶³ but mostly in proteins that can be found in oxidative surroundings (e.g. at the cell surface), and it is not obvious that disulfide bridges could be formed in the non-oxidative primitive terrestrial atmosphere. However, their abundance in thermophilic archaea suggests that they may have been important at the origin of life at least if it is to be sought in extreme conditions.⁶⁴ Second, cysteine exhibits antioxidant properties. The cysteine-cystine equilibrium is used for instance in the ubiquitous thioredoxins (Scheme 11),⁶⁵ when in peroxiredoxins the thiol function is oxidized to a sulfenic (SOH) group.⁶⁶,⁶⁷ Antioxidant processes, anyway, may well have been pointless in the primitive non-oxidative environment, which does not imply that the reductive properties of cysteine were of no use. For example, in a primitive metabolism, there had to be a way to reduce ribonucleotides into deoxyribonucleotides. In present biology, ribonucleotide reductase (RNR) uses a radical process to perform this transformation (Scheme 12).⁶⁸ Three classes of RNR exist of which type III has been proposed to be the most ancient.⁶⁹ In this class III RNR, the first intermediate thiol radical is produced from a glycil radical, itself derived from S-adenosylmethionine (SAM), thanks to an iron-sulfur protein. It might well be a simple coincidence, but it is noticeable: sulfur is everywhere in the process!

**Scheme 11** Redox equilibrium in thioredoxins

**Scheme 12** Principle of the reduction of ribonucleotides into desoxyribonucleotides thanks to RNR proteins

Reducing ribose to deoxyribose is making half of the way from RNA to DNA. Cysteine also participates in the methylation of uracil into thymine (Scheme 13). The involved process starts with the [1,4]-addition of the thiol function of a Cys residue of thymidylate synthase onto the C=C double bond of the uracil ring.⁷⁰ Even though alternative (older) prebiotic syntheses of deoxyribose and thymine, not using ribose and uracil, cannot be excluded, these observations in present biology argues for a cysteine dependent RNA to DNA transformation. In other words, if a RNA world ever existed, it would not have evolved into an RNA-DNA world in the absence of Cys-containing peptides.

**Scheme 13** Principle of the methylation of uracil to thymine using thymidylate synthase

Iron-sulfur proteins are used in a lot of processes, including electron transfer. They are ubiquitous, hence probably appeared early in the history of life, which implies that (at least small) Cys proteins existed. 4 Cys residues participate in 4Fe-4S clusters (Scheme 14) and in 2Fe-2S ferredoxin clusters (2 Cys + 2 Hys in Rieske proteins).⁷¹⁻⁷³ It has been proposed that UV light may have promoted the prebiotic synthesis of 2Fe-2S clusters from Fe²⁺ (the oxidation state of iron in the primitive ocean) and thiols.⁷⁴ Furthermore, a dodecapeptide consisting of alternated L and D amino acids bound to a 4Fe-4S cluster (with 2 L and 2 D Cys) has been shown to resist to hundreds of redox cycles,⁷⁵ and even the simple dipeptide Cys₂ is able to chelate Fe²⁺.⁷⁶ These results make plausible the early existence of iron-sulfur proteins.
**Scheme 14** Electrochemistry of 4Fe-4S clusters

Among their roles in contemporary biology, one of the most important is to participate in interactions with DNAS,<sup>77,78</sup> especially DNA primers<sup>79</sup> and RNAs.<sup>80,81</sup> Such nucleic acid-protein interactions also often involve zinc fingers, that is, units containing cysteine residues attached to a zinc cation (Scheme 15).<sup>82,83</sup> The most common type of these fingers is C2H2 (2 Cys and 2 Hist), but C4 and C6 fingers are also known.<sup>84</sup> The abundance of zinc in proteins (zinc fingers are coded in 3% of the genes in the human genome) is rather intriguing when compared to its overall rarity on Earth (the Fe/Cu/Zn ratio in the Earth crust is ca. 800/1/1, but is 2/0.03/1 in the human body).<sup>85</sup> As for iron-sulfur clusters, the ubiquity of zinc fingers<sup>86</sup> argues for their early introduction in the biological machinery. They are common in ribosomal proteins, and although these proteins may have aggregated in the ribosome at an already advanced stage on the way to its final shaping,<sup>87</sup> they are most probably very ancient.<sup>88,89</sup> They can contain a relatively large proportion of Cys residues. For example, the rat ribosomal protein S27 which consists of 84 residues (before removal of the N-terminal Met) contains 6 Cys (7%). Of the 56 residues of the rat ribosomal protein S29, 4 are Cys (also 7%).<sup>90</sup> Zinc fingers are present in ribosomal proteins of Archaea,<sup>91,92</sup> Bacteria<sup>93,94</sup> and Eukaryotes.<sup>95,96</sup> They are also part of aminoacyl-tRNA synthetases (e.g. AlaRS,<sup>97</sup> GluRS,<sup>98</sup> MetRS).<sup>99</sup> Cysteinyl-tRNA synthetase does not use an editing process to cleave erroneously produced aminoacyl adenylates. Despite this, it is very selective.<sup>100</sup> For instance, it activates serine at a rate one hundred million times slower than cysteine. This is explained by the presence in CysRS of a zinc cation, chelated to 2 Cys, 1 His and 1 Asp residues. The cysteine molecule to be activated first interact with this zinc cation forming a bipyramidal structure with 3 Cys, 1 His, 1 Asp around Zn<sup>2+</sup>. Such evolution is impossible with serine and any other amino acid. Interestingly, proteins containing both an iron sulfur cluster and a zinc finger are known and interacts with nucleic acids.<sup>101</sup>

**Scheme 15** Three types of zinc fingers

Iron and Zinc compounds of cysteine containing peptides probably played a major role at least when nucleic acids and proteins began to interact, and even before this for iron clusters. But other cysteine-metal compounds may also have been necessary. Cysteine is used in present biochemistry in connection with other metals, such as copper,<sup>53,54,102</sup> cadmium, nickel, manganese and molybdenum.<sup>103</sup> Added to their utility in catalytic triads (in which, however, serine and threonine have a similar role),<sup>104</sup> the examples reported above make it difficult to imagine starting life without cysteine.

**7 Prebiotic scenarios using sulfur amino acids**

Have cysteine, homocysteine and/or methionine been main players in a pre-RNA world? Did the sulfur atom they contain give them some specific advantage, in particular in the synthesis of the first peptide chains? Today some peptides are constructed not in the ribosome, thanks to tRNAs, but by the so-called non-ribosomal synthetic route, thanks to thiols (Scheme 16).<sup>105</sup>
**Scheme 16** Mechanism of peptide bond formation promoted by ACP proteins

The proteins (ACPs) which catalyze this process are complex, but its principle is simple and may be related to de Duve's thioester world.⁴⁰ Aminoacyl groups are engaged in thioester functions at the end of pantetheine linkers attached to serine residues in the main strand of the ACP protein via phosphate esters. Reaction of the amine of an aminoacyl group with the thioester function of another one then forms a peptide bond. However, the formation of thioesters is made possible thanks to prior activation of the incoming amino acids into aminoacyl phosphates, thanks to ATP. That is: even though it uses thioesters, the non-ribosomal synthesis of peptides is part of our phosphate world, and its introduction in a prebiotic scenario requires considering the use of a function capable of easily forming thioesters, without making use of a strong activating agent. This the case for nitriles which are known to undergo Pinner reaction with thiols, readily giving thioimidates and after hydrolysis, thioesters (Scheme 17).¹⁰⁶ Considering a primitive world rich enough in cyanide anions, it may be that the first amino acids were produced from the Strecker reaction of aldehydes. Aminonitriles are intermediates in such reactions. As nitriles are quite stable, it has been postulated that this aminonitriles may react with thiols before being hydrolyzed. When the used thiol is cysteine, thiazolines are formed which are then hydrolyzed into thiol-containing dipeptides (Scheme 18). Similar products are obtained starting from homocysteine. Mixtures of polypeptides and macrocyclic structures, such as **16** and **17**, were formed when starting materials were Cys-AACN dipeptides. Homocysteine thiolactone also reacts with aminonitriles (giving Hcy-AACN dipeptides). It was proposed that the common use of aminonitrile and thiolactones may have been at the origin of a thiol-rich peptide world.¹⁰⁷ In such a world, thioesters could be formed from the reaction of nitriles with the thiol functions of the multiple Cys and/or Hcy residues of these peptides. The possible formation of peptide bond in this way has been demonstrated, showing that thiol containing peptides possess “ACP like” aminoacyl transfer properties.

**Scheme 17** Synthesis of thioesters from nitriles (Pinner reaction)

**Scheme 18** Peptide bond formation via thiazolines and dihydrothiazines. Application of this procedure to the formation of oligomeric and polymeric structures including macrocycles

On the other hand, under oxidative conditions, thioacids reacts with aminonitriles, giving peptide nitriles which upon treatment with H₂S lead to enlarged thioacids (Scheme 19). This process therefore makes it possible to construct increasingly long peptides. Indeed, such peptide bond formation from nitrile is also feasible using under-stochiometric amounts of the simple N-acetylcysteine.²⁷,¹⁰⁸ This has been demonstrated on numerous examples in which yields were often very good.

**Scheme 19** Iterative elongation of thioacid peptides using an oxidative pathway

Such chemical pathways may have been used to form oligo- and polypeptides in the primitive ocean. The fact remains that their major drawback is that they use nitriles, and not carboxylic acids, i.e. they are very different from today's biochemistry (including non-ribosomal peptide biochemistry). This indeed calls into question their very relation to life (at least as we know it today).
The search for a kind of "prebiotic peptide chemical ligation" therefore remains very topical. This search is linked to the discovery of new ways of accessing thioesters, particularly those of cysteine, or cysteine residues in catalytic support peptides, at best directly from acids. Of course, in such chemistry, homocysteine can substitute for cysteine. In any case, it has the advantage of forming a 5-membered thiolactone, both fairly stable in water and reactive with amines.

## 8 Introduction of Cys and Met in the genetic code

It is likely that even in a pre-RNA world, peptides existed, but should these peptides be considered as ancestors of truly biological proteins?

Nucleic acids are produced from nucleic acids. All nucleic acids are copies of copies of copies... So that it seems logical to consider that there may still be some traces of the very first RNA (and/or DNA) strands in today's nucleic acids.

But today's proteins are produced on RNA scaffolds. They are all translations of the codon alphabet. Even non-ribosomal peptides are synthesized on protein scaffolds themselves synthesized using the genetic code. They are "translations of translations", indirect translations of a series of codons. In other words, the oldest imaginable memory traces of all proteins are to be looked for in nucleic acids.

One consequence of this is that it is possible that absolutely no trace of pre-RNA peptides survives in the proteins of today's living cells. Regardless of how these primitive peptides were synthesized, if it was not on tRNA's, there is no reason to consider them as precursors of today's proteins. Otherwise, it would be necessary to consider that some of the first nucleic acids would have adapted to pre-existing peptides (a reverse translation: they would have been constructed by putting the codons corresponding to the chain of amino acids end to end) and would have started to produce copies of these peptides.

Of course, such an apparent "dead end" does not prevent questions about the synthesis of ancestral peptides.

Methionine is thought to have been introduced late in the genetic code, just before the last introduced amino acid, tryptophan.¹⁰⁹ Does this mean that the introduction of a start codon / start amino acid pair appeared only lately in the chemical evolution process? Or, was this role assumed by another codon/amino acid pair? Since three of the four codons beginning with AU code for Ile, it could well be that AUG was doing the same before the introduction of methionine, which would lean towards isoleucine acting as the starting amino acid. However, as we already mentioned it, the start codon is not strictly invariable, and the use of rarer start codons, in conjunction with the more classical AUG, might indicate the primitive indifferent use of any of the three lipophilic residues Leu, Ile and Val.

Cysteine also is considered to be a late participant in the genetic code (ranked 16th in ref¹⁰⁸). Given the importance of Cys residues in catalysis, this may seem surprising. Today's codons for Cys are UGU and UGC. Adding that the stop codon UGA is also used by some organisms as the codon for the selenium analogue of cysteine, selenocysteine,¹¹⁰ and that UGG codes for tryptophan which is considered to be the probable last amino acid introduced in the genetic code, it is worth considering that the four codons beginning with UG once coded for Cys and that, in a hypothetical 2-letter genetic code, UG could code for Cys (or for no amino acid if Cys was not used).¹¹¹,¹¹² Should this be related to the fact that 4 of the 5 known start
codons (all coding for Met in this case) end with this UG pair? Or, is it only a mere coincidence?

## 9 Conclusion

There is an apparent discrepancy between the probable late introduction of Cys into the genetic code and its specificity which seems to make it essential for the development of biological chemistry. A similar discrepancy exists for Met, but it seems chemically less troublesome, with methionine being one nonpolar amino acid among others. It is however rather intriguing that the amino acid that today triggers the synthesis of all proteins was arguably not present in the very first proteins built on the ribosome. Even if the genetic arguments cannot be rejected, it is difficult to imagine a pre-RNA world without sulfur amino acids. They could have been part of small peptides (to speak of proteins before the establishment of the genetic code is probably excessive) fulfilling some catalytic functions impossible for ribosimes, or part of "co-factors" participating in the catalytic activity of ribosimes. If so, these co-factors and/or primitive peptides would have been later incorporated into proteins. This could have been the case for example with iron-sulfur clusters.<sup>113</sup> Small molecules such as glutathione (which contains a Cys residue) or SAM (which incorporates a methionine) may also have played a role very early on, unless a simplified form of SAM, perhaps as simple as S-methylmethionine,<sup>114</sup> has been used. As for the role of homocysteine, it remains uncertain. Its ability to form a thiolactone makes it a self-activating amino acid, which could have facilitated its introduction into primitive peptides (instead of cysteine and / or methionine?). What is certain is that today it is not a protein amino acid, and that too high a concentration of homocysteine in a living body is toxic.<sup>115,116</sup>

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