Prebiotic Peptides

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Prebiotic Peptides BERND M. RODE AND KRISTOF PLANKENSTEINER

ABSTRACT Based on modern geochemistry's view of the atmospheric and geological conditions on the primordial Earth -3.8-4 billion years ago, the most realistic scenario for the formation of amino acids and peptides in chemical evolution is discussed, including possible reasons for sequence preferences in early proteins and for biohomochirality. Arguments for a peptide/protein world as primary origin of life, preceding RNA/DNAbased evolution, are presented.

INTRODUCTION Looking at the astonishing variety and complexity of peptides and proteins in today's living organisms, the question of their origin and subsequent evolution is one of the most fundamental ones for chemists and biologists. Providing the pathways to these compounds long before life in its modern form developed, chemical evolution has become a most challenging research topic after Oparin's hypothesis [42] of a primordial soup, containing numerous organic compounds synthesized by nature under the conditions of the primitive Earth some 3.8 billion years ago, when the Earth had cooled down sufficiently to allow the existence of liquid water. As the key role of peptides in the origin of life [46, 51] is increasingly better understood, the question of how amino acids and their first polymers came into being became crucial. Was the formation of the first prebiotic peptides a matter of chance with arbitrary results, or did chemistry and the given scenario point the way to specific, almost compulsory syntheses? The main difficulty of prebiotic research is our limited knowledge about this scenario, the conditions prevailing on the primitive Earth. Fifty years ago, these conditions were, in the literal sense, still mostly terra incognita. Handbook of Biologically Active Peptides

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but since then specific and interdisciplinary research in physics, chemistry, and earth sciences has widely improved our knowledge. With this knowledge, it has become possible to achieve quite trustworthy reconstructions of the processes forming amino acids and subsequently linking them to peptides, almost ubiquitously and within a relatively wide variation of environmental conditions. Because we cannot get any direct access to remnants of that time, our only chance to obtain conclusive results is to attempt to experimentally repeat the evolutionary processes under laboratory conditions mimicking the primordial scenario. Any of these experiments can be scrutinized, therefore, by challenging the availability of necessary components, environmental conditions, and stability of products in the most likely scenario predicted by geochemical sciences for the primitive Earth.

THE PRIIMITIVE EARTH SCENARIO According to present geochemical knowledge, the Earth should have cooled down below the boiling point of water approximately 4 billion years ago, forming the first stable hydrosphere in its history. At that time, due to the weak gravitational field, the Earth had already lost its primary atmosphere of hydrogen and helium, and a secondary atmosphere was formed by gases of volcanic origin [18, 19, 22, 25, 40, 41, 59] that were stable enough to withstand the constant ultraviolet (UV) radiation of the sun and, thus, mainly consisted of CO2, N2, and water vapor, with smaller amounts of SO2 and possibly also some CO, H2S, and traces of noble gases [8, 23, 63]. Even certain amounts of oxygen formed by the decomposition of CO2 and water should have been present [5, 15, 24, 30, 48, 61]. Due to the high temperature, weather phenomena such as Copyright © 2006 Elsevier

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thunderstorms and rain must have been much more violent than today. For the same reason, evaporation and recondensation in the coastal regions of the primitive ocean and lakes must have been common and frequent processes. Many minerals—partly still identifiable in precambrian rocks—could have served as reactants and catalysts for chemical reactions, together with inorganic salts dissolved in the sea and lakes, where they could reach considerable concentrations. THE FORMATION OF AMINO ACIDS In 1953, Stanley Miller initiated the quickly expanding field of experimental simulations of prebiotic chemistry with his famous experiment investigating the possible formation of organic molecules in an assumed primitive atmosphere consisting of methane, hydrogen, ammonia, and water under the influence of electric discharges [35]. Soon some amino acids were identified among the products. However, after it had been realized that this reducing atmosphere could not have existed on the primordial Earth, the experiment had to be repeated, varying atmospheric composition and sources of energy [12, 16, 36, 43]. It was shown only very recently [46, 48] that amino acids can readily be formed in a neutral or even mildly oxidizing atmosphere of the type previously outlined as the most likely geochemical scenario of the primitive Earth. Up to now, numerous amino acids, such as Gly, Val, Ala, Ser, Pro, His, and Lys have been identified as products ([48] and unpublished results), and the fragments found by means of proton transfer reaction mass spectroscopy in the plasma of the artificial lightning in these experiments (unpublished results) point to a variety of synthetic pathways to these and other types of precursor molecules of today's biochemistry. THE FORMATION OF PEPTIDES Because the synthesis of a variety of amino acids on the primitive Earth thus appears to have been an easy and common process, the subsequent major evolution problem is the synthesis of peptides from these amino acids in aqueous solution. To form a peptide bond, water has to be removed, which makes the process thermodynamically very unfavorable. A second problem for peptide synthesis in water is the kinetic barrier because amino acids are rather unreactive concerning a nucleophilic substitution by an amino nitrogen at the carbonyl carbon. Therefore, the reaction partners would have to be activated somehow to lower the activation energy of the reaction [55]. When prebiotic chemistry research aimed at the formation of peptides, the main concern, therefore, was

to find ways to overcome both the thermodynamic and kinetic obstacles. The evaporation of water, condensation reagents, and catalysts were the main tracks along which solutions were researched. Fox and Harada proposed melts of dried amino acids to form peptides [13, 15, 46, 51]. At the melting temperatures of amino acid mixtures, all formed water evaporates and were thus removed from the reaction. However, a large excess of acidic a n d / o r basic amino acids is necessary to allow polymerization and to avoid decomposition. Because these amino acids are rarely synthesized in atmospheric processes and the temperature range between melting and decomposition is very narrow, the prebiotic feasability of this reaction appears highly questionable. Further, a more thorough analysis of the resulting polymers (initially termed proteinoids) showed that they are polymers that only occasionally contain peptide bonds [1]. Another approach to prebiotic peptide formation was the use of condensation agents to facilitate the reactions between amino acids [46, 51]. The substances used in these experiments included cyanamides, cyanates, trimetaphosphate, ATP, GTP, CTP, UTP, linear and cyclic inorganic polyphosphates, and imidazoles, all of which must have been rather scarce or unstable in a primordial Earth setting, due to their rapid hydrolytic decomposition and precipitation as insoluble salts. There is, therefore, only a very minor probability that these types of peptide synthesis contributed to chemical evolution. Under assumed hydrothermal vent conditions [62], amino acids can also condense to peptides, but this scenario would be restricted to specific locations in the deep sea and especially high partial pressures of carbon monoxide, and thus its relevance for large-scale prebiotic peptide formation is rather questionable. A further possibility is peptide formation on the surface of minerals such as clays, silica, and alumina [46, 51]. In principle, it is possible to form peptides from adsorbed amino acids, but the yields are very low and the applicability is apparently restricted to very few amino acids such as Gly and Ala. However, these adsorption processes are of great importance for prebiotic peptide formation because peptides formed in other processes can be protected from decomposition [2] on the surface of such minerals and, furthermore, short peptides can be elongated to longer chains by this mechanism [46, 51]. For example, the salt-induced peptide formation (SIPF) reaction [51, 55], which efficiently forms peptides, perfectly harmonizes with clay-catalyzed peptide elongation in a one-pot reaction [46,51]. This SIPF reaction is a special case among the many proposals for peptide formation under primordial Earth conditions [51]. It allows for peptide formation

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in aqueous solution directly from amino acids or from amino acids and short peptides, as long as sodium chloride, Cu(II) ions, and thermal energy are available. Sodium chloride has always been ubiquitously present [18, 19] in the oceans, in salt lakes, and in lagoons and puddles along the sea shore. In concentrations above 3M, which can be reached either in large-volume salt lakes or lagoons (simulated by the constant volume type [46, 51] of SIPF reaction with constantly high sodium chloride concentrations) or in evaporating small puddles and small lagoons (simulated by evaporation cycle experiments [46, 51] in which the active concentration is reached by evaporation and subsequent redilution), sodium ions do not have a saturated first hydration shell and can, therefore, act as a dehydrating agent to overcome the thermodynamical barrier of peptide formation in aqueous solution [55]. Computational investigations of sodium chloride solutions [46, 51, 55], showing this effect in detail, were the basis for the discovery of the SIPF reaction. Subsequent simulations and experiments showed that the complexation of amino acids to metal ions, in particular to Cu(II) [51, 55], can activate them and thus lower the activation energy for the peptide formation process. Because precambrian rock formations contain large amounts of Cu(II)-containing minerals [17, 38, 48], the availability of these ions on the primitive Earth appears secured and the proper oxidation state could have been reached easily under the assumed atmospheric conditions [39, 48]. Since the late 1980s, the SIPF reaction has been and still is being investigated in many areas [46, 51], continuously revealing new favorable aspects and proving its very general applicability for prebiotic peptide formation under the conditions of the primordial Earth scenario just outlined [46]. The ideal temperature for the SIPF reaction is between 60 and 90°C [46, 51], a very realistic condition for recently condensed oceans on the cooling surface of the Earth. The reaction prefers the biologically relevant a-amino acids to amino acids containing a more distant amino group [46, 51]. Amino acids such as glycine and histidine, but also small linear or cyclic peptides such as diglycine and diketopiperazine, show a yield-increasing catalytic effect in the reaction [44, 45, 46, 49, 51], which can produce peptides even with otherwise almost nonreactive amino acids, thus resulting in peptide formation with all amino acids investigated so far. The catalytic mechanism [49,51 ] apparently involves the intermediary formation of a peptide with the catalyst, followed by its condensation with another educt amino acid and the release of the catalyst by hydrolysis to the product peptide and the catalyst. Quite striking evidence for the prebiotic relevance of the SIPF reaction resulted from a comparison of the specific dipeptide yields of the reaction with the fre-

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quency of the respective peptide linkages in membrane proteins of Archaea and Procaryonta, some of the oldest still existing organisms on the Earth; a correlation of the preferred sequences was observed that could hardly have occurred only by chance [46, 51, 52], indicating an evolutionary pathway from the SIPF process to the proteins of the first living organisms. It is interesting that the (in)famous proteinlike prions show an even higher degree of similarity with the most preferred SIPF sequences (Gly-Gly > Gly-Leu > Ala-Ala > Ala-Gly > AlaVal > Pro-Pro > Pro-Gly > Val-Val > Gly-Ala > Val-Gly > Leu-Gly > His-Gly > Leu-Leu > Val-Ala > Gly-His > ValLys > Lys-Val), in particular the characteristic dominance of the Gly-Gly linkage, thus indicating an origin of the prions at a very early stage of evolution [53]. As mentioned previously, the oligopeptides formed in the SIPF reaction could have been stabilized and elongated to longer chains with the help of clay minerals such as montmorillonite or hectorite in the same environment [46, 51]. Thus, the role of clay minerals in peptide evolution could have been quite a significant one, not so much for the primary formation of peptides from amino acids but for the further development of larger and more complex peptides and for their survival against hydrolytic attack. Another quite important feature of the SIPF reaction refers to chirality-related aspects; during the reaction, the optical purity of the amino acids is almost fully conserved [46, 50], especially at low concentrations, as we would expect in a very dilute prebiotic soup. This preservation of chirality, albeit quite important, is not the only characteristic of the reaction with respect to optical activity. The active complex in the SIPF reaction [32, 46, 51, 60] has been found to be a copper(II) complex with one amino acid coordinated as a chelate, another amino acid or oligopeptide bound end-on via its carboxylic group, a chloride ion, and two water molecules at elongated distance (see Fig. 1). Quantum chemical calculations have predicted a pseudotetrahedral arrangement of the amino acid and chloride ligands [46], thus leading to a central chirality at the Cu(II) ion that seems to amplify the small inherent chirality of copper due to the parity violating effect, which because of its Z^'-dependence (Z being the atomic number) is considerably larger than in organic compounds. This chirality effect can lead in some cases, especially with simple aliphatic amino acids such as Ala and Val, to a preference of L-amino acids over D-amino acids in the SIPF reaction [44, 45, 46, 47, 50] and might be, together with yet unknown amplification mechanisms, a basis for the processes having led to the phenomenon of biohomochirality. Yield differences according to the chirality of the educt amino acids, differences in the complex formation constants of L- and D-alanine, and circular dichroism (CD) spectroscopy

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FIGURE 1. Ab initio geometry optimized structure of the tetrahedrally distorted SIPF complex [CuCI(alaH2)(H20)2r [46, 47].

have supplied independent indications toward this particular stereoselectivity in the SIPF reaction in favor of L-amino acids [52].

A PEPTIDE WORLD AS THE ORIGIN OF LIFE? Any discussion on the origin of life naturally involves a definition of a living system. It has become a convention to characterize life by the essential properties metabolism, replication, propagation of information, and the possibility of mutation, allowing evolution to adapted and more complex forms. Although a kind of metabolism can be assigned to a number of chemical systems, the other characteristics can be achieved according to our contemporary chemical knowledge by two types of biomolecules only, namely peptides/proteins and nucleic acids, which are the key players in all of today's living organisms. Nucleic acids are the modern information-carrying molecules and replicators, but peptides and proteins could have preceded them in that task because peptides can also work as carriers of information and can replicate, as shown recently [20, 29], albeit not as efficiently as nucleic acids. Under the harsh conditions of the primordial Earth, however, evolution of lifelike processes based on peptides would have had many favorable aspects. In addition to various problems with the formation of nucleic acids under primordial Earth conditions [11, 14, 56], the first argument to support this statement is the chemical and physical (in)stability of nucleic acids; in a hot salty ocean irradiated by the UV light of the sun, nucleic acids and their building blocks decompose very rapidly [6, 21, 27, 31, 33, 57, 58] and thus would not have had

time to pass on their information and to chemically evolve. Peptides, on the other hand, could be formed much more efficiently [46, 51] and, once formed, would be much more stable [3, 4, 26, 64] and well protected from UV damage by dissolved ions and other organic molecules in the prebiotic soup that would have absorbed a great part of the harmful UV radiation in the wavelength region below 220 nm [6]. Furthermore, peptides are far less prone to errors caused by mutations, because their informational content and their functionality are less harmed by an exchange of amino acids in the sequence. For example, a hydrophobic amino acid would mainly be replaced by another hydrophobic amino acid, which in most cases would not have a strong influence on the characteristics of the peptide, whereas the replacement of one or several nucleobases by another of the four possibilities could have dramatic effects, leading even to a total loss of function. Without the help of sophisticated enzymatic repair mechanisms (which demand the existence of sophisticated proteins!) nucleic acid evolution would quickly have reached a dead end. Peptides can deal with many more errors by themselves. Based on information theory, Freeman Dyson's model of chemical evolution [9] has predicted that, given the low autocatalytic efficiency of the first prebiomolecules, four basic building blocks, as in the case of nucleic acids, would be too few to reach a state in which the development of autocatalytic biochemical cycles would be possible; at least 8-10 would be required. This figure is realistic for early peptides, even considering a still limited number of amino acids formed during prebiotic chemical evolution. Finally, follow-up investigations on Eigen's RNA hypercyles [10] quickly showed through computational simulations [37] that such cycles would quickly be terminated by various catastrophic conditions and thus not provide the possible development of living systems. Therefore, an initial peptide/protein world [46], as opposed to an RNA world scenario, appears to be a much more likely starting point for life on Earth. Amino acids and peptides would have been formed first and then have chemically evolved to assume the function of primary information carriers and replicators. Nucleic acids could probably only have been synthesized and have chemically evolved after some kind of protection against environmental influences had become available, most probably in the form of peptide/protein membranes or shells [54]. Inside this protected environment, further evolution of the nucleic acids to nucleosides and nucleotides and the necessary enzymatic repair mechanisms could take place, passing over the functions of the first peptides and proteins to RNA and DNA and ultimately leading to the genetic apparatus of today's organisms. This later involvement of RNA/DNA in evolution was actually anticipated by

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