MS mass spectrum, a.m.u. atomic mass units, Rt retention time, ppb parts per billion (ng analyte per g Murchison), numbers shown in boldface are the main mass fragments. aGas chromatogram taken in deviation from the standard protocol with a specifically optimized temperature program of the chiral capillary column

MS mass spectrum, a.m.u. atomic mass units, Rt retention time, ppb parts per billion (ng analyte per g Murchison), numbers shown in boldface are the main mass fragments. aGas chromatogram taken in deviation from the standard protocol with a specifically optimized temperature program of the chiral capillary column acid, D,L-2,3-diaminopropanoic acid, and D,L-2,4-diaminobutanoic acid had been identified previously (Munoz Caro et al. 2002). Achiral 2,3-diaminobutanoic acid and 3,3'-diaminoisobutanoic acid were also detected in the carbonaceous chondrite.

Chiral diamino acids in the Murchison meteorite showed a racemic ratio as it is indicated in Table 8.1, accompanied by corresponding confidence intervals.

Attention was paid to exclude contamination with biological products. A serpentine sample, which was heated for 4 h at 500°C to entirely destroy thermolytically all eventually present amino acids and diamino acids, was taken as a blank, and subjected to the entire analytical protocol. In this context, serpentine has quite often been used as a blank sample since it possesses a sheet silicate structure with the potential to resorb amino acids and other organic compounds, like a sponge. As depicted in Fig. 8.3, no diamino acids were detected in the heated serpentine blank sample. These data show that the identified meteoritic diamino acids were not the result of contamination in the laboratory.

Diamino acids were quantified by comparison of the peak areas with external standards. The concentrations of the observed diamino acids were lower than those of monoamino acids in the Murchison carbonaceous chondrite (Cronin and Chang 1993; Cronin 1998). In general, the abundances of amino acids in carbonaceous chondrites decrease logarithmically with increasing carbon number. In the reported analyses of meteoritic material, the absolute abundance of amino acids generally is enhanced by previous 6 molar hydrochloric acid hydrolysis (see also Chap. 7; Cronin 1976). The concentration of free diamino acids in the Murchison meteorite was also observed to increase after hydrolysis by factors between 1.7 and 6.0 relative to the extraction with hot water at 100°C. After cold-water extraction, no diamino acids were detected. These data indicate that these products must have been originally formed as molecules of higher molecular mass, that release free diamino acids after hydrolysis.

For the past 40 years, the hydrolysis has been a reasonable and required step in any meteorite analysis protocol in order to make the incorporated amino acids visible. The amino acid structures including their molecular skeleton composed of carbon-carbon and carbon-nitrogen bonds are preformed in original molecules of higher molecular mass. The hydrolysis step is neither capable of forming a carboncarbon bond nor a carbon-nitrogen bond and this step can thus not be interpreted as a chemical reaction that "produces" amino acids. However, we may seriously ask the question concerning the chemical evolution of amino acids "where and under which environmental conditions do hydrolysis steps take place during chemical evolution releasing free (and chiral) amino acids?" Today, we assume that this hydrolysis step might have occurred over long time spans either in terrestrial liquid water, or - partly - in liquid water present in meteorite parent bodies. It is estimated that the parent body of the Murchison meteorite has reached temperatures of up to approximately 300 K due to radioactive heating.7 So do various meteorites in which fluid inclusions in salt crystals (Shock 2002) and specific mineralogical compositions show evidence of a water-rich past on their parent bodies indicating aqueous alteration at even much higher temperatures.

It is appropriate to make a comparison between the identified organic molecules such as amino acids and diamino acids in meteorites, and amino acids and di-amino acids obtained from UV-irradiation of circumstellar/interstellar ice analogues (Chap. 7). Such a comparison, however, is not necessarily straightforward, because interstellar ices are believed to be pristine while carbonaceous chondrites experienced a different history and underwent - possibly frequent - alterations through aqueous processing, thermal effects, or shock, which all may have affected their original composition (Kerridge 1999; Schwartz and Chang 2002). The Murchi-son meteorite is classified as a CM2-type chondrite, a type that is characterised by minimal alteration relative to other chondrites studied for organic components (Cronin and Chang 1993). The diamino acids described above have previously been identified as refractory products of photo- and thermal processing of circumstel-lar/interstellar ice analogues in the laboratory (Munoz Caro et al. 2002), and thus point to a correlation between the organic matter that might be present on icy grain mantles residing in star-forming regions (and hence likely also in circum-stellar disks) and carbonaceous chondrites. The exact pathways of formation of the amino acids and diamino acids in carbonaceous chondrites are hitherto unknown. Besides the well studied mono-a-amino acids, P-amino acids (Engel and Macko 2001; Ehrenfreund et al. 2001b), and diamino acids have been identified in both laboratory simulations of circumstellar/interstellar ice that was photolytically and thermally processed (Chap. 7), and - as presented here - in carbonaceous chondrites. The latter organic compounds could not have been formed by the known Strecker-cyanohydrin synthesis (Cronin 1998). The results suggest an alternative formation mechanism, namely, that amino acids and diamino acids as refractory products of experimental simulations of ice photoprocessing in circumstellar regions and in carbonaceous chondrites are synthesized photochemically via complex recombination processes of radicals.

7 Personal communication with Rainer Merk from Tel Aviv University in Mai 2007.

Why are diamino acids of immense interest for studies on the origin and the evolution of life on Earth? The function of diamino acids in the prebiotic development of polypeptide ^-structures was intensively studied by Brack and Orgel (1975) and Brack (1987, 1993). More recently, it became apparent that diamino acids might have contributed to the origin of life's genetic material as well.

8.2 Diamino Acids as Gene Trigger

The delivery of organic compounds by meteorites, interplanetary dust particles, or by comets (Oro 1961, Jessberger 1999) to the early Earth is one mechanism thought to have triggered the appearance of life on Earth (Chyba and Sagan 1992; Ehrenfreund 1999). Particularly the prebiotic formation of peptides and proteins on Earth by the use of amino acid monomers produced asymmetrically by interstellar photochemical processes is considered as an elegant model. However, to refer to one of the initial questions of this chapter, there is so far much less conclusive evidence on the origin of genes and the early development of genetic DNA material and its analogues.

In the course of the evolution of genetic material, it is widely accepted that today's "DNA-RNA-protein world" was preceded by a prebiotic system in which RNA oligomers functioned both as genetic materials and as enzyme-like catalysts (Gilbert 1986). RNA itself remains difficult to synthesize under prebiotic conditions. Furthermore, it is unstable in aqueous solution and was thus proposed to have been preceded by pre-RNA genetic material. Hitherto, the molecular structure of life's first genetic material is not understood and hence various world-leading research groups put much effort in elucidating its structure. This remains a rather challenging endeavour since life's first genetic material is probably no more present on Earth today. Even in biological organisms it might remain difficult - if not impossible -to detect its molecular relicts. Nevertheless, structural analogues of DNA and RNA can be synthesized in the laboratory and their solubility and stability as well as the capability of selected sequences to combine with DNA and RNA strands to form duplex structures can be studied including investigations of the physico-chemical properties of the duplexes.

After years of intensive international research, numerous molecular candidates have been proposed to serve as pre-RNA oligonucleotides. We will start our guided tour through all these candidates by having a closer look at one of the most intriguing structures, namely the peptide nucleic acid PNA. Peter Nielsen from the University of Copenhagen introduced PNA structures (Nielsen et al. 1991; Nielsen 1993). PNA is composed - as common oligonucleotides are as well - of a backbone of a regular pattern to which a sequence of the nucleotide bases adenine, guanine, cytosine, thymine, and uracil can be attached via appropriate spacer groups, eventually carrying life's first genetic code (Fig. 8.4). Two principal alternatives exist for the molecular architecture of the PNA backbone, letting us a priori distinguish between aegPNA and daPNA:

Fig. 8.4 Chemical structures of candidate precursors to RNA during the early history of life on Earth. a, Threose nucleic acid (TNA); b, peptide nucleic acid based on N-(2-aminoethyl)glycine monomers (aegPNA); c, peptide nucleic acid based on 2,4-diaminobutanoic acid monomers (daPNA); d, glycerol-derived nucleic acid analogue; e, d-^-ribopyranosyl-(2' ^ 4') oligonucleotide (p-RNA); f, ribonucleic acid (RNA). B, Nucleotide base

Fig. 8.4 Chemical structures of candidate precursors to RNA during the early history of life on Earth. a, Threose nucleic acid (TNA); b, peptide nucleic acid based on N-(2-aminoethyl)glycine monomers (aegPNA); c, peptide nucleic acid based on 2,4-diaminobutanoic acid monomers (daPNA); d, glycerol-derived nucleic acid analogue; e, d-^-ribopyranosyl-(2' ^ 4') oligonucleotide (p-RNA); f, ribonucleic acid (RNA). B, Nucleotide base

1. The PNA backbone is composed of N-(2-aminoethyl)glycine (aeg) monomers leading to aegPNA molecules. The monomer aeg was shown to be produced directly in electric discharge reactions from CH4, N2, NH3, and H2O (Nelson et al. 2000).

2. The PNA backbone is composed of diamino acids (da) as monomers leading to daPNA structures. Various structural analogues of diamino acids are known to form daPNA.

Different daPNA molecular structures are known to serve as DNA/RNA analogues. They consist of peptide backbones made of diamino carboxylic acids to which nucleotide bases are attached for example via carbonyl methylene linkers. Interestingly, the PNA backbone can be produced from diamino carboxylic acids by simple condensation reactions (Nielsen 1993)! The diamino acids 2,4-diaminobutanoic acid, and ornithine, which we detected in the Murchison meteorite as racemic pairs

(Meierhenrich et al. 2004), had been suggested to be essential constituents of PNA monomers (Nielsen 1993). The first identification of 6 molecular structures of di-amino carboxylic acids in simulated interstellar ice analogues is reported in Chap. 7 and Munoz Caro et al. (2002). A racemic mixture of 2,3-diaminopropanoic acid was synthesized by spark discharge experiments (Engel et al. 1995). Taken together, these results on the quasi omnipresence of diamino acids in relevant samples for the study of the origin of life might stimulate future research activities on appropriate reaction pathways from diamino carboxylic acids resulting in a variety of different PNA structures (Meierhenrich et al. 2002a). These data might then hold the key to fill the gap in our understanding between chemical evolution and biological evolution.

Besides peptide nucleic acids, other systems of oligonucleotides have been investigated and were proposed to have preceded RNA, just as RNA itself preceded DNA and protein. A systematic investigation of potentially natural nucleic acid analogues has led to the recognition of four candidate precursors to RNA.

1. The molecular structure of L-a-threo-furanosyl-(3'^ 2') nucleic acid (TNA) is based on L-a-threo-furanosyl units joined by 3',2'-phosphodiester linkages (Fig. 8.4 a) and able to form stable Watson-Crick base pairs with itself and with RNA (Fig. 8.4 f). In chemical evolution, TNA molecules are assumed to be more advantageous than RNA because of their relative chemical simplicity (Schöning et al. 2000).

2. In the uncharged peptide nucleic acid (PNA) the sugar-phosphate backbone of the RNA genome is replaced by a backbone composed of N-(2-aminoethyl)glycine units held together by amide bonds (Fig. 8.4 b). Nucleic acid bases are attached via methylenecarbonyl spacers to the PNA structure. PNA polymers of N-(2-aminoethyl)glycine form stable double helices with complementary molecules of RNA (Egholm et al. 1992, 1993b; Nelson et al. 2000). In template-directed reactions (Schmidt et al. 1997a, 1997b) information can be transferred from aegPNA to RNA, and vice versa, and aegPNA-DNA "chimeras" form readily on either DNA or aegPNA templates (Koppitz et al. 1998). The polycondensation reaction of diamino acids like 2,4-diaminobutanoic acid or ornithine yields daPNA structures of a corresponding constitution (Fig. 8.4 c), which also serve as potential pre-RNA oligonucleotides. The structural monomer 2,4-diaminobutanoic acid is clearly visible in the molecular daPNA structure given in Fig. 8.4.

3. Glycerol-derived nucleic acid analogues (Fig. 8.4 d) were proposed for pre-RNA self-replicating systems (Spach 1984; Joyce et al. 1987; Chaput and Switzer, 2000), although sufficient experimental support is missing to consider them as a strong candidate (Joyce 2002).

4. Members of the family of pentapyranosyl-(2'^ 4') nucleic acids (p-RNA, Fig. 8.4 e) were systematically synthesized in the laboratories of Albert Eschenmoser for the comparison with RNA with respect to those chemical properties that are fundamental to RNA's biological function (Eschenmoser 1997; Bolli et al. 1997). In these studies, the internal base-pairing-strength of the pentapyra-nosyl oligonucleotide systems was found to be higher than that of the corresponding pentafuranosyl RNA system (Beier et al. 1999).

The detection of diamino acids in samples of the Murchison meteorite enabled to explore new routes to interpreting chemical evolution. The results stated that (a) amino acid structures, the molecular building blocks of proteins and (b) molecular building blocks of pre-RNA oligonucleotides are present in interplanetary, interstellar, and circumstellar samples. The results are new clues for the assumption that organic ingredients of living systems have been delivered via (micro-) meteorites and comets to the early Earth from regions of the interstellar medium. After the transport these molecules were potentially participating in initial prebiotic reactions, which turned out to be of central importance for the origin of life on Earth.

The results would suggest that both early proteins and early genetic material were synthesized from molecular building blocks that had been delivered from interstellar/circumstellar space to the early Earth (see also Strasdeit 2005). We have outlined some guesses that life's genetic material might have arisen originally starting with the formation of diamino acids under interstellar condition followed by the formation of peptide nucleic acid structures by polycondensation reaction. The subsequent formation of RNA might have triggered the origin of today's DNA/protein world. The reader should be aware that this proposal on the evolutionary origin of DNA is only one hypothesis amongst others. It is not the standard model of the scientific community for the origin of the genetic material on Earth. Until now, such a standard model does not exist.

8.3 Survival of Organic Molecules After Impact on Earth

Often, the question arises whether organic molecules and particularly amino acids embedded in comets and/or meteoroids would survive an impact on Earth from outer space. For comets, the Greenberg-model suggested that organic molecules would at least partly survive such an impact (Greenberg et al. 1994). Greenberg expected that the survivability of cometary organic molecules depends on both the size of the comet and the scale height of the atmosphere as well as the comet density and morphological structure. Since comets are made up of aggregated interstellar dust particles (see next chapter) the heating of an impact would be concentrated at the surface because of their exceedingly low thermal conductivity. This would lead to rapid water (ice) evaporation and further fragmentation. This sequence would lead to smaller and smaller fragments whose deceleration in the atmosphere is progressively less and whose heating is consequently smaller. The general result is then, that a comet, rather than impacting as a single body with high kinetic energy, breaks up into smaller and smaller components, each of which is subjected to lower degrees of heating. The dissipation of the energy by impulsive evaporation of the volatiles can consequently lead to the survival of the refractory organic molecules during atmospheric entry (Greenberg 1993).

More recently, the hypothesis of the survival of organic molecules during atmospheric entry was strengthened for the case of meteorites by the STONE experiment. This experiment deserves attention, however at present it is not very well known:

An "artificial meteorite" was designed in order to investigate physical modifications to sedimentary rocks and chemical modifications to their organic ingredients during terrestrial atmospheric entry. Three different samples (basalt, dolostone, and an artificial rock simulating Martian soil) were doted with amino acids and fixed onto the heat shield of the recoverable Foton-12 spacecraft. The spacecraft was launched, taken in low Earth orbit followed by the atmospheric re-entry and recovery of the capsule. Since the basalt sample was lost during entry, mineralogical and isotopic studies were performed with the partly recovered dolostone and the simulated Martian regolith (Brack et al. 2002). The results indicated that intact organic molecules such as amino acids could be recovered from the artificial meteorite.

The delivery of organic molecules from interplanetary space to Earth might also have occurred via micrometeorites (Maurette 2006). These micrometeorites have a typical size of 20-500 |m and can be collected from deep-sea sediments, terrestrial sand, sedimentary rocks, Greenland lake sediments, and by melting large amounts of Antarctic ice and snow (Duprat et al. 2007). Micrometeorites are even assumed to be the dominant mass fraction of extraterrestrial material delivered to Earth today bringing to the Earth about 100 times more material than objects found outside this size range, including the much larger meteorites (Brinton et al. 1998). Enantioselec-tive analysis of a collection of micrometeorites revealed that they include terrestrial L-amino acid components making conclusions on eventual original enantiomeric excesses difficult. In one sample, the amino acid a-aminoisobutyric acid AIB was found to be present at a level significantly above the background blanks (Brinton et al. 1998). These data let us conclude that amino acids - at least in part - would survive an impact on Earth via micrometeorites.

8.4 Space Exploration and Chirality: What Next?

In future investigations the stable isotope compositions of hydrogen, nitrogen, and carbon in the carbonaceous chondrite's diamino acids need to be determined. The 52H, §15N, and §13C values of amino acids in Murchison generally lie outside the ranges of organic matter on Earth (Engel and Macko 2001; Pillinger 1984). A similar shift of the isotopic composition is expected for the diamino acids in the Murchi-son meteorite. Moreover, different constitutional isomers of diamino acids might be determined in other carbonaceous chondrites in the laboratory, in cometary samples by the GC-MS instrumentation onboard the ROSETTA mission and possibly by measurements to be performed by Mars mission ExoMars in the coming years (see Chap. 9). In addition, the synthetic, bio-organic chemical pathways of PNA structures, that start with the diamino acids like those reported here as present in meteorites for the first time, will be studied more intensely in order to elucidate possible origins of life's genetic material.

Chemical and analytical investigations of meteorites will most likely continue to attract the scientific interest in future times, since meteorites that originate from extraterrestrial environments contain molecular ingredients that can be assumed to have been present when life formed on the early Earth. On terrestrial samples, however, no such molecular relicts can be identified today since here the environment is 'contaminated' with life, throughout effacing information on its origin. One can thus hope for future experiments that the advancement in the development of analytical techniques might allow the identification of new families of organic compounds in carbonaceous chondrites; compounds that might be interesting in the context of the origin of life and its genome. Separation of organic molecules based on multidimensional GCxGC techniques and their identification with powerful time-offlight mass spectrometers or modern UPLC instruments in liquid chromatography will be applied to the analysis of meteorites in the future. These and other techniques might provide us with structural elucidation of the 'organic polymer' present in meteorites and in the end enables us to better understand the origin of life on Earth including its chiral asymmetry.

Meanwhile, other research teams might trace back biological evolution by, e.g., comparing RNA-sequences and advance in the understanding of the molecular structure of first self-replicating systems on Earth. Thereby, the gap in our understanding between chemical and biological evolution should become ever smaller.

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