Key to the Prebiotic Origin of Amino Acids

Amino acids are the crucial molecular components of life. They are the chiral building blocks for the molecular architecture of proteins (enzymes) and certainly played a key role in both the appearance of life and the emergence of biomolecular asymmetry (homochirality) on Earth. Proteins of living organisms are composed of 20 basic amino acids. It is thought that certain, if not all, amino acids had already been present on Earth nearly four billion years ago, long before the appearance of life. How did these chiral amino acids, the key molecular ingredients triggering biological evolution and life as we know it, originate?

Modern research distinguishes between three alternative models: (1) The formation of primordial amino acids might have occurred in the early Earth's atmosphere as successfully simulated in the laboratory by the well-known Urey-Miller experiment. (2) Hydrothermal systems are proposed to have the potential of generating a variety of amino acids before life started on Earth. And (3) The origins of amino acid formation may lay in the depths of space, since a pool of hitherto 89 amino acids has been identified in meteorites. Moreover, the foundation for the molecular asymmetry of amino acids may have taken place in well-defined regions of interstellar space: this scenario proposes that primordial amino acids were - frozen within tiny particles of ice - exposed to highly energetic, "asymmetric" ultraviolet radiation.

The aim of this chapter is to expose the current research activities on life's molecular building blocks and to present new data supporting the interstellar formation of amino acid structures including their asymmetry.

7.1 Amino Acid Formation Under Atmospheric Conditions

Based on the ideas of Alexander Oparin in Russia and Harold Urey in the United States in the first half of the 20th century, it was assumed that the atmosphere of the early Earth was strongly reducing and dominated by hydrogen H2, methane CH4, and ammonia NH3. These gases could have been captured from the primordial solar nebula. The giant planets like Jupiter and Saturn had retained these gases since that time, as they were too massive to release H2 into space. One concluded that

U. Meierhenrich, Amino Acids and the Asymmetry of Life. Advances in Astrobiology 125

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terrestrial planets, including Earth, had atmospheres of similar composition early in their histories before they had time to evolve to further stages.

Based on this supposition, Stanley L. Miller, member of Nobel laureate Harold Urey's research team at the University of Chicago, simulated such an atmosphere in the laboratory. The atmosphere composed of the above gases was processed by spark discharges imitating lightning. A 250 mL liquid water fraction taken from the simulation system showed the generation of a variety of amino acids and other organic molecules after several days of treatment (Miller 1953, 1998; Miller and Lazcano 2002). Here, a reducing atmosphere with methane (CH4) as the carbon-providing molecule was assumed. Several racemic amino acids were formed under these conditions as products of spark discharge, photoprocessing, or heat.

However, the dominant view in recent years has been that the primitive atmosphere consisted of a weakly reducing mixture of CO2, N2, and H2O, combined with lesser amounts of CO and H2. This is because the atmosphere of the early Earth is considered to be largely produced from volcanic outgassing. There is a considerable literature stating that the hydrogen emitted from volcanoes is released as H2O rather than H2, and most of the carbon is released as CO2 rather than CO or CH4 (Kasting and Brown 1998). The weakly reducing mixture of the early Earth's atmosphere and particularly the high partial pressure of CO2 of about 0.2 bar is consistent with the most likely mechanism for balancing the smaller solar luminosity at the time of formation of the earth, i.e., 30% lower than at present. This required an increased greenhouse effect in Earth's atmosphere, probably due to the accumulation of CO2 to a minimum amount of 0.2 bar (Walker et al 1981; Kasting 1992).

The problem with the Urey-Miller mechanism is that amino acids could not be generated in a weakly reducing atmosphere. The HCN intermediate, required for amino acid formation, forms only from spark discharges in gas mixtures containing N2 and CH4. However, it does not form in atmospheres containing predominantly N2 and CO2, in which the nitrogen atoms formed by splitting N2 almost invariably combine with oxygen atoms to yield NO (Kasting and Brown 1998). Stanley Miller himself reported that glycine was essentially the only amino acid synthesized in the spark discharge experiments with CO and CO2 (Miller 1998). Other research teams of Akiva Bar-Nun and Sherwood Chang investigated the problem and found that, at most, only traces of amino acids form under such realistic weakly reducing conditions (Bar-Nun and Chang 1983). As expected for Urey-Miller types of atmosphere simulating systems, the amino acids produced remained racemic, since no asymmetry was introduced via spark discharges or other carriers into the system.

Furthermore, the generation of amino acids under the simulated atmospheric conditions of the early Earth was performed with ultraviolet light as the energy source (Sagan and Khare 1971; Khare and Sagan 1971). In this case, the photochemical reaction of a gaseous mixture containing CH4, C2H6, NH3, H2O, and H2S yielded a variety of amino acids. However, the presence of ethane with its preformed carboncarbon bond was required as the main ingredient in the gas mixture. Hydrogen sulfide was also necessary as an acceptor for the long wavelength photons. Without ethane no amino acids were generated.

7.2 Amino Acid Formation Under Hydrospheric Conditions

Alternatively, hydrothermal systems were considered as an environment that could allow for the prebiotic formation of organic molecules (Hennet et al. 1992; Holm and Andersson 1998; Proskurowski et al. 2008). For the generation of hydrocarbons, lipids, amides, esters, and nitriles under such conditions, a Fischer-Tropsch type mechanism was proposed (Holm and Charlou 2001). However, the synthesis of amino acid structures in such an environment remains problematic. Under simulated "hydrothermal" conditions, selected starting compounds can react and lead to amino acids such as glycine, alanine, aspartic acid, serine, leucine, and others. However, the selected starting chemicals, i.e., formaldehyde, ammonia, and cyanide cannot be considered as being "prebiotic". Starting with these compounds of the Strecker synthesis was considered to be "equivalent to starting with glycine" (Miller 1998).

The team of Kensei Kobayashi at Yokohama National University tried to identify relicts of D-amino acids in different deep-sea hydrothermal systems pointing to their eventual abiotic origin. However, amino acids were only found in their L-configuration, suggesting their biogenic origin rather than any hints of abiotic chemical synthesis (Takano et al. 2003a, 2003c, 2004a).

Thus, by both atmospheric and hydrospheric simulation experiments, amino acids can obviously be synthesized under ideal, however not realistic, prebiotic conditions of the initial mixture of precursor molecules. This points to the need of formulating an alternative mechanism of prebiotic relevance for the spontaneous generation of amino acids.

7.3 Amino Acid Formation Under Interstellar Conditions

As nearly 90 chiral amino acids have been discovered in carbonaceous meteorites (see appendix; Kvenvolden et al. 1970; Cronin and Pizzarello 1997; Engel and Macko 2001; Ehrenfreund et al. 2001b; Meierhenrich et al. 2004), a significant contribution of extraterrestrial amino acids synthesized in interstellar environments under asymmetric conditions and delivered to Earth in the form of cometary and interplanetary dust offers an attractive alternative to a purely terrestrial (atmospheric and hydrospheric) origin of chiral organic molecules such as amino acids. Therefore, we remain particularly curious to topical research results and discussions on the formation of amino acids under interstellar conditions; these conditions need to be outlined.

7.3.1 Photochemistry in the Interstellar Medium

The interstellar medium and the circumstellar medium are harsh environments where predominantly simple gas molecules and interstellar dust particles are processed by the bombardment of hard ultraviolet photons and cosmic rays. Dense clouds in the interstellar medium possess kinetic temperatures as low as 10 K and densities from 103 - 106 particles cm~3. They are the birthplace of stars and planetary systems. As introduced in Chap. 1, solid dust particles in dense clouds, with a typical size of 0.1 |m, accrete an ice layer made of H2O, CO, CO2, CH3OH, and NH3 as the main components (Gibb et al. 2000, 2001, 2004). These ices undergo energetic processing by ultraviolet photons and cosmic ray particles (Mufioz Caro et al. 2001; Mennella et al. 2001). Ultraviolet irradiation is responsible for the dissociation of the ice molecules, creating free radicals, which recombine to form organic molecules (Greenberg 1986; Kerridge 1999; Schwartz and Chang 2002; Mufioz Caro and Schutte 2003; Munoz Caro et al. 2004).

In order to gain information on the chemical and chiral structure of interstellar organic molecules, interstellar processes can be (a) simulated in the laboratory on Earth. Such simulation experiments will be presented in this chapter. Furthermore, one may envisage supplementary chemical analysis of (b) meteorites, (c) comets, and (d) organic molecules on the surface and subsurface of the planet Mars. These ambitious projects, which are at the center of topical research activities of the European and the US Space Agencies, will be presented in the upcoming chapters always concentrating on the phenomenon of chirality.

7.3.2 Simulation of Interstellar Chemistry in the Lab

At Leiden Observatory for Astrophysics, The Netherlands, the research team of J. Mayo Greenberg specialized in the synthesis of simulated interstellar and circum-stellar ices. A specifically developed low pressure-low temperature chamber for the representative simulation of interstellar and circumstellar processes was installed there. A schematic view of this chamber is depicted in Fig. 7.1, a photo of the original instrument is given in Fig. 7.2 (a more detailed description can be found in Munoz Caro (2003)).

The chamber basically consists of a high vacuum chamber with a pressure of ap-prox. 1 -10-7 mbar. Inside this chamber, a gas mixture is deposited on a cold finger at 12 K, forming an ice layer, and irradiated. The system is pumped by a turbo pump backed up by a diaphragm pump. The low temperatures typical of dense clouds, down to 10 K, are achieved by means of a closed-cycle helium cryostat. The cold finger consists of a sample holder, in which an IR-transparent caesium iodide (Csl) window is mounted. The deposition and irradiation of the ice is monitored by means of infrared spectroscopy. Deposition of the molecules and subsequent photoprocess-ing occurrs on the surface of this cold finger. In the simulation experiment, the cold finger was simulating the interstellar dust particle.

After pumping and cooling the system down to simulated interstellar conditions, a gas mixture consisting of water, ammonia, methanol, carbon monoxide, and carbon dioxide (H2O, NH3, CH3OH, CO, and CO2) of molar composition 2:1:1:1:1 was deposited onto the cold finger's surface. The composition of the gas mixture

Fig. 7.1 Shedding light on interstellar processes: scheme of the low pressure-low temperature chamber for simulating the deposition and photoprocessing of interstellar and circumstellar ices by the adaptation to the physico-chemical interstellar and circumstellar conditions. 1 Deposition tube for gas molecules, 2 Vacuum UV lamp that emits mainly at Lyman-a (121 nm), 3 vacuum pump, 4 light source of IR spectrometer, 5 Csl window for sample condensation, 6 detector of IR spectrometer. Illustration: Stephane Le-Saint, Universite de Nice-Sophia Antipolis

Fig. 7.1 Shedding light on interstellar processes: scheme of the low pressure-low temperature chamber for simulating the deposition and photoprocessing of interstellar and circumstellar ices by the adaptation to the physico-chemical interstellar and circumstellar conditions. 1 Deposition tube for gas molecules, 2 Vacuum UV lamp that emits mainly at Lyman-a (121 nm), 3 vacuum pump, 4 light source of IR spectrometer, 5 Csl window for sample condensation, 6 detector of IR spectrometer. Illustration: Stephane Le-Saint, Universite de Nice-Sophia Antipolis

Fig. 7.2 Space simulation chamber at Leiden Observatory, The Netherlands. The ice sample is located inside the vacuum chamber on an Al-block, or Csl window, located on the tip of the cold finger at T = — 261°C and irradiated by vacuum ultraviolet photons. These conditions recreate interstellar ices present in the pre-solar nebula during the formation of the solar system is similar to that found close to protostellar sources (Gibb et al. 2001) and dense molecular cloud ices (Gibb et al. 2004). Interstellar ice abundances adopted for the starting ice mixture before irradiation were based on those derived from observations with the Interstellar Space Observatory (ISO) by Gerakines et al. (1999) and Ehrenfreund et al. (1999), excluding water ice which was increased by a factor of 2, as it is known that water is overabundant in comets. Two deposition lines were used for the gas mixture with a gas flow of 1016 molecules sec-1.

The system was separated by a MgF2 window from a UV-light source, i.e., a microwave-stimulated hydrogen discharge lamp, that emits 1.5 x 1015 photons sec-1 (Weber and Greenberg 1985) from 7.5 eV (165 nm) to 10.5 eV (117 nm) with the highest flux peaking at 9.8 eV (121 nm at the Lyman-a line) (Gerakines et al. 1995; Mufioz Caro and Schutte 2003). The typical irradiation and deposition time was 24 hours. Please note that the number of deposited molecules was higher than the number of photons induced into the system.

In order to follow the photochemical reactions during ice deposition, irradiation, and heating, infrared (IR) spectra were measured in situ with the help of a FTIR spectrometer.

After irradiation, the samples were heated at 1 Kmin-1 to 40 K and then at 4K min-1 up to room temperature. During the heating process, the photochemically formed radicals start to move in the simulated interstellar ice and can be assumed to recombine in order to form oligomers and polymers. After heating, the above-described samples to room temperature, a small amount of residue remains, showing a yellowish colour as it is depicted in Fig. 7.3.

Fig. 7.3 Producing a comet in the lab: yellowish residue obtained after simulating interstellar and circumstellar ices on the surface of the cold finger (here: an aluminium block of 30 • 40 mm surface). After extraction, hydrolysis, and chemical derivatization, a wide variety of different chiral amino acid structures was identified therein

Fig. 7.3 Producing a comet in the lab: yellowish residue obtained after simulating interstellar and circumstellar ices on the surface of the cold finger (here: an aluminium block of 30 • 40 mm surface). After extraction, hydrolysis, and chemical derivatization, a wide variety of different chiral amino acid structures was identified therein

In the residue, non-chiral saturated organic compounds containing carboxylic groups but also hexamethylenetetramine (HMT) (Agarwal et al. 1985; Briggs et al. 1992; Bernstein et al. 1995) and structurally related compounds such as methyl-HMT, hydroxy-HMT, and others (Meierhenrich et al. 2001f; Mufioz Caro et al. 2004) were identified. In addition, non-chiral N-heterocycles and amines were determined in the residues serving as potential precursors of biological cofac-tors (Meierhenrich et al. 2005a). Previous work claimed the identification of trace amounts of a small number of amino acids in photo-processed interstellar/ circumstellar ice analogues (Briggs et al. 1992; Kobayashi et al. 1999a). Except for glycine, however, such analytical results were not confirmed by isotopic labelling of the initial gas mixture, a necessary verification to exclude contamination with trace amounts of organic compounds.

7.3.3 Fresh in the Ice: Amino Acid Structures 7.3.3.1 Enantioselective Analysis

For the identification of chiral molecules, the irradiated samples of interstellar and circumstellar ice analogues were subjected to enantioselective analysis at the CNRS Centre de Biophysique Moleculaire in Orleans, France. An enantioselective gas chromatograph coupled with a mass spectrometer (GC-MS) was used. For each analyte, an individual sensitive analytical procedure was developed that consists of the sample's extraction, the hydrolysis, the chemical transformation of the an-alytes into suitable volatile derivatives, and the enantioselective capillary GC-MS separation and detection. The identities of the analytes obtained via the GC-MS technique were verified by comparing retention times (Rt) and mass spectra with literature data and external standards. Chiral analytes were resolved into enan-tiomers by an enantioselective GC-MS technique using Chirasil-L-Val capillary columns.

The obtained results demonstrated that carbon-to-carbon single bonds and carbon-to-nitrogen single bonds could be photochemically formed under simulated in-terstellar/circumstellar conditions. Even more surprisingly, 16 amino acids, six of which are among the 20 protein-constituent amino acids (i.e., glycine, alanine, va-line, proline, serine, and aspartic acid) were identified on the surface of simulated interstellar dust particles (Mufioz Caro et al. 2002). Figure 7.4 shows the chro-matogram of the residue obtained from the photo-processed interstellar ice analogue mixture. Most of the observed peaks were due to amino acids; three exceptions were identified as 2,5-diaminopyrrole, 2,5-diaminofuran, and 1,2,3-triaminopropane. The calculated signal-to-noise ratio was S/N > 10, even for the least abundant amino acids identified.

Glycine, the simplest amino acid, was the most abundant. For amino acids bearing only one amino group, species with higher molecular mass were less abundant. Aspartic acid was the only dicarboxylic compound found in the residues.

Fig. 7.4 Caught in the ice: Amino acid structures and other compounds in the gas chromatogram of an ice sample generated under simulated interstellar/circumstellar conditions. Data were obtained from enantioselective analysis of the room temperature residue of photo-processed ISM ice analogue. Chemical analysis was performed after extraction with ultra-pure water, 6 M HCl hydrolysis, and derivatization [ethoxy carbonyl ethyl ester (ECEE) derivatives taken from Huang et al. (1993) and Abe et al. (1996)]. A Varian-Chrompack Chirasil-l-Val coated capillary column 12 m x 0.25 mm inner diameter, layer thickness 0.12 |m was applied as chiral stationary phase with splitless injection, 1.5 mL min-1 constant flow of He carrier gas; oven temperature programmed for 3 min at 70°C, 5°C min-1, and 17.5 min at 180°C. The detection of total ion current was performed with the GC-MSD system Agilent 6890/5973. DAP, diaminopentanoic acid; DAH, diaminohexanoic acid; a.m.u., atomic mass units. The first publication of the illustration by Muñoz Caro et al. (2002) is acknowledged

Fig. 7.4 Caught in the ice: Amino acid structures and other compounds in the gas chromatogram of an ice sample generated under simulated interstellar/circumstellar conditions. Data were obtained from enantioselective analysis of the room temperature residue of photo-processed ISM ice analogue. Chemical analysis was performed after extraction with ultra-pure water, 6 M HCl hydrolysis, and derivatization [ethoxy carbonyl ethyl ester (ECEE) derivatives taken from Huang et al. (1993) and Abe et al. (1996)]. A Varian-Chrompack Chirasil-l-Val coated capillary column 12 m x 0.25 mm inner diameter, layer thickness 0.12 |m was applied as chiral stationary phase with splitless injection, 1.5 mL min-1 constant flow of He carrier gas; oven temperature programmed for 3 min at 70°C, 5°C min-1, and 17.5 min at 180°C. The detection of total ion current was performed with the GC-MSD system Agilent 6890/5973. DAP, diaminopentanoic acid; DAH, diaminohexanoic acid; a.m.u., atomic mass units. The first publication of the illustration by Muñoz Caro et al. (2002) is acknowledged

Decarboxylation is known as the main photo-damage of UV-exposed amino acids and probably competes with photoproduction (Johns and Seuret 1970).

Additional irradiation experiments involving a ten times higher H2O ice concentration and/or deposition at 80 K, monitored by infrared spectroscopy, showed that the residue production proceeded similarly, although the relative abundances of the main components, hexamethylenetetramine and carboxylic acids, varied somewhat (Munoz Caro et al. 2004; Nuevo 2005; Nuevo et al. 2007).

7.3.3.2 Isotopic Analysis

Changes of isotopic compositions of the initial gas mixture (12C/13C) combined with the mass spectroscopic isotope identification in the organic analytes provided clues of the reaction pathway. Did the amino acid structures really originate from the C-1

and N-1 units introduced in the system? Careful precaution was taken to eliminate risk of biological contamination: the reactants methanol, carbon monoxide, and carbon dioxide containing the usual 12C isotopes were replaced by their 13C isotopic counterparts. A similar chromatogram was obtained for the 13C isotopically labelled sample (molar concentration H2O:13CH3OH:NH3:13CO:13CO2 = 2:1:1:1:1). This isotope exchange resulted in increasing the mass of each amino acid's carbon atom by one. Thus, the synthesized amino acids were assumed to show a different mass, distinguishing them from biological amino acids of terrestrial origin by mass spectroscopy. The compounds corresponding to these mass increments were indeed identified as shown in Table 7.1.

To illustrate the measured isotopic mass shift, I will give an example for the enantiomers of the amino acid a-alanine: the mass spectrometric fragmentation of the 12C-isotope of the a-alanine ECEE-derivative is given by m/z = 189, 144, 116, and 88 atomic mass units (amu, parent peak in bold numbers). The mass spectra of the enantiomers D-Ala and L-Ala were, of course, identical. The 13C-isotopic derivatives of a-alanine were m/z = 147, 118, and 90amu. The ECEE-a-alanine mass spectrometric fragment of 147 amu contains three 13C atoms; the fragments of 118 and 90 amu contain two 13C atoms each. According to this fragmentation pattern, one can conclude that the detected amino acids were indeed formed by the photoprocessing of the initial gas mixture. Contamination of the system by amino acids of biological origin can definitely be excluded, since these amino acids would have been composed of 12C isotopes.

7.3.3.3 Hydrolysis and Meteoritic Samples

In the cases of reported analyses of meteoritic samples of carbonaceous chondrites, such as Murchison and others, the absolute abundances of the identified amino acids were enhanced by acid hydrolysis with 6 molar hydrochloric acid (Cronin 1976; Engel and Macko 2001). Similarly, in our case amino acids in the residues of simulated interstellar ices were identified in considerable amounts only after acid hydrolysis, which probably indicates that these products were originally formed as peptidic or oligomer molecules. The precise molecular structure of these oligomers and polymers is hitherto unknown; their structural elucidation is subject of numerous topical research activities: Matthews (1992, 2004), Minard et al. (1998) and Matthews and Minard (2006) expect that polymers could be major components of cometary matter; Kissel and Krueger (1987) and Kobayashi et al. (1998, 1999b) similarly suggest that complex organic macromolecules would make up the main organic ingredients. Atomic force microscopy images of similarly processed high-molecular-weight complex organic materials were recently published by Takano et al. (2007).

Free amino acids are released after hydrolysis of the polymer material. A gas chromatogram of photoprocessed simulated interstellar/circumstellar ices without hydrolysis is given in the literature (Meierhenrich et al. 2005a) showing minor quantities of the amino acid glycine among N-heterocyclic compounds. It should

Table 7.1 16 Amino acids, including 6 diamino acids, identified in simulated interstellar ices

Amino acid

Quantum yield.

MS 12C sample

MS 13C sample

Rt of analyte [min]

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