A Possible Prebiotic Origin on Volcanic Islands of Oligopyrrole-Type Photopigments and Electron Transfer Cofactors

Abstract

Tetrapyrroles are essential to basic biochemical processes such as electron transfer and photosynthesis. However, it is not known whether these evolutionary old molecules have a prebiotic origin. We have serendipitously obtained pyrroles, which are the corresponding monomers, in laboratory experiments that simulated the interaction of amino acid–containing seawater with molten lava. The thermal pyrrole formation from amino acids, which so far has only been reported for special cases, can be explained by the observation that the amino acids become metal bonded, for example in (CaCl₂)₃(Hala)₂·6H₂O (Hala=DL-alanine), when the seawater evaporates. At a few hundred degrees Celsius, sea salt crusts also release hydrochloric acid (HCl). On primordial volcanic islands, the volatile pyrroles and HCl must have condensed at cooler locations, for example, in rock pools. There, pyrrole oligomerization may have occurred. To study this possibility, we added formaldehyde and nitrite, two species for which plausible prebiotic sources are known, to 2,4-diethylpyrrole and HCl. We found that even at high dilution conjugated (oxidized) oligomers, including octaethylporphyrin and other cyclic and open-chain tetrapyrroles, were formed. All experiments were conducted under rigorously oxygen-free conditions. Our results suggest that primitive versions of present-day biological cofactors such as chlorophylls, bilins, and heme were spontaneously abiotically synthesized on primordial volcanic islands and thus may have been available to the first protocells. Key Words: Abiotic organic synthesis—Early Earth—Evaporites—Laboratory simulation experiments—Volcanism. Astrobiology 13, 578–595.

1. Introduction

A key question addressed by origin-of-life research is whether and how precursors of contemporary biomolecules could have formed abiotically on early Earth (Miller, 1998). In dealing with prebiotic syntheses, it seems useful to us to distinguish between formation in the primordial soup and protometabolic formation (Strasdeit and Fox, 2013). “Primordial-soup” (or “spontaneous”) synthesis means that an entire reaction sequence leading from inorganic starting materials to the final organic molecule proceeded independently of protocells [or “early cell ancestors” (Pohorille, 2009)]. “Protometabolic,” in contrast, refers to a situation where at least one reaction step relied on the existence of autocatalytic networks, that is, protocells. In the case of porphyrins, some early reports suggested a spontaneous prebiotic formation (e.g., Hodgson and Baker, 1967; Hodgson and Ponnamperuma, 1968). However, later it was concluded that no adequate prebiotic synthesis of porphyrins existed (Miller, 1998). The lack of experimental evidence for their abiotic formation has even led to the suggestion that porphyrins are ideal biomarkers (Suo et al., 2007). Recently, however, it has been found that porphyrinogens, the immediate precursors of porphyrins, form non-enzymatically from an α-aminoketone and a β-diketone or β-ketoester in water (Lindsey et al., 2009, 2011; Soares et al., 2012; Taniguchi et al., 2012). Based on current knowledge, it seems unlikely that the starting materials of this reaction could have formed spontaneously in sufficient concentration and purity on early Earth. On the other hand, their prebiotic synthesis in protometabolic small molecule networks (Shapiro, 2006) is conceivable. Thus, the aminoketone–diketone route to porphyrinogens may have existed already before polymeric catalysts evolved, but not before early cell ancestors.

As with any prebiotic synthesis, the simulation of a protocell-independent formation of porphyrins must be consistent with the geochemical and geophysical conditions on primitive Earth, as far as they are known (Strasdeit, 2010). Key steps of prebiotic chemical evolution took place during the first billion years of Earth's history, in the Hadean and early Archean eon (Brack, 2005). In this period, small volcanically active landmasses started to protrude from the ocean (Buick and Dunlop, 1990; Buick et al., 1995; Van Kranendonk et al., 2003, 2008; Westall, 2003; Nisbet and Fowler, 2004; Westall et al., 2006; Taylor and McLennan, 2009; Arndt and Nisbet, 2012). Thus, volcanic eruption clouds were probably a common phenomenon. Lightning in these clouds is thought to have produced organic compounds abiotically (Hill, 1992; Basiuk and Navarro-González, 1996; Navarro-González and Segura, 2004). This idea is supported by laboratory experiments demonstrating that several different amino acids are formed by spark discharges in steam-rich reducing gas mixtures (Miller, 1955; Johnson et al., 2008). Objects from space were additional sources of prebiotic amino acids (Pizzarello et al., 2006; Martins and Sephton, 2009), whereas the contribution of the bulk atmosphere is still unclear (Plankensteiner et al., 2004, 2006; Cleaves et al., 2008; Kuwahara et al., 2012).

It has been proposed that already in prebiotic times the ocean contained substantial concentrations of inorganic salts. In fact, the early seawater could have been even more saline than the modern one (Knauth, 1998, 2005; Izawa et al., 2010). This idea is consistent with the presence of highly saline fluid inclusions in ∼3.75 billion-year-old quartz (Appel et al., 2001); sodium chloride and potassium chloride in chondritic meteorites (Barber, 1981; Zolensky et al., 1999; Bridges et al., 2004); and evaporite minerals in ancient terrains on Mars (Gendrin et al., 2005; Osterloo et al., 2008; Glotch et al., 2010). Present-day observations show that large amounts of seawater evaporate when lava reaches the coast of a volcanic island and flows into the ocean. As a result, solid sea salt is formed and exposed to temperatures up to some hundred degrees Celsius (Edmonds and Gerlach, 2006). Organic molecules present in the seawater are embedded in and heated together with the sea salt crusts. It is reasonable to assume that these processes also occurred at the volcanic coasts of early Earth and that prebiotic amino acids that were dissolved in the ocean were involved.

In the present paper, we first explore the prebiotic chemistry associated with the interaction between seawater and lava. We present experimental results that show that pyrroles can form from amino acid–containing salt crusts. We further demonstrate that pyrroles form oligopyrroles, including porphyrins, under experimental conditions simulating primordial volcanic islands. Based on these results, a prebiotic reaction sequence is proposed whose individual steps proceeded spontaneously in different environments of volcanic islands. This sequence led from volcanic gases to potential photopigments and electron transfer molecules.

2. Materials and Methods

2.1. Chemicals and elemental analyses

Racemic isovaline was prepared as described before (Strasdeit et al., 2001). All other chemicals were purchased commercially and used without further purification. The ¹³C-substituted DL-alanines were obtained from Sigma-Aldrich (1-¹³C) and Eurisotop (2-¹³C and 3-¹³C), respectively. Biliverdin hydrochloride: 95% (Chemos GmbH); 2,4-diethylpyrrole: 98% (Frontier Scientific); formaldehyde: 16% aqueous solution, methanol free, Ultra Pure (Polysciences); hemin (ferriprotoporphyrin chloride): ≥98% (Roth); octaethylporphyrin: 97% (Aldrich). All metal salts used were of analytical grade. The water used throughout this study was distilled twice in a quartz apparatus.

CHN analyses were obtained from Mikroanalytisches Labor Pascher, Remagen, Germany. Cl analyses were performed by Mikroanalytisches Labor Pascher (compounds 2 and 3) and in our laboratory (compound 1 and thermolysis residue). Ca and Mg contents were determined by standard complexometric titration in our laboratory. The crystal water contents of 2 and 3 were measured thermogravimetrically on a Linseis L81-II thermal balance.

2.2. Gas chromatography–mass spectrometry

An Agilent 6890N/5973 GC/MSD system equipped with a DB-5MS capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness) was used. Measurement conditions: column temperature 50–200°C, helium carrier gas, inlet pressure 0.874 bar, inlet temperature 280°C, MSD operated in full-scan mode (15–350 amu, 2.36 scans s⁻¹, electron impact ionization at 70 eV, 2.5 min solvent delay). For quantification purposes, a flame ionization detector at 250°C was used instead of the MSD.

2.3. Spectroscopic measurements

The following instruments and measuring conditions were used. UV-visible absorption spectroscopy: Analytik Jena SPECORD 210 spectrometer, resolution 0.5 nm, scan speed 1 nm s⁻¹; the solutions were measured in a gas-tight quartz cuvette. IR spectroscopy: Thermo Nicolet 5700 FT-IR spectrometer, attenuated total reflection mode. Raman microscopy: Horiba Jobin Yvon LabRAM spectrometer, λ _excitation=633 nm. Proton nuclear magnetic resonance (¹H NMR) spectroscopy: Varian Unity Inova 300 spectrometer, 300 MHz.

2.4. High-resolution/high-accuracy mass spectrometry

The spectra were obtained with a Bruker LTQ Orbitrap XL mass spectrometer (ThermoScientific, Bremen, Germany) equipped with an electrospray ionization (ESI) ion source. An external calibration was performed according to the manufacturer's guidelines (calibration mixture: SDS, caffeine, sodium taurocholate, MRFA, and Ultramark 1621). The mass spectrometer was operated in positive ion mode with an ionization voltage of 4.5 kV. The capillary temperature was 275°C. N₂ served as sheath-gas (2 mL min⁻¹). The data were recorded in the 100–2000 Da mass range by the Orbitrap mass analyzer, which was operated with a target mass resolution of 30,000 (defined at m/z 400). Samples were introduced by direct infusion at a flow rate of 10 μL min⁻¹. The data were processed with Xcalibur software (version 2.0, ThermoScientific, Bremen, Germany).

2.5. X-ray crystallography

A suitable crystal of (CaCl₂)₃(Hala)₂·6H₂O (1) was grown by slow evaporation of a 1:1:1 MgCl₂–CaCl₂–alanine solution (Hala=DL-alanine). Single-crystal data were obtained on a Stoe IPDS diffractometer. Crystal data for compound 1: C₆H₂₆Ca₃Cl₆N₂O₁₀, M _r=619.23, crystal size 0.71×0.43×0.18 mm³, monoclinic, P2₁/n, a=8.9367(4), b=16.5768(9), c=9.0279(4) Å, β=108.335(5)°, V=1269.51(11) Å³, Z=2, ρ _calcd=1.620 g cm⁻³, μ(Mo_Kα)=1.321 mm⁻¹, λ=0.71073 Å, T=153(2) K, 2Θ _max=52.18°, 13,570 reflections collected, 2349 independent reflections used in refinement, R _int=0.0420, numerical absorption correction, structure solution by direct methods, refinement on F ², 176 parameters, R ₁[I>2σ(I)]=0.0176, wR ₂(all data)=0.0420, residual electron density+0.313/−0.207 e Å⁻³; programs used: SHELXS-97, SHELXL-97 (Sheldrick, 2008), DIAMOND (Crystal Impact, 2005). Complete crystallographic data of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number 656775).

Powder diffraction patterns were measured with Cu_Kα radiation with the use of a Bruker D8 Focus diffractometer equipped with a Sol-X energy dispersive detector.

2.6. Preparation of the salt–amino acid mixtures

NaCl (41.20 g, 705 mmol), KCl (1.12 g, 15 mmol), MgCl₂·6H₂O (16.26 g, 80 mmol), CaCl₂·2H₂O (2.21 g, 15 mmol), and the respective α-amino acid (10 mmol) were dissolved in water. The solution was evaporated at room temperature. The resulting salt crust was crushed, and adhering water was removed under vacuum. When amino acid mixtures were studied, between 1 and 6 mmol of each individual amino acid was employed.

2.7. Synthesis of the amino acid complexes

Preparation of (CaCl₂)₃(Hala)₂·6H₂O (1): CaCl₂·2H₂O (13.23 g, 90 mmol) and DL-alanine (5.35 g, 60 mmol) were dissolved in a sufficient amount of water. About half the water was then removed by rotary evaporation, and the solution was transferred into a crystallizing dish. After standing for a few days in the air (or in a vacuum desiccator over silica gel when the atmospheric humidity was too high), the solution dried. The remaining white crystalline solid of 1 was isolated and dried under vacuum for some minutes to remove adhering water. The product had the same IR spectrum as crystals that had been grown from only partly evaporated solutions. The X-ray powder diffraction pattern corresponded to the calculated one. 1 was found to be slightly hygroscopic and was therefore stored in tightly closed containers.

The compounds CaCl₂(Hala)₂·3H₂O (2) and MgCl₂(Hala)₂·4H₂O (3) were prepared from 50 mmol of CaCl₂·2H₂O and MgCl₂·6H₂O, respectively, and 100 mmol of DL-alanine. The procedure described for the preparation of compound 1 was used.

1: Yield: 17.9 g (96%). Elemental analysis (%) calculated for C₆H₂₆Ca₃Cl₆N₂O₁₀ (619.23): C 11.64, H 4.23, Ca 19.42, Cl 34.35, N 4.52; found: C 11.83, H 4.35, Ca 19.44, Cl 34.64, N 4.46.

2: Yield: 16.8 g (98%). Elemental analysis (%) calculated for C₆H₂₀CaCl₂N₂O₇ (343.22): C 21.00, H 5.87, Ca 11.68, Cl 20.66, N 8.16, H₂O 15.75; found: C 20.48, H 6.17, Ca 11.20, Cl 19.9, N 8.17, H₂O 16.1.

3: Yield: 16.4 g (95%). Elemental analysis (%) calculated for C₆H₂₂Cl₂MgN₂O₈ (345.46): C 20.86, H 6.42, Cl 20.53, Mg 7.04, N 8.11, H₂O 20.86; found: C 20.34, H 6.43, Cl 20.0, Mg 7.20, N 8.09, H₂O 20.5.

Infrared and Raman band positions of 1, 2, and 3 are given in Figs. 1 and 2.

FIG. 1.

Infrared spectra: (a) salt crust prepared by evaporating at room temperature an aqueous solution of NaCl (705 mmol), MgCl₂ (80 mmol), KCl (15 mmol), CaCl₂ (15 mmol), and DL-alanine (10 mmol); (b) (CaCl₂)₃(Hala)₂·6H₂O (1); (c) CaCl₂(Hala)₂·3H₂O (2); (d) MgCl₂(Hala)₂·4H₂O (3); (e) DL-alanine. Color images available online at www.liebertonline.com/ast.

FIG. 2.

Raman spectra: (a) salt crust prepared by evaporating at room temperature an aqueous solution of NaCl (705 mmol), MgCl₂ (80 mmol), KCl (15 mmol), CaCl₂ (15 mmol), and DL-alanine (10 mmol); (b) (CaCl₂)₃(Hala)₂·6H₂O (1); (c) CaCl₂(Hala)₂·3H₂O (2); (d) MgCl₂(Hala)₂·4H₂O (3); (e) DL-alanine. Color images available online at www.liebertonline.com/ast.

2.8. Thermolyses

The thermolysis apparatus used is shown in Fig. 3. Typically, 55 g of a salt–amino acid mixture, 10 g of a metal complex, or 1 g of ¹³C-substituted 1 were weighted into a quartz container. The container was placed into the quartz tube, which was 120 cm long and had a 4.0 cm inner diameter. Nitrogen (99.999% purity) was used as a protective gas and a carrier medium for the volatile products. After purging the apparatus for ∼48 h, the nitrogen stream was adjusted so that its velocity was 10 cm min⁻¹ in the quartz tube. The furnace was heated to the desired temperature. Then the sample was pushed into the heating zone. The thermolyses were conducted for 2 h. After this time, the formation of volatile products had ceased. Subsequently, the volatile product fractions that had condensed at T1–T3 (see Fig. 3) were separately dissolved in methanol or dichloromethane and immediately analyzed by gas chromatography–mass spectrometry (GC-MS). ¹H NMR spectra of thermolysis residues were measured in D₂O.

FIG. 3.

Two views of the thermolysis apparatus, which consists of a furnace (F), quartz tube (Q), glass rod (G) for placing the sample container (SC) into the heating zone (H), sample storage area (SA), Teflon-sealed connections (C), water-cooled (T1), and liquid nitrogen–cooled traps (T2, T3). Color images available online at www.liebertonline.com/ast.

To analyze for ammonia and carbon dioxide, the gas stream was split at the outlet and passed through hydrochloric acid and barium hydroxide solutions where NH₄Cl and BaCO₃, respectively, formed. The products were identified by IR spectroscopy. In these experiments, the traps T2 and T3 were cooled with ice.

The experimental setup of Fig. 3, with small variations, was also used to study the thermal stability of biliverdin hydrochloride, hemin, and octaethylporphyrin (OEP). An accurately weighed sample of the tetrapyrrole (1 mg) was held at a fixed temperature in a slow nitrogen stream for up to 24 h. Subsequently, the residue was extracted with methanol, N,N-dimethylformamide, and dichloromethane, respectively. The amount of undecomposed tetrapyrrole in the extract was determined spectrophotometrically. The following extinction coefficients were measured and used in the calculations: biliverdin hydrochloride, ɛ=50,200 L mol⁻¹ cm⁻¹ (376.5 nm, methanol); hemin, ɛ=75,300 L mol⁻¹ cm⁻¹ (399 nm, N,N-dimethylformamide); OEP, ɛ=177,200 L mol⁻¹ cm⁻¹ (398 nm, dichloromethane) [literature value: 176,000 L mol⁻¹ cm⁻¹ at 397 nm (Kinoshita et al., 1992)].

2.9. Synthesis of alkylpyrroles for identification purposes

General procedure: pyrrole or an alkylpyrrole (ca. 0.5 g) was dissolved in anhydrous tetrahydrofuran (10 mL) under an argon atmosphere. The solution was cooled to −20°C, and one equivalent of n-butyllithium was added dropwise. After the solution had warmed up to room temperature, it was again cooled to −20°C, and one equivalent of ethyl or methyl iodide was added. After stirring overnight at room temperature, a saturated solution of ammonium chloride was added. The product (mixture) was extracted twice with diethyl ether (10 mL each) and subjected to column chromatography (silica gel; hexane–ethyl acetate, 9:1, v/v). The absence or low intensity of ¹H NMR signals in the 3.1–4.0 ppm range (NCH ₃ and NCH ₂CH₃; CDCl₃ as solvent) indicated that N-alkylated products were neglectable.

By using the above procedure, pyrrole and several commercially available alkylpyrroles were C-alkylated. In some cases, only one product was possible, for example in the C-alkylation of 3-ethyl-2,4-dimethylpyrrole. Mostly, however, mixtures were obtained that often contained positional isomers. They were nevertheless useful in identifying the products of the thermolysis experiments. Positional isomers of alkylpyrroles exhibit virtually identical mass spectra. However, the GC retention times can be used to distinguish them. A straightforward example is shown in Fig. 4. When 2,4-dimethylpyrrole was monomethylated, both trimethyl isomers were formed. In contrast, the monomethylation of 2,5-dimethylpyrrole can only yield 2,3,5-trimethylpyrrole (t=9.8 min). The peak at t=10.5 min, which shows the same mass spectrum, must therefore belong to the 2,3,4-isomer. Thus, it was possible to unambiguously assign the corresponding peaks in the GC of the thermolysis products. In addition, the complete C-methylation of both 2,4-dimethylpyrrole and 2,5-dimethylpyrrole yielded 2,3,4,5-tetramethylpyrrole (t=13.3 min).

FIG. 4.

Gas chromatograms (total ion detection) of the products obtained by methylation of (a) 2,4-dimethylpyrrole and (b) 2,5-dimethylpyrrole with methyl iodide. Asterisks indicate impurities.

2.10. Estimation of the alkylpyrrole yield

A thermolysis experiment with 10.0 g of (CaCl₂)₃(Hala)₂·6H₂O (1) was performed at 350°C as described above. Three hundred forty milligrams of volatile products condensed at T1 (see Fig. 3). They were dissolved in anhydrous methanol in a 10.00 mL volumetric flask. Subtraction of the amount of water, which was determined by Karl Fischer titration, gave a total of 116 mg of organic products. The methanolic solution was analyzed by GC coupled with flame ionization detection. Addition of known amounts of 2,5-dimethylpyrrole allowed the quantification of the alkylpyrrole content, which was determined as 15.5 mg. Assuming a mean molecular mass of 123.20 g mol⁻¹, which corresponds to an ethyldimethylpyrrole, 0.126 mmol of alkylpyrroles had been formed. Based on the reaction mechanism (see below), it was further assumed that four alanine molecules are necessary to form one ethyldimethylpyrrole molecule. Ten grams of 1 contain 32.3 mmol of alanine. The percentage yield is therefore estimated at (4×0.126/32.3)×100%=1.6%. The true yield is almost certainly higher because, for technical reasons, losses were unavoidable during collecting the pyrrole-containing product fraction. Furthermore, additional 3-methylpyrrole was identified in the cool traps T2 and T3 but was not included in the yield calculation. No attempts were made to optimize the yield.

2.11. Oligopyrrole formation

A typical experiment was performed as follows. A solution of NaCl (705 mM), MgCl₂ (80 mM), CaCl₂ (15 mM), KCl (15 mM), and HCl (10 mM) was prepared. NaNO₂ (8.3 mg, 0.12 mmol) was dissolved in a 240 mL portion of this stock solution. The resulting solution was thoroughly degassed and transferred into a glove box filled with pure argon gas. Then we added 3,4-diethylpyrrole (16.5 μL, 0.12 mmol) and a 16% aqueous solution of formaldehyde (21.6 μL, 0.12 mmol HCHO). The reaction mixture was briefly shaken. After standing undisturbed for 5 days at 30°C, the mixture was analyzed by UV-visible spectroscopy.

For the ESI-Orbitrap MS measurements, it was necessary to conduct the above-described experiment without metal chlorides. The absence of these salts only slightly changed the electronic absorption spectrum, with the differences being mainly in the relative intensities of the absorption maxima. Immediately before measuring the mass spectra, we added acetonitrile to the reaction mixtures. The electronic spectra of the solutions with and without acetonitrile were virtually identical except for some minor shifts in band positions.

The total yields of the oligomers of 3,4-diethylpyrrole and 3-ethyl-2,4-dimethylpyrrole were estimated at 1–2% from the visible region of the absorption spectra. The relatively high molar extinction coefficients (in dichloromethane) of OEP (Kinoshita et al., 1992) and protonated 4,4'-diethyl-3,3',5,5'-tetramethyl-2,2'-dipyrromethene (Semeikin et al., 2003), respectively, were assumed for all products. This method inevitably underestimated the concentrations of the less-oxidized products, which have lower extinction coefficients or no absorption at all in the visible region. Consequently, the actual yields were probably higher.

2.12. Reaction between a volcanic rock and hydrochloric acid

We used unweathered picrobasalt sand that was rich in olivine [(Mg,Fe)₂SiO₄]. It had been collected near the coast at the Piton de la Fournaise volcano on the island of La Réunion. Five grams of the picrobasalt was added to 50.0 mL of hydrochloric acid of pH 2.0. The mixture was stirred in a closed measuring cell. The pH was monitored with a pH electrode. After 6 days, dissolved Mg²⁺ and Fe²⁺ ions were identified in the supernatant by precipitation of characteristic MgNH₄PO₄·6H₂O (struvite) crystals and Turnbull's blue, respectively.

3. Results

3.1. Amino acids in artificial sea salt crusts

To simulate the formation of primordial amino acid–containing sea salt crusts, we prepared aqueous solutions of NaCl, MgCl₂, KCl, CaCl₂, and an α-amino acid. At least ∼5 mmol of the amino acid was necessary for an unambiguous interpretation of the vibrational spectra and X-ray diffractograms. In most experiments, including the thermolyses, 10 mmol was employed, resulting in an amino acid content in the 1–2% range. Glycine, DL-alanine, DL-valine, DL-glutamic acid, α-aminoisobutyric acid, and DL-isovaline (2-amino-2-methylbutanoic acid) were used. In principle, the solids that remained after evaporation to dryness could have contained amino acid crystals; or, alternatively, the amino acids could have reacted with sea salt components. Raman and IR spectroscopy and X-ray powder diffraction clearly showed that amino acid crystals were absent. For alanine, for example, this can be easily seen by comparing the spectra a and e in Figs. 1 and 2. In fact, new compounds that contained magnesium chloride or calcium chloride had been formed. Glycine (Hgly), alanine (Hala), and α-aminoisobutyric acid (Haib), for example, were present as CaCl₂(Hgly)·H₂O (Yusenko et al., 2008), (CaCl₂)₃(Hala)₂·6H₂O (1), and MgCl₂(Haib)·6H₂O, respectively. To determine the binding form of the amino acids in the salt crusts, we used neat calcium and magnesium complexes for comparison. Most of these complexes were synthesized for the first time. This approach is exemplified for alanine in Figs. 1 and 2, which show that the vibrational spectra of the alanine-containing salt crust (spectrum a) and compound 1 (spectrum b) are virtually identical. Thus, alanine has selectively combined with calcium chloride from the salt mixture to form compound 1.

(CaCl₂)₃(Hala)₂·6H₂O (1) has been found to crystallize at roughly neutral pH and high CaCl₂-to-alanine molar ratios (between 1.5:1 and 10:1). In addition to 1, two other compounds have been isolated from aqueous solutions of Ca²⁺ and alanine. Also around pH 7 but at lower molar ratios (between 0.5:1 and 1:1), CaCl₂(Hala)₂·3H₂O (2) has been obtained. Near pH 12, the alaninate Ca(ala)₂·3H₂O formed (Fox et al., 2007). The latter pH value, however, is hardly prebiotically relevant and certainly not relevant to the neutral conditions under which our sea salt crusts were prepared. Interestingly, MgCl₂ strongly favored the formation of 1. For example, after addition of only 0.1 equivalents of this salt to a neutral 1:1 CaCl₂–alanine solution, compound 1 was isolated instead of 2, despite the low calcium-to-alanine ratio in the solution. Actually, the single crystal used in the crystal structure determination of 1 has been grown from an equimolar solution of MgCl₂, CaCl₂, and alanine. Even at much larger excesses of MgCl₂ over CaCl₂, no other calcium or magnesium compound crystallized. We also succeeded in preparing the magnesium compound MgCl₂(Hala)₂·4H₂O (3), which has the same metal-to-alanine ratio as 2. It crystallized in the absence of CaCl₂. In contrast to 1, compounds 2 and 3 have not been detected in the alanine-containing salt crusts (see spectra a, c, and d in Figs. 1 and 2).

From the composition and spectral properties of 1, it was possible to conclude that this compound is an “adduct” of alanine. The definition of a metal “complex,” however, requires that the alanine molecule acts as a “ligand,” that is, it must be directly metal bonded. The X-ray crystal structure analysis showed this to be the case. Crystalline 1 forms an infinite one-dimensional complex in which each alanine molecule is firmly bonded to three calcium ions (Fig. 5). This interaction seems to be crucial for the thermal behavior of alanine in sea salt crusts, which is strikingly different from that of the neat amino acid.

FIG. 5.

Crystal structure of (CaCl₂)₃(Hala)₂·6H₂O (1). A section of the infinite one-dimensional coordination polymer is shown with a Ca₃(μ ₃-Hala) moiety highlighted. The hydrogen atoms of the amino acid ligands have been omitted for clarity. Selected bond lengths (Å): Ca1–O1 2.3581(9), Ca2–O1 2.4870(9), Ca2–O2 2.7174(9), Ca2′–O2 2.3776(9).

3.2. Formation of pyrroles from salt-embedded amino acids

The amino acid–containing salt crusts described above were subjected to thermolyses at 350°C. A slow stream of nitrogen gas was used to simulate the non-oxidizing atmosphere and local atmospheric movements on a primordial volcanic coast. The volatile product fraction that condensed near room temperature was analyzed by GC-MS. It was found that the amino acids had formed several methylated and ethylated 1H-pyrroles. The only exceptions were glycine (no pyrroles) and glutamic acid (only 2-methylpyrrole detected). Salt-embedded amino acid mixtures [e.g., glycine (3 mmol), DL-alanine (2 mmol), DL-valine, DL-glutamic acid, α-aminoisobutyric acid, and DL-isovaline (1 mmol each)] similarly generated various alkylpyrroles. An example [glycine (6 mmol) and DL-alanine (4 mmol)] is shown in Fig. 6. The mass spectra reveal that the pyrroles were exclusively C-alkylated. In addition to pyrroles, a few alkylpyridines and alkylindoles were detected, but in much lower concentrations.

FIG. 6.

Gas chromatogram (total ion detection) of volatile products obtained by thermolysis of a glycine–DL-alanine mixture embedded in artificial sea salt. Composition of the salt crust (mmol): NaCl 705, MgCl₂ 80, KCl 15, CaCl₂ 15, glycine 6, DL-alanine 4. Thermolysis conditions: 350°C, N₂ atmosphere. In the following, abbreviations are used for the names of the pyrroles, e.g., 3-Et-2,4-Me₂ stands for 3-ethyl-2,4-dimethylpyrrole. Peak 1 : 2-Me; 2 : 3-Me; 3 : 2,5-Me₂; 4 : 2,4-Me₂; 5 : 2,3-Me₂; 6 : 3,4-Me₂; 7 , 8 : EtMe; 9 : 2,3,5-Me₃; 10 : 2,3,4-Me₃; 11 : 2-Et-3,5-Me₂; 12 , 13 , 16 : EtMe₂; 14 : 3-Et-2,4-Me₂; 15 : 2,3,4,5-Me₄; 17 , 18 , 21 : Et₂Me; 19 : 2-Et-3,4,5-Me₃; 20 : 3-Et-2,4,5-Me₃; 22 , 24 , 25 : Et₂Me₂; 23 : 2,4-Et₂-3,5-Me₂; 26 : Et₃Me.

In our study of alkylpyrrole formation, we focused on alanine, which is thought to be one of the prebiotically most relevant α-amino acids (Zaia et al., 2008). Under standard thermolysis conditions (350°C, N₂), neat alanine did not produce pyrroles. Instead, the amino acid mostly sublimed and partly formed the cyclic dipeptide (3,6-dimethylpiperazine-2,5-dione). In a mixture with NaCl (705 mmol) and KCl (15 mmol), obtained by evaporation, alanine (10 mmol) behaved in the same way. In agreement with this, vibrational spectra reveal that the mixture simply contained crystals of the pure amino acid. The same observations were made with α-aminoisobutyric acid, except that here the sublimation occurred without decomposition. These results showed that the alkylpyrrole formation was not caused by a physical effect of the salt crystals but by chemical interaction between the amino acids and MgCl₂ or CaCl₂ (see above).

In the artificial sea salt mixture, (CaCl₂)₃(Hala)₂·6H₂O (1) represented the only binding form of alanine. Thus, it was not surprising that neat 1 yielded almost the same set of pyrroles as the alanine-containing salt crusts. Since 1 was solely responsible for the formation of alkylpyrroles, it seemed appropriate to use this compound as a model for further studying the thermal behavior of the salt crusts. Alkylpyrrole formation from 1 was observed over a wide temperature range of 300°C to 800°C but mainly occurred between 350°C and 550°C. Temperatures such as these are frequently encountered in the vicinity of molten lava (see Discussion). The use of an N₂–CO₂ atmosphere (80:20, v/v; either dry or saturated with water vapor) instead of pure N₂ did not change the outcome of the thermolyses. Figure 7 shows a typical gas chromatogram (GC) of a volatile product fraction obtained from a thermolysis experiment at 350°C. The GC is characterized by a pronounced dominance of methylated and ethylated pyrroles. Thirty peaks could be unambiguously assigned to pyrroles by MS. In addition, several individual isomers were identified from their retention times by comparison with authentic samples. The latter were either commercially available or prepared by alkylating appropriate lithium pyrrolates with methyl or ethyl iodide. As with the alanine-containing salt crusts, no N-alkylated pyrroles were detected among the thermolysis products. On the other hand, all 14 possible Me_xEt_y combinations of C-alkylation (x+y=1, 2, 3, or 4) and almost 70% of their possible positional isomers were found. Thus, one can speak of a “random synthesis” that produces a library of C-methylated and C-ethylated pyrroles.

FIG. 7.

Gas chromatogram (total ion detection) of volatile products obtained by thermolysis of (CaCl₂)₃(Hala)₂·6H₂O (1). Thermolysis conditions: 350°C, N₂ atmosphere. Peak 1 : 2-Me; 2 : 3-Me; 3 : 2,5-Me₂; 4 : 2-Et; 5 : 2,4-Me₂; 6 : 2,3-Me₂; 7 , 8 : EtMe; 9 : 2,3,5-Me₃; 10 : 2,3,4-Me₃; 11 : 2-Et-3,5-Me₂; 12 : Et₂; 13 , 14 , 17 : EtMe₂; 15 : 3-Et-2,4-Me₂; 16 : 2,3,4,5-Me₄; 18 , 19 , 22 : Et₂Me; 20 : 2-Et-3,4,5-Me₃; 21 : 3-Et-2,4,5-Me₃; 23 : 2,3,5-Et₃; 24 , 26 , 27 : Et₂Me₂; 25 : 2,4-Et₂-3,5-Me₂; 28 , 29 : Et₃Me; 30: 2,3,4,5-Et₄. See the caption to Fig. 6 for the abbreviations of the pyrrole names.

Among the volatile products, a few mono-, di-, and trimethylpyridines were present in low concentrations as well as carbon dioxide and ammonia. Elemental analyses (Table 1) and ¹H NMR spectroscopy showed that the nonvolatile residue consisted almost entirely of calcium chloride and the cyclic alanine dipeptide. Under the reasonable assumption that calcium chloride remained completely in the residue, one can easily calculate from the values in Table 1 that at least 45% of the alanine molecules had been transformed into the cyclic dipeptide.

Table 1.

Elemental Analysis of the Nonvolatile Residue Obtained from the Thermolysis of (CaCl₂)₃(Hala)₂·6H₂O (1) at 350°C

Element	Found (%)	Calculated (%)
Ca	29.5	29.97
Cl	53.4	53.03
C	8.32	8.11
H	1.23	1.24
N	2.88	3.15

The values in column 3 were calculated for a mixture of 83% calcium chloride, 16% cyclic alanine dipeptide, and 1% water.

To elucidate the mechanism of pyrrole formation, we thermolized three isotopomers of compound 1 that were prepared from (1-¹³C)-, (2-¹³C)-, and (3-¹³C)alanine, respectively. After separation by GC, the mass spectra of the thermolysis products were analyzed for the number of ¹³C atoms incorporated. By comparing the data for different alkylpyrroles, it was possible to determine which carbon atom of the alanine contributed to which site in the alkylpyrroles. In the following, the line of reasoning is illustrated for 3-ethyl-2,4-dimethylpyrrole (kryptopyrrole). The three major MS peaks of this molecule correspond to the molecular ion and two ions that were formed by loss of one and two methyl groups. It can be seen from the first row of Table 2 that one C1 atom of the alanine molecule (M+1), three C2 atoms (M+3), and four C3 atoms (M+4) were incorporated into the 3-ethyl-2,4-dimethylpyrrole molecule. Only in the case of (3-¹³C)-substituted alanine, ¹³C-containing methyl groups were lost from the pyrrole [(M+4)→(M'+3)→(M”+2)]. Thus, the methyl groups of the pyrrole must have originated from the methyl group of alanine. Together with the results for other alkylpyrroles, the picture shown in Fig. 8 emerged. The main points are as follows: (i) The five-ring contained one C1, two C2, and one C3 atom of the alanine. (ii) Methyl carbon atoms were C3 atoms. (iii) Methylene carbon atoms originated from C2 atoms, that is, ethyl groups were (C2)H₂–(C3)H₃. These findings held for all alkylpyrroles whose mass spectra could be analyzed. The ring atom assignment in Fig. 8 is the one that follows from the formation mechanism (see below). For 3-ethyl-2,4-dimethylpyrrole, as for most other alkylpyrroles, two alternative connectivities of the alkyl groups to the ring C atoms are conceivable (see Fig. 8). However, experimentally it was not possible to distinguish between these alternatives.

FIG. 8.

Distribution of the alanine carbon atoms in the 3-ethyl-2,4-dimethylpyrrole molecule. Color images available online at www.liebertonline.com/ast.

Table 2.

m/z Values of the Three Major Ions of 3-Ethyl-2,4-Dimethylpyrrole Isotopomers Obtained from Compound 1 with Differently ¹³ C-Substituted Alanine

	Alanine isotopomer
Unsubstituted	1-¹³C	2-¹³C	3-¹³C
123 (M)	124 (M+1)	126 (M+3)	127 (M+4)
108 (M′)	109 (M′+1)	111 (M′+3)	111 (M′+3)
93 (M′′)	94 (M′′+1)	96 (M′′+3)	95 (M′′+2)

First row: molecular ion; second row: molecular ion minus one methyl group; third row: molecular ion minus two methyl groups.

3.3. Oligopyrrole formation under simulated volcanic island conditions

We investigated the possibility that pyrroles oligomerized at cooler locations on primordial volcanic islands, for example, in rock pools. Our simulated rock pool contents consisted of acidic artificial seawater (10 mM HCl≙pH 2) to which an alkylpyrrole, formaldehyde (HCHO), and an oxidant had been added. Three alkylpyrroles displaying different degrees of substitution were selected for the experiments, namely, 3-methylpyrrole, 3,4-diethylpyrrole, and 3-ethyl-2,4-dimethylpyrrole. Nitrite ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_2^ -$$ \end{document} ) and nitrate ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_3^ -$$ \end{document} ) were used as oxidants. We conducted our experiments at 30°C under strict exclusion of oxygen [c(O₂)≤5 ppm]. To avoid prebiotically implausible concentrations, the reaction mixtures were relatively dilute, containing typically 0.5 mM of alkylpyrrole, HCHO, and oxidant each.

Each of the three model pyrroles gave deeply colored products with HCHO and either or both of the oxidants \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_2^ -$$ \end{document} and \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_3^ -$$ \end{document} . Electronic absorption spectra indicate the formation of oxidized oligopyrroles. In the following, we focus on the reaction system 3,4-diethylpyrrole/HCHO/nitrite, which produced wine-red product mixtures. A visible absorption spectrum of such a mixture is shown in Fig. 9. Oxidant-free solutions of 3,4-diethylpyrrole and HCHO were only faintly colored. Addition of nitrate to these solutions caused no color change. This observation demonstrated that, in contrast to nitrite, nitrate was ineffective in oxidizing the initial condensation products. Oligopyrrole formation in the system 3,4-diethylpyrrole/HCHO/nitrite also occurred in the absence of metal chlorides. Thus, sea salt was required for neither the condensation step nor the subsequent oxidation.

FIG. 9.

Visible absorption spectrum of the products of the reaction system 3,4-diethylpyrrole/HCHO/NaNO₂ (dichloromethane extract of a metal chloride–free reaction mixture).

The electronic absorption spectrum of our test reaction system indicates that several products had been formed, among them the porphyrin (Soret band at 398 nm, Fig. 9). Further examination by ESI-Orbitrap MS revealed the presence of 38 oligopyrroles ranging from dimers to hexamers (Figs. 10 and 11). The elemental compositions of these molecules were calculated from the accurate masses (Table 3). The observed product diversity can, in part, be attributed to different degrees of double bond conjugation (i.e., oxidation) for a given oligomer size. This is exemplified for two series of tetramers in Fig. 12. One series consists of cyclic molecules, including OEP and octaethylporphyrinogen, the other of open-chain ones. Adjacent members of each series differ by two hydrogen atoms. We found that the tetrameric molecules showed the highest signal intensities, with OEP dominating the mass spectra. The intensity sequence was as follows: OEP>other cyclic tetramers≈open-chain tetramers>trimers≈pentamers>dimers≈hexamers. For 3,4-diethylpyrrole and 3-ethyl-2,4-dimethylpyrrole, the oligopyrrole yield was estimated at 1–2% from electronic absorption spectra. Given the experimental procedure, this value must be regarded as the lower limit of the yield (see Materials and Methods).

FIG. 10.

Structural formulas of the molecules with entry numbers 1 to 20 in Table 3. The configurations at the exocyclic double bonds of the open-chain structures are unknown and have been arbitrarily assigned. If tautomeric forms are possible, only one is shown here.

FIG. 11.

Structural formulas of the molecules with entry numbers 21 to 38 in Table 3. See the caption to Fig. 10 for further details.

FIG. 12.

High-resolution/high-accuracy mass spectra of (A) cyclic and (B) open-chain tetrapyrroles from a 3,4-diethylpyrrole/HCHO/nitrite reaction mixture. The numbers on the peaks correspond to the entry numbers in Table 3. The corresponding chemical structures are given in Fig. 10. The m/z values refer to the [M+H]⁺ ions. Asterisks denote ¹³C satellites.

Table 3.

High-Resolution/High-Accuracy Mass Spectrometric Identification of Oligopyrroles from the Reaction System 3,4-Diethylpyrrole/Formaldehyde/Nitrite

Entry no.	Elemental composition	[M+H]⁺ experimental	[M+H]⁺ calculated	Δ (ppm)
1	C₁₈H₂₄N₂O	285.19625	285.19614	+0.4
2	C₁₈H₂₆N₂O	287.21192	287.21179	+0.5
3	C₁₈H₂₂N₂O₂	299.17605	299.17540	+2.2
4	C₁₈H₂₄N₂O₂	301.19109	301.19105	+0.1
5	C₁₈H₂₆N₂O₂	303.20711	303.20670	+1.4
6	C₂₇H₃₅N₃O	418.28540	418.28529	+0.3
7	C₂₇H₃₇N₃O	420.30122	420.30094	+0.7
8	C₂₇H₃₉N₃O	422.31686	422.31659	+0.6
9	C₂₇H₃₃N₃O₂	432.26486	432.26455	+0.7
10	C₂₇H₃₅N₃O₂	434.28046	434.28020	+0.6
11	C₂₇H₃₇N₃O₂	436.29629	436.29585	+1.0
12	C₂₇H₃₉N₃O₂	438.31155	438.31150	+0.1
13	C₃₆H₄₆N₄	535.37936	535.37952	−0.3
14	C₃₆H₄₈N₄	537.39528	537.39517	+0.2
15	C₃₆H₅₀N₄	539.41073	539.41082	−0.2
16	C₃₆H₅₂N₄	541.42700	541.42647	+1.0
17	C₃₆H₄₆N₄O	551.37444	551.37444	±0.0
18	C₃₆H₄₈N₄O	553.39038	553.39009	+0.5
19	C₃₆H₅₀N₄O	555.40581	555.40574	+0.1
20	C₃₆H₅₂N₄O	557.42096	557.42139	−0.8
21	C₃₆H₄₄N₄O₂	565.35402	565.35370	+0.6
22	C₃₆H₄₆N₄O₂	567.36951	567.36935	+0.3
23	C₃₆H₄₈N₄O₂	569.38574	569.38500	+1.3
24	C₃₆H₅₀N₄O₂	571.40051	571.40065	−0.2
25	C₃₆H₅₂N₄O₂	573.41614	573.41630	−0.3
26	C₄₅H₅₇N₅O	684.46462	684.46359	+1.5
27	C₄₅H₅₉N₅O	686.48042	686.47924	+1.7
28	C₄₅H₆₁N₅O	688.49463	688.49489	−0.4
29	C₄₅H₆₃N₅O	690.51052	690.51054	±0.0
30	C₄₅H₆₅N₅O	692.52679	692.52619	+0.9
31	C₄₅H₅₅N₅O₂	698.44019	698.44285	−3.8
32	C₄₅H₅₇N₅O₂	700.45927	700.45850	+1.1
33	C₄₅H₅₉N₅O₂	702.47418	702.47415	±0.0
34	C₄₅H₆₁N₅O₂	704.48914	704.48980	−0.9
35	C₄₅H₆₃N₅O₂	706.50581	706.50545	+0.5
36	C₄₅H₆₅N₅O₂	708.52184	708.52110	+1.0
37	C₅₄H₆₈N₆O	817.55243	817.55274	−0.4
38	C₅₄H₇₀N₆O	819.56800	819.56839	−0.5

M is the molecular mass, and Δ is the difference between the experimental and the calculated [M+H]⁺ values. The corresponding structural formulas are given in Figs. 10 and 11.

To gain an impression of the thermal stability of OEP, we performed experiments where this compound was exposed to different temperatures in a nitrogen atmosphere. The results showed that OEP was stable for at least 24 h at 200°C and for 30 min at 240°C. Even after 30 min at 350°C, ∼13% of the OEP was still intact. It had survived mostly in the thermolysis residue but also by sublimation. For comparison, we also studied the biomolecule derivatives hemin (ferriprotoporphyrin chloride) and biliverdin hydrochloride. They proved to be less thermally stable than OEP. In 24 h experiments, hemin began to decompose at ∼150°C and biliverdin hydrochloride already at ∼100°C.

3.4. Role of hydrochloric acid

During the thermolysis of sea salt, hydrogen chloride is formed by decomposition of magnesium chloride hydrates. This reaction has been observed at present-day volcanic coasts (Edmonds and Gerlach, 2006) as well as in our pyrrole formation experiments where the aqueous product fractions were strongly acidic (pH≤1). Thus, if pyrroles were produced from salt crusts at primordial volcanic coasts, they were necessarily accompanied by hydrogen chloride. Therefore, we included hydrochloric acid in our oligomerization experiments with pyrroles. In these experiments, hydrochloric acid in fact turned out to be essential for oligopyrrole formation in the model system 3,4-diethylpyrrole/HCHO/nitrite. The reaction, which was usually conducted with 10 mM HCl, still proceeded when the HCl concentration was reduced to 1 mM but no longer when the acid was omitted.

Present-day biological oligopyrroles must often form metal complexes to be able to perform their functions. Prominent examples are chlorophylls (Mg²⁺ complexes) and hemes (Fe^2+/3+ complexes). Experiments on the possible prebiotic formation of metal–oligopyrrole complexes were not part of this study. However, we investigated whether dissolved metal ions, which are necessary for complex formation, could have been available in a volcanic island environment. To this end, we exposed a basaltic rock rich in olivine [(Mg,Fe)₂SiO₄] to 10 mM hydrochloric acid (pH 2.0). The solution pH increased to 3.9 within 24 h. A practically constant value of pH 4.8 was reached after 6 days. The pH increase from 2.0 to 4.8 was equivalent to the neutralization of 99.8% of the initial amount of acid. Under the experimental conditions, chloride did not form insoluble salts. Therefore, the loss of protons from the solution must have been compensated for by the release of an equivalent amount of metal ions in order to maintain electroneutrality. Accordingly, Mg²⁺ and Fe²⁺ were identified in the solution. The natural pH of water standing over the basaltic rock was 9.0.

4. Discussion

In the early Archean, volcanic islands provided various environments with different geochemical and geophysical conditions. The close mutual proximity of these environments facilitated the chemical exchange between them. Thus, prebiotic organic molecules could have been transferred from certain environmental conditions to other very different ones. In the chemical laboratory, such changes of reaction conditions are usually prerequisite for multistep syntheses of complex organic molecules. Alternatively, three or more components can react simultaneously to form a single product with no need for different conditions. These so-called “multicomponent reactions,” however, have relatively limited potential, in contrast to stepwise syntheses. These experiences from synthetic organic chemistry probably also applied to abiotic chemical syntheses on early Earth. Two volcanic island environments formed the basis for our simulation experiments: (i) the coasts where the interaction between seawater and molten lava produced and heated sea salt crusts and (ii) rock pools not in immediate vicinity of lava flows.

We started by preparing artificial amino acid–containing seawater. The six α-amino acids employed were chosen because of their high relative abundances in carbonaceous chondrites (Zaia et al., 2008; Martins and Sephton, 2009) and in product mixtures obtained in electric discharge experiments (Johnson et al., 2008). Lightning has been estimated to have produced a steady-state amino acid concentration of 0.3 mM in the primitive ocean (Miller, 1987). This number might be too high because the bulk atmosphere was probably not sufficiently reducing. However, besides extraterrestrial delivery, lightning in volcanic ash-gas clouds must also be considered (Hill, 1992; Basiuk and Navarro-González, 1996; Navarro-González and Segura, 2004). This source may have efficiently supplied amino acids to the surface ocean water near active volcanic islands. The exact composition of the early Archean seawater is unknown. Today the six ions Na⁺, Mg²⁺, K⁺, Ca²⁺, Cl⁻, and \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm SO}_4^{2 -}$$ \end{document} account for 99% of the mass of sea salt (Lide, 2003). The prevailing view is that the \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm SO}_4^{2 -}$$ \end{document} concentration in the early ocean was much lower than it is today, perhaps≤0.2 mM (Holland, 2004). We therefore chose a salt mixture consisting of Na⁺, Mg²⁺, K⁺, and Ca²⁺ in their present-day molar ratio and Cl⁻ as the only anion. However, we note that delivery by carbonaceous chondrites might have increased the \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm SO}_4^{2 -}$$ \end{document} concentration in the early ocean (Izawa et al., 2010).

The salt crusts that remained after evaporation of the amino acid–containing artificial sea salt solutions did not contain free amino acids. Instead, metal–amino acid compounds had been formed, for example (CaCl₂)₃(Hala)₂·6H₂O (1). Hala represents the neutral, zwitterionic form of alanine. There are geochemical arguments that the pH of the ancient ocean was not far from neutral (Sleep, 2010), probably ∼6.8 at the end of the Hadean (Morse and Mackenzie, 1998). Our sea salt–alanine mixtures had pH 6.5. A species distribution diagram, which was calculated for a 0.15 M NaCl solution at 37°C by using the IUPAC stability constants database (Pettit and Powell, 2001; protonation constants: Maeda et al., 1990), showed that at slightly acidic pH DL-alanine exists almost exclusively as the neutral molecule Hala, which is also present in artificial sea salt crusts. Even under more acidic prebiotic conditions, which may have been caused by hydrogen chloride (see below) or atmospheric carbon dioxide (Kua and Bada, 2011), Hala would have been the dominating species down to pH ∼2.4. Magnesium ions, even at high concentrations, did not compete with calcium for alanine binding in the solid state. Furthermore, compound 1 crystallized over a wide range of solution ratios of CaCl₂ to alanine. Thus, the formation of 1 seems compatible with a large variety of calcium, magnesium, and alanine concentrations in early Earth's ocean.

Heating of amino acid–containing artificial sea salt crusts produced pyrroles when the amino acids were DL-alanine, DL-valine, α-aminoisobutyric acid, or DL-isovaline (either alone or in amino acid mixtures). The model compound 1 likewise yielded pyrroles on heating. These observations were unexpected since, to our knowledge, alkylpyrrole formation has not been reported to be a common reaction of α-amino acids, though the thermal behavior of amino acids has been studied in detail (e.g., Basiuk et al., 1998, and references therein). Only in the special cases of serine and a serine–threonine mixture pyrrole formation has been previously observed (Baltes and Bochmann, 1987; Yaylayan and Keyhani, 2001). Obviously, the sea salt matrix allowed a new reaction pathway. Besides electronic effects caused by the Lewis acidity of the metal ions, the metal coordination means that the amino acids can no longer escape high temperatures by sublimation. Interestingly, pyrroles were not only obtained from the calcium compound 1 but also from MgCl₂(Hala)₂·4H₂O (3). This observation is prebiotically relevant because it suggests that a hypothetical very low calcium concentration in the early ocean would not have hampered the pyrrole formation at volcanic coasts. The temperatures where in our experiments pyrrole formation mainly occurred (350–550°C) are frequently encountered near molten lava on volcanic islands. On Kilauea volcano, for example, comparable temperatures have been measured for solid crusts on lava flows (425–750°C) and the inner levee walls of lava channels (300–650°C) (Pinkerton et al., 2002).

Mass spectrometric results obtained by using ¹³C isotopomers of alanine (see above) enabled us to suggest a possible mechanism of pyrrole formation (Fig. 13). In this mechanism, first a ketone is formed from two calcium-coordinated alanine molecules. This step is analogous to the long-known thermal decomposition of simple calcium carboxylates such as the acetate (Krönig, 1924; Lee and Spinks, 1953). Next, the ketone becomes α,β-unsaturated and undergoes intramolecular ring closure. Finally, aromatization and alkylation follow. Details of the alkylation step are unknown. Consistent with this mechanism, carbon dioxide and ammonia appeared as thermolysis products. It is noteworthy that the formation of pyrroles from serine proceeds via a completely different pathway (Yaylayan and Keyhani, 2001). From Figs. 8 and 3 it can be deduced that each alkylpyrrole molecule required between two and six alanine molecules for its formation, depending on the degree of substitution.

FIG. 13.

Possible mechanism of formation of pyrroles from salt-embedded alanine. Color images available online at www.liebertonline.com/ast.

Knowing the origin of every carbon atom allowed us to estimate the yield of pyrroles at ∼2%. For comparison, the yield of the main organic product, the cyclic alanine dipeptide, is at least 45%. The relatively low percentage yield of pyrroles, however, translates into large masses in a volcanic island scenario. During a larger eruption of an island volcano, more than 10⁸ m³ of lava can enter the sea and evaporate the same volume of seawater (Edmonds and Gerlach, 2006). If the amino acid concentration in the early ocean was 0.3 mM (see above), 3·10⁷ mol of amino acids would have been affected. We conservatively assume that only 10 mol % of these amino acids could, in principle, have produced pyrroles. Taking into account our experimental result that 32.3 mol of alanine yielded 0.126 mol of alkylpyrroles, ∼10⁴ mol (≈10³ kg) of alkylpyrroles would have been formed. Even if the oceanic amino acid concentration was 2 orders of magnitude lower, that is, in the micromolar range, still several kilograms of pyrroles could have been formed in a single volcanic eruption. Thus, substantial amounts could have accumulated over longer periods of time. Another interesting aspect is that the high local temperatures must have caused a spatial separation between the salt residue and the volatile products. The latter included not only the pyrroles but also hydrogen chloride from salt decomposition and huge amounts of water. Today, the steam clouds that originate from lava–seawater interaction predominantly move inland due to thermal convection. Its has been reported that such clouds, together with eruption plumes, caused acidic rain of pH<2 (Staudacher et al., 2009).

Prebiotic volatiles almost certainly condensed in cooler regions and accumulated in depressions (rock pools), analogous to the processes on modern volcanic islands. Therefore, we have experimentally addressed the question of how pyrroles could have reacted in rock pools. Formaldehyde (HCHO), which is generally regarded as a prebiotic molecule (Navarro-González, 1992; Cleaves, 2008), was employed as an additional organic component. The prebiotic bulk oceanic concentration of HCHO probably did not exceed ∼1 mM (Cleaves, 2008). Consequently, we have limited the HCHO concentration to 0.5 mM and made no attempts to optimize the product yields by increasing the concentrations of the starting materials. It is remarkable that even at this high dilution the condensation of alkylpyrroles and HCHO proceeded, albeit to a rather limited extent. The initial products, for example the porphyrinogen 16 (Fig. 10), are unstable in the sense that oligopyrrole formation is easily reversible unless an oxidation step follows. We therefore added the potential oxidants nitrite ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_2^ -$$ \end{document} ) and nitrate ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\rm NO}_3^ -$$ \end{document} ) to the simulated rock pool contents. On early Earth, an efficient pathway for the formation of both ions probably existed, as shown by theoretical and experimental studies (Mancinelli and McKay, 1988; Summers and Khare, 2007). When nitrite was present in an acidic 3,4-diethylpyrrole/HCHO solution, an intense color developed, indicating the oxidative formation of larger conjugated double-bond systems. Mass spectrometric analyses confirmed that several fully and partially oxidized oligopyrroles had been formed (Figs. 10 –12, Table 3).

The presence of hydrochloric acid proved essential for the formation of conjugated oligopyrroles. In fact, HCl has been reported to catalyze the condensation of unsubstituted pyrrole with HCHO in aqueous solution (Sobral et al., 2003). There are good reasons for assuming that hydrochloric acid was catalytically or stoichiometrically involved in the oxidation step, too. For example, it is well known that acidic conditions enhance the oxidizing power of nitrite (Wiberg, 2001). However, due to the low concentrations, we were unable to identify the reduction product(s) of nitrite and thus cannot propose an equation for the overall reaction. In contrast to HCl, the metal chlorides of the artificial seawater were not essential for oligopyrrole formation. This observation is prebiotically interesting because rock pools that were not situated in or near the intertidal zone could have been provided with sea salt only by aerosols. Thus, water in these rock pools may have contained little or no sea salt. In this context, we note the suggestion by Monnard et al. (2002) that life may have originated in a freshwater environment (see also Deamer, 2004).

Our results indicate that the prebiotic synthesis of oligopyrroles from amino acids gave relatively low yields. However, fully conjugated oligopyrroles, especially the aromatically stabilized porphyrins, are remarkably resistant compared to other presumably prebiotic oligomers such as peptides and oligonucleotides. For example, alkylporphyrins of biological origin are found in sediments hundreds of millions of years old (Callot and Ocampo, 2000). Our experiments showed that OEP withstood 200°C for several hours. It partially survived much higher temperatures for some minutes, which is in part due to its ability to sublime. Consistent with our findings, it has been reported that OEP vaporized without decomposition at 610°C under flash pyrolysis conditions (Gelin et al., 1996). Hence, it can be expected that, at least locally, substantial steady-state concentrations of abiotically formed porphyrins built up. Moreover, early cell ancestors may have benefited even from small, “catalytic” amounts of oligopyrroles.

Porphyrins are not the only oligopyrroles that can act as strong ligands toward metal ions. For example, the dipyrrins, which are “half-porphyrins,” also have a rich coordination chemistry (Wood and Thompson, 2007). The dipyrrin structural motif occurred in several reaction products of our 3,4-diethylpyrrole/HCHO/NaNO₂ model system, with compound 1 (Fig. 10) being the simplest representative. Metal complexes of porphyrins and dipyrrin-like open-chain oligopyrroles could have performed similar functions in early cell ancestors, for example, as electron transfer molecules. However, the availability of suitable metal ions was a prerequisite for complex formation. As part of the present study, we have experimentally demonstrated that Fe²⁺ and Mg²⁺ ions were released when hydrochloric acid reacted with picrobasalt. Picrobasalt, also referred to as oceanite, is an igneous rock found on ocean volcanic islands. On primordial volcanic islands, where hydrochloric acid must have been abundant, this reaction may have provided dissolved metal ions for the formation of Fe²⁺- and Mg²⁺-oligopyrroles. Our results suggest that in primordial volcanic environments hydrochloric acid was involved in (i) oligopyrrole synthesis (see above), (ii) solubilization of metal ions, and (iii) pH modification/regulation in rock pools. Thus, it seems that HCl is a more important prebiotic molecule than previously recognized.

5. Summary and Conclusions

Taken together, our results support a prebiotic two-step scenario in which oligopyrroles were spontaneously formed from α-amino acids: (i) near coastal lava flows, amino acids were trapped in solid sea salt and subsequently thermolyzed to pyrroles; (ii) the pyrroles and hydrochloric acid, which also appeared as a thermolysis product, condensed in rock pools where they reacted with formaldehyde and nitrite to give conjugated oligopyrroles, including porphyrins and open-chain tetrapyrroles. Plausible prebiotic sources are known for all starting materials. Furthermore, the scenario refers to a specific geological setting that can still be observed today on volcanic islands. It is interesting to note that contemporary biosynthetic pathways to tetrapyrroles also start from α-amino acids (Beale, 1999; Voet and Voet, 2011).

There is no reason to assume that the substitution patterns of modern biological tetrapyrroles existed from the beginning. In fact, basic concepts of evolution suggest that these patterns evolved from simpler ones (Strasdeit and Fox, 2013). Consistent with this idea, the methyl–ethyl substitution in some of our alkylpyrroles is already reminiscent of the methyl–vinyl and methyl–2-carboxyethyl substituent pairs in modern heme b. It is conceivable that in protometabolic reaction networks, oligopyrroles were able to perform rudimentary functions even without possessing well-defined, uniform substitution patterns. Initially, early cell ancestors may have utilized spontaneously formed oligopyrroles in redox reactions and for UV protection. Later on, they developed their own cyclic-tetrapyrrole syntheses, perhaps by starting from α-aminonitriles (Ksander et al., 1987) or aminoketones and diketones (Lindsey et al., 2009, 2011; Soares et al., 2012; Taniguchi et al., 2012). It has been proposed that UV protection by porphyrins and related pigments preceded light harvesting (e.g., Larkum, 2006). Protection against short-wavelength UV radiation was certainly necessary if early cell ancestors evolved in the subaerial environment of volcanic islands. Prebiotic porphyrins with only simple alkyl substituents were less vulnerable to thermal damage than modern biological ones so that they could accumulate despite occasionally hot conditions.

Finally, we note that key environmental prerequisites for our prebiotic volcanic island scenario probably also existed on Noachian Mars, namely, volcanoes (e.g., Xiao et al., 2012), standing bodies of water (e.g., Head et al., 1999), and soluble metal chlorides (Osterloo et al., 2008; Glotch et al., 2010). As a consequence, the usefulness of porphyrins as biomarkers in future Mars missions should be reevaluated (Suo et al., 2007).

Footnotes

Acknowledgments

We thank P. Guni, B. Hillers, I. Klaiber, F. Leißing, S. Ringer, M. Wälz, and M. Wagner for technical assistance; D. Haase and W. Saak for the X-ray diffraction data of compound 1; and T. Staudacher for providing the picrobasalt sample.

Author Disclosure Statement

The authors state that no competing financial interests exist.

Abbreviations

¹H NMR, proton nuclear magnetic resonance; ESI, electrospray ionization; GC, gas chromatogram, gas chromatography; Haib, α-aminoisobutyric acid; Hala, DL-alanine; Hgly, glycine; MS, mass spectrometry; OEP, octaethylporphyrin.

References

Appel

P.W.U.

, Rollinson

H.R.

, Touret

J.L.R.

2001. Remnants of an early Archaean (>3.75 Ga) sea-floor, hydrothermal system in the Isua Greenstone Belt. Precambrian Res, 112:27–49.

Arndt

N.T.

, Nisbet

E.G.

2012. Processes on the young Earth and the habitats of early life. Annu Rev Earth Planet Sci, 40:521–549.

Baltes

, Bochmann

1987. Model reactions on roast aroma formation. III. Mass spectrometric identification of pyrroles from the reaction of serine and threonine with sucrose under the conditions of coffee roasting. Z Lebensm Unters Forsch, 184:478–484.

Barber

D.J.

1981. Matrix phyllosilicates and associated minerals in C2M carbonaceous chondrites. Geochim Cosmochim Acta, 45:945–970.

Basiuk

V.A.

, Navarro-González

1996. Possible role of volcanic ash-gas clouds in the Earth's prebiotic chemistry. Orig Life Evol Biosph, 26:173–194.

Basiuk

V.A.

, Navarro-González

, Basiuk

E.V.

1998. Pyrolysis of alanine and α-aminoisobutyric acid: identification of less-volatile products using gas chromatography/Fourier transform infrared spectroscopy/mass spectrometry. J Anal Appl Pyrolysis, 45:89–102.

Beale

S.I.

1999. Enzymes of chlorophyll biosynthesis. Photosynth Res, 60:43–73.

Brack

2005. From the origin of life on Earth to life in the Universe. Lectures in Astrobiology, 1

Gargaud

, Barbier

, Martin

, Reisse

Springer: Berlin, 3–23.

Bridges

J.C.

, Banks

D.A.

, Smith

, Grady

M.M.

2004. Halite and stable chlorine isotopes in the Zag H3–6 breccia. Meteorit Planet Sci, 39:657–666.

10.

Buick

, Dunlop

J.S.R.

1990. Evaporitic sediments of early Archaean age from the Warrawoona Group, North Pole, Western Australia. Sedimentology, 37:247–277.

11.

Buick

, Thornett

J.R.

, McNaughton

N.J.

, Smith

J.B.

, Barley

M.E.

, Savage

1995. Record of emergent continental crust ∼3.5 billion years ago in the Pilbara craton of Australia. Nature, 375:574–577.

12.

Callot

H.J.

, Ocampo

2000. Geochemistry of porphyrins. The Porphyrin Handbook, 1

Kadish

K.M.

, Smith

K.M.

, Guilard

Academic Press: San Diego, CA, 349–398.

13.

Cleaves

H.J.

II . 2008. The prebiotic geochemistry of formaldehyde. Precambrian Res, 164:111–118.

14.

Cleaves

H.J.

, Chalmers

J.H.

, Lazcano

, Miller

S.L.

, Bada

J.L.

2008. A reassessment of prebiotic organic synthesis in neutral planetary atmospheres. Orig Life Evol Biosph, 38:105–115.

15.

Crystal Impact. 2005. DIAMOND—crystal and molecular structure visualization. Version 3.1, Crystal Impact. Bonn: Germany.

16.

Deamer

D.W.

2004. Prebiotic amphiphilic compounds: self-assembly and properties of early membrane structures. Origins: Genesis, Evolution and Diversity of Life. Seckbach

Kluwer: Dordrecht, 75–89.

17.

Edmonds

, Gerlach

T.M.

2006. The airborne lava–seawater interaction plume at Kīlauea Volcano, Hawai'i. Earth Planet Sci Lett, 244:83–96.

18.

Fox

, Büsching

, Barklage

, Strasdeit

2007. Coordination of biologically important α-amino acids to calcium(II) at high pH: insights from crystal structures of calcium α-aminocarboxylates. Inorg Chem, 46:818–824.

19.

Gelin

, Sinninghe Damsté

J.S.

, Harrison

W.N.

, Reiss

, Maxwell

J.R.

, De Leeuw

J.W.

1996. Variations in origin and composition of kerogen constituents as revealed by analytical pyrolysis of immature kerogens before and after desulphurization. Org Geochem, 24:705–714.

20.

Gendrin

, Mangold

, Bibring

J.-P.

, Langevin

, Gondet

, Poulet

, Bonello

, Quantin

, Mustard

, Arvidson

, LeMouélic

2005. Sulfates in martian layered terrains: the OMEGA/Mars Express view. Science, 307:1587–1591.

21.

Glotch

T.D.

, Bandfield

J.L.

, Tornabene

L.L.

, Jensen

H.B.

, Seelos

F.P.

2010. Distribution and formation of chlorides and phyllosilicates in Terra Sirenum, Mars. Geophys Res Lett, 37:L16202.

22.

Head

J.W.

III , Hiesinger

, Ivanov

M.A.

, Kreslavsky

M.A.

, Pratt

, Thomson

B.J.

1999. Possible ancient oceans on Mars: evidence from Mars Orbiter Laser Altimeter data. Science, 286:2134–2137.

23.

Hill

R.D.

1992. An efficient lightning energy source on the early Earth. Orig Life Evol Biosph, 22:277–285.

24.

Hodgson

G.W.

, Baker

B.L.

1967. Porphyrin abiogenesis from pyrrole and formaldehyde under simulated geochemical conditions. Nature, 216:29–32.

25.

Hodgson

G.W.

, Ponnamperuma

1968. Prebiotic porphyrin genesis: porphyrins from electric discharge in methane, ammonia, and water vapor. Proc Natl Acad Sci USA, 59:22–28.

26.

Holland

H.D.

2004. The geologic history of seawater. Treatise on Geochemistry: Volume 6: The Oceans and Marine Geochemistry. Elderfield

Elsevier-Pergamon: Amsterdam, 590.

27.

Izawa

M.R.M.

, Nesbitt

H.W.

, MacRae

N.D.

, Hoffman

E.L.

2010. Composition and evolution of the early oceans: evidence from the Tagish Lake meteorite. Earth Planet Sci Lett, 298:443–449.

28.

Johnson

A.P.

, Cleaves

H.J.

, Dworkin

J.P.

, Glavin

D.P.

, Lazcano

, Bada

J.L.

2008. The Miller volcanic spark discharge experiment. Science, 322:404.

29.

Kinoshita

, Tanaka

, Nishimori

, Dejima

, Inomata

1992. Synthesis of 2-(substituted methyl)-3,4-disubstituted pyrroles and their conversion into the corresponding porphyrins. Bull Chem Soc Jpn, 65:2660–2667.

30.

Knauth

L.P.

1998. Salinity history of the Earth's early ocean. Nature, 395:554–555.

31.

Knauth

L.P.

2005. Temperature and salinity history of the Precambrian ocean: implications for the course of microbial evolution. Palaeogeogr Palaeoclimatol Palaeoecol, 219:53–69.

32.

Krönig

1924. Über die Wärmezersetzung einiger reiner Metallacetate. Angewandte Chemie, 37:667–672.

33.

Ksander

, Bold

, Lattmann

, Lehmann

, Früh

, Xiang

Y.-B.

, Inomata

, Buser

H.-P.

, Schreiber

, Zass

, Eschenmoser

1987. Chemie der α-Aminonitrile, 1. Mitteilung: Einleitung und Wege zu Uroporphyrinogen-octanitrilen. Helv Chim Acta, 70:1115–1172.

34.

Kua

, Bada

J.L.

2011. Primordial ocean chemistry and its compatibility with the RNA world. Orig Life Evol Biosph, 41:553–558.

35.

Kuwahara

, Eto

, Kawamoto

, Kurihara

, Kaneko

, Obayashi

, Kobayashi

2012. The use of ascorbate as an oxidation inhibitor in prebiotic amino acid synthesis: a cautionary note. Orig Life Evol Biosph, 42:533–541.

36.

Larkum

A.W.D.

2006. The evolution of chlorophylls and photosynthesis. Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications. Grimm

, Porra

R.J.

, Rüdiger

, Scheer

Springer: Dordrecht, 261–282.

37.

Lee

C.C.

, Spinks

J.W.T.

1953. The mechanism of the ketonic pyrolysis of calcium carboxylates. J Org Chem, 18:1079–1086.

38.

Lide

D.R.

2003. CRC Handbook of Chemistry and Physics, 84 ^th. CRC Press: Boca Raton, FL, 14–17.

39.

Lindsey

J.S.

, Ptaszek

, Taniguchi

2009. Simple formation of an abiotic porphyrinogen in aqueous solution. Orig Life Evol Biosph, 39:495–515.

40.

Lindsey

J.S.

, Chandrashaker

, Taniguchi

, Ptaszek

2011. Abiotic formation of uroporphyrinogen and coproporphyrinogen from acyclic reactants. New Journal of Chemistry, 35:65–75.

41.

Maeda

, Okada

, Tsukamoto

, Wakabayashi

, Ito

1990. Complex formation of calcium(II) with amino acids under physiological conditions. Journal of the Chemical Society, Dalton Transactions, 2337–2339.

42.

Mancinelli

R.L.

, McKay

C.P.

1988. The evolution of nitrogen cycling. Orig Life Evol Biosph, 18:311–325.

43.

Martins

, Sephton

M.A.

2009. Extraterrestrial amino acids. Amino Acids, Peptides and Proteins in Organic Chemistry, 1

Hughes

A.B.

Wiley-VCH: Weinheim, 3–42.

44.

Miller

S.L.

1955. Production of some organic compounds under possible primitive Earth conditions. J Am Chem Soc, 77:2351–2361.

45.

Miller

S.L.

1987. Which organic compounds could have occurred on the prebiotic Earth? Cold Spring Harb Symp Quant Biol, 52:17–27.

46.

Miller

S.L.

1998. The endogenous synthesis of organic compounds. The Molecular Origins of Life, Assembling Pieces of the Puzzle. Brack

Cambridge University Press: Cambridge, 59–85.

47.

Monnard

P.-A.

, Apel

C.L.

, Kanavarioti

, Deamer

D.W.

2002. Influence of ionic inorganic solutes on self-assembly and polymerization processes related to early forms of life: implications for a prebiotic aqueous medium. Astrobiology, 2:139–152.

48.

Morse

J.W.

, Mackenzie

F.T.

1998. Hadean ocean carbonate geochemistry. Aquatic Geochemistry, 4:301–319.

49.

Navarro-González

1992. Role of formaldehyde in the origin of life. Proceedings of the Third International Conference on the Role of Formaldehyde in Biological Systems

Tyihák

Hungarian Biochemical Society, 93–100.

50.

Navarro-González

, Segura

2004. The possible role of volcanic lightning in chemical evolution. Origins: Genesis, Evolution and Diversity of Life. Seckbach

Kluwer: Dordrecht, 137–152.

51.

Nisbet

E.G.

, Fowler

C.M.R.

2004. The early history of life. Treatise on Geochemistry: Volume 8: Biogeochemistry. Schlesinger

W.H.

Elsevier-Pergamon: Amsterdam, 1–39.

52.

Osterloo

M.M.

, Hamilton

V.E.

, Bandfield

J.L.

, Glotch

T.D.

, Baldridge

A.M.

, Christensen

P.R.

, Tornabene

L.L

, Anderson

F.S.

2008. Chloride-bearing materials in the southern highlands of Mars. Science, 319:1651–1654.

53.

Pettit

L.D.

, Powell

K.J.

2001. The IUPAC stability constants database, SC-databaseVersion 5.18Academic Software: Otley, UK.

54.

Pinkerton

, James

, Jones

2002. Surface temperature measurements of active lava flows on Kilauea volcano, Hawai'i. Journal of Volcanology and Geothermal Research, 113:159–176.

55.

Pizzarello

, Cooper

G.W.

, Flynn

G.J.

2006. The nature and distribution of the organic material in carbonaceous chondrites and interplanetary dust particles. Meteorites and the Early Solar System II. Lauretta

D.S.

, McSween

H.Y.

Jr. The University of Arizona Press: Tucson, 625–651.

56.

Plankensteiner

, Reiner

, Schranz

, Rode

B.M.

2004. Prebiotic formation of amino acids in a neutral atmosphere by electric discharge. Angew Chem Int Ed Engl, 43:1886–1888.

57.

Plankensteiner

, Reiner

, Rode

B.M.

2006. Amino acids on the rampant primordial Earth: electric discharges and the hot salty ocean. Mol Divers, 10:3–7.

58.

Pohorille

2009. Early ancestors of existing cells. Protocells: Bridging Nonliving and Living Matter. Rasmussen

, Bedau

M.A.

, Chen

, Deamer

, Krakauer

D.C.

, Packard

N.H.

, Stadler

P.F.

MIT Press: Cambridge, MA, 563–581.

59.

Semeikin

A.S.

, Berezin

M.B.

, Chernova

O.M.

, Antina

E.V.

, Syrbu

S.A.

, Lyubimova

T.V.

, Kutepov

A.M.

2003. Alkyl-substituted dipyrrylmethenes and their oxa- and thia-analogs: “structure–solvation properties” correlations. Russian Chemical Bulletin, 52:1807–1813.

60.

Shapiro

2006. Small molecule interactions were central to the origin of life. Q Rev Biol, 81:105–125.

61.

Sheldrick

G.M.

2008. A short history of SHELX. Acta Crystallogr A, 64:112–122.

62.

Sleep

N.H.

2010. The Hadean-Archaean environment. Cold Spring Harb Perspect Biol, 2:a002527.

63.

Soares

A.R.M.

, Taniguchi

, Chandrashaker

, Lindsey

J.S.

2012. Primordial oil slick and the formation of hydrophobic tetrapyrrole macrocycles. Astrobiology, 12:1055–1068.

64.

Sobral

A.J.F.N.

, Rebanda

N.G.C.L.

, da Silva

, Lampreia

S.H.

, Ramos Silva

, Matos Beja

, Paixão

J.A.

, Rocha Gonsalves

A.M.d'A.

2003. One-step synthesis of dipyrromethanes in water. Tetrahedron Lett, 44:3971–3973.

65.

Staudacher

, Ferrazzini

, Peltier

, Kowalski

, Boissier

, Catherine

, Lauret

, Massin

2009. The April 2007 eruption and the Dolomieu crater collapse, two major events at Piton de la Fournaise (La Réunion Island, Indian Ocean) Journal of Volcanology and Geothermal Research, 184:126–137.

66.

Strasdeit

2010. Chemical evolution and early Earth's and Mars' environmental conditions. Palaeodiversity, 3,Supplement:107–116. http://www.palaeodiversity.org/pdf/03Suppl/Supplement_Strasdeit.pdf.

67.

Strasdeit

, Fox

2013. Experimental simulations of possible origins of life: conceptual and practical issues. Habitability on Other Planets and Satellites: The Quest for Extraterrestrial Life. de Vera

J.-P.

, Seckbach

Springer: Dordrechtin press.

68.

Strasdeit

, Büsching

, Behrends

, Saak

, Barklage

2001. Syntheses and properties of zinc and calcium complexes of valinate and isovalinate: metal α-amino acidates as possible constituents of the early Earth's chemical inventory. Chemistry, 7:1133–1142.

69.

Summers

D.P.

, Khare

2007. Nitrogen fixation on early Mars and other terrestrial planets: experimental demonstration of abiotic fixation reactions to nitrite and nitrate. Astrobiology, 7:333–341.

70.

Suo

, Avci

, Schweitzer

M.H.

, Deliorman

2007. Porphyrin as an ideal biomarker in the search for extraterrestrial life. Astrobiology, 7:605–615.

71.

Taniguchi

, Soares

A.R.M.

, Chandrashaker

, Lindsey

J.S.

2012. A tandem combinatorial model for the prebiogenesis of diverse tetrapyrrole macrocycles. New Journal of Chemistry, 36:1057–1069.

72.

Taylor

S.R.

, McLennan

S.M.

2009. Planetary Crusts: Their Composition, Origin and Evolution. Cambridge University Press: Cambridge, 233–274.

73.

Van Kranendonk

M.J.

, Webb

G.E.

, Kamber

B.S.

2003. Geological and trace element evidence for a marine sedimentary environment of deposition and biogenicity of 3.45 Ga stromatolitic carbonates in the Pilbara Craton, and support for a reducing Archaean ocean. Geobiology, 1:91–108.

74.

Van Kranendonk

M.J.

, Philippot

, Lepot

, Bodorkos

, Pirajno

2008. Geological setting of Earth's oldest fossils in the ca. 3.5 Ga Dresser Formation, Pilbara Craton, Western Australia. Precambrian Res, 167:93–124.

75.

Voet

, Voet

J.G.

2011. Biochemistry, 4 ^th. Wiley: Hoboken, NJ, 1019–1087.

76.

Westall

2003. Le contexte géologique de l'origine de la vie et les signatures minérales de la vie fossile. Les Traces du Vivant. Gargaud

, Despois

, Parisot

J.-P.

, Reisse

Presses Universitaires de Bordeaux: Pessac, France, 319–342.

77.

Westall

, de Ronde

C.E.J

, Southam

, Grassineau

, Colas

, Cockell

, Lammer

2006. Implications of a 3.472–3.333 Gyr-old subaerial microbial mat from the Barberton greenstone belt, South Africa for the UV environmental conditions on the early Earth. Philos Trans R Soc Lond B Biol Sci, 361:1857–1875.

78.

Wiberg

2001. Holleman-Wiberg Inorganic Chemistry. Academic Press: San Diego, CA, 1762.

79.

Wood

T.E.

, Thompson

2007. Advances in the chemistry of dipyrrins and their complexes. Chem Rev, 107:1831–1861.

80.

Xiao

, Huang

, Christensen

P.R.

, Greeley

, Williams

D.A.

, Zhao

, He

2012. Ancient volcanism and its implication for thermal evolution of Mars. Earth Planet Sci Lett, 323–324:9–18.

81.

Yaylayan

V.A.

, Keyhani

2001. Elucidation of the mechanism of pyrrole formation during thermal degradation of ¹³C-labeled L-serines. Food Chem, 74:1–9.

82.

Yusenko

, Fox

, Guni

, Strasdeit

2008. Model studies on the formation and reactions of solid glycine complexes at the coasts of a primordial salty ocean. Zeitschrift für anorganische und allgemeine Chemie, 634:2347–2354.

83.

Zaia

D.A.M.

, Zaia

C.T.B.V.

, De Santana

2008. Which amino acids should be used in prebiotic chemistry studies? Orig Life Evol Biosph, 38:469–488.

84.

Zolensky

M.E.

, Bodnar

R.J.

, Gibson

E.K.

Jr. , Nyquist

L.E.

, Reese

, Shih

C.-Y.

, Wiesmann

1999. Asteroidal water within fluid inclusion-bearing halite in an H5 chondrite, Monahans (1998) Science, 285:1377–1379.