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New Insights into the Characterization of Insoluble Black HCN Polymers

by Marta Ruiz-Bermejo* a ), Jose L. de la Fuente b ), Celia Rogero a ), Cesar Menor-Salvan a ), Susana Osuna-Esteban a ), and Jose A. Martn-Gago a ) c ) ) Centro de Astrobiologa (Consejo Superior de Investigaciones Cientficas Instituto Nacional de Tecnica Aeroespacial (CSIC-INTA)), Carretera Torrejon-Ajalvir, Km 4, E-28850 Torrejon de Ardoz, Madrid (phone: 34-91-520-6402/6458; fax: 34-91-520-6410; e-mail: ruizbm@inta.es) b ) Instituto Nacional de Tecnica Aeroespacial (INTA), Carretera Torrejon-Ajalvir, Km 4, E-28850 Torrejon de Ardoz, Madrid c ) Instituto de Ciencia de Materiales de Madrid (CSIC), C/Sor Juana Ines de la Cruz, 3, E-28049 Cantoblanco, Madrid
a

The data presented here provide a novel contribution to the understanding of the structural features of HCN polymers and could be useful in further development of models for prebiotic chemistry. The interpretation of spectroscopic and analytical data, along with previous results reported by other authors, allowed us to propose a mechanism for the aqueous polymerization of HCN from its primary and simplest isolated oligomer, the diaminomaleonitrile (DAMN) tetramer. We suggest that insoluble black HCN polymers are formed by an unsaturated complex matrix, which retains a significant amount of H2O and important bioorganic compounds or their precursors. This polymeric matrix can be formed by various motifs of imidazoles and cyclic amides, among others. The robust formation of HCN polymers assayed under several conditions seems to explain the plausible ubiquity of these complex substances in space.

Introduction. HCN is ubiquitous in the universe and is a significant product in prebiotic simulation experiments [1 4]. HCN Polymers may be the major components of dark matter, which could be present in objects such as asteroids, moons, planets, and, in particular, comets [1] [5 7]. It has been proposed that the reddish haze (tholins) present in the atmosphere of Titan, the largest moon of Saturn, could be due to the presence of HCN polymers [8]. In addition, it has been suggested that HCN polymers may be important substances in the first stages of the chemical evolution of life [9]. This hypothesis is based on the fact that HCN polymers are precursors of important bioorganic compounds such as purines, pyrimidines, and amino acids, as well as other biological compounds such as oxalic acid and guanidine [10 13]. HCN can spontaneously polymerize in the presence of bases such as NH3 and free radicals from ionizing radiation, and occurs over a wide range of temperatures and pressures in both polar (water) and non-polar (hydrocarbon) solvents and surfaces [1]. The HCN polymers, also known as HCN oligomers, azulmic acid or azulmin, are heterogeneous solids ranging in color from yellow or orange to brown or black, depending on the degree of polymerization and/or cross-linking processes. The structures of HCN polymers have not been fully characterized and remain controversial due to their complex and heterogeneous nature.
 2012 Verlag Helvetica Chimica Acta AG, Zrich

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Several models attempting to explain the complex structure of HCN polymers have been proposed [14 19]. Scheme 1 compiles these models.
Scheme 1. Different Models Proposed in the Literature for HCN Polymers

During the polymerization of HCN in aqueous environments, a H2O-soluble and an insoluble solid product are formed. Our study is focused on the insoluble solid, commonly named insoluble black HCN polymers or black azulmic acids. These polymers were prepared from solutions of equimolar amounts of NH4Cl and NaCN in pure H2O at concentrations of 1 and 10m using different reaction times. To further

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elucidate the structure of this polymer, we used the methodology we developed previously for the characterization of tholins [20] [21]. Data from the elemental analysis, FT-IR, 13C-CP-MAS-NMR spectra, and GC/MS analysis of the insoluble black HCN polymers were compared to results previously reported in the literature. Additionally, we used X-ray photoelectron spectroscopy (XPS) to quantify various chemical species in powder samples [20 22]. Additionally, to obtain complementary information, the insoluble black HCN polymers were acid hydrolyzed. The hydrolyzed supernatants and black insoluble residues were also analyzed. Scheme 2 outlines the general treatment of the samples, the separation in fractions and the techniques used for this study.
Scheme 2. Reaction Conditions, General Treatment of Samples, Fractionation, and Techniques Used

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The results presented herein complete the data reported in a previous paper [20] where some spectroscopic data were shown. The strength of HCN polymerization reactions in an aqueous medium and the proposed structures for insoluble black HCN polymers are discussed. Results. The results of the elemental analyses of the insoluble black HCN polymers are compiled in Table 1. Our results are comparable to those reported by Eastman et al. [23] and Labadie et al. [24] for short reaction times (1 10 d). For longer reaction times (30 d), black insoluble solids richer in oxygen were obtained. However, a longer reaction time did not have a significant influence on the yield of these insoluble products, 29 4%. In this work, the yields were calculated from the initial amount of C in the NaCN used as a reactant. The estimated empirical formulae, C3H4N3O and C5H6N4O2 , indicate the formation of highly unsaturated structures with a backbone based on C and N. The elemental analyses of the insoluble black residues after acid hydrolysis provided the following results: C, 36.6 0.6%; H, 4.1 0.2%; N, 33.2 0.8%; and O, 22.3 0.9%. Thus, the estimated empirical formula for the hydrolyzed residues was C2H3N2O.
Table 1. Experimental Conditions, Yields, and Elemental Analyses for the Series of Insoluble Black HCN Polymers Prepared in This Work and by Other Authors. The yields in this work were calculated from the initial amount of carbon in the initial NaCN. N.R., not reported; c, initial concentration; T, reaction temp.; t, reaction time. Reactants c [m] T [8] t Yield [%] C H N O Empirical Ref. [%] [%] [%] [%] formula N.R. N.R. N.R. 31 N.R. 51 31 25 27 33 36.5 35 39.6 38.77 44.5 40.2 36.2 36.5 36.5 36.2 4.2 39.2 N.R. 38 4.0 3.96 4.0 3.8 4.3 4.4 4.4 3.9 46.1 40.85 52.5 41.8 40.8 40.8 41.9 39.6 15.7 12 10.4 16.42 N.R. N.R. 17.7 17.4 17.2 19.2 C3H4N3O [23] [25] C5H6N5O C3H4N3O C3H4N3O C3H4N3O C3H4N3O C5H6N4O2 [16] [24] [18] [19] This work This work This work This work

KCN (aq) NH4Cl(aq) 1 1.5 70 HCN(l) NH3(aq) Et3N N.R. r.t. HCN(g) NH3(aq) HCN(aq) NH4OH HCN(l) NH3(l) HCN(aq) NH3(aq) NaCN(aq) NH4Cl(aq) NaCN(aq) NH4Cl(aq) NaCN(aq) NH4Cl(aq) NaCN(aq) NH4Cl(aq) 12.5 1 N.R. N.R. 10 1 1 1

80 h 3 d, after water 30 d r.t. 4d 90 16 h r.t. 30 d 40 50 5 h 38 3d 38 3d 38 10 d 38 30 d

Determination of Functional Groups. The FT-IR spectra of the insoluble black HCN polymers synthesized in our laboratory were similar, with no significant differences among them (Fig. 1, c). These spectra highly resemble those reported in the literature for HCN polymers prepared under different experimental conditions. Quirico et al. [8] reported an IR spectrum of an HCN polymer prepared from pure NH3 and liquid HCN that was similar to the IR spectrum in this study. Similar IR data were reported by Liebman et al. [26] and Umemoto et al. [16] for HCN polymers obtained from liquid HCN and Et3N in MeCN, and from gaseous HCN and aqueous NH3 , respectively. The IR spectra do not provide sufficient information to identify the

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Fig. 1. Transmission FT-IR spectra of a) hydrolyzed supernatant, b) hydrolyzed insoluble black residue, and c) insoluble black HCN polymers

differences between HCN polymers obtained in H2O solutions and those prepared in aprotic mediums or without solvents. Therefore, the IR spectra must be deconvoluted to identify the functional groups in the HCN polymers. The features in the IR spectra of our insoluble black HCN polymers can be assigned to N-containing groups: primary and secondary amines (3444 cm 1, NH stretch; 3330 cm 1, NH2 antisym. stretch, and 3191 cm 1 NH2 sym. stretch), CN and carbodiimide groups (2187 cm 1, CN and

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NCN), amides, urea and triazines (1655 cm 1, CO stretch (amide I band); 1560 cm 1, NH deformation (amide II band) plus ring stretch in triazine compounds; 1395 cm 1, CN stretch (amide III band)), and azoxy groups (1490 cm 1, NNO antisym. stretch). All of the insoluble black hydrolyzed residues presented similar bands in their IR spectra (Fig. 1, b). These IR bands were assigned to the following functional groups: 3084 cm 1 (very broad), primary and secondary amines (OH groups of carboxylic acids, and CH in aromatic and unsaturated hydrocarbons may overlap); 1725 and 1665 cm 1, CO stretch in carbonyl and amide compounds; 1590 cm 1, NH2 primary alkyl amide; 1515 cm 1, NH in secondary amides and triazine compounds; and 1420 cm 1 OH in carboxylic acids and CN in primary amides. The IR spectra of the soluble hydrolyzed materials exhibit bands that may correspond to the NH cation (3050 and 1410 cm 1), CH3 and CH2 groups in 4 aliphatic compounds (2895 cm 1), and CO in carbonyl, carboxylic, and urea compounds (1735 and 1670 cm 1). As in the FT-IR analysis, the 13C-CP-MAS-NMR spectra of all of our samples were similar and resembled spectra obtained by Garbow et al. [25] for the water-insoluble fraction as well as spectra reported by Mamajanov and Herzfeld [14] [15] for an HCN polymer prepared from gas HC15N and Et3N. The deconvoluted solid-state 13C-NMR spectrum of our insoluble black HCN polymers displayed the following resonances: i) resonance at 168 ppm, which may correspond to amide groups (CONH2 ); ii) resonance at 159 ppm, which may correspond to imine and/or heterocyclic groups (CN); iii) a group of resonances at 154, 149, and 139 ppm, which may correspond to C-atoms of heterocyclic compounds containing N; iv) a group of low-intensity resonances between 130 and 100 ppm, which may correspond to alkenes ( CC ) and nitriles (C  N); and v) a third group of unresolved resonances between 100 and 60 ppm, which may correspond to C-atoms bound to a heteroatom, such as C(N) of amines (Fig. 2, b). The 13C-CP-MAS-NMR spectra of the insoluble hydrolyzed residues (Fig. 2, a) exhibit resonances similar to those of the insoluble black HCN polymers, but with a well-defined signal at 171 ppm, which may correspond to carboxylic acids, as was observed in IR analysis. XP Spectra were recorded to obtain further structural data for the HCN polymers. XP Spectra of the prepared samples recorded from 1200 to 0 eV provided an overview of the main elements in the insoluble black HCN polymers, as well as any trace contaminants (data not shown). As expected, the HCN polymers consisted mainly of C and N with a lower contribution of O. It is important to note that C and O were the main contaminants in samples prepared in air and were also the main contaminants of the KBr pellets prepared. On the other hand, the C bonds were relatively well-defined by 13C-CP-MAS-NMR and IR spectra. Therefore, only the high-resolution N 1s core level spectra were relevant for elucidation of the structures of the controversial Ncontaining functional groups in the insoluble black HCN polymers, which can be considered the distinctive fingerprint of the possible structures of the insoluble black HCN polymers. Fig. 3 and Table 2 present the binding energies of the different components of the N 1s core level peak, as well as their assignment and quantitative results. The two main components are centered at 397.6 and 398.7 eV, and can be associated with the NC
=
=

=
=

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Fig. 2.

13

C-CP-MAS-NMR Spectra of a) hydrolyzed insoluble black residue and b) insoluble black HCN polymers

bonds (imine and/or heterocyclic groups) and the CON group (amides), respectively. The small contribution detected at higher binding energies, 400.5 eV, corresponds to the NH groups in pyrrolic structures, as well as CN and amine groups. This assignment of the binding energies for the N 1s core level peak was checked against standard samples prepared in our laboratory [21]. The functional groups identified by XPS were in agreement with the IR and 13C-NMR assignments . The advantage of the XPS technique is that it provides quantitative information about each N-containing group in the insoluble black HCN polymers. Additionally, the XP spectra of the insoluble hydrolyzed residues were recorded (Table 2). For the insoluble hydrolyzed residues, the absence of a component signal at 400 eV, the increase in
=

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signals for amide groups in the XP spectra, and the presence of resonances assigned to carboxy C-atoms in the 13C-NMR spectra indicated that the component with the signal at 400.5 eV in the insoluble black HCN polymers can be assigned to nitrile groups.

Fig. 3. High-resolution N 1s core level spectra of insoluble black HCN polymers. The dots correspond to experimental data, the solid line corresponds to the fit, and the filled gray-scale curves correspond to the curve components used for deconvolution of the spectra. Table 2. X-Ray Photoelectron Spectroscopy ( XPS) Data of the N 1s Core Level, Indicating the Nitrogen Chemical Environments in Black HCN Polymers and in the Hydrolyzed Insoluble Residue Group BE [eV] BE (% of the component) Insoluble black HCN polymers N (imines)/NC (heterocycles) CON (amides) NH (amines)/C  N (nitriles)/pyrrolicNH
=
=

Hydrolyzed insoluble residues 398.0 (51.9) 399.4 (48.1)

397 398 399 400

397.6 (47.4) 398.7 (47.4) 400.5 (5.1)

Additionally, the UV/VIS spectra were recorded, and one sample is shown in Fig. 4. The UV/VIS spectra of the all samples showed two shoulders at ca. 345 and ca. 465 nm that can be related to N-heterocyclic macromolecular systems with p-extended conjugation [27] [28]. Identification of Small Molecules Absorbed in the Insoluble Black HCN Polymers. The ability of the insoluble black HCN polymers to retain H2O and other lowmolecular weight molecules was confirmed by preliminary thermogravimetric (TG) and GC/MS analyses. The TG measurements (Fig. 5) indicated that the amount of adsorbed H2O was 8 10% depending on the reaction times. Long reaction times led to a greater amount of adsorbed H2O in the matrix of the insoluble black HCN polymers, which was consistent with data from the elemental analysis. A complete thermal study of these

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Fig. 4. UV/VIS Spectrum of HCN polymers in DMSO

Fig. 5. Thermogravimetry (TG) Curve for HCN polymers. Heating rate was 108/min under N2 .

samples was described in an other publication, dealing with the structureproperty relationships in this complex polymeric system [29]. About 30 40% of the insoluble black HCN polymers was released as H2O-soluble organic material after acid hydrolysis. This is consistent with the results reported by Labadie et al. [24]. The dried residues of the hydrolyzed supernatants were analyzed for polar organic compounds by GC/MS (Scheme 2). The chromatograms of all the samples showed similar profiles (Fig. 6) independent of the reaction times. In general,

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Fig. 6. GC/MS Chromatogram showing the profile of organic molecules absorbed by the matrix of the insoluble black HCN polymers. These compounds are released after acid hydrolysis and were detected in the hydrolyzed dried supernatants independently of the initial reaction conditions. The GC/MS analyses of the insoluble black HCN polymers before hydrolysis and of the hydrolyzed insoluble residues were negative; no small polar molecules were detected.

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all samples contained large amounts of oxalic acid, urea, and glycine, as expected [13] [24]. These three compounds comprised ca. 10 15% of the soluble hydrolyzed material. The purines, such as adenine, hypoxanthine, and guanine, and the pyrimidines, such as uracil, 5-hydroxyuracil, 5-aminouracil, and orotic acid, were previously identified by Miyakawa et al. [10] in a low-temperature experiment at the eutectic point of HCN. Negron-Mendoza et al. [30] quantified the dicarboxylic, malonic, succinic, and maleic acids in experiments involving the irradiation of NH4CN. However, to the best of our knowledge, these acids were not previously reported in H2O polymerization experiments with HCN. Furthermore, for the first time, the organic compounds pyrazine-2,5-diol, hydroxymalonic acid, aminomalonic acid, 2,4,5trihydroxypyrimidine, and 2,4,7-trihydroxypteridine were detected in HCN polymerization experiments. Further analyses are in progress to identify organic compounds in the HCN polymers, because the detection of these molecules seems to depend on sample preparation and analytical tools rather than experimental conditions of polymerization [31]. In summary, we can conclude that insoluble black HCN polymers are formed by a highly unsaturated CN matrix, which can absorb H2O and low-molecular-weight polar molecules. This matrix likely consists of macrostructures with a high content of Ncontaining heterocycles and non-hydrolyzable amides. Temperature and reaction times did not seem to affect the synthesis of insoluble black HCN polymers in an aqueous medium. Therefore, the formation of insoluble black HCN polymers is robust and is expected to occur under diverse environmental conditions. Discussion. Proposal of Structures for Insoluble Black HCN Polymers. The initial steps in the oligomerization of HCN are well-understood [9]. The rate-determining step is the nucleophilic attack of the CN ion at the C  N bond of HCN, which leads to the formation of imino-acetonitrile. This step is followed by the stepwise condensation of HCN to form aminomalonitrile (AMN) and diaminomaleonitrile (DAMN). DAMN is readily formed at room temperature in 0.1 1.0m aq. HCN solutions [32]. The lowestorder oligomer is isolated from aqueous solutions. The mechanism of formation of higher order HCN oligomers is much less clear. In this study, we propose a possible formation mechanism and structures for the insoluble black HCN polymers starting with DAMN (Scheme 3; new structures are framed). DAMN is a weakly basic amine, and its chemistry has been explored in detail [33]. This reagent has been used extensively in the preparation of heterocyclic molecules, such as dicyanoimidazoles, dicyanopyrazines, and purines. Furthermore, Johnson et al. have used DAMN in the preparation of different polymeric systems [34] [35]. DAMN is an A2B2 monomer. The linear polyamine shown in Scheme 3, particularized as a 12-mer, could be obtained by an addition reaction between the amine groups, the nucleophilic agent, and the CN groups of the other molecule of this reactive monomer, which acted as the electrophilic agent. Subsequent elimination reactions, such as decyanation (Pathway a) and/or deamination (Pathway b), for this polyamine may allow formation of new extended-conjugation macrostructures. The formation of stable five- or six-membered rings is possible from the decyanation of this linear polyamine. Pathway a-1 is consistent with the recent work of Mamajanov and Herzfeld [14], who have studied the solid-state reaction of crystalline DAMN (Scheme 1). The

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Scheme 3. Mechanistic Proposal for the Formation of Insoluble Black HCN Polymers from DAMN. The framed structures are indicated for the first time.

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linear polyamide (Pathway a-2) displayed a high tendency to form cyclic components, because the polymerization reactions in solution favor intramolecular cyclizations. For the first time, the resulting cyclic polyamide was considered as a motif of the insoluble black HCN polymers. Taking into account the quantitative results of XPS, this cyclic polyamide system appeared to be prevalent in the insoluble black HCN polymers. On the other hand, the formation of five- and six-membered rings would also be expected by a deamination reaction (Pathway b) from the polyamine and intramolecular cyclization between CN groups, which may yield the heterocyclic polymer indicated in Scheme 3. This is, to the best of our knowledge, the first time that this novel macrostructure based on pyrrole and dihydropyrazine rings has been proposed for HCN polymers. These heterocyclic macrostructures indicate the presence of a non-hydrolyzable matrix for the insoluble black HCN polymers, according to the spectroscopic data presented above. This polymeric matrix can be formed by different motifs of imidazoles and cyclic amides, as well as other groups. Astrobiological Implications of HCN Polymers. The study of HCN polymers is relevant to prebiotic chemistry and astrobiology. It has been suggested that primitive Earth may have been covered by water and carbonaceous materials originating from bolide bombardment or from photochemical reactions in the atmosphere, including HCN polymers, which would have supplied essential components for establishing protein/nucleic acid life [1]. As mentioned above, it is well-known that HCN polymers release nucleic acid bases and amino acids. In addition to those species, dicarboxylic acids were detected in this work. Some of these acids could have been involved in a primordial variant of the Krebs cycle [36]. Metabolic-type reactions could have played a central role in the processes that led to the origin of life. Although the hypothetical existence of self-organizing proto-metabolic cycles is controversial [37], our preliminary GC/MS analysis of the insoluble black HCN polymers demonstrated that the materials required for the development of a proto-metabolic system, and structural and informational materials could be synthesized simultaneously. Recently, polycyclic aromatic hydrocarbons (PAHs) have been suggested as energy transduction elements [38] in possible proto-metabolic systems, because they absorb light in the near-UV and blue region, and can capture light energy either by donating electrons to produce molecules with higher chemical potential or by generation of ionic gradients. Thus, HCN polymers may also be capable of capturing or transducing energy due to the unsaturated nature of their matrix (Scheme 3). Conclusions. This study offers new insights into the structural characterization of HCN polymers based on the complementary use of several spectroscopic and analytical tools. Based on the data reported here, we conclude that insoluble black HCN polymers are formed by an unsaturated complex matrix, which can retain H2O, and important bioorganic compounds or their precursors. We propose that this insoluble matrix is composed of several polyheterocyclic motifs, mainly imidazoles and cyclic polyamides. HCN Polymerization in H2O yields many compounds considered to be potential life precursors. Furthermore, insoluble black HCN polymers can be obtained under several environmental conditions, which explains their ubiquity in a large diversity of space environments.

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The authors used the research facilities of Centro de Astrobiologa (CAB) and were supported by the Instituto Nacional de Tecnica Aeroespacial Esteban Terradas (INTA), and the projects AYA2009-13920 C02-01 and BIO2007-67523 of the Ministerio de Ciencia e Innovacion (Spain). We thank M. T. Fernandez for recording the IR spectra, I. Sobrados from ICMM (Instituto de Ciencia de Materiales de Madrid, CSIC, Spain) for recording the 13C-NMR spectra, and S. Veintemillas and G. Munoz-Caro for useful comments.

Experimental Part Preparation of Insoluble Black HCN Polymers. Insoluble black HCN polymers were prepared according to a method described by Borquez et al. [31] using solns. of equimolar concentrations of NH4Cl and NaCN in pure H2O (MilliQ grade) at concentrations of 1 and 10m. The 1m solns. were left to stand for 3 d, one week, and four weeks, and the 10m soln. was left to stand for 3 d. All of the solns. were heated to 388 for the duration of the reaction for comparison to previous work concerning the formation of tholins under a CH4 atmosphere and spark discharges [20] [21] [39]. After the reaction, the samples were filtered using a glass fiber filter (0.25 mm) and washed with dist. H2O (4 ) to collect the black solids, which were dried under reduced pressure. The results of the elemental analyses and the XPS recordings indicated the absence of salts in all of the samples. NH4Cl and NaCN were obtained from SigmaAldrich. Hydrolysis of the Insoluble Black HCN Polymers. For hydrolysis, 6n HCl at 1108 for 24 h was used. The hydrolyzed samples were centrifuged. The insoluble black residues were collected, washed with H2O (3 ), dried under reduced pressure, and studied by the same techniques as the insoluble black HCN polymers. The supernatants were collected, freeze-dried, and analyzed. Standard Spectroscopy Techniques. UV/VIS Spectra were obtained using an Agilent 8453 spectrophotometer. All spectra were recorded in DMSO. All samples were partially soluble. IR Spectra were obtained using a Nexus Nicolet FT-IR spectrometer. The spectra were obtained in CsI pellets in the reflectance mode and were recorded from 4000 to 500 cm 1. 13C-CP-MAS-NMR Spectra were obtained using a Bruker Advance 400 spectrometer and a standard cross-polarization pulse sequence. Samples were spun at 10 kHz, and the spectrometer frequency was set to 100.62 MHz. A contact time of 1 ms and a period of 5 s between successive accumulations were used. The number of scans was 5000, and the chemical shift values were referenced to TMS. Elemental Analysis. Elemental C, H, and N analyses were performed using a LECO CHNS-932 elemental analyzer. For the elemental O analyses, a LECO VTF-900 analyzer was used. XP Spectroscopy. XP Spectra were collected in an ultra-high vacuum (UHV) system equipped with a multi-channeltron hemispherical electron energy-analyzer (Phoibos, Specs GmbH) using an Al Ka X-ray source, and recorded at normal emission (incident angle 458). For the XPS experiments, 25% KBr pellets were prepared for all the samples analyzed. During the recording, the samples showed a small charge effect induced by the X-ray radiation. Therefore, to maintain the energy position of the first scan, the peaks were energy-corrected. High-resolution N 1s, C 1s, and O 1s core level spectra were recorded with an estimated resolution of 0.9 eV. The spectra were well described by the superposition of several DoniachSunjic curve components. Data were analyzed with CASAXPS and FittXPS software. The intensities of the XPS core levels were evaluated by the peak areas after subtraction of the standard background area according to the Shirley procedure. Assignment of the binding energy was performed using standard spectra from the  Handbook of X-ray Photoelectron Spectroscopy [40] according to previous experimental data for which reference samples were used [21] [22] [41]. Organic-Compound Analysis by GC/MS. To identify polar organic compounds of biological interest in hydrolyzed supernatants, the following protocol was used: i) hydrolyzed samples were freeze-dried to remove H2O and HCl for 48 h; ii) ca. 6 mg of the dried residues in 100 ml of BSTFA TMCS (N,Obis(trimethylsilyl)trifluoroacetamide with Me3SiCl (Thermo Scientific)) were maintained at 608 overnight to obtain the corresponding TMS derivatives; iii) the derivatized samples were analyzed by GC/MS using the following GC oven program: 608 (initial temp.) held for 1.5 min, heating to 1308 at 58/min and holding for 11 min, heating to 1808 at 108/min and holding for 10 min, heating to 2208 at 208/min and holding for 15, and heating to 3008 at 108/min and holding for 10 min. An injection volume of 2 ml was

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used for each sample. The temp. of the injector was 2208, and the injections were performed in splitless mode. The detector temp. was 3008. The flow rate was 9.2 psi. The GC/MS analyses were performed in full-scan mode on a 6850 network GC system coupled to a 5975 VL MSD with triple-axis detector operating in electron-impact (EI) mode at 70 eV (Agilent), using an HP-5 MS column (30 m 0.25 mm i.d. 0.25 mm film thickness) and He as a carrier gas. Identification of the GC/MS peaks was confirmed by comparison of retention times and mass spectra with those of external standards purchased from SigmaAldrich and Fluka. Thermogravimetry (TG) Analysis. A Perkin-Elmer TGA-7 was used for TG measurements. The instrument was calibrated both for temp. and weight by standard methods. Non-isothermal experiments were performed over a temp. range of 25 10008 at a heating rate of 108/min. The average sample weight was ca. 10 mg, and the dry N2 flow rate was 100 cm3/min.

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