A Comparison of Peptide Bond Formation and

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on Mineral Surfaces – A Comparison of Peptide Bond ... In contrast, the equilibrium of AMP .... Thus, for inorganic phosphates (Pi)/SiO2 and AMP/SiO2.

Orig Life Evol Biosph DOI 10.1007/s11084-013-9345-2 ASTROBIOLOGY

Formation of Activated Biomolecules by Condensation on Mineral Surfaces – A Comparison of Peptide Bond Formation and Phosphate Condensation Thomas Georgelin & Maguy Jaber & Houssein Bazzi & Jean-François Lambert

Received: 20 February 2013 / Accepted: 15 August 2013 # Springer Science+Business Media Dordrecht 2013

Abstract Many studies have reported condensation reactions of prebiotic molecules, such as the formation of peptide bonds between amino acids, to occur to some degree on mineral surfaces. We have studied several such reactions on the same divided silica. When drying steps are applied, the equilibria of peptide formation from glycine, and polyphosphate formation from monophosphate, are displaced to the right because these reactions are dehydrating condensations, accompanied by the emission of water. In contrast, the equilibrium of AMP dismutation is not significantly favored by drying. The silica surface plays little role (if any) in the thermochemistry of the condensation reactions, but is does play a significant kinetic role by acting as a catalyst, lowering the condensation temperatures with respect to bulk solids. Of course, the surface also catalyzes the inverse hydrolysis reactions. Keywords Amino acids . Polymerization . Nucleotides . Phosphorylation . Silica . Catalysis

Introduction Interesting results have appeared since the 1970s involving “interfacial” prebiotic scenarii, where two molecules or more are made to react together after adsorption on a mineral surface, yielding activated molecules such as peptides from amino acids ((Lambert 2008) and references therein). However, progress in this field has been irregular, largely a hit-or-miss affair, and fundamental questions raised early about these scenarii remain unanswered (de Duve and Miller 1991). This is in part because the thermodynamic and kinetic aspects of the problem are not clearly distinguished and we will therefore start by recalling a few basic facts. Many thermodynamically uphill prebiotic reactions are in fact condensation reactions accompanied by the elimination of one water molecule, of the general form: Paper presented at the 12th European Workshop on Astrobiology “EANA’12” in Stockholm, Sweden (October 15 to 17, 2012). Editors Axel Brandenburg and Nils Holm.

T. Georgelin : M. Jaber : H. Bazzi : J. 0 (remember that K ¼ e RT ). Now this conclusion of course depends on what thermodynamic phase the reaction is occuring in. Biochemists and geochemists usually have in mind reactions in aqueous solution so that reaction (1) should be specified as: Aaq þ Baq ¼ A−Baq þ H2 Oliq


Why would things be different at the interface? The first suggestion that comes to mind is that some or all of the molecules involved may have different energy (or rather, Gibbs free energy) levels than in the solution. Without putting too fine a point on it, it might be acceptable to define an “adsorbed phase” (subscript “ads”), in which the corresponding reaction: Aads þ Bads ¼ A−Bads þ H2 Oads


is thermodynamically favored, i.e. (ΔrG°)ads ≠ (ΔrG°)aq, and (ΔrG°)ads < 0. This is perfectly conceivable. It will be the case if the products interact with the surface significantly more strongly than the reagents, and/or if higher effective concentrations are achieved in the adsorbed phase as compared to the bulk solution. Presumably, it was the original idea of Bernal who first proposed a prebiotic role of mineral surfaces (Bernal 1951). The question of whether the reaction products of (3) are more stabilized by adsorption than its reagents was clearly stated by de Duve and Miller in 1991 (de Duve and Miller 1991). However, it is only in 2010 that the idea has been rigorously tested—with a negative result. Marshall-Bowman et al. (2010) studied the equilibrium between glycine and diglycine (A = B = Gly, as well as related oligomerization equilibria) in aqueous solution with and without added minerals, and found no significant difference between the reaction equilibria in the “adsorbed phase” and the solution. The dispersed solids tested included several of the most likely prebiotic minerals, among them montmorillonite clays and silica. This begs the question of why many studies observed significant polymerization in the same systems (Gly on silica and Gly on montmorillonite), even up to the observation of pentapeptides. In fact, all of these studies involve wetting-and-drying cycles, first used by Lahav et al. (1978): for significant times in the procedure, the Gly-mineral samples are activated at rather high temperatures in the dry state, i.e. the interface is not between the mineral and an aqueous solution, but between the mineral and the gas phase. In these conditions water may desorb and the reaction has to be rewritten as: Aads þ Bads ¼ A−Bads þ H2 Ogas


This makes a difference, because the Gibbs free energy of reaction has both an enthalpic and an entropic part (ΔrG° = ΔrH°−T ΔrS°). Even in the hypothesis that the reaction enthalpy is exactly the same in the adsorbed phase as in solution, reaction (4) may become favorable because its standard entropy will be very positive if water has a low activity in the gas phase, i.e. if the conditions are very dry. More intuitively, this is an application of Le Chatelier’s

A Comparison of Peptide Bond Formation and Phosphate Condensation

principle: eliminating H2O, one of the reaction products, drives the equilibrium to the right. Numerically, to keep the reaction quotient constant: Qeq ¼

aAB aH 2 O aA aB

while decreasing aH2O, one has to increase the dimer/monomer ratio. Thus, the mineral surface most likely plays little role in reaction thermodynamics. The same argument would be valid if A and B were precipitated in a bulk solid phase instead of being adsorbed—and indeed studies of prebiotic reactions in the solid state have drawn renewed interest recently (Kolb 2012). When it comes to kinetics the mineral surface does matter however. Polymerization reactions may be theoretically favored in bulk solids but they are certainly slow since e.g. bulk glycine is indefinitely stable at room temperature. The fact that they have been reported to occur at a measurable rate for adsorbed glycine at temperatures as low as 80 °C means that some surface groups can have a significant catalytic effect on condensation reactions. Due to a basic principle of kinetics, they must also have a catalytic effect on the inverse reaction, namely the hydrolysis of A–B (in fact, this has been evidenced in the already mentioned study (Marshall-Bowman et al. 2010) that excluded a significant thermodynamic effect of interfaces). In what follows we intend to illustrate the above general considerations by their application to three different systems of prebiotic interest: glycine/silica, monophosphate/silica, and AMP/silica. In previous studies, high-surface amorphous silica has proved to constitute a good test material for prebiotic condensation studies (Meng et al. 2004; Lambert et al. 2009; Lopes et al. 2009; Bouchoucha et al. 2011)—not only do molecules deposited on this mineral support show interesting reactivity, but their reactions can be characterized rather easily by thermogravimetry and NMR for technical reasons. Thus, they were selected for a comparison of the reactivity of different biomolecules on the same support. This eliminates a source of variability that is often neglected: the differences between the surfaces of several minerals with the same bulk composition may be much more important than is usually realized.

Experimental Materials Potassium dihydrogenophosphate (KH2PO4), 5′ adenosine monophosphate sodium salt (AMP), 5′ adenosine diphosphate disodium salt (ADP) and 5′adenosine triphosphate disodium salt (ATP), were purchased from Sigma-Aldrich. The mineral phases used for deposition were commercial fumed silicas that are quite wellknown because they are often used in heterogeneous catalyst preparation studies. Aerosil 380 was provided by Evonik; it has a BET surface aera of 380 m2/g. Aerosil 150 was provided by Degussa and has a surface area of 150 m2/g. Deposition Procedure The deposition procedures used in the present paper included both impregnation and selective adsorption. In an impregnation procedure aqueous solutions of the relevant (bio)molecules are contacted with the solid silica support, and after some equilibration time the whole system is

T. Georgelin et al.

dried without previous phase separation, so that the entire amount of introduced biomolecules remains in the final solid sample. Thus, for inorganic phosphates (Pi)/SiO2 and AMP/SiO2 preparation, 10 mL of either KH2PO4 or AMP solution of the desired concentration were added dropwise to 400 mg of silica (Aerosil 150 in the first case, Aerosil 380 in the second case). The mixture was a fluid dispersion; it was stirred for 30 min at RT and dried overnight in an oven at 70 °C. A similar procedure was used to prepare 6 % Gly-Gly/SiO2. However the Gly/SiO2 samples discussed in this paper was prepared by a selective adsorption procedure. A 0.03 M solution of glycine isotopically enriched in 13C on the carbonyl was contacted with Aerosil 380 (silica concentration 30 g.L−1). After 4 h under stirring, the solid phase was separated by centrifugation. Our previous results indicated that the nature of the deposition procedure is not crucial; samples having the same Gly content exhibit similar behaviors, irrespective of whether they were prepared by impregnation or selective adsorption. Solid samples were stored in a desiccator containing silica gel before analysis, except when the objective was to test the effect of high water activity. In this case they were stored in a sealed container containing a beaker full of distilled water. Thermogravimetric Analyses Thermogravimetric analysis (TGA) of the samples was carried out on a TA Instruments Waters LLC, with a SDT Q600 analyzer, using variable heating rates β (standard: 5 °C/min), under dry air flow (100 mL/min). Solid state-NMR Solid-state 13C MAS NMR spectroscopy was carried out at room temperature on a Bruker Avance 400 spectrometer operating with a field of 9.4 T, equipped with a 4 mm MAS probe with a spinning rate of 12 kHz. Spectra were recorded using a proton cross-polarization (CP) sequence with a pulse length of 3 μs (for a π/2 pulse of about 5 μs), a data acquisition time of 154 ms, and a recycle delay of 5 to 10s. The chemical shifts were determined by reference to an external adamantane sample (δ=+38.52 ppm), Solid-state 31P MAS NMR spectra were recorded at room temperature with a Bruker Avance 500 spectrometer with a field of 14.0 T, equipped with a 4 mm MAS probe with a spinning rate of 10 kHz. We used a simple 1-pulse sequence sequence with a pulse length of 1.25 μs, a data acquisition time of 30 ms, and a recycle delay of 5 s. MALDI-TOF Mass spectra were generated using a 4700 Proteomic Analyzer MALDI-TOF/TOF (Applied Biosystems) fitted with a Nd:YAG laser (λ=355 nm, pulse duration 4 ns, repetition rate 200 Hz). The organic matter contained in the activated Gly/SiO2 samples was desorbed with distilled water, and 1 μL of the desorption solution was mixed with 1 μL of matrix (CHCA, alpha-cyano-4-hydroxycinnamic acid, ~5 mg/mL in 1/1 acetonitrile/water 0.1%TFA) All MALDI-TOF spectra, resulting from the average of a few tens or hundred laser shoots, were obtained in positive and negative ion reflector mode in the (10–4000) m/z range. The final solution was vortexed for 1 min. at high speed prior to deposition on the MALDI plates. It was checked that the desorption procedure used was quantitative for these samples, i.e., no residual adsorbed organic matter was detected by TG.

A Comparison of Peptide Bond Formation and Phosphate Condensation

Results Glycine on Silica Silica is among the supports that have been studied in early work on amino acids polymerization using wetting and drying cycles, including the simplest AA, glycine (Gromovoy et al. 1991; Bujdák and Rode 1997, 1999). The reaction that takes place upon moderate temperature activation of these systems is actually a little different from Eqs. (1–4) stoechiometrically, since it is a cyclodimerization yielding the heterocycle diketopiperazine or DKP (Basiuk et al. 1991, 1992; Basiuk and Gromovoy 1993) with the elimination of two water molecules: Glyads þ Glyads ¼ DKPads þ 2H2 Ogas


However this does not fundamentally modify the basic arguments put forward in the introduction. Reaction (5) is easily observed on silica by thermogravimetry—differential thermoanalysis (TG-DTA) and its product has been identified by several spectroscopic techniques (Meng et al. 2004). Gas phase elimination of water molecules formed along this reaction is detected around 150–160 °C (in the fast heating conditions we used; see Fig. 1a, b). It is markedly endothermic, indicating that the enthalpic part of ΔrG° remains unfavorable in the adsorbed state (Fig. 1c, d). Yet the reaction is possible because of the highly favorable entropic contribution as discussed above. As regards the kinetics, we must underline that bulk α-glycine also dimerizes to DKP—but at much higher temperatures, i.e., 240 °C in the same conditions. At these temperatures dimerization is immediately followed by decomposition to illcharacterized polymers. Thus, the silica surface is quite efficient for catalyzing reaction (5).

Fig. 1 DTG/DTA of glycine on silica under air flow, β=5 °C/min, zoom on the region corresponding to the thermal condensation to DKP. Trace a: differential thermogravimetry (DTG) of 5 % Gly/Aerosil 380; trace b: corresponding blank (DTG of bare Aerosil 380); trace c: heat flow signal for 5 % Gly/Aerosil 380; trace d: corresponding blank

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Our TG experiments were conducted under dry air, and in these conditions quantification of the DTG signal of water usually indicates (at least for low Gly loadings,

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