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Selectivities in Adsorption and Peptidic Condensation in the (Arginine and Glutamic Acid)/Montmorillonite Clay System Maguy Jaber,*,†,‡ Thomas Georgelin,§,∥ Houssein Bazzi,§,∥ France Costa-Torro,§,∥ Jean-François Lambert,*,§,∥ Gérard Bolbach,⊥,# and Gilles Clodic⊥,# †

Sorbonne Université, Univ Paris 06, UMR 8220, LAMS, Case courrier 225, 4 place Jussieu, 75005 Paris, France CNRS, UMR 8220, LAMS, 4 place Jussieu, 75005 Paris, France § Sorbonne Université, Univ Paris 06, UMR 7197, LRS, Case courrier 178, 3 Rue Galilée, 94200 Ivry-sur-Seine, France ∥ CNRS, UMR 7197, LRS, 94200 Ivry-sur-Seine, France ⊥ Sorbonne Université, Univ Paris 06, UMR 7203 and plate-forme Spectrométrie de Masse et Protéomique-IBPS/FR3631, Case courrier 41, 7-9 Quai St Bernard, 75005 Paris Cedex 05, France # CNRS, UMR 7203, 75005 Paris Cedex 05, France ‡

S Supporting Information *

ABSTRACT: The present study examines the selective adsorption and polymerization of two amino acids, glutamic acid (Glu) and arginine (Arg), on a cationic clay mineral, montmorillonite (Mt). Two experimental procedures were used: selective adsorption and wet impregnation. In the first case, an adsorption selectivity is observed based on pH-dependent speciation of the amino acids. At natural pH, arginine is positively charged and thus extensively exchanges the cations in the interlayer space of the montmorillonite whereas glutamic acid is negatively charged and adsorbed in weak amounts, probably on the clay edges. In contrast, incipient wetness impregnation forces equivalent quantities of both amino acids to be deposited. After moderate thermal activation, combined characterization techniques, especially solid-state NMR and matrix-assisted laser desorption ionization time-of-flight analysis, highlight a peptidic condensation between the amino acids and hint at a selective polymerization yielding preferably heteropeptides (e.g., cyclo(Glu-Arg)) rather than homopeptides.

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disfavored in aqueous solution2 and (ii) the corresponding reaction, and the reverse one, are kinetically slow.4,5 It was first proposed in the 1950s by Bernal6 that primeval amino acid polymerization could have occurred in the “adsorbed phase”, in which the interaction of biomolecules with mineral surfaces could alter both the reaction thermodynamics and its kinetics. We have underlined elsewhere the importance of these two factors and the necessity of studying both carefully.7,8 Over the years, a number of studies have been devoted to testing Bernal’s hypothesis, and the polymerization of adsorbed or “supported” amino acids has been observed experimentally. The field has been reviewed at least twice.9,10

INTRODUCTION The adsorption of amino acids onto mineral surfaces has been the topic of a large number of studies as it plays an important role in a wide range of areas, e.g., low-temperature aqueous geochemistry, origins of life studies, or the study of natural and artifical biomaterials such as bones.1 In particular, the study of the formation of amino acids and subsequent condensation to form peptides and proteins is important for prebiotic chemistry because amino acids, peptides, and proteins are one of the key components of living cells. It has been hypothesized that oligopeptides were the first biomolecules to rise in complexity,2,3 in a “peptide/protein world” scenario rivaling the better-known “RNA world”. Such a scenario has to overcome both thermodynamic and kinetic difficulties, such as (i) the condensation of amino acids monomers to form an amide bond is thermodynamically © 2014 American Chemical Society

Received: July 22, 2014 Revised: October 14, 2014 Published: October 14, 2014 25447

dx.doi.org/10.1021/jp507335e | J. Phys. Chem. C 2014, 118, 25447−25455

The Journal of Physical Chemistry C

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Several mineral surfaces have been tested,7,11,12 but clay minerals have been the object of particularly sustained interest. Some studies have shown their ability to polymerize amino acids and/or elongate peptides when starting from activated amino acids: amino acids adenylates,13−15 glutamic acid,16 or αthioglutamic acid17 with EDAC, via an NCA intermediate. In this case, the effect of the mineral surface is catalytic and therefore addresses mostly the polymerization kinetic problem, as the activation of the monomer eschews the thermodynamic problem (this is also the case in well-known studies of RNA formation by Ferris et al.18). But peptides can also be formed from chemically unactivated, adsorbed amino acids, provided a thermal activation step is applied, as in wetting-and-drying cycles.19−28 In this case the thermodynamic driving force is provided by drying,8 perhaps with the cooperation of saltinduced peptide bond formation.29 The problem is complex, and some amino acids like L-DOPA do not exhibit peptide formation.30,31 Demonstrating the possibility of oligopeptide formation in the adsorbed phase is not sufficient to obtain a realistic prebiotic scenario. The problem remains of how significant amounts of a given well-defined functional polypeptide could be obtained, starting from a more or less random mixture of different amino acids. This is possible only if the adsorption and/or the subsequent polymerization process exhibit a strong selectivity. The importance of selectivity in both processes has been recognized by early researchers.32 As regards adsorption selectivity, experimental evidence is scarce and rather inconclusive33 (the question of enantioselectivity34,35 is important and related to the present one but will not be treated in this paper). In particular, and strangely enough, no coadsorption experiments have been carried out to evaluate practical adsorption selectivity from amino acid mixtures in solution. In regard to polymerization selectivity, on the other hand, a few tantalizing data are available on the composition of peptides obtained from heating mixtures of amino acids supported on inorganic minerals. On montmorillonite clay, Bujdák and Rode26 observed the preferential formation of the tripeptide H-Ala-Gly-Gly-OH from a mixture of adsorbed Ala and GlyGly and from a mixture of Gly and Tyr, short mixed peptides (e.g., H-Gly-Tyr-OH and H-Gly-Gly-Tyr-OH) were formed on montmorillonite, but the production of mixed oligomers was considerably lower on a Cu-hectorite clay. The preferential formation of specific amino acid sequences has been studied in more detail on another mineral support, nonporous alumina:36 for instance, in the (Gly+Val)/alumina system, 10 times more H-Gly-Val-OH than H-Val-Gly-OH was formed. Also relevant are some early studies on nonsupported, solid-state amino acid mixtures.37−39 Reports on thermal polymerization selectivity in these systems had been met with some disbelief, but they have seen renewed interest recently, both from a theoretical40−42 and an experimental43,44 point of view. The present work was undertaken to investigate adsorption and polymerization selectivity in a model system, (glutamic acid and arginine) on montmorillonite clay, abbreviated as (Glu +Arg)/Mt. Montmorillonite, the most common smectite-group clay mineral on the Earth’s surface, has a 2:1 layer structure which consists of an octahedral sheet intercalated between two tetrahedral sheets. The two amino acids selected have very different aqueous speciation properties (see Experimental Section), leading one to expect adsorption selectivity;

furthermore, interesting polymerization selectivity was evidenced in solution studies of the corresponding system.45 Different amino acid deposition protocols were used to study selective adsorption and selective polymerization. Highperformance liquid chromatography (HPLC) of the contact solutions, X-ray diffraction (XRD), thermogravimetric analyses, 13 C solid- state NMR of the solids, and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of the desorption solutions were used to characterize the successive steps of adsorbed amino acid reactions.

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EXPERIMENTAL SECTION Montmorillonite Synthesis. Reagents were mixed in the following order: deionized water, hydrofluoric acid, sodium fluoride, and sources of interlayer cation, Mg, Al and Si. The use of fluoride allows us to obtain a better crystallinity of the final product.46 The hydrogels were matured over a 2 h period at room temperature and then introduced into a poly(tetrafluoroethylene)-lined stainless steel autoclave. The autoclaves were heated at 200 °C for 72 h. After reaction, autoclaves were cooled to room temperature under a stream of water. Run products were then recovered by filtration, washed thoroughly with distilled water, and dried at 60 °C for 12 h. The ideal formula per half unit cell is Na0.2[Si4Al1.8Mg0.2O10(OH,F)2] with a theoretical octahedral substitution rate (i.e., the number of Mg2+ to Al3+ substitution per half-cell) of 0.2, yielding a cation exchange capacity of 0.382 mequiv g−1. Adsorption Procedure. We used L-glutamic acid and Larginine from Aldrich (99% purity). Glutamic acid can exist as four different species, which will be denoted as H3Glu+, H2Glu± (zwitterion; the neutral form H2Glu is not stable in water solutions), HGlu− (monoanion), and Glu 2− (dianion), connected by acid−base equilibria with pKa values of 2.1, 4.1, and 9.5 in the aqueous phase. The abbreviation Glu will be used for glutamic acid as a component when there is no need, or no possibility, to determine its speciation. Glu has a pI of 3.2. In the same way, arginine can exist as four different species, connected by acid−base speciation (H3Arg2+, H2Arg+, HArg±, and Arg−) with pKa values around 1.7, 9.0, and 12.6. The abbreviation Arg will be used for arginine as a component.47 In the selective adsorption (SA) procedure, 10 mL of (glutamic acid and arginine) solution in distilled water with the requested concentration was prepared, and the solid powder was immediately dispersed into it with a mass concentration of 30 g L−1 under stirring. After 4 h of equilibration, a small amount of the supernatant was collected for HPLC analysis; the solid phase was then separated from the solution by filtering and dried at 70 °C overnight. In the wet impregnation procedure, 5 mL of amino acid (AA) solution (Glu and Arg) were mixed with 1 g of Mt and dried at 70 °C overnight. No separation step was carried out between the solid phase and the solution: the solid obtained contained equal weight loadings (8%) of both amino acids, i.e., 0.46 mmol of Arg for 0.54 mmol of Glu. X-ray powder diffraction was carried out on the final solids with a Bruker D8 Avance diffractometer using Cu Kα radiation (wavelength, λ = 1.5404 Å). XRD patterns were recorded between 3 and 70° 2θ with a step size of 0.05°. Thermogravimetric analysis (TGA) of the samples was carried out on a TA Instruments SDT Q600 analyzer with a heating rate of 5 °C min−1 under dry air flow (100 mL min−1). 13C MAS NMR spectra were obtained on a Bruker Avance 300 spectrometer operating at ωL = 300 MHz (1H) and 60.37 MHz 25448



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(13C). Proton cross-polarization (CP-MAS) was applied with a contact time of 1 ms. Samples were spun at the magic angle at a frequency of 10 kHz. The 13C pulse length was 5 ms (close to π/2), and the recycle delay was 5 s. Quantification of amino acids adsorption was carried out by HPLC after centrifugation. The column used was a XDBC8ZORBAX from Agilent (4.6 × 150 mm) packed with octadecylsilica; the eluant was an aqueous solution of KH2PO4 (10−2 mol L−1) and hexanesulfonate (5.10−3 mol L−1) whose pH was adjusted to 2.5 with orthophosphoric acid. After the preparation of samples by wet impregnation method, and according to previous work,26 the solids were activated thermally at 200 °C for different durations. Alternatively, seven cycles of drying and wetting were applied to the samples. This procedure was carried out to see if the “wetting-and-drying cycles” activation procedure applied in many amino acid polymerization studies resulted in a different outcome from simple heating. The organic matter contained in the activated samples was then desorbed: 0.1 g of the solid sample were dispersed in 15 mL of 0.5 M CaCl2 solution under stirring during 3 h. The solid was then filtered on a Buchner and washed with 1.5 mL of CaCl2 1 M solution. This desorption procedure was used by Bujdák and Rode in similar systems25 and indeed results in quantitative desorption of the organic molecules, as witnessed by the fact that a control TG of the remaining solid material after CaCl2 treatment did not show any peaks attributable to organic matter combustion. A 1 μL sample of the desorption solution was mixed with 1 μL of the matrix solution (CHCA, αcyano-4-hydroxycinnamic acid, ∼5 mg mL−1 in 1/1 acetonitrile/water 0.1% TFA). Mass spectra were generated using a 4700 Proteomic Analyzer MALDI-TOF/TOF (Applied Biosystems) instrument fitted with a Nd:YAG laser (λ = 355 nm; pulse duration, 4 ns; repetition rate, 200 Hz). All MALDI-TOF spectra, resulting from the average of a few tens of or a few hundred laser shoots, were obtained in positive and negative ions using the reflector mode in the m/z range of (10−4000). The final solution was vortexed for 1 min at high speed prior to deposition on the MALDI plates. Complementary experiments using a electrospray ionization mass spectrometry instrument (ESI-MS; 2000 Q-Trap, ABSciex) and its coupling with μLC (Dionex U3000) were also implemented. It was checked that the desorption procedure used was quantitative for these samples, i.e., no residual adsorbed organic matter was detected by TG. The MALDI-TOF spectra of the samples were compared to that of the matrix deposit in the same conditions of laser fluence with no ion signal saturation. The mass spectrum of the chosen matrix (CHCA) does not exhibit peaks that could interfere with the various expected amino acids or linear or cyclic peptides. Mass spectra internal calibration was performed using the matrix ions (mH+ and (2m+H)2+).

a strong preference for the adsorption of arginine was manifest. The arginine adsorption isotherm, shown in Figure 1, is not

Figure 1. Arginine adsorption isotherm on montmorillonite from equimolar Arg+Glu solutions, estimated from HPLC (solid circles) and from TG (open squares). Error bars are presented for only one set of data but are estimated to be of constant values. They represent a 95% confidence interval based on several controls of reproducibility.

Langmuirian as it does not appear to reach a plateau but keeps increasing almost linearly with the arginine concentration in solution. Indeed, it seems to follow a Frumkin dependency, which is often encountered for ion exchange with lateral interaction between the adsorbed ions.48 Note that the maximum adsorbed amount of Arg over the studied concentration range is slightly superior to that of the clay CEC (0.40 versus 0.38 mequiv g−1). Almost total ion exchange of Na+ is confirmed by the elemental analysis of sample (Glu +Arg)/Mt 0.05 M, in which the amount of Na+ fell to 1.5% of its initial value after contact with the amino acid solution. TG analyses of the separated solids, shown in Figure 2, can also yield information on the total amount of adsorbed organic molecules. The DTG of raw montmorillonite exhibits a main peak at T < 100 °C corresponding to the loss of physisorbed water (weight loss, 4%) and two peaks, at about 420 and 660 °C which are usually attributed to dehydroxylation of the clay matrix.46 The DTG curves of (Glu+Arg)/Mt samples show the peaks of montmorillonite plus some specific signals with maxima at about 240 and 400 °C, which are assigned to the progressive elimination of the organic matter. These additional signals have been quantified by subtracting the contribution of the blank in the 140−600 °C region. Supposing that all adsorbed molecules are arginine gives the data shown as squares in Figure 1. The correspondence with HPLC is quite good, although adsorbed amounts estimated by TG are systematically a little superior to those measured by HPLC (except at the lowest loading). As we will see later, this may be the result of the presence of a minor amount of adsorbed Glu molecules at high loadings, which are not detected by HPLC. The XRD pattern of raw montmorillonite exhibits hkl bands at 4.45−4.48 Å (020, 110 reflexions), 2.52−2.58 Å (130, 200 reflexions) and 1.67−1.70 Å (210 reflection), which are typical of smectites. The (001) reflection shows an interreticular distance of 1.26 nm, typical of the “one-water layer” form (Figure 3). After contact with the Arg/Glu solution, the d001 of montmorillonite increases to 1.33 nm, which does not correspond to a stable spacing for hydrated raw montmorillonite. This must be ascribed to intercalation of arginine in the

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RESULTS (Glu+Arg)/Mt by Selective Adsorption: Characterization after Adsorption. The amount of adsorbed amino acids was obtained using two methods: HPLC of the supernatant and TG of the solid sample. HPLC analysis revealed that significant amounts of arginine were adsorbed in the experimental conditions investigated (RT; natural pH 5) while the amounts of glutamic acid adsorbed were too low to be evaluated with this method, even though the initial concentrations of both amino acids were the same. Thus, 25449



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Figure 4. TEM micrograph of (Glu+Arg)/Mt SA 0.05 M.

Figure 2. DTG traces of (Glu+Arg)/Mt samples prepared by selective adsorption from solutions containing (a) 0.01 M, (b) 0.02 M, (c) 0.03 M, (d) 0.04 M, and (e) 0.05 M of each amino acid. Trace f is the blank (Mt without adsorbed amino acids).

Figure 5. 13C CP-MAS NMR spectra of (a) bulk arginine; (b) unheated Glu+Arg/Mt SA 0.05 M; (c) same, heated 30 min at 200 °C; and (d) same, heated 2 h at 200 °C.

in the guanidinum group, is typical of arginine, while the other carbons resonate in the 20−60 ppm range: C2 at 55 ppm, C3 at 28 ppm, C4 at 24 ppm, and C5 at 42 ppm. The spectrum of (Glu+Arg)/Mt SA with the highest loading globally exhibits the same peaks, compatible with the overwhelming presence of arginine molecules. Some shifts are observed with respect to bulk Arg, however. The signals corresponding to C3 and C4 overlap. A single intense signal attributable to the carboxylate function of Arg is observed at 174 ppm, displaced upfield by 6 ppm with respect to bulk Arg; the direction and magnitude of this shift are consistent with a protonation from HArg± to H2Arg+.50 MALDI-TOF MS experiments were performed on the solution obtained by desorption from the solid samples (see Experimental Section). Only protonated molecules derived from Arg and Glu are detected and assigned within 2−5 ppm accuracy (Table 1). We did not use an internal standard for quantification; thus, the intensities cannot be used for a precise evaluation of the amounts of Arg or Glu. The fact that the signal for Arg overwhelmingly predominates over that for Glu is in agreement with the results of the other characterization methods. Nevertheless, the presence of Glu was not observed at all by 13C NMR, nor could it be inferred from HPLC of the supernatant, likely due to the small amount adsorbed and to the sensitivity limits of these techniques.

Figure 3. XRD patterns of Mt before and after selective adsorption of Glu+Arg.

interlayer space because only arginine is significantly adsorbed. The observed spacing probably corresponds to arginine molecules lying flat, parallel to the layers, as the spacing between the clay layers, i.e., 3.6 Å, does not leave enough space for another disposition. Transmission electron microscopy (TEM) micrographs show layered structures with alternate dark and bright fringes with a repeat length of 1.32 nm (Figure 4), confirming the XRD results. 13 C CP-MAS NMR spectra are depicted in Figure 5. The solid-state 13C NMR spectrum of bulk Arginine contains three sharp peaks in the CO region (177−183 ppm) corresponding to several chemically distinct carboxylate environments in the crystals.49 The peak at 157 ppm, attributable to carbon C6 25450



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Table 1. Assignement of the Different Signals Observed in MALDI-TOF (m/z of the First Isotope)a m/z of MH+ 130.09 148.06 157.11 175.12 286.15 313.19 331.21 442.26 644.01

molecular species (M) pyroGlu Glu cyclo-Arg Arg cyclo(Glu-Arg) or PyroGlu-Arg cyclo(Arg-Arg) Arg-Arg cyclo(Arg-Arg-Glu) Arg4

SA without heating * ****

Scheme 1. Formation of Cyclo(Arg-Arg) from Two Arg Molecules

SA with heating * ** **** ** *** ** **** *

a

Ion signal abundance are scaled from the highest (****) to the lowest (*). Intensities are reported in Supporting Information, Table S1.

Scheme 2. Formation of the Lactam, Cyclo(Arg)

(Glu+Arg)/Mt by Selective Adsorption: Thermal Treatments. The main purpose of studying the thermal reactivity of theses samples was to determine if peptidic condensation could be evidenced, as in other supported amino acids systems.7 TGA-DTA indicated that the thermal event with Tmax = 240 °C was endothermic; in view of this, and its relative intensity, it might be the consequence of the elimination of water molecules resulting from an amide condensation reaction (R1-NH2 + HOOC-R2 = R1-NH-OC-R2 + H2O). This event is present even in the low-loading samples that contain only Arg and might therefore be ascribed to the condensation of two Arg molecules. A smaller DTG peak also appeared at 160 °C for the samples prepared from high amino acid concentrations (0.03 and 0.05 M), i.e., those that were hypothesized to contain a minor amount of Glu. It is tempting to ascribe this peak to condensation reactions involving Glu molecules. We submitted the highest AA loading sample to spectroscopic characterization after heating at 200 °C for various durations; at this temperature, the event with Tmax = 240 °C should occur with a slow kinetics. After the sample is heated at 200 °C for 30 min, a NMR signal at 181.3 ppm becomes conspicuous and keeps increasing when heating at the same temperature for longer times (2 h), as shown in Figure 2. It would be coherent to assign this signal to a carbonyl in the product of the peptide condensation reaction. The main condensation product should involve Arg molecules because these are predominant in the original sample. MALDI-TOF shows that after 2 h, the protonated molecule of Arg is still the most abundant species, but several new species are detected (Table 1); all of them can be rationalized as oligopeptides, or other condensation products, formed from the initial monomers. Thus, a peak at m/z = 313.1 is attributed to the formation of the cyclic dimer cyclo-Arg-Arg, which can be considered as substituted diketopiperazine (DKP). The formation of the later may occur according to Scheme 1; it is not surprising because analogous reactions can be observed for other amino acids.7 A smaller peak is present at m/z = 157.1, that is, 18 amu lower than arginine. We are probably witnessing a cyclization reaction (Scheme 2) leading to a lactam, cyclo(Arg), with the elimination of one water molecule. Finally, two other peaks with m/z = 286.13 and 442.2 may be assigned to mixed oligomers containing Arg and Glu, cyclo(Glu-Arg) and cyclo(Arg-Arg-Glu), respectively. Appa-

rently, they formed at the expense of the minor amount of Glu that was present before heating. To be sure that the detected signals were not spurious, the MALDI characterization was repeated at higher laser fluence. The same peaks were observed, with the same relative intensities. We also tried to use a C18 microcolumn ZipTip (Millipore) to purify and concentrate the solution before analysis. Here again, the results were in good agreement with the basic experiment. Thus, the formation of oligopeptides appears obvious. In particular, the condensation of two Arg molecules to yield the substituted DKP, cyclo(Arg-Arg), proceeds within a few minutes at 240 °C, and in longer times (a few hours) at 200 °C. Such temperatures are rather high, about 70−80 °C higher than amino acid cyclodimerization temperatures reported for other systems such as Gly/SiO2. Of course, as usual, temperature may be traded for time: after the sample was dried at 70 °C for several weeks, a minor amount of cyclo(ArgArg) was observed by MALDI and a 13C NMR peak at 180 ppm was present as a shoulder. Yet the dimerization of Arg appears to be sluggish, and this is certainly correlated with the fact that arginine is adsorbed as positively charged H2Arg+ cations, which should repel each other electrostatically. As we have seen, it is quite possible that the condensation between Arg and Glu, which does not suffer from this drawback, occurs at the significantly lower temperature of 160 °C. However, this sample, in which the initial amounts of the two amino acids were very dissimilar, is not the best-suited one for studying possible polymerization selectivities. For instance, not much can be drawn from the fact that Glu molecules form polymers only with Arg and not among themselves; in fact, this is expected from a statistical point of view because Glu are a minority species. Therefore, we carried out the same type of study on a sample in which Arg and Glu were deposited by incipient wetness impregnation (IWI), which allows the control of the amounts deposited. (Glu+Arg)/Mt IWI. This sample was prepared in order to contain comparable amounts of both amino acids, namely 8 wt % of each. This corresponds to 0.46 mmol of Arg and 0.54 mmol of Glu per gram of montmorillonite, i.e., to an Arg:Glu 25451



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Figure 6. Speciation diagrams of Arg and Glu in aqueous solution as a function of pH.

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DISCUSSION The basic idea of this paper was to study the nature of the interaction between amino acids and the montmorillonite support and to determine if there is any selectivity in adsorption and polymerization. Selectivity of Adsorption. The pH of the amino acids and clay suspensions had values between 5.4 and 5.8. In this range, as shown by the speciation curves in Figure 6, arginine is overwhelmingly positively charged (≥99% H2Arg+) whereas glutamic acid is mostly negatively charged (90% HGlu−, the rest being H2Glu±). There is, therefore, a clear physicochemical basis for discrimination between the two amino acids in the adsorption process: among the species present in the solution, H2Arg+ is the only one to have a favorable electrostatic interaction with the clay layers, and it is expected to replace at least part of the native compensating sodium cations by ion exchange. This is confirmed by the detection of H2Arg+ as the majority species in the final solid by 13C NMR. In addition, XRD and TEM analyses indicate that the latter ions are present in the interlayer in sufficient quantities to impose the interlayer spacing. The nonlangmuirian adsorption isotherm means that the adsorption process cannot be characterized by a single equilibrium constant. Indeed, lateral repulsive electrostatic interactions are operative between H2Arg+ ions in the interlayer, so that the adsorption energetics is not fully described by cation−surface sites interactions. However, a broad idea of the montmorillonite clay affinity for H2Arg+ may be conveyed by the fact that at equilibrium, about 98.5% of the CEC is saturated by the latter ion in sample (Glu+Arg)/Mt 0.05 M, in which the total amount of arginine cations initially introduced in solution amounted to 4 times the CEC. In other words, in the overall system (solution and clay), H2Arg+ amounted for only 80% of the total monovalent cation system. Thus, the clay lattice prefers H2Arg+ over Na+. Regarding glutamic acid, its adsorption should not be favored as the majority species is an anion (which should be electrostatically repelled by the clay layers) with a minor amount of zwitterions. Indeed, MALDI-TOF analysis indicates only weak Glu adsorption, while the other two techniques are not even able to detect the adsorbed Glu molecules, proving that a strong adsorption selectivity exists between the two amino acids that were present in equimolar concentrations in the adsorption solution. Mass spectrometric techniques are sensitive enough to reveal a small amount of Glu species in the sample equilibrated in the presence of the highest amount of amino acids. This amount

molar ratio of 0.85. The arginine content in the solid phase was therefore quite close to that of the (Glu+Arg)/Mt SA 0.05 M sample discussed above, while the glutamic acid content was several times higher. The (Glu+Arg)/Mt IWI samples were activated by heating at various temperatures. In an alternate thermal activation procedure, an aliquot was submitted to seven cycles consisting of heating to 200 °C for 30 min and rehydrating by exposure to water overnight. XRD patterns of the sample after 70 °C drying exhibit an increase of the d001 value from 1.21 to 1.32 nm, a value close to that observed for SA samples and probably indicating the intercalation of cationic H2Arg+ molecules in the interlayer space of the montmorillonite. The sample submitted to seven wetting−drying cycles did not undergo any noticeable changes of the d001 with respect to a single heating. TGA showed a profile quite comparable to that of (Glu +Arg)/Mt SA 0.05 M, except that the peak at 160 °C was significantly more intense. The MALDI-TOF spectrum of (Glu+Arg)/Mt IWI heated at 200 °C for 30 min showed several signals attributable to AAs and their condensation products, several of which had already been observed for (Glu+Arg)/Mt SA (see Table 1). The most intense signal remained that of the H2Arg+ monomer, but a strong signal of the dimer cyclo(Glu-Arg) was also present, as well as minor signals for monomeric Glu, cyclo(Arg-Arg), and cyclo(Arg-Arg-Glu). Finally, a very minor peak at 644.1 could correspond to Arg4. These data indicate that polymerization of amino acids occurs upon heating at 200 °C. A limited number of polymerization products are observed, and the arginine-rich products cyclo(Arg-Arg) and cyclo(Arg-Arg-Glu) are present in quantities much smaller than those for the Glu-poor (Glu +Arg)/Mt SA. It should especially be noted that no polymeric species containing only Glu are observed. After seven cycles of drying-wetting at 200 °C, the same peaks are present with only a relative weakening of the monomeric Arg. In contrast, when heating at lower temperature (85 or 125 °C) even for 24 h, or applying several wettingdrying sequences at these low temperatures, no polymerization was detected. Microchromatography coupled with mass spectrometry (μLC-MS) was used to confirm these results. Using μLCESI-Q-Trap, different ions were detected at m/z values 175.1, 286.3, and 313.1, corresponding to H2Arg+, cyclo(Glu-Arg), and cyclo(Arg-Arg), respectively. Thus, μLC-ESI-MS essentially confirms the results obtained by MALDI-TOF. 25452



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illonite). In the latter, low glycine loading samples, in which most of the glycine was present in the interlayer space, exhibited only high-temperature thermal events (Tmax > 250 °C). Higher glycine loading samples, in which glycine started to saturate edge sites, exhibited an additional peak at 214 °C which was assigned to peptidic condensation on edge sites. In the same way, if we are correct in assuming that Glu molecules are selectively adsorbed on the edges in (Glu+Arg)/Mt, the event at 160 °C in the latter systems would correspond to the formation of mixed Glu-Arg polymers in the region where the two amino acids may meet, i.e., the clay edges. (Glu+Arg)/Mt IWI: Polymerization in a System Containing Similar Amounts of Arg and Glu. Globally, this sample has the same organization as (Glu+Arg)/Mt AS; in particular, the interlamellar distance indicates that after amino acid deposition, Arg-derived cations are present in the interlayer space. The rather large amount of Glu-derived species deposited does not precipitate as a separate phase, as indicated by the absence of the peaks of bulk glutamic acid in the diffractogram (see Figure S1 in Supporting Information). Can we suppose that they are adsorbed on the clay edges? If the latter represent 30 m2 g−1, the density of Glu molecules would be about 10 nm−2, well over a monolayer. The most likely hypothesis is then that Glu species are present as an amorphous deposit surrounding the clay particles, with a significant percentage interacting with the clay edges. The SM methods that we used indicate the formation of the following peptides after heating at 200 °C: Arg-Arg, cyclo(ArgArg), cyclo(Arg-Glu), cyclo(Arg-Arg-Glu), and Arg4, while significant amounts of the monomers are still present. It seems logical to believe that cyclo(Arg-Arg) formation results from interlayer condensation of Arg species, which should happen very much in the same way as for the (Glu+Arg)/Mt SA sample. At the same time, in the clay edge region where both amino acids can meet, mixed polymers (heterodimers and trimers) can form. If the dimers were formed in a perfectly random manner between all amino acids in the sample, a basic probabilistic reasoning indicates that the following molar ratios should be expected for the three distinct cyclic dimers: 0.21 for cyclo(ArgArg), 0.29 for cyclo(Glu-Glu), and 0.50 for cyclo(Arg-Glu). While MS methods cannot easily provide quantitative analyses, the homodimer cyclo(Glu-Glu) is not observed at all. Therefore, a significant polymerization selectivity for heterodimers is evidenced in this system. This selectivity is all the more striking if we recall that many of the Arg molecules are located in the interlayer, unavailable for reaction with Glu; in parallel, Glu molecules are concentrated outside the clay layers where they should have more opportunities to react together than to react with Arg, yet no detectable amount of Glu-Glu homodimers is formed. One can only speculate on the reasons for this selectivity. Quite possibly, it is characteristic of the amino acid couple we selected and independent of the interaction with the Mt matrix. Indeed, in homogeneous solutions, the copolymerization of Ncarboxyanhydrides of Arg and Glu preferentially yields polymers with equal proportions of the two amino acids,45 and this might be due to the more favorable electrostatic interactions between the side chains of the oligopeptide when positively charged Arg and negatively charged Glu residues alternate. A longer peptide, Arg4, was also evidenced, but Arg3 was not observed. That could be explained by the condensation of two Arg2 peptides to form directly Arg4.

cannot be accurately quantified, but a rough estimate is that adsorbed Glu should be at most 10 times less than adsorbed Arg, otherwise it would be detected by HPLC as well. While there is no direct evidence for their location, we believe that they are adsorbed on the edge of the clay particles, which do not bear any significant electric charge in our pH range but exhibit silanol and other OH groups capable of H-bonding. We do not favor the alternative possibility of coadsorption with Arg in the interlayer region on the basis of energetics considerations. The interaction of the negatively charged Glu species with the clay layers that also bear a negative charge is repulsive; thus, it is hard to envisage a sufficient driving force for Glu intercalation. Furthermore, we have observed before that amino acids adsorbed on clay edges exhibit thermal dehydration at a temperature lower than those intercalated in the interlayers,7 and this would be in keeping with assignment of the TG peak at 160 °C in Figure 6 to dehydration of Glu on clay edges. Selectivity of Polymerization. Upon heating, peptidic condensation occurs as in many other supported amino acids systems, but is extensive only at high temperatures of the order of 200 °C. Arginine-Rich Sample (Glu+Arg)/Mt SA. At 200 °C, the event which is manifested in TGA by a peak at 240 °C occurs with a rather slow kinetics. Polymerization is incomplete even after 2 h at this temperature, and its main outcome is a molecule characterized by a NMR signal in the carbonyl region at 181.3 ppm. According to MALDI-TOF data, the most likely outcome of polymerization is a cyclic dipeptide, cyclo(ArgArg), which may be viewed as a substituted diketopiperazine. At first sight, this reactivity may seem quite natural as DKP formation has been observed in several other supported amino acid systems.11 Upon further examination, this result raises interesting questions. Scheme 1 represents the condensation of two neutral Arg molecules into a neutral cyclo(Arg-Arg), and this does not correspond to the actual acid−base speciation. As we have noticed before, Arg molecules are intercalated in the montmorillonite interlayers as H2Arg+ ions. These species should repel each other electrostatically, which should result in a high activation energy; this is in line with the high reaction temperature, 230 °C (as compared, e.g., to 165 °C in the same apparatus for the reaction of two globally uncharged glycine zwitterions on silica11). Some questions remain open, however, regarding the exact reaction mechanism. Peptide condensation usually involves a nucleophilic attack of the −NH2 group of one AA residue on the −COOH of the other one. Does it mean that H2Arg+ must first be deprotonated before they can react? If so, what happens to the protons, and what is the protonation state of the final cyclic dipeptide? Obviously, a deeper theoretical study is needed to answer these questions. While the majority of oligopeptides in this arginine-rich sample are the homodimers cyclo(Arg-Arg), the minor amount of Glu residues initially present also condense, and the final sample contains the mixed species cyclo(Glu-Arg) and cyclo(Arg-Arg-Glu). In this sample, the absence of homodimers (Glu-Glu) would be expected anyway because an encounter between two Glu molecules would be unlikely. This is discussed further in the following paragraph. We believe that the heterogeneous condensation between Glu and Arg may correspond to the low-temperature event discernible at 165 °C in the higher-loading samples (traces d, e, and f in Figure 2). This is based on a comparison with the thermal reactivity of the related system Gly/laponite7 (laponite is a phyllosilicate clay mineral with similarities to montmor25453



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CONCLUSION

AUTHOR INFORMATION

Corresponding Authors

The amino acids−mineral support selected for this study demonstrates unmistakable adsorption selectivity and very likely polymerization selectivity. Regarding the first contention, montmorillonite efficiently selects Arg over Glu from equimolar aqueous solutions at natural pH. This can readily be rationalized in terms of an electrostatic adsorption mechanism, taking into account the opposite effects of acid−base speciation on the two amino acids. Arginine is overwhelmingly present as H2Arg+ cations that replace the native Na+ in the interlayer by ion exchange. Conversely, glutamic acid is mostly present as HGlu− anions that are electrostatically repelled by the negative charge of the clay layers. In addition to the adsorption selectivity that exists between Arg and Glu, the clay actually shows selectivity for Arg cations over Na+. Thus, selective adsorption is a plausible scenario to concentrate AAs from a dilute solution and “choose” some of them from a mixture. At high concentrations, glutamic species are adsorbed too, but only in minor amounts. We believe that they are most probably adsorbed at the clay edges by weak interactions such as H-bonds. This would mean that there is a spatial segregation of the two amino acids. Upon thermal activation, the formation of peptide bonds between amino acids is observed without significant backbone degradation. Montmorillonite does a poorer job than other supports such as silica. In fact, the intercalated H2Arg+, held by electrostatic interactions, are rather unreactive and need prolonged heating at 200 °C to condense; the more weakly adsorbed Glu species react more easily. The immediate product of these condensation reactions are mostly cyclic dimers and trimers. When sufficient Glu-derived species are initially present, mixed hetero-oligomers are favored over homooligomers, at least in part for thermodynamic stability reasons. Thus, polymerization selectivity is observed as well as adsorption selectivity. Once again, this is interesting from a prebiotic chemistry perspective because it raises the possibility that amino acid polymerization may be oriented preferentially toward some sequences rather than others, helping to overcome the apparent low probability of obtaining welldefined amino acid sequences in peptides, which appears to be a prerequisite for further evolution. In our clay-based system, the presence of spatial segregation of the two amino acids (with Arg-derived species concentrated in the interlayer region, Glu-derived species outside it) is a mixed blessing from a prebiotic point of view. On the one hand, it is an interesting feature as it constitutes a source of structuration, a kind of natural chromatographic separation from a homogeneous mixture. On the other hand, it complicates the interpretation of polymerization selectivity results. Therefore, in a forthcoming publication we intend to compare the results obtained for (Glu and Arg) on montmorillonite with those on a silicate support that does not cause spatial segregation, namely, Aerosil-type amorphous silica.

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*E-mail: [email protected]. *E-mail: [email protected]. Laboratoire de Réactivité de Surface (UMR 7197 CNRS), UPMC Univ Paris 06, Case courrier 178, 3 Rue Galilée, 94200 Ivry-sur-Seine, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.B. was supported by a doctoral grant from the French Ministery of Research (MESR)

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ABBREVIATIONS Arg: arginine (independent of acid−base speciation) Glu: glutamic acid or glutamate (independent of acid−base speciation) DKP: diketopiperazine SA: selective adsorption IWI: incipient wetness impregnation REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Diffractogram of sample (Glu+Arg)/Mt IWI; intensity of MALDI-TOF peaks. This material is available free of charge via the Internet at http://pubs.acs.org. 25454



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