Glutamic Acid Adsorption and Transformations on Silica - The Journal

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Glutamic Acid Adsorption and Transformations on Silica Meryem Bouchoucha,†,‡ Maguy Jaber,†,‡ Thomas Onfroy,†,‡ Jean-Franc-ois Lambert,*,†,‡ and Baiyi Xue§ †

Laboratoire de Reactivite de Surface, UPMC Univ Paris 06, UMR 7197, Tour 54-55, 2eme etage - Casier 178, 4, Place Jussieu, F-75252 Paris CEDEX 05, France ‡ Laboratoire de Reactivite de Surface, CNRS, UMR 7197, Tour 54-55, 2eme etage - Casier 178, 4, Place Jussieu, F-75252 Paris CEDEX 05, France § Institut Parisien de Chimie Moleculaire, UPMC Univ Paris 06, UMR 7201, Equipe Spectrometrie de Masse, F-75252 Paris 05, France

bS Supporting Information ABSTRACT: We report here on the adsorption and deposition of L-glutamic acid (Glu) on an amorphous silica and its subsequent thermal transformations. When adsorbed from aqueous solutions, Glu only has a low affinity for the silica surface but at high concentrations the adsorbed molecules serve as nuclei for the formation of small glutamic acid crystallites. As long as the Glu loading remains inferior to a saturation limit (about 0.5 molecule nm 2), the thermal behavior of adsorbed Glu is significantly different from that of bulk glutamic acid. Two successive condensation steps are observed upon mild thermal activation, and their products have been identified by a combination of techniques including thermogravimetry, in situ IR, solid-state NMR, and ESI-MS. The first step at about 120 °C is a lactam ring closure quantitatively yielding pyroglutamic acid in a first-order reaction. The second step, at 150 °C, probably results in the formation of a highly activated tricyclic imide, PyroGluDKP, and is easily reversible in the presence of water. These reactions have implications for prebiotic peptide formation and for synthetic chemistry.

’ INTRODUCTION The adsorption of amino acids on inorganic surfaces has been widely investigated in recent years.1 Among these small biomolecules, L-glutamic acid ((2S)-2-aminopentanedioic acid, abbreviated as Glu or E) is one of the most widespread biological amino acids and has given rise to a huge number of studies due to its fundamental interest and applications in biochemistry, medicine,2 and the food and drug industries; together with Laspartic acid, it is one of the two amino acids to bear a side chain that is negatively ionized at physiological pH from its carboxylic acid moiety in the γ-position. The adsorption of glutamic acid on oxide surfaces has been studied experimentally by several authors: on alumina,3 on hydroxyapatites (HAP4 where glutamic acid can model protein interaction with the mineral matter of bones), into layered double hydroxides (LDH) by Reinholdt,5,6 on TiO2 by RoddickLanzilotta7 and later by Jonsson et al.8 who also compared rutile with hydrated ferric oxide.9 Relevant data can also be found in more general, comparative studies of amino acids adsorption on clays,10,11 SiO2 (quartz,12 mesoporous silica SBA1513), and zeolite (BEA14). A comparative molecular modeling study of the affinity of different amino acids, including Glu, on amorphous silica is found in.15 These studies reveal a wide diversity of behaviors and adsorption mechanisms depending on the surface under consideration. On HAP, Glu specifically interacts with the (001) face with a strong interaction energy on the order of 400 kJ/mol16 as r 2011 American Chemical Society

revealed by molecular modeling and the effects on crystal growth. On titania (rutile), the carboxylate moieties coordinatively bind to exposed Ti4+ ions (surface complexation8). Several different modes of coordinative bonding (bridging, chelating, monodentate+chelating) can even be identified, as is also the case on ferric oxohydroxides.9 Coordinative binding is also observed on alumina,3 while on LDH-type anionic clays, which have a permanent negative layer charge, glutamate anions are retained by ion exchange.5,6 Another conclusion to be drawn is that complementary aspects of the adsorption process can be revealed by macroscopic modeling of the adsorption data,8 spectroscopic studies, chiefly vibrational (ATR,7,9 or SFG17) and solid-state NMR,5,6 and molecular modeling.16,18 20 A sound approach to the study of Glu adsorption should combine several of these techniques. After their preparation, adsorbed Glu/SiO2 systems may undergo specific transformations on gentle heating. It is wellestablished that heating amino acids in the presence of some inorganic oxides such as silica, alumina, or clay minerals results in the “clean” formation of peptide bonds at relatively low temperatures in the absence of any activating agents. This question is of great importance in prebiotic chemistry studies because thermal polymerization in the “adsorbed phase” is one of Received: July 21, 2011 Revised: September 12, 2011 Published: September 20, 2011 21813

dx.doi.org/10.1021/jp206967b | J. Phys. Chem. C 2011, 115, 21813–21825

The Journal of Physical Chemistry C

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Scheme 1. Possible Condensation Reactions of Glutamic Acid: Intramolecular Cyclization Pathway As Compared To Intermolecular Cyclizationa

a

Carbon numbering for the Glu molecule is also provided.

the scenarios for the initial formation of oligopeptides in the absence of the elaborate machinery of the cell. We have argued elsewhere1 that thermal peptide formation from adsorbed amino acids is mostly a thermodynamic effect due to the possibility of working under low water activity conditions: because the amino acid condensation reaction yields water as a product, lowering water activity draws this reaction to the right. However, a catalytic effect of the surface is present as well because the condensation occurs at considerably lower temperatures than in bulk amino acids. Glutamic acid and its derivatives would constitute particularly interesting starting molecules for this kind of study (see Discussion) but also to understand the thermal behavior of amino acids for other applications. Indeed the existence of the γ-carboxylic acid function in Glu allows many interesting condensation reactions in addition to the classical peptide bond formation. One of the possible condensation products is pyroglutamic acid (L-5-oxopyrrolidine2-carboxylic acid, abbreviated as PyroGlu), as illustrated in Scheme 1. Pyroglutamic acid, the result of monomolecular lactam cyclization, is a chiral synthon and a chiral auxiliary in catalytic reactions on metal surfaces.21 Scheme 1 also illustrates an alternative reaction route, similar to the one observed for simpler amino acids such as glycine, that goes through the intermolecular condensation of two Glu to a substituted diketopiperazine (GluDKP). Substituted DKPs have a lot of potential applications and seem particularly adept at forming supramolecular structures (references quoted in ref 22). Finally, further condensation may proceed from the latter molecules to the formation of imide groups, yielding either the polycyclic compound sometimes called PyroGluDKP (1,7diazatricyclo[7.3.0.0]dodecane-2,6,8,12-tetrone or 3,5,8,10-tetraketoperhydrodipyrrolo[a,d]pyrazine23), or possibly a linear polyglutarimide. Scheme 1 does not exhaust the possibilities offered by the condensation of Glu molecules. Other products could be formed from Glu such as γ-polyglutamate, a degradable linear polymer

with acidic side groups,24 or a polyimide could be formed from PyroGlu. In the present work, we have studied the adsorption and thermal reactions of glutamic acid adsorbed from water solutions on a high-surface, nonporous fumed silica powder that had earlier proved to yield easily interpretable information concerning the adsorption and reactivity of other amino acids, especially glycine.25 28

’ EXPERIMENTAL SECTION We used L-glutamic acid from Aldrich (99% purity), 97 99% 15 N and 13C5 isotopically enriched L-glutamic acid, and a fumed silica with BET surface area of 380 m2/g provided by Evonik Industries (Aerosil 380), with a pH (PZC) of about 3.5.29 In the following, silica or SiO2 will refer to this particular support unless otherwise specified. Additional experiments were carried out using the following supports: Ludox HS40 colloidal silica (provided by Sigma-Aldrich as a 40w% dispersion in aqueous solution with pH = 9.35), Laponite (synthetic phyllosilicate with formula Na+0.7[(Si8) (Mg5.5 Li0.3)O20(OH)4] 0.7, provided by Rockwood Additives Limited), Na-montmorillonite (trioctahedral phyllosilicate synthesized according to a previously published procedure30), and MCM-41 (mesoporous silica synthesized according to ref 31). Glutamic acid can exist as four different species connected by acid base equilibria, which will be denoted as H3Glu+, H2Glu( (zwitterion), HGlu (monoanion), and Glu2 (dianion), with pKas of 2.1, 4.1, and 9.5 in the aqueous phase. It has a pI of 3.2. H2Glu will refer to the uncharged species, as opposed to the zwitterion; H2Glu is not stable in water solutions but may be considered in other conditions. Finally, the abbreviation Glu will be used for glutamic acid as a component when there is no need, or no possibility, to determine its speciation. Glutamic acid adsorption on silicic materials was carried out using two procedures inspired from the field of catalyst preparation. 21814

dx.doi.org/10.1021/jp206967b |J. Phys. Chem. C 2011, 115, 21813–21825

The Journal of Physical Chemistry C In the selective adsorption (SA) procedure, an amount (generally 10 or 50 mL) of glutamic acid solution in distilled water with the requested concentration was prepared, and the solid silica powder was immediately dispersed into it with a mass concentration of 15 g L 1 under stirring. After 4 h 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 60 °C overnight. Because of the application of a solid/liquid separation step, the amount of glutamic acid retained in the final solid was determined by the equilibrium of adsorption from the solution and therefore not known a priori. In the incipient wetness impregnation (IWI) procedure, the solid powder was impregnated with a volume of the glutamic acid solution sufficient to produce a homogeneous paste but not to observe the formation of a separate liquid phase (this volume is equal to 10 mL per g of solid in the case of Aerosil 380); after a waiting time of 5 min, the paste was dried at 60 °C overnight. In this case, the amount of glutamic acid in the final powder is known because it is equal to the total amount introduced. The amounts of Glu in the solid samples will be indicated as loadings, in mmol g 1 (millimole of Glu per gram of silica support, not per gram of sample, although the difference is significant only at high loadings). For reference, a loading of 0.1 mmol g 1 corresponds to 1.41 wt % (of Glu, related to SiO2), or to 0.158 molecules per square nanometer on Aerosil 380. It is not particularly important to estimate the loading corresponding to a physical monolayer because, as will be seen later, Glu has no tendency to adsorb as a monolayer. HPLC analysis was carried out on an Elite-Lachrome system (VWR-Hitachi) fitted with a diode array detector, using an octadecylsilica column maintained at 40.0 °C. The mobile phase was an aqueous solution of sodium hexanesulfonate (5 10 3 mol L 1) + K2HPO4 (10 2 mol L 1) adjusted to pH 2.5 with H3PO4. IR spectra of solutions and solution silica suspensions were recorded in the ATR mode. One milliliter aliquots of the solution or suspension were transferred to a cell fitted with a ZnSe ATR crystal at the bottom, and FT-IR spectra were recorded on a Nicolet-Magna 5700 FT-IR spectrometer equipped with a liquid nitrogen-cooled MCT detector, by adding 256 scans, with a nominal resolution of 8 cm 1. IR of solid samples was recorded in the transmission mode on self-supported pellets in a cell fitted with KBr windows. Samples may have two positions in the cell, in the oven, allowing in situ thermal treatments under vacuum or various atmospheres, and room-temperature recording of spectra without reexposure to air. FT-IR spectra were recorded on a Bruker-Vector 22 FT-IR spectrometer equipped with a DTGS detector, having a nominal resolution of 4 cm 1, by adding 128 scans. X-ray powder diffraction (XRD) 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° 2 θ with a step size of 0.05° 2 θ. Thermogravimetric analysis (TGA) of the samples was carried out on a TA Instruments Waters LLC, SDT Q600 analyzer with a heating rate of 5 °C min 1 under dry air flow (100 mL min 1). 13 C MAS NMR spectra were obtained on a Bruker Avance 300 spectrometer operating at ωL = 300 MHz (1H) and 60.37 MHz (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

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Figure 1. Amounts of Glu (mmol g 1) adsorbed on Aerosil 380 silica as a function of the equilibrium Glu concentration in the aqueous solution, estimated from HPLC (triangles) and from TG (squares). The dotted curve is an optical guide; the vertical dashed line indicates the change of behavior discussed in the text, and the vertical bold line is the solubility limit.

π/2), and the recycle delay was 5 s. 15N MAS NMR spectra were obtained on a Bruker Avance 500 spectrometer operating at ωL = 500 MHz (1H) and 50.705 MHz (15N), also with proton CP. The following parameters were used in this case: pulse length, 3.2 μs; recycle delay, 5 s; cross-polarization contact time, 5 ms. The external chemical shift reference was bulk α-glycine with a signal at 346.6 ppm. ESI MS experiments were performed using an electrospray source combined with a LTQ-Orbitrap XL hybrid instrument (ThermoFisher Corporation, San Jose, CA). Twenty microliters of sample was injected via a LC system (ThermoFischer) without column and eluted with H2O containing 0.1% formic acid. The ESI ion source was operated at 3.8 kV. The temperature of the ion transfer capillary was maintained at 275 °C. Full-scan mass spectra with a mass range of 110 1000 Da were acquired by Orbitrap with high mass resolution of 60 000 (full width at halfmaximum as defined at m/z = 400).

’ RESULTS Characterization of the Adsorption at the Silica Water Interface. ATR has been shown to constitute an effective tool

for the in situ investigation of amino acids speciation upon adsorption.32,33 Both the starting Glu solutions and the Glu/ SiO2 suspensions have been examined by ATR. Contrary to what is observed, for example, on TiO2,7 contact with the silica particles did not significantly modify the Glu speciation. At natural pH (about 3), the spectra of both the starting solution and the silica suspension are dominated by bands at 1400 1403 cm 1 (νsym CdO), 1508 (δsym HNH), 1558 1562 (νasym CdO), 1610 1620 (δas HNH), and 1720 cm 1 (νCdO in side-chain-protonated COOH), which indicate the predominant species to be the H2Glu( zwitterion. If the solution is adjusted to pH 8 to 9 by NaOH, the band of protonated COOH is absent as expected, following the basic titration of side-chain COOH. The predominant species is then the anion HGlu , and once again contact with the silica surface does not alter this speciation. Figure 1 shows the amounts of Glu adsorbed at the silica water interface as estimated from HPLC in the SA series of samples (pH 3). They are in rather good agreement with the amounts of organic matter as deduced from the TG traces of the 21815



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Figure 2. TG and DTG signals of bulk Glu (β = 5 °C min 1).

resulting solids. On fundamental grounds, one might object to calling these data adsorption isotherms because neither the pH nor the ion strength of the solution was fixed (this is because the addition of other species, whether electolytes or an acid base couple for buffering, often leads to complications due to coadsorption). However, the experimental pH was essentially the same for all adsorption points at 3.1 to 3.3, i.e., close both to the PI of Glu and the PZC of the silica support. Two domains can be distinguished in Figure 1: below an equilibrium concentration of about 0.39 mol L 1, adsorbed quantities seem to follow a type III isotherm according to the IUPAC classification. (This classification, and the BET treatment of adsorption data, are most commonly used for adsorption from the gas phase, but they are also found in geochemistry studies when dealing with multilayer adsorption and surface precipitation; see for example, ref 34). This would indicate a low affinity between aqueous Glu and the silica surface; indeed the molar enthalpy of adsorption for Glu/SiO2 has been reported as slightly positive at +3 kJ mol 1,35 and the predominant species is the H2Glu( zwitterion which has no net electrostatic interaction with the silica surface, almost neutral itself (pH close to PZC). On the other hand, multilayer growth on the few adsorbed Glu molecules would be more favorable thermodynamically, and the Glu multilayers formed at higher concentrations might be viewed as nuclei for surface-induced precipitation. Above 0.39 mol L 1, data points are more scattered and the adsorbed amount would seem to decrease with increasing solution concentrations, which is impossible on the basis of a simple adsorption equilibrium. Probably at these concentrations quite close to the nominal solubility limit of glutamic acid (about 0.5 mol L 1), more complicated phenomena occur, possibly with slow kinetics; we did not investigate this point further. XRD. For the IWI series (on Aerosil 380), the peaks of β-Glu become visible when the Glu loading exceeds 0.35 mmol g 1 (β-Glu must of course be understood as the β-polymorph of solid crystalline glutamic acid and does not refer to a β-amino acid.) Thus, the bulk phase that precipitates in the presence of silica is the same that would be obtained from a concentrated aqueous solution of glutamic acid; this can be contrasted to the case of glycine, where the presence of the same silica surface favored the precipitation of the β polymorph, instead of the normally more stable α form.28

Figure 3. Thermograms (DTG) of Glu/Aerosil IWI samples. The figure next to each thermogram denotes the Glu loading (mmol g 1). The blank refers to an Aerosil powder that was contacted with distilled water and dried at 60 °C.

TG. Figure 2 shows the thermogram of bulk glutamic acid.

Three thermal events are observed, with a global shape very similar to that reported by Nunes and Cavalheiro36 (the peak maxima are only shifted to higher temperatures in their work because of their use of a faster temperature ramp). The first one at 197 °C is very sharp, endothermal (coincident with sample melting), and corresponds to the elimination of one water per Glu molecule. We can assign it to the formation of PyroGlu according to the first reaction in Scheme 1. The formation of PyroGlu after the sharp thermal event could be confirmed by IR (PyroGlu bands at 1717, νCdO in COOH, and 1654 cm 1, amide band) and NMR (δ 13C = 31.1, 56.6, and 175.6 ppm) when heating was carried out more slowly and stopped after partial transformation. It also corresponds to the conclusions of a recent in-depth study of the thermal evolution of glutamic acid.37 It was impossible however to separate this event from the pyrolytic degradation of PyroGlu that starts immediately afterward (large, almost athermal DTG peak at 233 °C, followed by a second, exothermal event at 483 °C). The TGs of several Glu/SiO2 samples are illustrated in Figure 3. Up to a loading of at least 0.35 mmol g 1, the thermograms of Glu/Aerosil exhibit two well-defined peaks at low temperatures, about 110 120 and 150 160 °C, respectively. They can be quantified after subtraction of the support contribution; up to 0.35 mmol g 1, within experimental error, each one corresponds to the elimination of one water molecule per Glu (see Figure 1 in Supporting Information) They are well separated from further thermal degradation events that peak above 300 °C and lead to total elimination of the organic matter. These higher temperature events may be interesting in their own respect, but we will not be concerned with them in further discussion, except as a means to quantify total adsorbed Glu in the SA procedure. The thermograms of higher-loading samples gradually become similar to those of bulk glutamic acid. It must be noted that no significant difference in the shape of the TGs was observed 21816



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Figure 4. Effect of temperature ramp (heating rate, β) on DTG maxima. Left: Effect of β on maximum temperature (Tmax) for events 1 and 2. Symbols: inverted triangle, Glu/SiO2 IWI 0.04 mmol g 1; diamonds, 0.10 mmol g 1; open squares, 0.20 mmol g 1; circles, 1.0 mmol g 1 Right: Kissinger analysis for samples Glu/SiO2 IWI 0.10 mmol g 1 and 0.20 mmol g 1 .

between the IWI and SA deposition procedures; what matters is the overall Glu loading in the solid sample. Under the same conditions and on the same silica support, other amino acids including Gly, Ala, Leu, and Phe show a single low-temperature thermal event at about 165 °C that has been assigned to the formation of substituted DKPs (diketopiperazines, cyclic dimers).25,27 As was mentioned in the introduction, the possibilities of thermal condensation are more diverse with Glu because of the additional carboxylic acid function in the side chain. Several assignments of the two thermal events are compatible with the quantification, and the available evidence will be discussed below. At any rate, it must be emphasized that the thermal transformations of adsorbed Glu occur at a much lower temperature than in bulk glutamic acid. Therefore, there exists a population of glutamic acid molecules which can be activated by the silica surface, and it saturates between 0.3 and 0.35 mmol g 1. This loading corresponds to the one beyond which β-Glu crystals become visible in the XRD. When it is exceeded, the TG traces start to ressemble those of bulk β-Glu crystals. In order to better characterize these thermal events, we have investigated the effect of variable heating rates β (Figure 4, left). Both peaks are expectedly displaced to higher temperatures with increasing β. The lower-temperature DTG peak maximum (Tmax 1) shows the same behavior irrespective of the loading, while for the second peak, Tmax 2 depends on the loading, in a counterintuitive way because the peak occurs at higher temperatures when the loading is increased. The independence of Tmax 1 upon loading suggests that it corresponds to a kinetically first-order process. To check this, we subjected the data to a Kissinger analysis, plotting ln(β/(Tmax)2) as a function of 1/Tmax38,39 (see refs 40 and 41 for recent applications in the field of amino acids and peptides). The resulting plot is linear, and the slope corresponds to an activation energy of 136.5 kJ mol 1 for this event. We also checked the possibility of reproducing the observed thermal events outside the TG apparatus. The question is important because some of the characterization techniques we used (e.g., solid-state NMR) could only be conveniently applied after ex situ heating and reexposure to the atmosphere, certainly a noninnocent step for dehydration equilibria which should be sensitive to the water vapor pressure. Therefore, portions of

sample Glu/Aerosil IWI 0.2 mmol g 1 were heated to 130 °C or 175 °C in a drying oven under dry nitrogen and subjected to thermogravimetric analysis. TG traces showed that the first thermal event (denoted as T1 above) was selectively suppressed after ex situ heating to 130 °C. After ex situ heating to 175 °C, the response was not so clear. No definite peak was apparent at either T1 or T2, but the TG trace still showed a mass loss significantly superior to that of the blank ( Figure 2 in Supporting Information). We conclude that reexposure to ambient atmosphere at RT did not cause a significant reversibility of the first thermal transformation, while the second one could be partially reversible. Finally, it must be mentioned that the first thermal event can be suppressed in another way. When the starting solution was titrated with NaOH up to pH 9, where the monoglutamate species HGlu predominates, prior to IWI deposition on silica, the T1 event was not present on the TG of the dried solid (see Figure 3 in Supporting Information). On the other hand, titration with HCl down to pH 1.8, where the cation H3Glu+ predominates, did show the T1 event but at a higher temperature than usual (131 °C instead of 118 °C). NMR. NMR of the Unactivated Glu/SiO2. At first sight, as shown in Figure 5, the natural abundance solid-state 13C spectrum of a typical Glu/SiO2 IWI is very similar to that of bulk glutamic acid. Both exhibit five peaks that can be readily assigned to the five nonequivalent carbons of the Glu molecule. In solution, the chemical shifts of the corresponding peaks are very sensitive to the acid base speciation.42 The peak positions for crystalline Glu do not precisely correspond to any of the acido-basic species of the aqueous solution as indicated in Table 1; this is certainly due to the existence of a specific network of H-bonds between the individual molecules, that can shift the resonances by several ppm. This would constitute an interesting research topic in itself as evidenced by the many studies of these effects for the simplest amino acid, glycine (refs 43 and 44 and references therein). Thus, at first sight, the main species in the IWI samples would be a form close to bulk β-glutamic acid. However, upon a closer look, a small additional component appears to be present as a shoulder in the methylene region, at 30.5 ppm. To check this, we have recorded the NMR spectrum of a much lower loading Glu/ SiO2 IWI (0.027 mmol g 1), which necessitated the use of 13Cenriched Glu to obtain spectra in a reasonable accumulation 21817



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Figure 5. 13C CP-MAS NMR spectra of bulk glutamic acid (β form, bottom), Glu/SiO2 IWI 0.2 mmol g 1, natural abundance (middle), and Glu/SiO2 IWI 0.027 mmol g 1, 97% enriched in 13C (top). Left: carbonyl region; right: methylene region.

Table 1. Species

13

C Peak Maxima (ppm) for the Different Carbon Atoms of Glu and Supported Glu, Compared with Those for Aqueous

sample

C° carboxylic

bulk β-Glu Glu/SiO2 0.2 mmol g

1

Glu*/SiO2 0.027 mmol g

1

Cα

Cβ methylene

Cγ methylene

Cδ carboxylic

178.2

54.85

26.1

28.2

180.2

178.2

54.85

26.05

28.1 + sh.

180.2 180.1

178.2, 173.95

54.85

25.95

27.95, 30.5

HGlu , aq

175.0

55.4

27.6

34.2

181.7

H2Glu(, aq H3Glu+, aq

174.2 172.0

54.85 53.0

26.3 25.75

30.9 30.3

177.7 176.75

Figure 6. 13C CP-MAS NMR spectra in the carbonyl region of Glu*/ SiO2 IWI 0.027 mmol g 1, 97% enriched in 13C, unactivated (bottom), heated under nitrogen up to 130 °C (middle), and heated under nitrogen up to 175 °C (top).

time. The signal previously present as a shoulder is now predominant in the CH2 region, and a new signal is present in the carboxylate region at 173.95 ppm, while the other signals are almost unaffected. These new solid-state signals are closer to the resonance positions of the H2Glu( zwitterions in water solution than in bulk crystalline Glu. We interpret this as evidence for two Glu populations: a small amount of zwitterionic Glu molecules adsorbed on the silica surface (minority species), which beyond a certain saturation loading give way to small β-Glu crystallites. Deconvolution of the signals would indicate the saturation limit as about 0.016 mmol g 1, i.e., 0.025 molecules nm 2, but this figure must

be taken with caution because the spectra were obtained using CP-MAS which is not necessarily quantitative. Recall that in the SA series of samples, it was supposed that a very small number of adsorbed Glu served as nucleation sites for crystallites. We will come back to the interpretation of the minority species in the discussion. The 15N NMR spectrum shows for Glu/SiO2 a single signal at 336.0 ppm corresponding to the terminal ammonium of the Glu zwitterion, quite close to the position observed in bulk glutamic acid ( 337.6 ppm), considering the large chemical shift range of this nucleus. NMR after Thermal Treatments. When Glu/SiO2 IWI 0.2 mmol g 1 is heated (ex situ) to 120 °C, i.e., above the temperature of the first TG event, the initial 13C NMR lines gradually transform to give new signals at +182.3 (weak), 177 (very weak), 56.7, 29.3, and 24.7 ppm. These positions are close to those of PyroGlu, and the assignment was independently checked by depositing the latter molecule on the SiO2 support. The weakness of the two signals in the carbonyl region is an artifact due to inefficient 1H 13C polarization transfer in the CP experiments; indeed, a single-pulse spectrum showed similar intensities for all five lines, but it needed long recycle delays and correspondingly long accumulation times. The thermal evolution of the low-loading, isotopically enriched Glu*/SiO2 IWI 0.027 mmol g 1 was similar as illustrated in Figure 6 for the carbonyl region. Figure 6 also shows that ex situ heating to 175 °C, which should be above the temperature of the second TG event, does not induce any significant modification of the 13C spectrum. We also recorded the 15N NMR spectra of the same sample. Upon heating to 120 130 °C, the signal of the terminal ammonium 21818



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Figure 7. Transmission mode IR spectra of a self-supported pellet of Glu/SiO2 IWI 0.2 mmol g 1, in vacuum. Left: after successive heating in situ at 50 °C (a), 80 °C with immediate interruption (b), 80 °C for 5 min (c) and 15 min (d), 90 °C for increasing times up to 17 min (unlabeled). Right: Same pellet after further heating at 120 °C with immediate interruption (a) and then 120 °C for 20 min, 140 °C and 160 °C (unlabeled).

Figure 8. Transmission mode IR spectra of a self-supported pellet of Glu/SiO2 IWI 0.2 mmol g 1, in vacuum (continuation of Figure 6), in the two regions containing the amide and imide bands after heating at (a) 140 °C for 20 min, (b) 180 °C for 15 min, and (c) 250 °C for 5 min.

at 336 ppm was replaced by a thin line at 256.7 ppm, i.e., in the amide region. The spectrum of the same sample subjected to further heating up to 175 °C was not significantly different (single sharp line at 255.8 ppm). IR. We recorded the transmission IR spectra of a selfsupported Glu/SiO2 in situ after heating under vacuum at various temperatures (it was later checked that a pellet heated under dry air exhibited exactly the same evolution). They are shown in Figure 7. The original spectrum (50 °C) is quite similar to that obtained by ATR for Glu at the silica/water interface (cf. supra), with bands at 1407 (νsym CdO), 1507 (δsym HNH), 1634 (δas HNH), and 1721 cm 1 (νCdO of COOH). The νasym CdO is not visible for adsorbed Glu, and the δas HNH interferes with the bending mode of adsorbed water if the latter is not completely removed, but the spectrum still remains compatible with H2Glu( zwitterions at this point. When the sample was heated up to 80 °C, most of the above bands disappeared in about 15 min to be replaced by a new pattern dominated by bands at 1682 and 1745 cm 1, with a sharp feature at 1465 cm 1 and a weaker band at 1420 cm 1; outside the range represented, a medium band was

also present at 3420 cm 1. These features may be attributed to PyroGlu molecules.45,46 We independently checked this attribution by depositing PyroGlu on the silica support (breathing mode at 1469, amide band at 1688, νCdO of the COOH at 1751, νN H at 3420 cm 1). Upon further heating, the only change observed is the progressive disappearance of the band at 1510 cm 1 which is complete at 120 °C. At this point all the observed bands may be attributed to PyroGlu modes. Conversely, at no time during the heating process did we observe the bands of the linear dipeptide Glu-Glu. Heating the same sample ex situ in a drying oven (under dry air flow) at 120 °C for 24 h resulted in a spectrum almost superimposable to the final spectrum in Figure 7, with intense bands at 1739 and 1675 cm 1, and smaller features at 1467 and 1420 cm 1 (see Figure 4 in Supporting Information). Further treatments of the self-supported pellet used for Figure 7 at higher temperatures led to a gradual transformation of the IR spectrum, a few stages of which are shown in Figure 8. The PyroGlu bands, including the N H stretching and amide vibrations, disappear while new features become apparent at 21819



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Table 2. Main Diagnostic Peaks in ESI-MS of Desorption Solution from Glu/Aerosil 0.2 mmol g m/z, expt

m/z, calcd

deviation

130.04978

130.04987

0.00009

148.06067

148.06043

+0.00024

152.03160

152.03187

0.00027

241.08124

241.08190

0.00066

259.09303

259.09246

+0.00057

281.07473

281.07441

+0.00032

Scheme 2. The PyroGlu PyroGlu Covalent Dimer

1712 and 1380 cm 1, with a less well-resolved increase in intensity in the 1780 1800 cm 1 region. These features are close to those reported for the imide function in succinimide: νCdO at 1793 (weak) and 1718 (strong) cm 1, C N C stretch (tentative) at 1390 cm 1.47 49 These changes occur at somewhat higher temperatures and/or more slowly than the thermal transformation in TG, but this may be an effect of the very different experimental setup. In 1985, Macklin and White50 studied by IR the evolution of PyroGlu deposited on a silica powder and heated to 200 °C. They observed changes in the IR spectra which were quite similar to those reported here, but interpreted them as the consequence of a covalent, acyl-type bond formation with the silanols of the silica surface (R (CdO) O Si). This interpretation has however been invalidated by further research on supported amino acids, including molecular modeling which showed that such a reaction should be thermodynamically unfavorable51,52 unless special defects such as D2 four-membered rings are present,53 which is unlikely for hydrated silicas. Desorption after Thermal Treatments. We have attempted to analyze the organic matter in the thermally treated samples by desorption followed by HPLC of the resulting solutions. It appears that quantitative desorption is not easy to obtain, because TGs of the solid samples after the desorption treatments still show the presence of organic matter. This holds both for desorption with distilled water and with CaCl2 solutions, the procedure used by Bujdak and Rode in several papers on the thermal reactivity of supported amino acids.54 61 We must keep in mind therefore that some of the species formed in the thermal treatments might not be found in the desorption solutions. With this cautionary note in mind, when samples heated to 120 °C or 130 °C were contacted with a 0.1 M CaCl2 solution for 30 min, pyroglutamic acid desorbed in significant amounts, as was expected from the results of solid-state characterization. It is noticeable that if desorption was prolonged up to 5 h, both pyroglutamic and uncondensed glutamic acid were observed in the solution, as though slow opening of the lactam ring occurred in the silica suspension. This was unexpected because the PyroGlu molecule is indefinitely metastable in aqueous solutions.

1

IHN Heated at 175 °C

species C5H7NO3H+ (PyroGluH+) C5H9NO4H+ (GluH+, corresponding to species noted H3Glu+ in aqueous solution) C5H7NO3Na+ (PyroGlu 3 3 3 Na+) C10H12N2O5H+ (PyroGlu PyroGlu 3 3 3 H+) C10H14N2O6H+ (PyroGlu 3 3 3 H+ 3 3 3 PyroGlu)

C10H14N2O6Na+ (PyroGlu 3 3 3 Na+ 3 3 3 PyroGlu)

When the same treatment was applied to the sample heated to 180 °C, the main component of the desorption solution was still PyroGlu, but there was a minor HPLC peak at high retention times that did not correspond to any of the available references (in particular, it is not the linear dimer Glu-Glu). DESI and ESI Mass Spectrometry. Attempts were made to characterize the adsorbed molecules directly on the solid samples by DESI using a 80:20 (vol) water methanol mixture as the desorbing fluid. These were unfruitful because only very weak intensity signals were obtained in this way. Therefore, we decided to carry out desorption ex situ in the same way that was done prior to HPLC analysis. The desorption solution from the 180 °C heated sample was analyzed by ESI. The ultrahigh resolution that can be reached using the orbitrap FT/MS instrument allows us to obtain molecular mass measurements accuracy better than 5 ppm. The diagnostic peaks displayed by the ESI mass spectra are presented in Table 2 together with their attribution. The gas-phase formation of sodium adducts is a well-known artifact in ESI, the presence of Na+ cations being due to traces of salts naturally present in the solution, or in the glassware. We could verify that (PyroGlu 3 3 3 Na+ 3 3 3 PyroGlu) (peak at around 281.07) is a nonspecific dimer, because the intensity of the corresponding peak decreased much upon dilution of the solution.62 Two possible structures could be proposed for this dimer, the first one involving un-ionized PyroGlu, the second one involving a zwitterionic salt form (see Figure 5 in Supporting Information). In opposition, PyroGlu PyroGlu (peak at around 241.08, Scheme 2) corresponds to a species that was actually present in the desorption solution. Interestingly, the mass defaults observed are similar for the three diagnostic peaks assigned to PyroGlu species. Note also that the PyroGlu PyroGlu covalent dimer appears as a protonated species rather than being cationized. This difference may be attributed to the close proximity of the carboxylic acid function to the nitrogen atom of the lactam ring which does not allow salt formation. Additional Experiments on Different Supports. We only undertook a complete characterization of supported products on Aerosil silica, but because the TG traces showed a simple fingerprint of the two thermal transformation steps, we checked if the latter could also be observed on other inorganic supports (see Figure 6 in Supporting Information). This was indeed the case on the mesoposous silica MCM-41, but the quantification of the second thermal transformation was significantly inferior to one water molecule per Glu. On the phyllosilicate clay mineral montmorillonite, and possibly also on Laponite (dubious due to the strong peak of physisorbed water elimination), apart from the thermal degradation peaks at T g 300 °C, only a single weak and broad thermal event was observed at a comparatively high temperature (182 °C on montmorillonite). 21820


The Journal of Physical Chemistry C When Glu was deposited on Ludox colloidal silica, starting from the commercial solution that had a pH of 9, the TG was similar to that of Glu/Aerosil pH 9, with a single peak at 158 °C. It was expected therefore that titrating the initial suspension to a low pH, similar to the natural pH of Glu/Aerosil suspensions, would also give the familiar TG profile with two low-T events. In fact this was not the case: the low-T event at around 120 °C that we assigned to PyroGlu formation was never observed on Ludox (Figure 7 in Supporting Information).

’ DISCUSSION Initial Interaction of Glu with the Silica Support. The

available data do not all refer to the same state of the Glu/SiO2 system. Quantitative adsorption data from HPLC (Figure 1) in the SA deposition procedure concern the silica/aqueous solution interface. They indicate a weak affinity between the Glu species and the hydrated silica surface, in keeping with the finding by Churchill et al.12 that quartz (a silica polymorph) only adsorbs amino acids strongly when pH(PZC) and pI differ significantly, which is not the case here. In the absence of significant net electrostatic interactions, the Glu species, predominantly H2Glu( at natural pH, probably interact through hydrogen bonding. They are in competition with water molecules for adsorption on silica sites, and they are little modified by the adsorption process as shown by ATR. Multilayer adsorption starts at moderate concentrations, forming nuclei for Glu crystallites. The total amount adsorbed at the silica/water interface never exceeds 0.3 mmol/g. The other solid-state characterization techniques could only be applied after a drying step (oven drying at 60 °C). In particular, samples obtained through the IWI procedure were only characterized after this step. In this procedure, where all the introduced Glu is forced to remain in the final solid samples, large bulk Glu crystals are observed for loadings above 0.35 mmol g 1. On the other hand the Glu nuclei postulated for samples obtained by the SA procedure, which should not be altered by drying, are not detected by XRD, probably because their organization is imperfect. Thus, in both procedures, there seems to exist a limiting loading of 0.3 to 0.35 mmol g 1 which represents the maximum amount of Glu that can be in significant interaction with the silica surface. Any amount in excess of this will result in bulk Glu precipitation during the drying step, either independently of the silica or on the preexisting nuclei. Solid-state NMR provides interesting complementary information. For a 0.2 mmol g 1 loading, the majority species has 13C chemical shifts indistinguishable from bulk β-Glu. Since 13C chemical shifts are good indicators of the H-bonding state of amino acids,28,43 this means that Glu molecules in the nuclei locally form the same type of H-bond network as in β-Glu crystals, even though they are less well-organized. However experiments with 13C-enriched Glu also revealed a minority species which we estimated to about 0.016 mmol g 1. Its chemical shifts are very close to those of H2Glu( in aqueous solution, and one can wonder if this is coincidental. In fact, highsurface silica powders may retain very high amounts of water, depending on the drying conditions, that can amount to several water layers on the surface; indeed one can sometimes speak of a supported water solution phase even though the sample appears as a dry powder, and in the Glu-LDH system a large amount of Glu was observed as quasifree species in a surface fluid film.5 However, in our samples, after drying at 60 °C, TG indicates that

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Figure 9. Different species of Glu that can be postulated under saturation loading. Above saturation, large Glu crystallites form in addition.

the amount of retained water is limited to 5 8% of the dry mass, corresponding to four to six molecules of water per nm2, i.e., less than one physical monolayer of water. Furthermore, if the total amount of water in the oven-dried samples was assimilated to a Glu-saturated solution, this would account for only 2% of the minority species. These rough calculations lead us to believe that the latter really corresponds to an adsorbed species, directly interacting with the silica surface (and probably also with a few water molecules). If the minority species corresponds to Glu directly interacting with the surface, it is tempting to hypothesize that the majority species are the Glu molecules in the multilayer, i.e., in the nuclei. There would then be 10 to 12 of them per nucleus, a figure that is of course still speculative. Figure 9 depicts this view of Glu adsorption. Thermal Reactivity. Glu/SiO2 is an interesting system where two well-defined thermal condensation events can be induced in separate temperature intervals, around 110 120 °C and around 150 160 °C, respectively. The first thermal event is undoubtedly the formation of PyroGlu through an internal cyclization reaction. The overwhelming presence of PyroGlu in thermally activated solid samples is evidenced by IR, solid-state 13C NMR, and analysis of the desorption solution. The first product of Glu/SiO2 thermal activation is therefore PyroGlu, as is also the case for bulk glutamic acid, but the kinetics of the Gluf PyroGlu transformation reaction is different. It is not surprising that it should obey a first-order kinetics because it has a monomolecular ratedetermining step. Its formation, like other previously observed thermal condensation reactions of amino acids, is activated by the silica surface in the sense that it occurs at moderate temperatures and is therefore well-separate from pyrolysis reactions. However, the term activation is rather vague and deserves some comments. In water solutions, the cyclization of glutamic acid to PyroGlu is thermodynamically unfavorable and does not occur to any measurable extent irrespective of the presence or absence of a catalyst. It does happen however both in bulk crystalline Glu and when the molecule is supported on silica. In both cases, the activity of water can be very low, and the dehydrating condensation reaction is simply driven to the right by virtue of LeChâtelier’s rule; it is noticeable that whatever the temperature ramp, PyroGlu formation is observed on TG traces immediately after completion of the elimination of physisorbed water. The cyclization occurs at considerably lower temperatures in SiO2-supported Glu (118 °C) as compared to bulk Glu (197 °C). Therefore, the silica surface also exerts a kinetic effect on the reaction by working as a catalyst. In other words, one energy barrier in the reaction mechanism has to be lowered by the silica surface. Our data would indicate that the effective Arrhenius activation energy for amide condensation from glutamic acid in the presence of silica is 136.5 kJ mol 1. 21821


The Journal of Physical Chemistry C Scheme 3. Two-Step Reaction Mechanism for Gas-Phase Glutamic Acid Condensation to PyroGlu

The nonbiological formation of peptide bonds has been studied in the gas phase, especially with respect to the relative likelihood of concerted vs two-step reaction mechanisms.63 Theoretical activation energies in the 160 180 kJ.mol 1 range were reported. Scheme 3 is an adaptation of the well-known two-step peptide formation mechanism to the case of PyroGlu formation. The mechanism involves a nucleophilic attack by the amine group, and thus it is written starting from a neutral form of the amino acid (unprotonated amine, protonated carboxylic acid functions) which is indeed the most stable isomer in the gas phase. In water solutions, on the other hand, the zwitterion (H2Glu(, with ammonium and carboxylate groups) is more stable. Unsurprisingly, the corresponding reactions have been little studied in aqueous solutions because they are thermodynamically unfavorable. However, the inverse reaction, amide hydrolysis, has been the object of several mechanistic studies64 69 which should be relevant because of the principle of microreversibility. A complicated picture emerges, with different reaction mechanisms in neutral, basic, and acidic conditions, and the possibility of assisted mechanisms where the water molecules play an important role in stabilizing reaction intermediates and/ or transition states. Because of this complexity, in the present state of knowledge, it is difficult to reason from first principles, but we can still review the available evidence regarding the mechanism of silica catalysis. First, the catalytic condensation seems to necessitate globally neutral Glu species because it was completely inhibited with samples prepared at basic pH where HGlu is the predominant species and more sluggish with samples where H3Glu+ is the predominant species. Under what form are the Glu molecules when they condense to PyroGlu: zwitterion H2Glu( or uncharged H2Glu? In fact, in bulk crystalline glutamic acid, the species present are H2Glu( zwitterions, and no condensation occurs as long as Glu remains in the solid state. When the melting temperature is reached, the particular lattice of H-bonds that stabilized the zwitterionic form breaks down and the neutral form H2Glu becomes predominant. It is observed that condensation to PyroGlu immediately starts upon melting. One could then surmise that the increase in reactivity of the adsorbed glutamic acid molecules is due to their transition from zwitterionic to neutral form upon dehydration, the neutral molecules being the reactive species: for the related glycine/silica system, it is quite possible that the removal of physisorbed water causes such a transition. Dehydration would then play a 2-fold role, thermodynamic (making the reaction favorable) but also kinetic (providing a new reaction pathway with a smaller activation barrier). However, things are probably not as simple. The possibility of low-barrier cooperative mechanisms involving the participation

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of water molecules, already mentioned earlier for solution studies, has been confirmed in the adsorbed state by Rimola et al.70 Thus, complete dehydration might actually have a detrimental kinetic effect. Of course there is a definite possibility that silanol groups directly act on the electron density of the adsorbed Glu molecule to facilitate nucleophilic attack. Further molecular modeling studies would be in order to choose between these different possibilities. Data available from DFT molecular modeling on the apparently simple (glycine + glycine)/ aluminosilicate system70 should serve as a warning against any premature conclusions on the detailed mechanism, because different Br€onsted acidic catalyzed amide bond formation pathways considered in this case had activation barriers that differed by a factor of 3. For the second thermal event occurring at about 160 °C, its identification is not so clear-cut. The PyroGlu molecules formed in the first step could in principle undergo several different further condensations. TG results consistenly indicate the loss of one water molecule per Glu (or per PyroGlu) in the second step as well. As shown in Scheme 1, this stoichiometry could correspond to the formation of the dimer PyroGluDKP, but formation of a linear polyimide would hardly be distinguishable on this basis. What is surprising is the lack of any noticeable difference in the NMR spectra of the samples activated at 120 °C and 175 °C (except for a small shift in 15N NMR). NMR is an ex situ technique: prior to NMR characterization, the samples have to be reexposed to ambient air with a non-negligible water vapor pressure. This may lead to a reversal of the dehydration reaction, especially if the silica surface acts as a catalyst. The TGs of the ex situ heated sample used for NMR did not show a thermal peak as clear as that of the pristine sample, but neither were they superimposable with the blank, so the evidence was mixed. In situ IR, on the other hand, did show some bands attributable to the imide function (Figure 7). This may not be fully convincing because there is some temperature lag with regard to the TG event considered (the temperatures of imide band appearance remained, however, inferior to those of the high-temperature pyrolysis/degradation events in TG). The results of desorption followed by analysis in the liquid phase yield partial support to the imide hypothesis because a dimer with an imide function was observed by ESI (PyroGlu PyroGlu). This molecule is a minority species in the solution but probably because it is hydrolyzed at the water silica interface as demonstrated by the disappearance of the corresponding HPLC peak upon standing. In fact, the TG quantification suggests that PyroGlu PyroGlu could itself be the result of a very quick hydrolysis of PyroGluDKP. The sequence of reactions upon rehydration after drying at 170 180 °C would be as described in Scheme 4. Some comments are in order. First, the supposition of complete thermal transformation to PyroGluDKP relies only on TG quantification. The other techniques only demonstrate, later, more hydrolyzed stages, and furthermore desorption may privilege some species over others in HPLC and ESI-MS. The observation of PyroGlu signals only by NMR suggests that liquid water is not required for the first two steps, which can be caused also by water vapor from the atmosphere. Second, the choice of PyroGluDKP as the initial species after drying is somewhat arbitrary because the available information would also be compatible with a long-chain linear polyimide. We chose PyroGluDKP because (i) the hydrolysis of the linear polymer would probably give other oligomers in addition to PyroGlu PyroGlu and because (ii) the formation of a long chain polymer would be 21822



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Scheme 4. Reaction Sequence upon Reexposure to Water of the 175 °C Activated Samples

expected to become more facile as the Glu loading is increased, while the TG study shows the opposite. A definite identification of the imide formed would require some sensitive analysis technique to be applied to the dry samples without reexposure to water, perhaps MALDI-TOF using the silica support itself as a matrix. The synthesis of PyroGluDKP has previously been carried out in strongly dehydrating conditions.23 A yield of 60% was obtained in a 5:1 acetic anhydride: pyridine mixture at 110 °C,22 so that its obtention by a simple medium-temperature activation of Glu on silica would be interesting. If one accepts the initial formation of PyroGluDKP, the next question is the nature of the ring-opening reaction that occurs upon rehydration. Parrish and Mathias22 have observed the opening of the 6-ring as well as of the 5-rings upon attack by various nucleophiles according to the experimental conditions. The addition of water in strongly acidic solutions led to preferential opening of the 5-rings, while on silica the 6-ring is preferentially opened. In view of the synthetic interest of the resulting products,71 these solid-state ring-opening pathways will deserve further investigation. Finally, the experiments on different solid supports shed some light on the nature of the active sites that are important for catalysis. TG traces indicate that Glu condensation at low temperatures is facilitated on Aerosil, Ludox, and MCM41 supports, which are covered with silanol groups. In opposition, the events assigned to Glu condensation are less intense and occur at higher temperatures on the two T-O-T phyllosilicates. The latter also exhibit silanols but in limited amounts and only on the particle edges, not on the basal plane. It is reasonable to deduce that Glu activation requires silanols, but not all silanols exhibit the same reactivity as indicated by the different behaviors of Aerosil, Ludox, and MCM41. The surface of silica is in fact more chemically diverse than usually realized as we have stressed in the context of heterogeneous catalyst preparation.72 Both PyroGlu and the hypothesized PyroGluDKP may be considered as activated forms of the initial amino acid. As early as 1985, Macklin and White50 expressed an interest in PyroGlu, where the amine group is protected by internal cyclization, to obtain nonrandom condensation as compared to other amino acids. It has been proposed from bulk copolymerization studies73 76 that glutamic acid could have played a special role in prebiotic thermal peptide formation. The particular role of pyroglutamyl moieties as peptide chain initiators was mentioned as a factor to explain nonrandom thermal polymerization in bulk (molten) amino acid mixtures,74 and recently the growth of regularly alternating Gly-Glu polypeptides was studied in amino acid melts in the presence of alumina.77 These findings show the interest of thermally activated Glu in prebiotic syntheses. In fact, the main thermodynamic problem of abiotic amino acid polymerization is how to provide the free

enthalpy for endergonic amide bond formation. In PyroGlu, there is one amide (lactam) bond per amino acid molecule, i.e., the same amount as in an infinite linear peptide. In PyroGluDKP, there are actually two such bonds per amino acid monomer. This may explain the relative success of wetting-anddrying cycles that mimic climatic variations,78 to obtain linear polypeptides. Pascal et al. have proposed the concept of amino acid overactivation by cyclization79,80 in the frame of a different scenario that involved N-carboxy anhydride obtained by reaction with cyanate. This phrase might actually also apply to what happens to glutamic acid upon drying on the surface. The next step for further study would of course be to determine what happens to overactivated Glu in the later exposure to amino acid solutions, and we are planning to conduct such experiments. The formation of linear peptides is a definite possibility in view of the results reported by Parrish and Mathias22 on the reaction of glycine with PyroGluDKP. The latter reference also shows that PyroGluDKP might be a useful chemical intermediate in organic syntheses, independent of the validity of prebiotic scenarios.

’ CONCLUSION Glutamic acid (Glu) can interact with the silanol groups present in amorphous silica and other related oxides to give weak but specific adducts. At the aqueous solution/oxide interface, competition with water molecules leads to limited Glu adsorption, but this may be followed by nucleation of small multilayer aggregates up to a maximum loading of 0.3 mmol g 1. When the surface is dehydrated by mild thermal treatment, condensation reactions become thermodynamically favorable and two separate condensation reactions are indeed observed at low temperatures, in two well-separated consecutive steps. Both are catalytically activated, no doubt by the effect of the interaction with surface silanols. In the first step, pyroglutamic acid (PyroGlu) forms by internal condensation (lactam formation). Depending on the heating conditions, this may occur at temperatures as low as 85 °C. The second step results in further condensation with imide groups formation. The final product is somewhat hypothetical, but it may be the tricyclic dimer PyroGluDKP. Upon reexposure to higher water activities, the second dehydration step is easily reversible, as the final condensation product may be hydrolyzed by simple exposure to water vapor in ambient air. The reversal of the first step (PyroGlu hydrolysis to Glu) is more sluggish and requires liquid water. PyroGlu, and even more PyroGluDKP, may be considered as activated molecules that could lead to further controlled reactivity in the field of prebiotic chemistry and more generally in organic syntheses. 21823



’ ASSOCIATED CONTENT

bS

Supporting Information. Additional TG traces, IR spectra, and MS schemes as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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