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Adsorption and Thermal Condensation Mechanisms of Amino Acids on Oxide Supports. 1. Glycine on Silica Ming Meng,† Lorenzo Stievano, and Jean-Franc¸ ois Lambert* Laboratoire de Re´ activite´ de Surface, UMR CNRS 7609, Universite´ Pierre et Marie Curie, case courrier 178, 4 place Jussieu, 75252 Paris Cedex 05, France Received July 23, 2003. In Final Form: October 29, 2003 Glycine was adsorbed on the surface of a well-defined silica from aqueous solutions of variable concentrations and pHs. The adsorbed molecules were characterized using middle-IR and UV-vis-NIR spectroscopies. Except at the lowest pH (2.0), they were predominantly present on the surface as zwitterions. Two successive deposition mechanisms were evidenced with increasing glycine concentration. At low concentrations, glycine is specifically adsorbed on silica surface sites, probably through its NH3+ moiety. The pH dependence suggests that these sites may be silanolate groups (=Si-O-). At higher concentrations, specific adsorption sites are saturated and surface-induced precipitation of β-glycine is observed. The thermal reactivity of adsorbed/deposited glycine was then investigated by thermogravimetric analysis, in situ diffuse reflectance IR spectroscopy, and thermoprogrammed desorption coupled with mass spectrometry. Adsorbed glycine molecules react to form peptide bonds at a temperature considerably lower than that for bulk crystalline R-glycine. The main reaction product is the cyclic dimer diketopiperazine, with no evidence of the linear dimer. The activation mechanism is not diffusionally limited; the formation of “surface acyls”, previously proposed for related systems, has not been evidenced here. These findings are of relevance for the evaluation of prebiotic peptide synthesis scenarios.
1. Introduction The adsorption of amino acids on the surface of metals or oxides has attracted a lot of attention for several decades,1-6 as it is of great significance for a number of applications, such as solid-phase peptide synthesis,7 development of organic mass spectrometry,8 medical implants, and biomedical sensors.9,10 Moreover, the study of the mechanism of peptide bond formation and chain elongation on silica, alumina, or clay contributes to a better understanding of prebiotic chemical evolution. In fact, clays and other oxides were present in large amounts on the prebiotic earth crust after the formation of the hydrosphere and may have played an important role in the process of chemical evolution. Glycine, because it is the simplest amino acid, is often regarded as a model for the study of these systems. Several studies have been performed on the glycine/silica system, where adsorption was carried out either from aqueous solutions or from the gas phase, by chemical vapor * Corresponding author. Tel: (33) 1 44 27 55 19. E-mail:
[email protected]. Fax: (33) 1 44 27 60 33. † Current address: Department of Catalysis Science & Technology, School of Chemical Engineering, Tianjin University, Tianjin 300072, P. R. China. (1) White, D. H.; Kennedy, R. M.; Macklin, J. Origin Life 1984, 14, 273-278. (2) Bujda´k, J.; Le Son, H.; Yongyai, Y.; Rode, B. M. Catal. Lett. 1996, 37, 267-272. (3) Lo¨fgren, P.; Krozer, A.; Lausmaa, J.; Kasemo, B. Surf. Sci. 1997, 370, 277-292. (4) Bujda´k, J.; Rode, B. M. React. Kinet. Catal. Lett. 1997, 62, 281286. (5) Kalra, S.; Pant, C. K.; Pathak, H. D.; Mehta, M. S. Indian J. Biochem. Biophys. 2000, 37, 341-346. (6) Munsch, S.; Hartmann, M.; Ernst, S. Chem. Commun. 2001, 19781979. (7) Merrifield, R. B. Angew. Chem., Int. Ed. Engl. 1985, 24, 799810. (8) Benninghoven, A.; Kempken, M.; Kluesener, P. Surf. Sci. 1988, 206, L927-L933. (9) Thull, R. Med. Prog. Technol. 1982, 9, 119-128. (10) Lundstro¨m, I.; Salaneck, W. R. J. Colloid Interface Sci. 1985, 108, 288-291.
deposition (CVD).1,4,11,12 One of the first investigations was carried out by Rohlfing and co-workers,13 who observed the condensation of amino acids on the bare surface of silica at temperatures higher than 100 °C. Later on, it was reported that during a cyclic heating/ drying/wetting process of glycine solutions in the presence of dispersed oxides, some oligopeptides, such as glycylglycine (Gly-Gly), the cyclic dimer diketopiperazine (DKP), and triglycine, are produced on silica and alumina.4,12,14-17 The formation of DKP from the monomer appears to be easier on silica, while on alumina chain elongation seems to take place more efficiently. Basiuk and co-workers have also obtained oligopeptides and other products after amino acid condensation from vapors on silica or alumina.18-23 Generally, the nature of the condensation products strongly depends on reaction conditions, such as the reaction temperature, the amount of water, the reaction time, and the chemical nature of the adsorbate and adsorbent. Although the effects of several reaction parameters on the activity and selectivity for amino acid polymerization (11) Macklin, J. W.; White, D. H. Spectrochim. Acta, Part A 1985, 41A, 851-859. (12) Bujda´k, J.; Rode, B. M. J. Mol. Evol. 1997, 45, 457-466. (13) Rohlfing, D. L.; McAlhaney, W. W. BioSystems 1976, 8, 139145. (14) Bujda´k, J.; Eder, A.; Yongyai, Y.; Faybikova, K.; Rode, B. M. J. Inorg. Biochem. 1996, 61, 69. (15) Bujda´k, J.; Le Son, H.; Rode, B. M. J. Inorg. Biochem. 1996, 63, 119-124. (16) Bujda´k, J.; Rode, B. M. Origin Life Evol. Biosph. 1999, 29, 451461. (17) Bujda´k, J.; Rode, B. M. J. Inorg. Biochem. 2002, 90, 1-7. (18) Basyuk, V. A. Russ. J. Theor. Exp. Chem. 1990, 26, 89-93. (19) Basyuk, V. A.; Gromovoi, T. Y.; Glukhoi, A. M.; Golovaty, V. G. Origin Life Evol. Biosph. 1991, 21, 129-144. (20) Basiuk, V. A.; Gromovoy, T. Y.; Glukhoy, A. M.; Golovaty, V. G. Origin Life Evol. Biosph. 1991, 21, 129-144. (21) Basiuk, V. A.; Gromovoy, T. Y.; Chuiko, A. A.; Soloshonok, V. A.; Kukhar, V. P. Synthesis 1992, 449-451. (22) Basiuk, V. A.; Gromovoy, T. Y. Collect. Czech. Chem. Commun. 1993, 59, 461-466. (23) Basiuk, V. A.; Gromovoy, T. Y. React. Kinet. Catal. Lett. 1993, 50, 297-303.
10.1021/la035336b CCC: $27.50 © 2004 American Chemical Society Published on Web 01/08/2004
Adsorption of Amino Acids on Oxide Supports
have been carefully studied, the condensation mechanism is not known with certainty. It has been proposed that the formation of an amino acid ester with silanol groups from the silica surface (tSi-O-CO-R) plays an important role in the activation of peptide bond formation. Except in special systems,1,4,11 however, the implication of such esters in the mechanism of condensation remains hypothetical. In the glycine/silica system for instance, the information on interactions was obtained in most cases at a macroscopic level, which means that little or no spectroscopic data are available. In addition, the effect of all the adsorption parameters has not been yet systematically investigated: for example, only limited work has been carried out on the adsorption of glycine on silica from aqueous solution at variable pH.2 In the present work, we performed a series of experiments of glycine adsorption on silica from aqueous solutions at different initial concentrations and pHs. To elucidate the interaction between glycine and silica during adsorption and thermal condensation, several techniques, such as differential thermogravimetry (DTG), X-ray diffraction (XRD), thermoprogrammed desorption and mass spectrometry (TPD-MS), Fourier transform infrared (FT-IR) spectroscopy, UV-vis-NIR spectroscopy, and in situ diffuse-reflectance infrared Fourier transform spectroscopy (DRIFTS) were employed. The collected data are discussed in order to clarify the role of silica in the process of peptide bond formation. 2. Experimental Section 2.1. Adsorption Procedure. In a typical adsorption experiment, 1.5 g of silica (Aerosil 380 from Degussa, BrunauerEmmett-Teller (BET) surface area of 380 ( 20 m2/g from N2 physisorption at 77 K, PZC ) 2) was immersed in 50 mL of an aqueous solution of glycine of the suitable concentration and stirred at room temperature for 2 h. After the adsorption, the solid was separated by centrifugation, washed once with 50 mL of distilled water for 5 min, and finally dried under a vacuum at room temperature. For the experiments carried out at variable pH, the pH of the solution was adjusted using appropriate amounts of aqueous solutions of HNO3 (1.2 M) or NaOH (2 M). In general, the solid samples will be noted as Gly/SiO2-xMpHy, where x refers to the glycine concentration in the adsorption solution (in mol L-1; specification omitted when x ) 0.5 M) and y refers to the adsorption pH (specification omitted when y ) 6). In parallel, and to identify the products of transformation of adsorbed glycine, the linear dipeptide Gly-Gly and the cyclic dimer DKP were adsorbed on silica from aqueous solutions. The resulting solid samples are denoted Gly-Gly/SiO2 and DKP/SiO2. 2.2. Powder XRD. X-ray powder diffraction patterns were collected on a Siemens D 500 X-ray diffractometer (Cu KR, wavelength ) 1.5406 Å). The scanning range was set between 10° and 70° (2θ) with a step size of 0.02°. 2.3. DTG. Differential thermal gravimetry experiments were performed in air or dinitrogen flow (120 mL/min) and with a heating rate of 5 °C/min on a TG/DTA 220 thermal analyzer (Seiko Instruments Inc.) For each experiment, 6-8 mg of sample was used. Before the measurement, the sample was purged in the flow for about 1 h in order to eliminate most physisorbed water and obtain a stable baseline. 2.4. UV-Vis-NIR. The UV-vis-NIR spectra were recorded on a Cary 5E UV-vis-NIR spectrometer, which permits the measurement of solid samples by a diffuse-reflectance light path. The samples were first ground to a fine powder and then installed into the sample cell (a 5 cm diameter disk), and their surface smoothened. The measurements were carried out in air at room temperature. For samples needing pretreatment at higher temperatures, the heating was performed ex situ in air flow (120 mL/min) with a heating rate of 5 °C/min, and the sample was kept at the required temperature for 10 min. 2.5. TPD-MS. TPD-MS measurements were carried out on a HPR 20 mass spectrometer (Hiden Co.) in a flow of pure oxygen
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Figure 1. DTG profiles of glycine/silica as a function of initial concentration in the adsorption solution: (a) raw SiO2 support, (b) Gly/SiO2-0.01M, (c) Gly/SiO2-0.03M, (d) Gly/SiO2-0.08M, (e) Gly/SiO2-0.20M, and (f) Gly/SiO2-0.50M (adsorption pH ) 6). or helium, with a flow rate of 120 mL/min from room temperature to 600 or 700 °C and a heating rate of 5 °C/min. For each measurement, 150 mg of sample was employed, and the signals corresponding to the (m/z) ratios from 2 to 75 were recorded simultaneously. The main MS peaks were observed at m/z ) 2 (H2+), 17 (OH+, possibly NH3+), 18 (H2O+), 27 (HCN+), 28 (CO+, H2CN+), 29 (CHNH2+), 30 (+CH2-NH2), and 44 amu (CO2+).24,25 The signals of m/z ) 52 ((CN)2+) and 75 (glycine molecular ion) are usually very weak. 2.6. FT-IR. FT-IR spectra were recorded in air at room temperature on a Vector 22 infrared spectrometer (Bruker). The spectra were measured in KBr pellets with a concentration of the sample in the pellet of a few percent by weight. The absorption of a pellet of pure KBr was used as the background. 2.7. In Situ DRIFTS. Diffuse-reflectance FT-IR measurements were carried out on an IFS 66v spectrometer (Bruker), using a sample cell (Spectratech Co.) allowing in situ treatments in gas flow up to 800 °C. The sample was mixed with ultrafine diamond powder26 in a ratio of 1% of sample by weight (pure glycine) or 10 wt % (Gly/SiO2 samples). Before the measurement, the cell was purged in air flow until the band at 2300-2500 cm-1, typical of the absorption of CO2, totally disappeared. The heating was performed in air flow with a heating rate of 5 °C up to 460 °C. The heating ramp was interrupted by several plateaus of about 20 min at constant temperatures during which the spectra were recorded. The absorption of pure diamond at each temperature was used as the background.
3. Results 3.1. Adsorbed Amounts from Thermogravimetric Analysis. The most accurate way to estimate the amounts of adsorbed glycine is by thermogravimetry under air flow. The DTG patterns of glycine/silica samples show rather sharp peaks that are absent in the trace of the naked support and can be quantified by integration. In addition, all organic matter is eliminated when heating to 600 °C, as confirmed by elemental analysis. Figure 1 and Figure 2 show the DTG signals of glycine/silica prepared from solutions at different concentrations and different pHs, respectively. Several peaks are visible in two separate regions: a first, sharp endothermic peak appears at ∼140220 °C, whereas a broad group of peaks show up in the ∼240-400 °C region. The first peak is absent in high-pH samples (Figure 2d,e). (24) Junk, G.; Svec, H. J. Am. Chem. Soc. 1963, 85, 839-845. (25) Lo¨fgren, P.; Krozer, A.; Chakarov, D. V.; Kasemo, B. J. Vac. Sci. Technol. 1998, A16, 2961-2966. (26) TeVrucht, M. L. E.; Griffiths, P. R. Talanta 1991, 38, 839-849.
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Figure 2. DTG profiles of glycine/silica as a function of pH in the adsorption solution: (a) Gly/SiO2-pH2, (b) Gly/SiO2-pH4, (c) Gly/SiO2-pH6, (d) Gly/SiO2-pH9, and (e) Gly/SiO2-pH10.5.
Figure 3. Adsorption isotherm of glycine on silica at room temperature and natural pH (pH ) 6).
Table 1. Amounts of Glycine Adsorbed on Silica as a Function of Initial Concentration in Solution Gly/SiO2 sample
weight loss for DTG peaks (g/g SiO2) peak 1
peak 2
C ) 0.5 M 0.092 (195)a 0.088 (287) C ) 0.2 M 0.025 (178) 0.060 (271) C ) 0.08 M 0.005 (158) 0.012 (271) C ) 0.03 M 0.002 (155) 0.005 (281) -4 C ) 0.01 M
