Experimental Evidence of a Spontaneous Condensation of Amino Acids and Hydroxy Acids in Aqueous Ethanol Mieczysław Sajewicz, Dorota Kronenbach, Monika Gontarska, Teresa Kowalska Institute of Chemistry, University of Silesia, Katowice, Poland INTRODUCTION It is generally believed that condensation of amino acids and hydroxy acids (resulting in peptides and poly(hydroxy acids), respectively), and also of profens is rather difficult because energetically unfavourable, as it needs a considerable energetic input in order to split one water molecule from each pair of binding compounds [1]. This conviction affects many present-day presumptions regarding, e.g., prebiotic condensation of amino acids resulting in formation of peptides coupled through peptide bonds (NH-C=O). Hence, the experiments are still devised which involve ion irradiation of amino acid solutions to imitate the presumable prebiotic conditions of peptide formation [2]. Moreover, computational simulations are carried out to prove that energetically, polycondensation of amino acids (and hydroxy acids) would be more favourable, if carbon, oxygen, and/or nitrogen atoms in amino acid and hydroxy acid molecules were replaced by their respective analogues, i.e., silicon, sulphur, and phosphorus atoms [3]. AIM In this study, an experimental evidence is provided to prove that condensation with certain low-molecular-weight profens, amino acids and hydroxy acids carried out at ambient temperature can be effortless, if it is carried out in aqueous ethanol (with predominant proportion of alcohol), or in the course of chromatographic procedure on microporous surface of silica gel, evidently due to dehydrating properties of both, ethanol and silica gel. Further it seems that energetically effortless condensation of profens, amino acids and hydroxy acids is inseparably linked with an ability of these acids to undergo a spontaneous oscillatory chiral conversion, first described in papers [4-6]. TLC Demonstration of the phenylglycine ageing product immobile on the starting point of the chromatogram, suggesting a considerable molecular weight change Again, TLC proves to be a simple yet invaluable analytical technique which can provide a decisive experimental evidence to demonstrate that the aged samples of the investigated profens, -amino acids, and - and -hydroxy acids not only undergo chiral conversion: (+)-enantiomer  enol  (-)-enantiomer but some other more profound structural changes as well. BIURET TEST Experimental evidence of peptization of phenylglycine in 70% aq. EtOH Fig. 1. Densitograms showing concentration profiles of R-phenylglycine, S-phenylglycine, and racemic R,S-phenylglycine, depending on storage period with the respective amino acid solutions in 70% aqueous ethanol. Fig. 2. 3D densitogram of R-phenylglycine, S-phenylglycine, and the racemic and scalemic phenylglycine mixtures. Lanes 1 and 2: R-phenylglycine; lanes 3 and 4: racemic R,S-phenylglycine; lanes 5 and 6: scalemic phenylglycine (R:S, 0.75:0.25); lanes 7 and 8: scalemic phenylglycine (R:S, 0.25:0.75); lanes 9 and 10: S-phenylglycine. (a) (b) (c) Fig. 3. Test tubes showing the colour outcome of the biuret test with 70% ethanol solutions of (a) R-phenylglycine, (b) S-phenylglycine, and (c) racemic R,S-phenylglycine after storage for three days. 13C NMR spectroscopy Experimental evidence of condensation of selected carboxylic acids in solutions Ketoprofen monomer condensate Fig. 4. 100 MHz 13C NMR spectrum of L-(+)-lactic acid first dissolved and stored for ten days in pure ethanol, and then recorded in CDCl3 at 25 ºC. Fig. 5. 100 MHz 13C NMR spectrum of S-(+)-mandelic acid first stored for ten days in pure ethanol, and then recorded in CDCl3 at 25 ºC. (a) Schematic presentation of the combined process, involving chiral conversion cum condensation of profens Profens (two parallel reaction steps) (b) Fig. 6. Aliphatic range of the 13C NMR spectra for (a) ketoprofen condensate and (b) ketoprofen monomer. Methyl line at 18.26 ppm in (b) is accompanied by two additional lines at 18.61 and 18.81 ppm in (a). These new lines originate from the methyl groups in the repeating units of the condensate. The signal of methine carbon at 45.31 ppm in pure ketoprofen (b) is flanked by a new small line at 45.55 in (a) coming from the end groups. The new lines at 68.03 and 68.51 ppm in (a) originate from new quaternary carbons formed in the main chain of the polymer. α- and β-Hydroxy acids (two parallel reaction steps) α-Amino acids (two parallel reaction steps) (a) (a) (b) (b) Chiral conversion cum condensation involving enol formation Fig. 7. Aromatic range of the 13C NMR spectra for (a) ketoprofen condensate and (b) ketoprofen monomer. In spectrum (a) two small new peaks appear at 129.09 and 131.65 ppm, respectively, which can be attributed to C-2’ and C-6’, respectively, in the new molecular environment of ketoprofen condensate (as compared with pure ketoprofen, spectrum (b)). Chiral conversion cum condensation involving enol formation REFERENCES [1] A.B. Meggy, J. Chem. Soc., 1444–1454 (1956) [2] J.M. Chiaramello et al., Int. J. Astrobiol., 4, 125-133 (2005) [3] W. Wang, H. Yuan, X. Wang, Z. Yu, Adv. Space Res., 40, 1641-1645 (2007) [4] M. Sajewicz, R. Piętka, A. Pieniak, T. Kowalska, Acta Chromatogr., 15, 131-149 (2005) [5] M. Sajewicz et al., J. Liq. Chromatogr. Relat. Technol., 31, 1986-2005 (2008) [6] M. Sajewicz, M. Gontarska, D. Kronenbach, T. Kowalska, Acta Chromatogr., 20, 209-225 (2008) CONCLUSION We provide an inventive experimental evidence on condensation of selected profens, amino acids and hydroxy acids obtained, e.g., by means of thin-layer chromatography, 13C NMR spectroscopy, and biuret test. The work of two of the authors (M.G. and D.K.) was partially supported by PhD scholarships granted to them in 2008 within the framework of the ‘University as a Partner of the Economy Based on Science’ (UPGOW) project, subsidized by the European Social Fund (EFS) of the European Union.