Spontaneous condensation of amino acids in binary mixtures M. Sajewicz1, M. Dolnik2, T. Kowalska1, and I.R. Epstein2 1Institute of Chemistry, University of Silesia, 9, Szkolna Street, 40-006 Katowice, Poland 2Department of Chemistry, MS015, Brandeis University, Waltham, MA 0245-9110, U.S.A. I. Introduction In our earlier liquid chromatographic studies, we have demonstrated that single amino acids can undergo an oscillatory chiral conversion (e.g., [1,2]} and an oscillatory condensation (e.g., [3,4]). This experimental evidence has been obtained with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). II. Aim The aim of this study was to investigate if two amino acids could exert mutual influence on the process of peptidization and if heteropeptides could emerge. III. Oscillatory chiral conversion – HPLC evidence Oscillatory chiral conversion has been experimentally demonstrated multiple times by means of TLC {e.g., [1,2]), but we managed to demonstrate this process by means of HPLC in a semi-continuous way only once (in a binary L-Phg and D-Phg mixture in solution) [5]. The reason is that the chiral HPLC stationary phase dedicated to the enantioseparation of amino acids (with D-penicillamine packing) is both very expensive and chemically very unstable. The results obtained are summarized in Fig. 1. IV. Oscillatory condensation Oscillatory peptidization has been investigated with aid of three amino acid pairs dissolved both in aqueous and non-aqueous solvents, as listed below. L-Pro – L-Hyp (dissolved in 70% aqueous MeOH); L-Pro –L-Phe (dissolved in pure acetonitrile); L-Hyp- L-Phe (dissolved in pure acetonitrile). Experiments performed with each amino acid pair provided evidence of mutual interactions in the course of condensation and of heteropeptide formation. Selected examples are given below of mutual interactions and heteropeptide formation. Scheme 1. Parallel chiral conversion and condensation of an amino acid Figure 1. L-Phg and D-Phg peak heights as function of sample storage time for the chiral HPLC–DAD chromatograms of the L-Phg–D-Phg solution in the mixture of 2 mM copper(II) sulfate–water–2-propanol (95:5, v/v). Time ranges of the enantiomer peak separation and the enantiomer peak coalescence are illustrated by the chromatogram insets. Evidence of mutual amino acid interactions in the condensation process A: HPLC evidence Evidence of mutual interaction is seen in the synchronized non-linear changes of the two amino acids’ concentrations, as expressed by the corresponding changes of the respective chromatographic peak heights B: HPLC evidence C: HPLC evidence Figure 2. (a) Chromatographic concentration profile of L-Pro–L-Hyp in 70% aqueous MeOH registered at 22oC with an ELSD detector (qualitatively, the chromatogram remained unchanged throughout the experiment); (b) Time series of chromatographic peak heights for L-Pro and L-Hyp in 70% MeOH at 22oC. Plots are for peaks at tR = 6.90 min (L-Hyp) and 7.30 min (L-Pro) Figure 3. Time series of chromatographic peak heights for L-Pro–L-Phe in acetonitrile (registered with ELSD detector); 1, L-Pro; 2, L-Phe; 3, the main peptidization product. . Figure 4. Time series of chromatographic peak heights for L-Hyp–L-Phe in acetonitrile (registered with ELSD detector); 1, L-Phe; 2, L-Hyp; 3, the main peptidization product. . Evidence of heterocondensation product formation A: HPLC evidence B: HPLC evidence C: MS evidence Figure 7. Chromatogram of an L-Pro–L-Hyp solution taken with the LC/MS system after 7 days storage in 70% aqueous MeOH. Insets show mass spectra registered at the maxima of the respective chromatogram peaks. Peaks corrresponding with m/z values higher than molar weights of Pro and Hyp demonstrate the abundant yields of the condensation products. Figure 5. Time window showing a synchronized drop in concentrations / peak heights of (1) L-Pro and (2) L-Phe, and a rise in concentration / peak height of (3) the main condensation product Figure 6. Time window showing synchronized rise in concentrations / peak heights of (1) L-Phe and (2) L-Hyp, and a drop in concentration / peak height of (3) the main condensation product. V. Theory Oscillator 1: n11P1 → E1 rate = k01P1 (oligomerization) n21E1 → M1 rate = ku1E1 (uncatalyzed aggregation) 2M1 + n21E1 → 3M1 rate = ka1M12E1 (catalyzed aggregation) M1 → products rate = kb1M1 (decomposition) Table 1. Parameters used for simulation of plots Parameter 6 0scillator 1 Oscillator 2 P0 (initial conc., M) 0.1 0.06 n1 5 3 n2 8 6 k0(s-1) 1.5 x 10-5 2.5 x 10-5 ku(s-1) 5.0 x 10-5 8.0 x 10-5 ka(M-2s-1) 2.5 x 105 kb(s-1) 5.0 x 10-3 7.0 x 10-3 kc(M-2s-1) 2.5 x 104 or 0 .5 x 104 or 0 Oscillator 2: n12P2 → E2 rate = k02P2 (oligomerization) n22E2 → M2 rate = ku2E2 (uncatalyzed aggregation) 2M2 + n22E2 → 3M2 rate = ka2M22E2 (catalyzed aggregation) M2 → products rate = kb2M2 (decomposition) Cross catalysis: 2M1 + n22E2 → 2M1 + M2 rate = kc1M12E2 (1 catalyzes 2) 2M2 + n21E1 → 2M2 + M1 rate = kc2M22E1 (2 catalyzes 1) (a) (b) Pi = monomer of amino acid I, Ei = oligomer of amino acid I, Mi = catalytically active aggregate of amino acid I, VI. Conclusions Condensation reactions in binary solutions of amino acids naturally raise the question of mutual interactions taking place in such processes and of the possible formation of heteropeptides. In semi-continuous HPLC experiments, synchronization among the concentration changes of the two amino acids and the main condensation product were observed, which allows us to conclude that mutual interactions really take place and that heteropeptides are effectively formed. Additional evidence is furnished by mass spectrometry, which supports the notion of heteropeptide formation by allowing us to ascribe particular mass values to the hetero-oligopeptides. A theoretical model has been developed that allows for varying degrees of interaction between a pair of amino acids undergoing oscillatory oligomerization. Even though oversimplified, it qualitatively mirrors the predominant features of the amino acid pair behavior observed in our experiments. (c) (d) VII. References [1] M. Sajewicz, R. Piętka, A. Pieniak, T. Kowalska, Acta Chromatographica, 15, 131-149 (2005) [2] M. Sajewicz, R. Piętka, A. Pieniak, T. Kowalska, Journal of Chromatographic Science, 43, 542-548 (2005) [3] M. Sajewicz, M. Gontarska, D. Kronenbach, M. Leda, T. Kowalska, I.R. Epstein, Journal of Systems Chemistry, 1:7 (2010) [4] M. Sajewicz, M. Matlengiewicz, M. Leda, M. Gontarska, D. Kronenbach, T. Kowalska, I.R. Epstein, Journal of Physical Organic Chemistry, 23, 1066-1073 (2010) [5] M. Sajewicz, M. Gontarska, T. Kowalska, J. Chromatogr. Sci., DOI:10.1093/chromsci/bmt033 Figure 8. Simulations of oligomer concentrations in a solution initially containing two monomers that undergo oligomerization, uncatalyzed and catalyzed aggregation. Parameters are given in Table 1. (a) No cross catalysis (kc1 = kc2 = 0); The two species behave independently, equivalent to separate solutions. (b) 1 catalyzes 2 (kc2 = 0); (c) 2 catalyzes 1 (kc1 = 0); (d) mutual cross-catalysis. [E1] is upper curve, [E2] is lower curve.