Oligopeptidization oscillations of binary amino acid mixtures in solution M. Sajewicz1, M. Dolnik2, M. Matlengiewicz1, T. Kowalska1, and I.R. Epstein2 1Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland 2Department of Chemistry, MS 015, Brandeis University, Waltham, MA 02454-9110, USA

Aims Providing experimental evidence of spontaneous oscillatory chiral conversion of L- and D-Phg obtained by chiral HPLC/DAD. Providing experimental evidence (by means of non-chiral HPLC/DAD and HPLC/ELSD) of spontaneous oscillatory peptidization of L-Phg, L-Phe, and L-Phg – L-Phe dissolved in 70% aqueous EtOH and DCM. Devising a theoretical model that reflects basic features of peptidization dynamics.

(+) enantiomer enolate ion (-) enantiomer Physicochemical background Chiral conversion of amino acids can occur according to the following reaction mechanisms: In aqueous solution: (+) enantiomer enolate ion (-) enantiomer P. Belanger, J.G. Atkinson, and R.S. Stuart, Chem. J. Chem. Soc. D: Commun.. 1067-1068 (1969) (b) In non-aqueous solution: (+) enantiomer enol (-) enantiomer Y. Xie, H. Liu, and J. Chen, Int. J. Pharm., 196, 21-26 (2000) Parallel chiral conversion and peptidization can occur according to the following scheme:

Physicochemical background of enolization and polymerization Step 1: Initiation of the free radical reaction chain - ENOLIZATION Step 2: Propagation of the free radical reaction chain Step 3: Termination of the free radical reaction chain – higher molecular weight polymerization products

Physical evidence of enolization with L-Phg and L-Phe as an intermediary step toward chiral conversion 1 mm Irradiation of L-Phg and L-Phe solutions with UV light (λ= 254 nm) results in the appearance of multiple microscopic blinking spots that emit visible light. This fluorescence seems to be connected with the presence of the conjugated phenyl – enol π-electron system of L-Phg and L-Phe, thus confirming the relative stability of the enol form Fig. 1. Multiple blinking spots on the surface of the L-Phg solution in 70% EtOH Fig. 2. Probable mechanism of generating fluorescence, which involves the conjugated phenyl – enol π-electron system of L-Phg and L-Phe

Experimental evidence of the spontaneous oscillatory chiral conversion of L- and D-Phg obtained by chiral HPLC/DAD Apparatus: Gyncotek liquid chromatograph (Gyncotek, Macclesfield, UK) equipped with a Gyncotek Gina 50 model autosampler, Gyncotek P 580A LPG model pump, Gyncotek DAD UVD 340U model diode array detector, and Chromeleon Dionex v. 6.4 software for data acquisition and processing Stationary phase: Chirex 3126 (D)-penicillamine column (1504.6 mm i.d.; Phenomenex, Torrance, CA, USA) Mobile phase: 2mM copper(II) sulfate / water – 2-propanol (95:5, v/v) at a flow rate of 1.0 mL min-1 Single analytical run: 15 min Fig. 3. (a) L- and D-Phg peak height changes as a function of the sample storage time for the chiral HPLC/DAD chromatograms of the L-Phg–D-Phg solution in the 2mM copper(II) sulfate / water – 2-propanol (95:5, v/v) mixture. (b) The time changes of the peak height ratios (hD/hL) for the separated L-Phg and D-Phg peaks as a function of the sample storage time. Time ranges of the enantiomer peak separation and enantiomer peak coalescence are additionally illustrated by the respective chromatogram insets

The first question answered: The HPLC confirmation of correctness of the upper branch mechanism The second question: How about the correctness of the lower branch mechanism?

Experimental evidence of the spontaneous oscillatory peptidization of L-Phg obtained by achiral HPLC/ELSD (b) (a) Fig. 4. Superposition of selected achiral HPLC/ELSD chromatograms registered in the time intervals indicated in the respective figures for solutions of L-Phg in (a) 70% aqueous EtOH and (b) DCM (a) (b) Fig. 5. Peak height changes for the achiral HPLC/ELSD chromatograms of the L-Phg solution (a) in 70% aqueous EtOH and (b) DCM. Retention times (tR) of the peaks are indicated in the respective figures

Experimental evidence of the spontaneous oscillatory peptidization of L-Phe obtained by achiral HPLC/ELSD (b) (a) Fig. 6. Superposition of selected achiral HPLC/ELSD chromatograms registered in the time intervals indicated in the respective figures for solutions of L-Phe in (a) 70% aqueous EtOH and (b) DCM (a) (b) Fig. 7. Peak height changes for achiral HPLC/ELSD chromatograms of the L-Phe solution (a) in 70% aqueous EtOH and (b) DCM. Retention times (tR) of the peaks are indicated in the respective figures

Experimental evidence of the spontaneous oscillatory peptidization of L-Phg – L-Phe obtained by achiral HPLC/ELSD (a) (b) Fig. 8. Superposition of selected achiral HPLC/ELSD chromatograms registered in the time intervals indicated in the respective figures for the solutions of L-Phg– L-Phe in (a) 70% aqueous EtOH and (b) DCM (a) (b) Fig. 9. Peak height changes for achiral HPLC/ELSD chromatograms of the L-Phg–L-Phe solution (a) in 70% aqueous EtOH and (b)DCM. Retention times (tR) of the peaks are indicated in the respective figures

Possible oligopeptide structures Dipeptides: LL LD DL DD Tripeptides: LLL LLD LDL DLL LDD DLD DDL DDD Tetrapeptides, pentapeptides, … etc.

Experimental evidence of the spontaneous oscillatory peptidization of L-Phg obtained by achiral HPLC/MS (b) (a) (d) (c) Fig. 10. Achiral HPLC chromatogram with MS inserts for L-Phg; (a) a freshly prepared sample dissolved in DCM; (b) an aged sample dissolved in DCM; (c) a fresh sample dissolved in 70% aqueous EtOH; and (d) an aged sample dissolved in 70% aqueous EtOH

Experimental evidence of the spontaneous oscillatory peptidization of L-Phe obtained by achiral HPLC/MS (b) (a) (d) (c) Fig. 11. Achiral HPLC chromatogram with the MS inserts for L-Phe; (a) a freshly prepared sample dissolved in DCM; (b) an aged sample dissolved in DCM; (c) a fresh sample dissolved in 70% aqueous EtOH; and (d) an aged sample dissolved in 70% aqueous EtOH

Experimental evidence of the spontaneous oscillatory peptidization of L-Phg – L-Phe obtained by achiral HPLC/MS (a) (b) (c) (d) Fig. 12. Achiral chromatogram with MS inserts for L-Phg– L-Phe; (a) a freshly prepared sample dissolved in DCM; (b) an aged sample dissolved in DCM; (c) a fresh sample dissolved in 70% aqueous EtOH; and (d) anaged sample dissolved in 70% aqueous EtOH

The 1H NMR spectroscopic evidence of spontaneous peptidization ( ) n (b) 3.6 3.4 3.2 3.0 ppm Fig. 13. The 400 MHz 1H NMR spectra of the protons in the CH2 groups adjacent to the nitrogen atom in the (hydroxy)proline ring recorded (a) for the freshly prepared L-Pro–L-Hyp in ACN-d3 – D2O (70:30, v/v), and (b) for the same mixture after 7 days storage time

Experimental evidence of supramolecular aggregation Fig. 15. Raman spectra collected from the center (‘‘white pupil’’) of the concentric pattern shown in Fig. 14a, the dark rim of the pattern, and outside the pattern. Outside: superposed signals of EtOH S(+)-ketoprofen Rim: also superposed signals of EtOH S(+)-ketoprofen, but in lower concentration Center: Pure water. Fig. 14. Microscopic structures registered in the aged samples of S(+)-ketoprofen solution in 70% aqueous EtOH after 1 year storage period. Experimental proof of the emulsion formation M. Sajewicz, R. Wrzalik, M. Gontarska, Ł. Wojtal, D. Kronenbach, M. Leda, I. R. Epstein, T. Kowalska, J. Liq. Chromatogr. Relat. Technol., 32, 1359 (2009)

Theory n1P → E rate = k0P (oligomerization) n2E  →  M                    rate = kuE                    (uncatalyzed aggregation) 2M + n2E  →  3M        rate = ka M2E              (catalyzed aggregation) M  →  products           rate = kbM                   (decomposition) where: P: precursor (e.g., monomeric molecule of amino acid) E: a short oligomer derived from the P M: an aggregate, possibly a micelle, aggregate of several molecules E A more detailed model might include a sequence of elementary aggregation steps to form M: E + E    →  E2 k1 E2 + E    →  E3 k2 … Ei + E    →  Ei+1 ki ... En2-1 + E    →  M kn2-1 …and another set of elementary steps for the formation of the oligomer E from P monomers. M. Sajewicz, M. Dolnik, D. Kronenbach, M. Gontarska, T. Kowalska, I.R. Epstein J. Phys. Chem. A, 115, 14331-14339 (2011)

Fig. 15. Simulated oscillations. Initial concentration of P = 0. 02 M Fig. 15. Simulated oscillations. Initial concentration of P = 0.02 M. Parameter values: n1 = 5, n2 = 8, k0 = 1.5 x 10-5 s-1; ku = 5 x 10-5 s-1; ka= 2.5 x 105 M-2s-1, kb = 5 x 10-3 s-1.  M. Sajewicz, M. Dolnik, D. Kronenbach, M. Gontarska, T. Kowalska, I.R. Epstein J. Phys. Chem. A, 115, 14331-14339 (2011)

Discussion Surprisingly, the spontaneous oligopeptidization of amino acids in neutral abiotic (aqueous and non-aqueous) systems has not attracted the attention of amino acid, peptide, and protein researchers. Also, HPLC analysts typically avoid direct (enantio)separation and quantification of underivatized amino acids. Many arguments, e.g., problems with three ionized amino acid forms, cationic, anionic, and zwitter-ionic, have been offered to justify this omission. Therefore the spontaneous behaviour of amino acids in solution has largely (and perhaps intentionally) been neglected by most researchers. Thus, it is difficult to decide if (and to what extent) commercial amino acid samples are or can be (e.g., in the solid state) contaminated with oligopeptides, which distort amino acid studies and the results thereof. A simple model allowing for oligomerization, micelle formation and micelle-catalyzed aggregation qualitatively reproduces the amplitude and period of the observed oscillations in lactic acid. None of the kinetic parameters of the model have yet been determined experimentally. Extension of the model to the mixtures of amino acids is planned. In our view, the work presented here is important not only from a chemical but also from an evolutionary perspective.

Thank you for your kind attention!