1. e-mail: agnieszka-godziek@wp.pl
Investigation of the peptide nanofibers and nanospheres formation by chromatographic and microscopic techniques  
Agnieszka Godziek1, Anna Maciejowska1, Ewa Talik2, Teresa Kowalska1, and Mieczysław Sajewicz1 1Institute of Chemistry, University of Silesia, 9 Szkolna Street, Katowice, Poland 2Department of Physics of Crystals, University of Silesia, 4 Uniwersytecka Street, Katowice, Poland

2. Aims
The main aim of the research project is to investigate, if the mixtures of amino acids (L-Phg-L-Cys; L-Phe-L-Cys; L-Pro-L-Cys) can undergo the oscillatory chiral conversion combined with oscillatory peptidization
Further, it will be investigated, if the spontaneously formed simple peptides can self-organize in peptide nano- and microstructures.
Finding the correlations between chemical structures of the amino acids involved and the peptide structures obtained (peptide nanofibers, nanospheres, nanodiscs, and nanotubes).

3. Oscillatory reaction a) b) Concentration Time Time
Figure 1. Comparison of temporal change of concentration of the substrate [S] and the product [P] in the classical chemical reaction (a) and intermediate products[X], [Y] in the oscillatory reaction (b) [1]
[1] Orlik M., Reakcje oscylacyjne porządek i chaos, Wydawnictwa Naukowo – Techniczne, Warszawa 1996

4. Mechanism of the parallel oscillatory chiral conversion and oscillatory peptidization
Figure 2. Mechanism of the parallel oscillatory chiral conversion and oscillatory peptidization of phenylglycine and cysteine in aqueous media.

5. HPLC-ELSD -results 1mg ∙mL L-Phg + 1mg ∙mL L-Cys
Dissolved in 70 % aquenous methanol
HPLC-ELSD -results
The Varian (model 920) liquid chromatograph;
C-18 column (ThermoQuest Hypersil, 150 mm × 4.6 mm i.d., 5 µm particle size);
ELSD detector,
10-μL aliquots of the investigated amino acid solution;
mobile phase: methanol-water (50:50, v/v);
flow rate: 0.8 mL min-1;
Time of analyses: 15 min
Figure 3. Selected snapshots showing chromatograms of L-Phg – L-Cys (after 1.98, 78.98, , and h storage time), registered with use of the ELSD detector; 1- Cys, 2-Phg, 3-polycondensation product,
Figure 4. Time series of the chromatographic peak heights for L-Phg–L-Cys in aquenous methanol (registered with ELSD detector);

6. Turbidimetry - resultsa) b) c)
Turbidimetry - results
1mg ∙mL L-Phg +
1mg ∙mL L-Cys
Dissolved in 70 % aquenous methanol
Turbidity: Turbidity measurements were carried out with use of the turbidity sensor (TRB-BTA, Vernier Software & Technology, Beaverton, OR, USA);
15 mL aliquots of the amino acids solution was freshly prepared and placed in the instrument vial;
Figure 5. Turbidity changes (in nephelometric turbidity units, NTU) for the mixtures of amino acids solution; a) L-Phg –L-Cys; b) L-Phe –L-Cys; c) L-Pro –L-Cys; 6

7. aged L-Phg – L-Cys solution
HPLC-MS - results
HPLC-MS - results b) b)
m/z]
Proposed structure
-CO-NH
-S-S-
aged L-Phg – L-Cys solution 1.
148
[Cys7 +5H]5+ 6
263
[Cys-Cys +Na]+ 1
279
[Cys16+6H]6+ 14 9
301
[Cys6+Phg2+3H]3+ 7
531
[Cys5]+ 4
976
[Cys9+H]+ 3
1436
[Cys6+Phg6+H]+ 12
1795
[Cys17+H]+ 2.
151
[Phg]+ -
174
[Phg+Na]+
325
[Phg12+5H]5+ 11
542
[Phg8+2H]2+ 3.
205
[Cys6+Phg6+7H]7+
648
[Cys6]+
1972
[Cys10+Phg7+H]+ 16
1mg ∙mL L-Phg +
1mg ∙mL L-Cys
Dissolved in 70 % aquenous methanol a)
The Varian MS-100 mass spectrometer (ESI-MS scan from m/z 100 to 2000, positive ionization, spray chamber temperature 50oC, drying gas temperature 350oC, drying gas pressure 25 psi, capillary voltage 50 V, needle voltage 5 kV);
Figure 6. Mass spectrum recorded for the 70% aqueous methanol solution of the: (a) aged L-Phg – L-Cys solution; (b) possible structures attributed to certain MS signals. 7 7

8. 1mg ∙mL L-Phg +
1mg ∙mL L-Cys
Dissolved in 70 % aquenous methanol
Scanning electron microscope (SEM): The Jeol JSM-7600F SEM; recording of a series of micrographs for the spheres suspended in solution (upon evaporating the solvent to dryness); the two weeks lasting sample ageing period;

9. Nanostructures of L-Phe–L-Pro and L-Cys
A B
Figure 7. Series of micrographs recorded for the (A) Pro-Phe sample after one year ageing period. (a)-(b): Scanning electron micrographs of fibers filtered off from the solution; (c)-(d): scanning electron micrographs of fibers suspended in solution, upon evaporating the solvent to dryness. (a) X800; (b) X13000; ( (c) X1900; (d) X In (b-d), diameters of certain fibers are indicated; (B) Cys sample after two weeks ageing period. (a)-(d) Scanning electron micrographs of the Cys microstructures from solution residue evaporated to dryness; (a) X1000; (b) X3000; (c) X2300; (d) X3300. Size bars make insets in each individual picture.

10. Nanostructures of L-Phg-L-Cys, L-Phe-L-Cys, and L-Pro-L-Cys
Figure 8. Series of micrographs were recorded for the (a-c) Phg-Cys, (d-e) Phe-Cys, (g-i) Pro-Cys samples after two weeks ageing period. Scanning electron micrographs of the amino acids microstructures from solution residue evaporated to dryness; magnification of (a) X500; (b) X1100; (c) X1100; (d) X14000; (e) X8000; (f) X 3500; (g) X5000; (h) X3000; (i) X900;. Size bars make insets in each individual picture.

11. Conclusions
Selected pairs of amino acids (L-Phg-L-Cys; L-Phe-L-Cys; L-Pro-L-Cys) can undergo the oscillatory chiral conversion combined with oscillatory peptidization, what was confirmed by HPLC-ELSD and HPLC-MS
Simple peptides formed by oscillatory peptidization reaction have the ability to self-organize in the peptide nano- and microstructure what was confirmed by microscopic studies.
It was also proved that the cysteine determines the shape of the resulting nanostructures, and the dynamics of self-organization of peptides.

12. Thank you for your kind attention !
Acknowledgement
One author (A.G.) acknowledges the financial support of the DoktoRIS project, co-financed by the European Union within the European Social Found.