INTRODUCTION: For centuries it is known that silver possesses antimicrobial properties. It was regularly used for the treatment of burns, wounds and several bacterial infections, but with the emergence of antibiotics it was nearly forgotten for almost 50 years^1,2. Nowadays, due to the rising concern regarding infectious diseases induced by multidrug-resistant bacteria, silver has made a remarkable comeback as a potential antimicrobial agent². Therefore, we study the antimicrobial effects of a dipeptide, Boc(βala)₂N₂H₃, that has four potential silver coordination sites and the capability, due to the hydrazine end group, to reduce silver ions to silver nanoparticles (AgNPs). METHODS: To obtain the Boc(βala)₂N₂H₃ dipeptide, a standard liquid phase synthesis was used³. The dipeptide was characterized by ¹H- and ¹³C-NMR, MS-ESI, thermal analysis (TGA, DSC), FT-IR and XRD measurements. Crystals were obtained in DMSO. ¹H-NMR and MS-ESI titrations were performed with Boc(βala)₂N₂H₃ and AgNO₃ in DMSO d⁶ or D₂O. AgNP formation with Boc(βala)₂N₂H₃ and AgNO₃ were recorded overnight by UV-Vis spectroscopy and analysed with TEM, MS-ESI and FT-IR. Different temperature conditions where hereby investigated. Antimicrobial tests were performed with E.coli (ATCC 25922) and Boc(βala)₂N₂H₃. The OD₆₀₀ was taken over a time period of 24 h at 37 °C. RESULTS: Figure 1a represents the time-resolved formation of AgNP at pH 7 and 60 °C, while 1b depicts the obtained AgNPs. Figure 1c shows the crystal structure of Boc(βala)₂N₂H₃. A first trial of silver complexation with Boc(βala)₂N₂H₃ by NMR titration gave a high-field shift for the hydrazine end group of 1.026 (NH₂) and 0.591 ppm (NH). Antimicrobial tests revealed an OD₆₀₀ of 0.7 for just E.coli, 0.68 for E.coli with 1 mg/ml Boc(βala)₂N₂H₃, and 0.55 for E.coli with 2 mg/ml Boc(βala)₂N₂H₃ after 24 h of growth. DISCUSSION & CONCLUSIONS: The peptide crystallizes in the orthorhombic space group P2₁2₁2₁. The packing reveals three H‑bonds of 2.03–2.13 Å, which connect the peptide molecules into 1D-ribbons, which are themselves connected in an anti-parallel fashion via H-bonds between the hydrazine groups to form dimers of ribbons. These dimers of ribbons are arranged in zigzag into layers, which are themselves assembled through short interactions into a 3D structure with a mean distance of ~ 2.3 Å. First tests showed that Boc(βala)₂N₂H₃ is capable to form AgNPs at 40, 50 and 60 °C but not at RT. For 60 °C the highest absorbance was observed meaning that the reaction is temperature dependent. AgNP formation occurred over several hours, reaching in case of 60 °C sizes of 30 to 90 nm. The NMR titration showed that the interaction with silver takes place mainly at the hydrazine end group. The antimicrobial tests indicated a slight decrease in bacterial growth for 2 mg/ml of Boc(βala)₂N₂H₃.

Figure 1: a) UV-VIS of AgNP formation with 1.25 mM Boc(βala)₂N₂H₃ and 1.25 mM AgNO₃ at pH 7 and 60 °C; b) TEM of 30-90 nm big AgNP formed, scale 2 µm; c) Crystal structure of Boc(βala)₂N₂H₃, R = 3.61 %

[1] Sonja Eckhardt et al., Chem. Rev., 2013, 113 (7), 4708–4754 [2] Mahendra Rai, Alka Yadav, Aniket Gade, Biotechnology Advances, 2009, 27, 76–83 [3] Peter S. Petrie, Lee H. Horsley, United States Office, 1958, US2834781 A