Amino Acid Functionalized Imdazolium Salts and Their Silver(I) and Gold(I) N-Heterocyclic Carbene Complexes Tina H.T. Hsu, Ivan J.B. Lin Department of Chemistry, National Dong Hwa University, Shoufeng, Hualien 974, Taiwan. Abstract: The amino acid functionalized imdazolium salts (Cn,gly,im-X) and their silver(I) and gold(I) NHC complexes [M(Cn,gly,imy)2][X] (n = 6, 16 ; M = Ag, Au ; X = Cl, NO3 ) have been synthesized and characterized. Subsequently, the antibacterial activity of imdazolium salts Cn,gly,im-Cl (n = 6, 16) was evaluated against three Gram negative bacteria (A. baumannii, P. aeruginosa and E. coli) and three Gram positive bacteria (S. aureus, S. msrcesenceSS1, and V. parahaemolyticus93 ). Here, we found that the imidazolium salt (C16,gly,im-Cl) possessing excellent antibacterial activity against A. baumannii, E. coli, S. aureus, and V. parahaemolyticus93 strains bacteria. In general, these imidazolium salts possess excellent, broad spectrum antimicrobial activity against Gram positive and Gram negative bacteria dependency on length of the alkyl substituent for activity. However, antibiofilm experiment results show that these imidazolium salts cannot inhibit the biofilm formation. Introduction Bacterial biofilms can cause urethritis, prostatitis, kidney stones, otitis media, dental caries, periodontitis, bad breath and other diseases. They repeatedly will often manifest suddenly, and extremely difficult to cure thoroughly. Under the natural condition, the bacteria have planktonic and the biofilm two kind of growth condition existence. Environment for the resistance to various unfavorable factors, like antibiotics sterilization, per acid either alkali environment, phagocytosis by the host immune cells, single or multiple clumps of bacteria convergence integrated to form a single walk-state cells with the corresponding biofilm. In bacterial biofilms, the bacteria itself represents less than 1 / 3 the size of the remaining space is secreted by the bacteria, "extracellular matrix" of the sticky substance occupies. It is these viscous substances linked to thousands of bacteria. According to the U.S. Centre for Disease Control and Prevention experts estimate that more than 65% of human bacterial infections have relations with bacterial biofilms. Biofilm bacteria for antibiotic resistance with a natural, it's very different resistance mechanisms with a single bacterial. Biofilm formation is associated with the virulence of pathogenic bacteria, and cells included within a biofilm are generally more resistant (up to 1000-fold) to antibiotics and disinfectants than free-living bacteria.1 Biofilms are a major concern in medicine and in medical environments, but also in all domains where their growth constitutes a source of contamination for humans or animals (food industry, cooling towers, water pipes, etc) or leads to economic losses (biofouling of boats and immersed structures, material biocorrosion, etc). The development of antibiofilm strategies is major interest and currently constitutes an important field of investigation. Recently, a great deal of effort has been made toward imidazolium salts directly due to their wide range of potential applications in the chemistry fields, such as ionic liquids, 2 , 3 ionic liquid crystals, 4 N-heterocyclic carbenes, 5 medicinal chemistry, 6,7,8,9,10,11 …etc. Several types of antimicrobial compounds, a series of imidazolium compounds have been reported and they showed significant antibacterial and antifungal activities. 12,13,14,15,16,17,18,19,20,21,22,23 It was found that the type of substituent and the length of the alkyl chain have an important effect on the antimicrobial activities. In recent years, Sedden and et al. reported the antibiofilm activities of 1-alkyl-3-methylimidazolium salts. It catches our attention, because these imidazolium salts have potential applications in antibiofilm martial. However, no reports were focused on amino acid imidazolium cation salts for antimicrobial. Therefore, in this work, we study the antibacterial and antibiofilm Properties of different type functionalized imidazolium salt. Result and Discussion Imidazolium salts The compounds [Cn-im-amino][Cl], the specific notation of En is designated for type E compounds with n numbers of 16. (Scheme 5-1) Scheme 5-1. Structures of imidazolium compounds and their NHCs In this study, we used two methods to measure the minimum inhibitory concentration (MIC) values, method 1 is improve from filter paper disc agar diffusion method and method 2 is common used method. The antibacterial property of these imidazolium salts was tested on three Gram negative bacteria (A. baumannii, P. aeruginosa and E. coli) and three Gram positive bacteria (S. aureus, S. msrcesenceSS1, and V. parahaemolyticus93 ) Antibacterial assay: Method 1 As show in Table 5-1, the MIC values for the compound En of n = 16 is 83 µM for A. baumannii, 2648 µM for P. aeruginosa, 662 µM for E. coli, 83 µM for S. aureus , 1324 µM for S. msrcesenceSS1, 41 µM for V. parahaemolyticus93, respectively. The data No data available means the compounds have no antibacterial effect at the highest concentrations (0.1g/10 mL). Most of these imidazolium salts with longer alkyl chains have significant antibacterial activity, the reason may due to the lipophilicity and bacterial compatibility of the imidazolium salts. We find most of mono-cation imidazolium salts have no antibacterial effect toward P. aeruginosa and S. msrcesenceSS1, and di-cation imidazolium salts have better antibacterial effect for more bacterial. Table 5-1. MIC values for [C16-im-amino][Cl] in µM Bacteria [C16-im-amino][Cl] Gram negative A.baumannii MIC 83 P. aeruginosa MIC 2648 E. coli MIC 662 S. aureus MIC 83 S. msrcesenceSS1 MIC 1324 Gram positive V. parahaemolyticus93 MIC Antibiofilm experiment result 41 Recently, Sedden and et al. reported the antibiofilm activities of 1-alkyl-3methylimidazolium salts. They measure the minimum biofilm eradication concentration. It means in this concentration, the biofilm would be destroying. However, in our purpose we want to know these imidazolium salts can inhibition the biofilm formation or not. After the biofilm formation assays and observation, beside MBC, other concentrations of imidazolium salts have both formation biofilm. Therefore, these imidazolium salts cannot inhibit the biofilm formation. Conclusion In summary, these imidazolium salts possess excellent, broad spectrum antimicrobial activity against Gram positive and Gram negative bacteria dependency on length of the alkyl substituent for activity. We strongly believe that, the amino acid functionalized in imidazolium salts may improve hydrophilicity and possible interactions with proteins and genes to be a bioactive molecule, or increase degradability for imidazolium salts. Further, the anticancer studies of amino acid functionalized NHC complexes are under investigation. Reference 1 D. J. Musk and P. J. Hergenrother, Curr. Med. Chem., 2006, 13, 2163–2177. 2 S. J. Zhang and X. M. Lu in Ionic Liquids: From Fundamentals to Applications, Science Press, Beijing, 2006, pp.149 –193. 3 T. L. Greaves and C. J. Drummond, Chem. Rev., 2008, 108, 206–237. 4 K. Binnemans, Chem. Rev., 2008, 105, 4148–4204. 5 A. M. Voutchkova, L. N. Appelhans, A. R. Chianese and R. H. Crabtree, J. Am. Chem. Soc., 2005, 127, 17624–17625. 6 P. Rajakumar, K. Sekar, V. Shanmugaiah and N. Mathivanan, Bioorg. Med. Chem. Lett., 2008, 18, 4416–4419. 7 D. Demberelnyamba, K. S. Kim, S. Choi, S. Y. Park, H. Lee, C. J. Kim and I. D. Yoo, Bioorg. Med. Chem., 2004, 12, 853–857. 8 M. Hindi, T. J. Siciliano, S. Durmus, M. J. Panzner, et al., J. Med. Chem. 2008, 51, 1577–1583. 9 X. D. Yang, X. H. Zeng, Y. L. Zhang, C. Qing, et al., Bioorg. Med. Chem. Lett. 2009, 19, 1892– 1895. 10 C. G. Fortuna, V. Barresi, G. Berellini and G. Musumarra, Bioorg. Med. Chem. 2008, 16, 4150– 4159. 11 Q. L. Li, J. Huang, Q. Wang, N. Jiang, C. Q. Xia, H. H. Lin, J. Wu and X. Q. Yu, Bioorg. Med. Chem., 2006, 14, 4151–4157. 12 J. Ranke, A. Skrzypczak, G. Lota and E. Frackowiak, Chem. Eur. J., 2007, 13, 3106–3112. 13 J. Pernak and P. Chwala, Eur. J. Med. Chem., 2003, 38, 1035–1042. 14 J. pernak, M. Smiglak, S. T. Griffin, W. L. Hough, T. B. Wilson, A. pernak, J. Zabielska-Matejuk, A. Fojutowski, K. Kita and R. D. Rogers, Green Chem., 2006, 8, 798–806 15 J.Cybulski, A. Wiśniewska, A. Kulig-Adamiak, L. lewicka, A. Cieniecka-Rostonkiewicz, K. Kita, A. Fojutowski, J. Nawrot, K. Materna and J. Pernka, Chem. Eur. J., 2008, 14, 9305–9311. 74 16 A. Busetti, D. E. Crawford, M. J. Earle, M. A. Gilea, B. F. Gilmore, S. P. Gorman, G. Laverty, M. McLaughlin and K. R. Seddon, Green Chem., 2010, 12, 420–425. 17 J. Pernak and P. Chwala, Eur. J. Med. Chem., 2003, 38, 1035–1042. 18 J. Pernak, K. Sobaszkiewicz and J. Foksowicz-Flaczyk, Chem. Eur. J., 2004, 10, 3479–3485. 19 J. Pernak, K. Sobaszkiewicz and I. Mirska, Green Chemistry., 2003, 5, 52–56. 20 K. M. Docherty and C. F. Kulpa, Green Chem., 2005, 7, 185–189. 21 J. Pernak, I. Goc and I. Mirska, Green Chem., 2004, 6, 323–329. 22 L. Carson, P. K. W. Chau, M. J. Earle, M. A. Gilea, B. F. Gilmore, S. P. Gorman, M. T. McCann and K. R. Seddon, Green Chem., 2009, 11, 492–497. 23 S. Kanjilal, S. Sunitha, P. S. Reddy, K. P. Kumar, U. S. N. Murty and R. B. N. Prasad, Eur. J. Lipid Sci. Technol. 2009, 111, 941–948. 75