## 2 Results
### NMR Analysis of the Solution Behavior of Tritrpticin  
As has been shown previously, the \({}^{1}\)H NMR spectra of Tritrp are complex because in aqueous solution, the peptide exists with several slowly interconverting conformations. In our experience, this is most easily detected without serious peak overlap by studying the imino-protons around 10 ppm of the three Trp indole groups in the peptide [30; 36; 37]. This \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline  & \multicolumn{6}{c}{**PAO1 (BM2)**} \\ \cline{2-7}  & \multicolumn{2}{c}{**MIC at 600 nm**} & \multicolumn{2}{c}{**MBIC (CV at 595 nm)**} & \multicolumn{2}{c}{**MBEC (CV at 595 nm)**} \\ \cline{2-7}  & **LD50** & **LD90** & **LD50** & **LD90** & **LD50** & **LD90** \\ \hline Tritrp-Lys & 32 & 32 & 32 & 32 & 128 & 128 \\ \hline Tritrp-Orn & 16 & 16 & 16 & 32 & 64 & 128 \\ \hline Tritrp-Dab & 16 & 16 & 16 & 16 & 32 & 64 \\ \hline Tritrp-Dap & 8 & 16 & 8 & 16 & 16 & 64 \\ \hline \hline \end{tabular} Note: MIC: minimum inhibitory concentration; MBIC, minimum biofilm inhibitory concentration; MBEC, minimum biofilm eradication concentration.  
\end{table} 
Table 6: The activities measured by the crystal violet staining method for the Tritrp-Lys derivatives against _P. aeruginosa_ PAO1. Figure 6: The MIC (**A**), MBIC (**B**) and MBEC (**C**) values for the tritrpticin-Lys derivatives against _P. aeruginosa_ PAO1 were assessed by the crystal violet (CV) assay for the measurement of inhibition of biofilm formation and eradication of biofilm cells, respectively. The biofilm cells were rinsed, stained with 0.1% crystal violet for 15 min and then rinsed again. The CV was released from biofilm cells by the addition of 95% ethanol and quantified by reading on a spectrometer at 595 nm. Three replicates were run for each sample, the values were averaged and the error bars represent the standard deviation (\(\pm\)SD). complexity disappeared when the peptide was bound to membrane mimetics, suggesting that a single conformer is found for the membrane-bound AMP. Similar results for Tritrp binding to various membrane mimetics have been reported by other researchers [54]. As tritrp- and Arg-rich peptide there is the potential that the formation of cation-pi interactions, which are strongly favored to occur for Trp and Arg sidechains [55; 56; 57], could lead to forming dimeric or multimeric peptide species. Therefore, we wanted to investigate if such intermolecular interactions occurred and contributed to the spectral complexity. \({}^{1}\)H NMR spectra were recorded at four different peptide concentrations (shown in Figure 7) to determine if there were any concentration-dependent changes in the spectral appearance. They all show complex spectra with multiple peaks, and it is noteworthy that the ratio of the peak intensities is maintained--as are the peak linewidths--suggesting that it is unlikely that such intermolecular complexes are formed. We also recorded \({}^{1}\)H NMR spectra for the Lys-substituted form of the peptide, as Lys-Trp cation-pi complexes are highly unlikely to form in aqueous solution [56] These spectra showed similar multiple conformations and peak distributions as the Arg-containing peptide (for details see [37]), further supporting this notion. To study if some of the Trp amino-NH peaks showed exchange behavior, we subsequently recorded the 2D ROESY spectra (see Figure 8). These results clearly show that there are interconverting species, which are related to Pro cis-trans isomerism. As can be seen, the three most intense peaks (labeled A, B and C) all have a partner peak that is positioned downfield (by \(<0.1\) ppm) from these main peaks. Because these six peaks collapse into three when Pro9 is substituted by Ala [37], we can ascribe these to the trans and cis peptide backbone structures of the Trp-Pro-Phe bond, where about 70% is in the trans form and 30% is in the cis form. The ratio of cis to trans around a regular X-Pro bond in a linear peptide is normally between 6 and 10%, and hence we have a much higher ratio.