Next, we studied the impact of SNBD fluorogenic labeling in the physicochemical and bioactivity properties of the three CPPs. We confirmed minimal differences between the clogP values of all unlabeled vs labeled peptides (Figure ) and compared their secondary structures by circular dichroism (CD) spectroscopy (Figure ). In these experiments, unlabeled TAT showed a CD spectrum with a negative minimum at ∼190 nm, which is characteristic of random coil structures. This profile was also observed for TAT-SNBD, suggesting that both peptides are unstructured in solution. The CD spectra of penetratin and penetratin-SNBD were both indicative of the presence of α-helical structures, with the characteristic mean residual ellipticity (MRE) minima at 208 and 222 nm. The calculated helicities were 61% for penetratin and 51% for penetratin-SNBD. The unlabeled sC18 peptide and its fluorogenic analogue sC18-SNBD also showed profiles indicative of α-helical structures with calculated helicities of 70% and 78%, respectively. These results suggest that the incorporation of SNBD fluorophores did not substantially alter the secondary structure of the CPPs, which is critical for their cell internalization mechanisms. We also evaluated the biological properties of all peptides by performing haemolysis assays as a bioactivity feature of most CPPs. The three fluorogenic TAT-SNBD, penetratin-SNBD, and sC18-SNBD peptides showed haemolytic activities in the same range as their unlabeled counterparts (i.e., EC50 values for the different peptides: TAT=79±6 µM and TAT-SNBD=53±2 µM; penetratin=34±5 µM and penetratin-SNBD=27±3 µM; sC18=70±4 µM and sC18-SNBD= 43±3 µM) (Figure ). Overall, these results indicated that the physiochemical properties of CPPs were largely retained after labeling with Sbridged benzodiazoles, confirming that this strategy provides a suitable means to directly visualize the cellular uptake and trafficking of native-like peptide structures.