An NBD tertiary amine is a fluorescent quencher and/or a weak green-light fluorophore in HS-specific probes.

The thiolysis of NBD piperazinyl amine (NBD-PZ) is highly selective for HS over GSH and has been widely used for the development of many HS fluorescent probes. Whether the NBD amine in HS-specific probes could be a fluorescent quencher should be further clarified, because NBD amines have been used as environment-sensitive fluorophores for many years. Here, we compared the properties of NBD-based secondary and tertiary amines under the same conditions. For example, the emission of NBD-N(Et)2 is much smaller in water and less responsive to changes in polarity than that of NBD-NHEt. The emission of NBD-PZ-TPP is also smaller than that of NBD-NH-TPP both in aqueous buffer and in live cells. In addition, confocal bioimaging signals of NBD-PZ-TPP with excitation at 405 nm and 454 nm are much weaker than that at 488 nm. Based on these results as well as the previous work on NBD-based probes, we discuss and summarize the design strategies and sensing mechanisms for different NBD-based HS probes. Moreover, NBD-PZ-TPP may be a useful tool for reaction with and imaging of mitochondrial HS in live cells. This work should be useful for clarification of the roles of NBD in HS-specific fluorescent probes as well as for facilitating the development of future NBD-based probes.