Azaphthalocyanines: Red Fluorescent Probes for Cations

Fluorescent indicators are important tools for the analysis of biological cations. For example, these probes are useful for the quantification of blood electrolytes, assaying iontransport channels, and monitoring of lithium-drug levels. While the interactions of metal cations with phthalocyanines (Pcs) have previously been studied using absorption, magnetic circular dichroism, and NMR spectroscopy, none of these methods is suitable for in vitro and in vivo studies, owing primarily to a lack of sensitivity, and/or to the incompatibility of the instrumentation with biological systems. In this context, we describe the development of new aza analogues of Pc, azaphthalocyanines (AzaPcs), which upon binding to cations fluoresce in the red region of the visible spectrum. The development of probes that absorb and emit red light at wavelengths greater than 630 nm is highly desirable, especially for in vivo applications, because this increases the depth that the excitation light penetrates, and reduces the effects of light scattering and the autofluorescence of endogenous chromophores that often occurs at shorter wavelengths. Recently, we confirmed that alkylamino-functionalized AzaPcs undergo an ultrafast intramolecular charge transfer (ICT) upon excitation. The ICT relaxation pathway of the excited states results in the quenching of the AzaPc fluorescence (i.e., produces the OFF state). Their fluorescence can be switched ON by protonation of the donor center (i.e., of the amino substituent), if it is sufficiently basic. This property led us to develop a new type of AzaPc sensor based on the coordination of cations to the donor center. In a proof of concept study, an aza[15]crown-5 moiety, which is used as the recognition element, was attached to the periphery of the AzaPc macrocycle. This molecule was further functionalized with bulky tert-butylsulfanyl substituents to eliminate AzaPc aggregation. During the synthesis of the sensors, we observed that their fluorescence quantum yields (FF) are sensitive to substituent groups in the ortho position to the crown ether. Therefore, a series of model compounds with a diethylamino donor center (1a–f, Scheme 1) was syn-