Small-Scale Preparation of Fluorescently Labeled Chemical Probes from Marine Cyclic Peptides, Kapakahines A and F

Abstract

A number of bioactive marine natural products have been isolated so far, but it is still difficult to disclose their modes of action. In this study, we prepared fluorescently labeled chemical probes from the cytotoxic marine cyclic peptides kapakahines A (1) and F (2) to visualize their localization as the first step of the study of their modes of action. We used fluorescent dyes 3a or 3a/b (a 1:1 mixture of 3a and 3b) whose terminal N-hydroxysuccinimide (NHS) group can react with the free amino groups of kapakahines. The fluorescently labeled kapakahine A (Kap A-5-FL, 5a) stained P388 murine leukemia cells and HeLa human cervical cancer cells, while cells treated with fluorescently labeled kapakahine F (Kap F-5-FL, 6a) only weakly stained them. Further analysis of the confocal images of the stained cells with higher magnification (×100) indicated the localization of Kap A-5-FL (5a) in the cells. In this paper, we report the small-scale preparation and a new delivery method of fluorescent probes, as well as the application of these procedures to cell staining.

Preparation and Application of a Chemical Probe for Identifying the Targets of the Marine Cyclic Peptide Kapakahine A

“…In our previous report [ 10 ], the fluorescently labeled kapakahine A in HeLa cells was not found to subcellularly localize with the endoplasmic reticulum or the Golgi apparatus; however, a fluorescent probe of kapakahine E was shown to localize with the Golgi apparatus [ 3 ]. To investigate the subcellular localization of the kapakahine A fluorescent probe in P388 cells—towards which kapakahine A shows cytotoxicity—co-staining with Kap A-5FL ( 2 ) and MitoTracker was carried out.…”

“…Rocha et al synthesized a fluorescent probe comprising kapakahine E conjugated with coumarin and found that the fluorescent probe localized to the Golgi apparatus in PC-3M prostate carcinoma cells [ 9 ]. In a previous study, we prepared a fluorescent probe of kapakahine A [Kap A-5FL ( 2 )] by direct coupling with an FITC derivative with an active ester linker [ 10 ]. Co-localization analysis of Kap A-5FL ( 2 ) and Golgi-binding lectin HPA, or concanavalin A (endoplasmic reticulum marker) in HeLa cells, showed that Kap A-5FL does not localize in the Golgi apparatus or endoplasmic reticulum.…”

The tryptophan connection: cyclic peptide natural products linkedviathe tryptophan side chain

“…Fluorescently labelled kapakahine A has been used to stain P388 murine leukemia cells and HeLa human cervical cells. 131…”

Comprehensive review of α-carboline alkaloids: Natural products, updated synthesis, and biological activities

“…Kapakahines (structures not shown), a large family of cyclic peptides containing an α-carboline skeleton, were isolated from Cribrochalina olemda (marine sponge) (Yeung et al, 1996;Nakao et al, 2003). They were often studied as fluorescently labeled chemical probes (Rocha et al, 2015;Kamihira and Nakao, 2021) or anti-malarial agents (Goto et al, 2021).…”