CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum

Abstract

Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool

“…The design of the quantification platform has two components: the tagging of secreted polyketides through engineered biosynthesis with an alkyne moiety, followed by a click reaction with an azido fluorogenic probe (Scheme 1). Fluorogenic probes are endowed with a functionality that suppresses fluorescence 17–20 . Its transformation during the click reaction creates a new functionality that no longer quenches the fluorescence of the underlying system, resulting in a fluorescence enhancement 17–20 .…”

“…The fluorescence signal of the XZ1 culture was ~2-fold higher than that of the medium-only control (Table S4), demonstrating the success of the fluorogenic reaction albeit with a limited response range. We then evaluated the performance of additional azido fluorogenic probes, including di-pegOF ( 4 ), CalFluors 580 ( 5 ), and CalFluors 647 ( 6 ) 17 (Figure 1b). Compared to 3 , 4 – 6 showed significant fluorescence enhancement (>10-fold) upon the incubation with XZ1 supernatant (Table S4).…”

Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation

“…2B). Using the biorthogonal probe ω-alkyne palmitoyl coenzyme A (alk–palm–CoA), we were able to detect and quantify N-Ras palmitoylation using a novel fluorogenic probe CalFluor 4883334, which is essentially non-fluorescent until it participates in 1, 3-dipolar cycloaddition (click) reaction with an alkyne-containing moiety (Fig. S1).…”

Modern Synthetic Avenues for the Preparation of Functional Fluorophores

“…Bertozzi and co‐workers described the synthesis of “Calfluor” fluorogens with emission maxima covering the entire visible spectrum (Figure 7 A) 54. Conveniently, Calfluors are internally quenched by azide groups so that their fluorescence emission is turned on after a click reaction with suitable alkynes.…”

“…Right: Gel analysis of BSA–DIBAC and OVA–Nor after incubation with either Syd‐630, Tz‐504, both reagents simultaneously, or no reagent (−). Reproduced with permission from the American Chemical Society54 (A), Wiley‐VCH56 (B), and the Royal Society of Chemistry57 (C).…”