Excitation and emission maximum wavelengths of 6-Carboxyfluorescein (6-FAM) and its synthesis

6-Carboxyfluorescein (6-FAM; CAS 3301-79-9) is a high-purity (98% HPLC) fluorescent dye. It exhibits maximum excitation and emission peaks at 492 nm and 517 nm, respectively. 6-FAM features a fluorescein core with a carboxyl group at the 6-position and a hydroxyl group at the 7-position; it differs from 5-carboxyfluorescein solely in the position of the carboxyl group (6-position vs. 5-position).

Applications

6-FAM serves as a fluorescent labeling probe for DNA termini, enabling the detection of AGT-DNA binding through changes in fluorescence anisotropy and intensity. In studies involving 6-FAM-labeled DNA, AGT binding to saturation resulted in an approximately 40% reduction in fluorescence intensity, regardless of whether the FAM moiety was located at the 5′ or 3′ end of the DNA. 6-FAM binds directly to the active site of AGT, competitively inhibiting both its DNA-binding activity (IC₅₀ ≈ 76 μM) and DNA-repair activity (IC₅₀ ≈ 63 μM), thereby representing a novel class of reversible, non-covalent AGT inhibitors. Furthermore, 6-FAM, alongside other polycyclic aromatic compounds, can serve as a lead compound for the development of AGT inhibitors designed to enhance the efficacy of chemotherapy[1].

Method of Use

6-FAM is covalently attached to the 5′ or 3′ end of an oligonucleotide via chemical methods (such as NHS ester reaction) to generate 6-FAM-labeled DNA. The labeled DNA is then incubated with AGT protein (for 1 hour at 4°C) in a buffer solution (10 mM Tris-HCl, pH 7.6, 50 mM KCl, 5 mM 2-mercaptoethanol), and binding is monitored via changes in fluorescence anisotropy or intensity. Additionally, the fluorescence intensity of 6-FAM is sensitive to pH and polarity; upon binding to AGT, the emission intensity decreases by approximately 40%, and the dye becomes protected against acrylamide quenching, indicating its entry into an internal protein site.

Preparation of 6-FAM

6-Carboxyfluorescein is prepared by the condensation of resorcinol with 4-carboxyphthalic anhydride in methanesulfonic acid, followed by isolation via recrystallization from methanol or an ethanol-hexane mixture[2]. The specific reaction route is as follows:

References

[1] MANANA MELIKISHVILI; Michael G F; David W Rodgers. 6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O6-alkylguanine DNA alkyltransferase[J]. DNA Repair, 2011, 10 12: Pages 1193-1202. DOI:10.1016/j.dnarep.2011.09.007.

[2] Y. UENO; K. B; G Jiao. Preparation of 5- and 6-Carboxyfluorescein[J]. Synthesis-Stuttgart, 2004, 28 1: 2591-2593. DOI:10.1055/S-2004-829194.