Efficient and Scalable Synthesis of 4-Carboxy-Pennsylvania Green Methyl Ester: A Hydrophobic Building Block for Fluorescent Molecular Probes

Abstract

Fluorinated fluorophores are valuable tools for studies of biological systems. However, amine-reactive single-isomer derivatives of these compounds are often very expensive. To provide an inexpensive alternative, we developed a practical synthesis of 4-carboxy-Pennsylvania Green methyl ester. Derivatives of this hydrophobic fluorinated fluorophore, a hybrid of the dyes Oregon Green and Tokyo Green, are often cell-permeable, enabling labeling of intracellular targets and components. Moreover, the low pK _a of Pennsylvania Green (4.8) confers bright fluorescence in acidic cellular compartments, such as endosomes, enhancing its utility in chemical biology investigations. To improve access to the key intermediate 2,7-difluoro-3,6-dihydroxyxanthen-9-one, we subjected bis(2,4,5-trifluorophenyl)methanone to iterative nucleophilic aromatic substitution by hydroxide on scales in excess of 40 grams. The key intermediate was used to prepare over 15 grams of pure 4-carboxy-Pennsylvania green methyl ester in 28% overall yield without the use of chromatography. This compound can be converted into the amine-reactive N-hydroxysuccinimidyl ester in essentially quantitative yield for the synthesis of a wide variety of fluorescent molecular probes.

Key words

fluorophore - bioorganic chemistry - chemical biology - molecular probes - fluorine - conjugation

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