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 report 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 pKa of Pennsylvania Green (4.8) confers bright fluorescence in acidic cellular compartments such as endosomes, enhancing its utility for 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 of > 40 g. This intermediate was used to prepare over 15 grams of pure 4-carboxy-Pennsylvania Green methyl ester in 28% overall yield without requiring 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.

Hydrophobic resorufamine derivatives: Potent and selective red fluorescent probes of the endoplasmic reticulum of mammalian cells

“…8 However, to our knowledge, only one analogue has been described in a peer-reviewed journal that replaces the phenol of resorufin with a nitrogen atom (in the form of the azide). 30 We previously used Buchwald-Hartwig amination chemistry to convert the fluorophore Penn-sylvania Green 31–33 to N -alkyl rhodols such as 6 , and we envisioned using a similar approach to access N -alkyl resorufamines for studies of the subcellular distribution of these structurally related compounds.…”

Groebke–Blackburn–Bienaymé multicomponent reaction: emerging chemistry for drug discovery

“…Fluorescent dyes have been widely used in the photovoltaic cells, optical sensors, light-emitting diodes (LEDs), fluorescent colourants and biological labels [86,87]. The latter are most often used to prepare various bioconjugates for immunochemistry and histochemistry via modification of antibodies, amino acids, peptides, oligonucleotides, nucleic acids, carbohydrates and other biological molecules [88][89][90].…”

Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells

“…To synthesize these compounds, the fluorophore Pennsylvania Green ( 5 , Scheme 1), [20-21] prepared from 2,7-difluoro-3,6-dihydroxyxanthene-9-one, [22-23] was transformed into iodoarene 6 . This iodoarene was converted to rhodols 3 and 8 via Buchwald-Hartwig cross-coupling with microwave irradiation.…”