A concise synthesis of the Pennsylvania green fluorophore and labeling of intracellular targets with O6-benzylguanine derivatives

We report improved syntheses of the Pennsylvania Green and 4-carboxy-Pennsylvania Green fluorophores; the latter compound was prepared from methyl 4-iodo-3-methylbenzoate in a three-pot process (32% overall yield). Chinese hamster ovary cells expressing O-6-alkylguanine-DNA alkyltransferase fusion proteins were treated with Pennsylvania Green and Oregon Green linked to O-6-benzyiguanine (SNAP-Tag substrates). Analysis of living cells by confocal microscopy revealed that Pennsylvania Green derivatives exhibit substantially higher cell permeability than analogous Oregon Green-derived molecular probes.