Synthesis of 7-aminocoumarin by Buchwald-Hartwig cross coupling for specific protein labeling in living cells.

The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. To enable minimally invasive studies of proteins in their native context, it is desirable to tag proteins with small, bright reporter groups. Recently, our lab described PRIME technology (for PRobe Incorporation Mediated by Enzymes) for such tagging [1-3]. An engineered variant of Escherichia coli lipoic acid ligase (LplA) is used to covalently attach a fluorescent substrate, such as 7-hydroxycoumarin, onto a 13-amino acid peptide recognition sequence (called LAP, for Ligase Acceptor Peptide) that is genetically fused to a protein of interest (POI) (Figure 1A). The targeting specificity is derived from the extremely high natural sequence specificity of LplA [4]. PRIME was used to label and visualize various LAP-tagged cytoskeletal and adhesion proteins in living mammalian cells. One limitation of the 7-hydroxycoumarin probe used in our previous study is its pH-dependent fluorescence. The 7-OH substituent has a pK a of 7.5 [5] , and the fluorophore is only emissive in its anionic form. Proteins labeled by PRIME with 7-hydroxycoumarin (on the extracellular or luminal side) therefore cannot be visualized in acidic compartments of the cell such as the endosome (pH 5.5-6.5 [6]), where >90% of 7-hydroxycoumarin is expected to be neutral and therefore non-fluorescent. This problem prevents the use of 7-hydroxycoumarin for imaging receptor internalization and recycling, for example. A potential solution is to use 6,8-difluoro-7-hydroxycoumarin (Pacific Blue [5] , Figure 1B), which has a reduced 7-OH pK a of 3.7. We found, however, that our engineered 7-hydroxycoumarin ligase, the W37V mutant of LplA, did not ligate an isosteric Pacific Blue substrate onto LAP efficiently [1]. It is likely that the ligase active site prefers neutral and hydrophobic substrates and therefore rejects Pacific Blue, which is predominantly anionic at physiological pH. An alternative coumarin structure is 7-aminocoumarin, shown in Figure 1B. In contrast to 7-hydroxycoumarin and Pacific Blue, 7-aminocoumarin is expected to be both neutral and highly fluorescent at a wide range of pH values. We also predicted that it would be a substrate for W37V LplA, since it is sterically similar to 7-hydroxycoumarin and is uncharged at physiological pH. The synthesis of the 7-aminocoumarin substrate 6 required a novel route, however. Previous synthetic routes to 7-aminocoumarin derivatives have used either Pechmann [7] or Perkin [8] condensation. The Pechmann reaction condenses aminoresorcinol with β-ketoesters and unavoidably produces 4-alkyl substituted aminocoumarins. Based on …