Aqueous oxidative Heck reaction as a protein-labeling strategy

Abstract

An increasing number of chemical reactions are being employed for bio-orthogonal ligation of detection labels to protein-bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium-catalyzed oxidative Heck reaction as a new and robust bio-orthogonal strategy for linking functionalized arylboronic acids to protein-bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells. Advantageously, this reaction proceeds under aerobic conditions, whereas most other metal-catalyzed reactions require inert atmosphere.

Keywords: Heck reaction; cross-coupling; homogeneous catalysis; palladium; protein modifications.

Scheme 1

Oxidative Heck reactions with alkenes linked to the protein 4-OT R61C as shown in Table 1.

Scheme 2

Oxidative Heck reactions with small-molecule alkenes and the isomers formed as shown in Table 2.

Figure 1

Fluorescent labeling of protein 4-OT R61C-1 with the aid of the oxidative Heck reaction. A) Mass spectrum demonstrating full conversion of pure 4-OT R61C-1. B) Mass spectrum demonstrating the conversion of 4-OT R61C-1 mixed with a complex protein mixture from cells (ratio 1:1). C) Fluorescence imaging on SDS-PAGE of labeled 4-OT R61C-1. 1) 5 μg of unlabeled protein 4-OT R61C (the dimer is also visible), and 2) 2 μg, and 3) 3 μg of labeled 4-OT R61C-1. D) Coomassie Brilliant Blue staining of C. E) Fluorescence imaging on SDS-PAGE of 4-OT R61C-1 labeled in the presence of a cell lysate (protein ratio 1:1). Reaction 1) in the presence of 4-OT R61C-1, and 2) in the absence of 4-OT R61C-1. F) Coomassie Brilliant Blue staining of E. G) Fluorescence imaging on SDS-PAGE of 4-OT R61C-1 labeled in the presence of a cell lysate (protein ratio 1:10). Reaction 1) in the presence of 4-OT R61C-1, 2) in the absence of 4-OT R61C-1, and 3) in the presence of an equivalent amount of fluorescently labeled 4-OT R61C after direct coupling with N-[2-(dansylamino)ethyl]maleimide. H) Coomassie staining of G.