Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases

Abstract

ATP analogues containing a CXY group in place of the α,β-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a d-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of α,β-CHF-ATP and α,β-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of α,β-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to α,β-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of α,β-CHF-γ-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes.

Figure 1

Co-crystal structures of Src and ATP analogs. (A) Co-crystal structure of Src/4a-2 establishes the absolute configuration of the nucleotide and reveals its interaction with the K295 of Src. Stick model of α, β-CHF-ADP overlaid with |2F_o − F_c| electron density (blue mesh, contoured at 1σ). (B) Co-crystal structure of Src/4b-2. (C) Structural overlay reveals that the bisphosphonate moiety of (R)-α, β-CHF-ADP (green) assumes a similar conformation to that of γ-thiol-ATP (cyan) bound to Src (3DQW). (D) Overlay of the α, β-CHX nucleotide bound Src structures with the previously solved active (3DQW) and inactive (2SRC) Src structures reveal different conformations of the glycine-rich loop.

Figure 2

Enzymatic approach to resolve a mixture of α, β-CHX-ATP diastereomers.

Figure 3

Utilization of γ-S-ATP, (R)-α, β-CHF-γ-S-ATP and (S)-α, β-CHF-γ-S-ATP by various kinases to phosphorylate substrate proteins in cell lysates. All γ-S nucleotides were used at 0.25 mM, and thiolphosphorylation was detected using a monoclonal antibody.

Scheme 1

Synthesis of Individual α, β-CHX (X = F, Cl) ATP Diastereomers