Alternative synthetic tools to phospho-specific antibodies for phosphoproteome analysis: progress and prospects

Abstract

Signal transduction cascades in living systems are often controlled via post-translational phosphorylation and dephosphorylation of proteins. These processes are catalyzed in vivo by kinase and phosphatase enzymes, which consequently play an important role in many disease states, including cancer and immune system disorders. Current techniques for studying the phosphoproteome (isotopic labeling, chromatographic techniques, and phosphospecific antibodies), although undoubtedly very powerful, have yet to provide a generic tool for phosphoproteomic analysis despite the widespread utility such a technique would have. The use of small molecule organic catalysts that can promote selective phosphate esterification could provide a useful alternative to current state-of-the-art techniques for use in, e.g., the labeling and pull-down of phosphorylated proteins. This report reviews current techniques used for phosphoproteomic analysis and the recent use of small molecule peptide-based catalysts in phosphorylation reactions, indicating possible future applications for this type of catalyst as synthetic alternatives to phosphospecific antibodies for phosphoproteome analysis.

Keywords: Kinase mimetic; Kinase mimic; Nucleophilic catalysis; Phosphoproteome; Phosphorylation; Phosphospecific antibody.

Fig. 1

Different binding modes of phosphorylated peptides (1), non-phosphorylated peptides (2), and 2,5-dihydroxybenzoic acid (3) to a TiO₂ surface (taken from Engholm-Keller et al. 2011) [22, 24, 26]

Fig. 2

Potential approach to the tagging of a phosphorylated protein

Scheme 1

Enantioselective synthesis of d-myo-inositol-1/3-phosphate (taken from Miller et al. 2001/2002) [37, 38]

Scheme 2

Proposed mechanism for asymmetric phosphorylation (adapted from Miller et al. 2001) [37]

Scheme 3

Low selectivity of 4- (cf. 6-) phosphorylation of inositol (adapted from Sculimbrene 2004) [42]

Scheme 4

Proposed catalytic cycle for tetrazole-based catalysts (adapted from Miller et al. 2010) [41]

Scheme 5

Hayakawa’s general strategy for oligonucleotide synthesis via tetrazole-based catalysis [44]

Scheme 6

Sculimbrene’s phosphorylation of alcohols via tetrazole-based catalysis [46]

Scheme 7

Pyridine N-oxides in phosphotriester bond formation (adapted from Efimov et al. 1985) [51]