Synthesis and Use of a Phosphonate Amidine to Generate an Anti‐Phosphoarginine‐Specific Antibody

Abstract

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) have impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate-amidines and sulfonate-amidines as isosteres of pArg and then use these mimics as haptens to develop the first high affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation.

Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation

“…Recently, we showed that arginine phosphorylation occurs in B. subtilis wildtype cells that were stressed with diamide (Fuhrmann et al, 2015b). However, the underlying molecular mechanism of the induction of arginine phosphorylation is not known.…”

“…Using a pArg specific antibody (Fuhrmann et al, 2015b), we observed that YwlE efficiently dephosphorylates McsB (Figure 5B). However, arginine phosphorylation of McsB can be detected under oxidative stress, indicating that YwlE is inactivated while McsB is active during these conditions.…”

“…To test the influence of YwlE and YwlE inhibition on McsB arginine phosphorylation, 0.5 μM YwlE was added in the absence or presence of CuSO 4 , H 2 O 2 or diamide. Arginine phosphorylation was monitored by dot blot analysis using the recently generated anti-pArg antibody (Fuhrmann et al, 2015b). Protein samples were directly spotted onto the nitrocellulose membrane and tested for the presence of pArg as described in the supplemental information.…”

“…Although the existence of protein N-phosphorylation has been known for decades, the scope and importance of these PTMs, especially in eukaryotic organisms, is only slowly emerging. The lack of studies in this area likely relates to the fact that these PTMs are more difficult to study because, in contrast to the O–P bond, the N–P bond is highly unstable under the strongly acidic conditions that are usually employed during phosphopeptide analysis (Engholm-Keller and Larsen, 2013; Fuhrmann et al, 2009; Fuhrmann et al, 2015b; Kee and Muir, 2012; Kowalewska et al, 2010; Schmidt et al, 2013a). …”

The McKenna reaction – avoiding side reactions in phosphonate deprotection

“…Usually the reaction is carried out for 1-3 days [26,27], in a solution of DCM or acetonitrile (ACN), although it can also be performed in neat BTMS. As far as the reaction temperature is concerned it can be performed either at room temperature, in refluxing CH 2 Cl 2 [19,28,29], at higher temperatures [1,30,31], and also under microwave conditions [32][33][34]. At lower temperatures or shorter reaction times an incomplete transesterification was reported [35].…”