Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison between polymer brush coating and surface functionalization.

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe(3+), were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an α(S1), α(S2) mixture and ß casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the α(S1), α(S2) casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe(3+) IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).

Palladium-catalyzed stereoconvergent formylation of (E/Z)-ß-bromo-ß-fluorostyrenes: straightforward access to (Z)-α-fluorocinnamic aldehydes and (Z)-ß-fluorocinnamic alcohols.

We report here the stereoconvergent formylation of (E/Z)-ß-bromo-ß-fluorostyrene mixtures with carbon monoxide and sodium formate catalyzed by palladium. Optimization of reaction conditions leads to the corresponding pure (Z)-α-fluorocinnamaldehydes in good yields. The reaction was extended to styrenes bearing electro-attracting or electro-donating groups. The obtained α-fluoroaldehydes were smoothly reduced to the corresponding (Z)-ß-fluorocinnamic alcohol by NaBH(4). The reaction could be performed on functionalized substrates as demonstrated by the access to the glucoside of ß-fluoroconiferyl alcohol, (Z)-ß-fluoroconiferin, a strong inhibitor of lignin polymerization.