Positional Scanning Peptide Libraries for Kinase Substrate Specificity Determinations: Straightforward and Reproducible Synthesis Using Pentafluorophenyl Esters

Abstract

An efficient method to synthesize positional scanning synthetic combinatorial libraries (PS-SCLs) for studying the specificity of protein kinases is presented. Isokinetic ratios for pentafluorophenyl esters were determined iteratively using a new approach incorporating high performance liquid chromatography (HPLC) quantification and statistical experimental design. In the development process a large amount of work was put in to find efficient ways of screening for new isokinetic mixtures and to optimize the process of PS-SCL synthesis. The newly developed methods for the screening of isokinetic mixtures could be used for the screening of other interesting mixtures, but more importantly, the isokinetic ratios determined for the preactivated pentafluorophenyl esters were incorporated into a new efficient protocol. This straightforward protocol allows for a convenient synthesis of high quality PS-SCLs regardless of previous experience in solid phase synthesis.

Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates

“…The PS-SCL used was synthesized as described in [21] and consisted of 162 peptide mixtures, each with one of 18 amino acids fixed on one of nine positions in relation to the central phosphorylation residue (serine). Before the assay, the kinases were activated by incubation in activation buffer [50 mM Tris/HCl, 150 mM NaCl, 5 mM MnCl 2 , 10 mM MgCl 2 , 10% glycerol, 1 mM DTT (dithiothreitol), 100 μM ATP and 100 μM Na 3 VO 4 ] at 30 • C for 20 min.…”

A Novel Method for the Determination of Isokinetic Ratios and Its Application in the Synthesis of Two New Positional Scanning Libraries

“…Several methods have been reported for determining the isokinetic ratio for a variety of different reagent classes such as amino acids, carboxylic acids, and aldehydes {REF 9, REF 10, REF 11, REF 12, REF 13}. These methods include the use of amino acid analysis as well as binary and iterative HPLC analysis, and MALDI-TOF.…”

Positional Scanning MUC1 Glycopeptide Library Reveals the Importance of PDTR Epitope Glycosylation for Lectin Binding

“…Active esters, in particular pentafluorophenyl esters of Fmocprotected amino acids, were already proven as the most suitable option in the synthesis of PS-SCL for studying the specificity of protein kinases. 44 The simple mixture of two components, Fmoc-Ser/Thr(Tn)-OPfp (9/10) and Fmoc-Thr/Ser(OtBu)-OPfp (5/6), was incorporated in different ratios at randomized positions (X). To increase the reaction rate, couplings were performed in the presence of 1hydroxybenzotriazole (HOBt).…”