Journal of Peptide Science J. Pept. Sci. 2008; 14: 989–997 Published online 14 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.1032 Short Communication Mechanistic studies of amide bond scission during acidolytic deprotection of Pip containing peptide CHIARA RUBINI, ALESSIO OSLER, ANDREA CALDERAN, ANDREA GUIOTTO and PAOLO RUZZA* Institute of Biomolecular Chemistry of CNR, Padua Unit, 35131 Padua, Italy Received 19 November 2007; Revised 1 February 2008; Accepted 11 February 2008 Abstract: Unusual TFA catalyzed cleavage reaction is reported for peptide containing pipecolic acid residues. Although the use of TFA under standard cleavage conditions is sufficiently mild to prevent degradation of the desired products, the amide bond between consecutive pipecolic acid residues is unexpectedly hydrolyzed by standard TFA treatment. The hydrolysis is proposed to proceed via an oxazolinium ion intermediate. This mechanism is supported by H/D exchange as observed by ESI-MS and NMR experiments. Copyright  2008 European Peptide Society and John Wiley & Sons, Ltd. Supplementary electronic material for this paper is available in Wiley InterScience at http://www.interscience.wiley.com/jpages/ 1075-2617/suppmat/ Keywords: pipecolic acid; TFA cleavage; peptide degradation; SH3 domain The nonproteinogenic amino acid pipecolic acid (so named pipecolinic acid or 2-piperidine carboxylic acid) is a proline homolog which contains a six-membered ring. It is found in several important natural products [1] and it has been extensively used as a proline sub- stitute in several syntheses of peptidomimetics [2]. In order to probe the role of proline ring size in peptide recognition by proline recognition domains (PRDs) and in particular by SH3 domains, we designed and synthe- sized a compound which contains (Pip) (named peptide 2) in place of all proline residues into the suitable SH3 ligand peptide of sequence H-Pro-Pro-Pro-Leu-Pro-Pro- Lys-Pro-Lys-Phe-OH (named peptide 1), corresponding to a Pro-rich peptide from HPK1 kinase [3]. Pipecolic rich peptide was synthesized manually on solid phase (SPPS) using an Fmoc/t -Bu-based strategy with stepwise coupling. Coupling reactions were performed using HATU as coupling reagent. Cleavage from the resin and side-chain deprotection were carried out by acidolysis with TFA using H 2 O and triisopropylsilane (TIS) as scavengers [4], and the crude peptides were precipitated by addition of cold ethyl ether. Surprisingly, RP-HPLC analysis of the crude compound showed the presence of three components (named 2a, 2b, and 2c, respectively) in the crude peptide (Figure 1(A)). ESI-MS analysis of the individual components revealed that the MH + of component 2a was 644.390 mass units, corresponding to the fragment * Correspondence to: Paolo Ruzza, Institute of Biomolecular Chemistry of CNR, Padua Unit, via Marzolo 1, 35131 Padua, Italy; e-mail: paolo.ruzza@unipd.it 6–10 of the parent Pip-rich peptide. The other two compounds 2b and 2c showed identical MH + values of 576.349 mass units as determined with an accuracy of ±0.05 by calibration against a mixture of neurotensin, angiotensin, and bradykinin at a concentration of 1 pmol/μl as external standard, corresponding to the fragment 1–5 of the Pip-rich peptide. The presence of two fragments with identical MH + value (compound 2b and 2c) indicated that an amino acid residue racemized during the amide bond cleavage reaction. The amino acid involved in epimerization is the Pip5 residue, which is equilibrated to give a 3 : 2 ratio of L/D as determined from the ratio of HPLC peaks (Figure 1(A)). The identity of the L- and D-isomers was established by the synthesis of authentic H-Pip-Pip-Pip-Leu-(L- or D-)Pip-OH isomers, respectively. The resin-bound peptide was alternatively treated with TFA and 1,2-ethanedithiol (EDT) as scavengers. Also, in this case the whole Pip-rich peptide was not detected by ESI-MS analysis, while two distinct compounds corresponding to the fragment 6–10 (2a) and the C-terminal 2-thioethyl thioester of fragment 1–5 (2d) (Scheme 1) have been detected and characterized by the ESI-MS analysis (Figure S1). These evidences indicated that the observed peptide fragmentation is due to an unexpected TFA catalyzed amide bond cleavage reaction under the standard cleavage/deprotection conditions used in SSPS carried out on acid labile Wang resin (Scheme 1). Since peptides are routinely treated with strong acids such as TFA and anhydrous HF without loss Copyright  2008 European Peptide Society and John Wiley & Sons, Ltd.