Slow tert-Butyl Ester Acidolysis and Peptide 3 10 -Helix to a-Helix Transition in HFIP Solution Alessandro Moretto, 1 Marco Crisma, 1 Fernando Formaggio, 1 Bernard Kaptein, 2 Quirinus B. Broxterman, 2 Timothy A. Keiderling, 3 Claudio Toniolo 1 1 Department of Chemistry, Institute of Biomolecular Chemistry, CNR, University of Padova, 35131 Padova, Italy 2 DSM Research, Life Sciences, Advanced Synthesis and Catalysis, Geleen, MD 6160, The Netherlands 3 Department of Chemistry, University of Illinois at Chicago, M/C 111, Chicago, IL 60607-7061 Received 26 October 2006; revised 13 December 2006; accepted 19 December 2006 Published online 10 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20680 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION T he 3 10 -helix constitutes a small but significant per- centage of secondary structural elements in globular proteins. 1,2 According to a general survey of protein structures, about 10% of all helical conformations are 3 10 -helices, in which an intramolecular H-bond is formed between the C¼¼O group of the ith amino acid resi- due and the NH group of the (i þ 3) residue (i þ 4 for the closely related a-helix). 3 The 3 10 -helices are mainly observed at the termini of a-helices, in loops, and as connectors between b-strands, with an average length of 3.3 residues. They play important functional roles in several proteins. 4–8 Some transmembrane channel-forming antibiotics, such as peptaibols, have a significant content of 3 10 -helices. 9–12 Moreover, the 3 10 -helix structure has been proposed as an intermediate in the folding of a-helices and observed as pico- second intermediates in simulation studies of a-helix melt- ing. 13–18 To fully understand peptide helix formation pro- cesses, including nucleation of nascent helices, theoretical findings should be combined with experimental techniques that have a high sensitivity to the subtle structural differences between 3 10 - and a-helices. In this connection, we have reported CD and NMR studies of terminally protected Z (or Ac)-[L-(aMe)Val] 8 -OtBu [Z, benzyloxycarbonyl; Ac, acetyl; (aMe)Val, C a -methyl valine; OtBu, tert-butoxy], which have shown that these two homo- octapeptides undergo an intriguing phenomenon, namely an unexpectedly slow and irreversible conversion from 3 10 -helix to a-helix in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) solu- tion 19,20 [this conformational transition does not seem to take place, at least within 3 weeks time, in 2,2,2-trifluoroetha- nol (TFE) or in CHCl 3 ]. Therefore, the terminally protected- [L-(aMe)Val] 8 -was the only peptide sequence based exclu- sively on C a -tetrasubstituted a-amino acids known before this Slow tert-Butyl Ester Acidolysis and Peptide 3 10 -Helix to a-Helix Transition in HFIP Solution Correspondence to: C. Toniolo, Institute of Biomolecular Chemistry, CNR, Depart- ment of Chemistry, University of Padova, 35131 Padova, Italy; e-mail: claudio. toniolo@unipd.it Presented at 43rd Japanese Peptide Symposium/4th Peptide Engineering Meeting. ABSTRACT: We have already shown by CD and NMR techniques that the terminally protected homo-octapeptides Z (and Ac)- [L-(aMe)Val] 8 -OtBu undergo a slow and irreversible 3 10 -helix to a-helix transition when dissolved in 1,1,1,3,3,3-hexafluoroisopropanol. In the present work, we find by HPLC and CD that under the aforementioned experimental conditions, a slow acidolysis of the tert- butyl ester functionality does take place affording the corresponding octapeptide free acids. The results of our combined chromatographic and spectroscopic experiments are confirmed by a comparison with the properties of independently synthesized and chemically characterized authentic compounds. # 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 233–238, 2007. Keywords: circular dichroism; helix-to-helix transition; HPLC; peptide helices; C a -tetrasubstituted a-amino acid V V C 2007 Wiley Periodicals, Inc. PeptideScience Volume 88 / Number 2 233