Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4.

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant beta-sheeted structures in water that convert progressively into alpha-helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches.

Most synthesized peptides are nowadays produced using solid-phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF-acetate) salts. However, TF-acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF-acetate exchange by another counter-ion is often required. Up to now, the classical procedure has consisted of freeze-drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa approximately 0): generally HCl (pKa = - 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF-acetate counter-ion from the dicationic octapeptide lanreotide: (i) reverse-phase HPLC, (ii) ion-exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF-acetate counter-ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF-acetate counter-ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19F-NMR, 1H-NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT-IR). We also show that ATR-IR can be used to monitor the TFA removal. The counter-ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF-acetate by any other counter-ion.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.