Mineko Tominaga

Preparation of Partially Protected Peptide Thioesters Containing a Cysteine Residue and Their Segment Condensation.

Methods for the preparation of partially-protected cysteine-containing peptide segments were developed. The peptide thioester, obtained via a solid-phase method of Boc chemistry, was used for segment condensation with an amino component peptide in the presence of silver ions and N-hydroxysuccinimide to give a desired product in good yield.

TFA/HCl mixture as an alternative to the standard propionic acid/HCl mixture for aminoacyl- and peptidyl-resin hydrolysis

For decades, the propionic acid/12 N HCl (1:1, v/v) mixture at 130 °C [1] or 160 °C [2] has been used as the standard solution for aminoacylor peptidyl-resin hydrolysis. However, no further systematic study has been carried out concerning the applicability of this hydrolysis protocol to other resins introduced later in the SPPS field. In this regard, we have recently demonstrated that much longer hydrolysis time than reported in the literature is necessary for quantitative cleavage of aminoacylor peptidyl-groups from most resins used in the Boc-strategy [3]. The hydrolysis times necessary for complete cleavage of Phe resin are 100 h (at 130 °C) or 30 h (at 160 °C) for benzhydrylamine-resin (BHAR) and 30 h (at 130 °C) or 18 h (at 160 °C) for p-methyl-benzhydrylamine(MBHAR) and 4(oxymethyl)-phenylacetamidomethyl-resin (PAMR). In order to propose a more accelerated hydrolysis protocol, the present report describes a comparative time-course hydrolysis study of aminoacyland dipeptidyl-resins using a trifluoroacetic acid (TFA)/12 N HCl hydrolysis mixture in different proportions. The Gly and Phe residues and the corresponding dipeptide sequences (Ala-Gly and Ala-Phe) coupled to PAMR, BHAR, MBHAR and also to chloromethyl-resin (CMR) were studied as models for this alternative hydrolysis condition at 160 °C.

Structure analysis of the second transmembrane segment of the rat bradykinin receptor in solution and in micelles by CD and fluorescence spectroscopies

In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthesizing and purifying a long and hydrophobic transmembrane peptide sequence (Ac-CTVAEIYLGNLAGADLILASGLPFWAITANNFDNH2 , denoted TM2-34) corresponding to the (64–97) region of the second transmembrane segment of the rat BKB2 receptor [2]. Improved yield of synthesis was obtained by changing the solvent system during the coupling step, accordingly to our conjugated resin solvation-polarity of the medium strategy [3]. We also performed a preliminary conformational analysis of this 34-mer peptide and its minor fragment (74–94), denoted TM2-24, by circular dichroism (CD) in aqueous solution containing different amounts of hexafluoroisopropanol (HFIP). In this solvent, a well-known α-helical structure inducer, we have found a α-helical content lower than expected for this type of transmembrane fragment. Hence, to better investigate structural properties of these two peptides, taken as model of transmembrane fragment, CD and fluorescence spectroscopic methods were applied in aqueous solution, varying the pH and the proportion of trifluoroethanol (TFE), and an other secondary structure inducing solvent. This structural investigation was also carried out in presence of dodecylsulp h a t e ( S D S ) a n d N -hexadecyl-N,N,dimethyl-3-ammonium-1 propylsulphate(HPS)-type micelles. Owing to the low solubility of TM2-34 analog, most of experiments in aqueous solution were performed with the TM2-24 transmembrane fragment.