For decades, the propionic acid/12 N HCl (1:1, v/v) mixture at 130 °C [1] or 160 °C [2] has been ... more 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.
The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetrameth... more The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity.
The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT 1... more The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT 1A receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the ...
For decades, the propionic acid/12 N HCl (1:1, v/v) mixture at 130 °C [1] or 160 °C [2] has been ... more 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.
The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetrameth... more The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity.
The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT 1... more The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT 1A receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the ...
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