Comparative evaluation of the synthesis and purification of transmembrane peptide fragments - Rat bradykinin receptor fragment 64-97 as model

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1997-04-01
Autores
Oliveira, E.
Miranda, A.
Albericio, F.
Andreu, D.
Paiva, ACM
Nakaie, C. R.
Tominaga, M.
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The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C-4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of cc-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor. (C) Munksgaard 1997.
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Journal Of Peptide Research. Copenhagen: Munksgaard Int Publ Ltd, v. 49, n. 4, p. 300-307, 1997.
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