XXXV Reunião Anual da SBBqResumoID:8537


strategies to synthesize and purify cyclic fragments of AT1 angiotensin II receptor and their conformational studies through CD spectroscopy

 


Lopes, D.D.1; Souza, S.E.G.1; Cuvero, J.H.1; Schreier S.2; Paiva, A.C.M.1 and Nakaie, C.R.1

 



1Dept. of  Biophysics, Universidade Federal de São Paulo; 2Dept.of Biochemistry,  

                                Chemistry Institute, Universidade de São Paulo

 


According to previous report [1], the N-terminal portion comprising the 13-27 segment (RIQDDCPKAGRHSYI or P15) linked to the 266-278 region (IQLGVIHDCKISD or P13) present in the third external loop of the angiotensin II (AII) AT1 receptor might form a combined structure that seems to mimic the putative region of the AII binding. Thus the present work intended to start examining the feasibility of synthesizing different analogues produced by linking of these two receptor regions. To better evaluate the distance and spacer-type necessary for binding these fragments and characterized by the structure P15-X-P13, the following peptides were initially tested towards possible synthetic and purification  difficulties : P15, P13, P13 containing the Npys Cys-side chain protecting group [2] for use in alternative cyclization procedure, P15-P13, P15-C11-P13 and finally P15-(C11)2-P13. The C11 residue corresponds to the aminoundecanoic acid and the Npys group was located at P15 or P13 segment during the synthesis for further cyclization step. As expected, the results indicated pronounced difficulties in either the synthesis procedure -large amount of recoupling reactions were necessary for amino acid insertion - or even in the HPLC purification step, due to low solubility of most of cyclic peptides obtained. The oxidation reaction was also problematic and as a general rule for the S-S formation, the use of Npys as Cys protecting group was more advantageous than the standard cyclization by bubbling of oxygen in solution. For reasons not yet clarified, it was not possible to obtain the P15-C11-P13. In this case the failure seemed to occur at the oxidation step no matter the strategy applied (O2, Nyps at P13 or at P15 fragments). Alternative experimental protocols are currently been tested to overcome this problem. Lastly, CD conformation study was initiated with the P15, P13, P15-P13 and P15-(C11)2-P13 fragments varying the solution pH and the amount of secondary structure inducing TFE. All compounds depicted extended and flexible conformations but of note, only the latter cyclic fragment depicted a differentiated random coiled profile in the CD curves, regardless of the approach applied (variation of pH or of TFE amount). Different experimental approaches are planned to further verify the degree of AII binding to each of these receptor fragments. [1] Prot. Eng. (1999) 12, 1087; [2] J. Am. Chem. Soc. (1990) 112, 5345. Supported by Fapesp, CNPq and Capes.