Process for preparing cardiodilatin fragments; highly purified cardiodilatin fragments and intermediate products for the preparation of same

Abstract

The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same.

1 . A process for preparation of cardiodilatin fragments of formula I 
       R 1 -ANP(105-121)-R 2   (I), having a chain length of 17-37 amino acid in total, wherin ANP(105-121) represents the amino acid sequence [SEQ ID NO. 1],    R 1  represents an amino acid chin of sequence ANP(90-104) [SEQ ID NO. 2] or fragments thereof having a chain length of 0-15 amino acids, and    R 2  represents an amino acid chain of sequence ANP(122-126) [SEQ ID. 3] or fragments thereof having a chain length or 0-5 amino acids, characterized in that synthesis is effected via condensation of at lease three partial fragments, the condensation of said partial fragments to give the cardiodilatin fragment of formula I being carried out between the amino acid positions Gly  108  and Arg 109  and amino acid positions Gly 120  and Cys 121 .    
     
     
         2 . The process according to  claim 1 , wherein 
 (a) in a first step, condensation of the partial fragments is effected between the amino acid positions Gly  120  and Cys 121  from the partial fragments ANP(109-120and Cys 121 -R 2 , and    (b) in a second step, condensation of the partial fragments is effected between the amino acid positions Gly 108  and Arg 109  from the partial fragment ANP(109-121)-R 2  obtained according to step (a) and the partial fragment R 1 -ANP(105-108).    
     
     
         3 . The process according to one of claims  1  or  2 , wherein R 2  represents the amino acid sequence ANP(122-126), characterized in that in a first step, the fragment ANP(109-126)-OtBu is prepared by condensation of the fragment Fmoc-ANP(109-120)-OH, which is synthesize on a solid support phase according to the Merrifield process and removed therefrom, with the fragment H-ANP(121-126)-OtBu, and subsequently, the Fmoc protecting group is removed from the resulting fragment Fmoc-ANP(109-126)-OtBu.  
     
     
         4 . The process according to one of claims  1 - 3 , wherein R 1  represents the amino acid sequence ANP(95-104), characterized in that the cardiodilatin fragment of formula I is prepared by condensation of the fragment Boc-ANP(95-108)-OH, which is synthesized on a solid support phase according to the Merrifield process and removed therefrom, with the fragment H-ANP(109-126)-OtBu, and subsequently, the protecting groups are removed from the resulting fragment Boc-ANP((95-126)-OtBu.  
     
     
         5 . The process according to one of claims  1 - 4 , characterized in that when forming the three partial fragments R 1 -ANP(105-108), ANP(109-120) or ANP(121)-R 2  according to the Merrifield process, bonding to the solid support material is effected by means of a super-acid-sensitive linker.  
     
     
         6 . The process according to one of claims  1 - 5 , characterized in that the amino and hydroxy protecting groups are removed from the obtained fully protected cardiodilatin fragment R 1 -ANP(105-121)-R 2 , forming the fragment protected by the protecting group Acm at Cys 105 , and subsequently, the protecting group Acm is removed from the thus obtained fragment and thereafter, the cardiodilatin fragment is cyclized by oxidation.  
     
     
         7 . The process according to one of claims  1 - 6 , characterized in that R 1  represents the amino acid sequence selected from the group of ANP(95-104), ANP(99-104) and ANP(102-104).  
     
     
         8 . The process according to one of claims  1 - 7 , characterized in that R 2  represents the amino acid sequence selected from the group of ANP(122-125) and ANP(122-126).  
     
     
         9 . A process for the preparation of high-purity cardiodilatin fragments R 1 -ANP(105-121)-R 2  having a chain length of 17-37 amino acids in total, wherein R 1  represents an amino acid chain of sequence ANP(90-104) or fragments thereof having a chain length of 0-15 amino acids, and R 2  represents an amino acid chain of sequence ANP(122-126) or fragments thereof having a chain length of 0-5 amino acids, characterized in that purification of the crude product is performed using a reversed-phase HPLC column, and the cardiodilatin fragment is eluted with a buffer system containing triethylammonium phosphate and acetonitrile.  
     
     
         10 . The process according to  claim 9 , characterized in that the elution is performed at a pH value of 2-5, more specifically of 2-3.  
     
     
         11 . The process according to one of claims  9  or  10 , characterized in that the reversed-phase HPLC column is equilibrated with a triethylammonium phosphate buffer, thereafter the concentrated crude product of the cardiodilatin fragment is applied and subsequently, the cardiodilatin fragment is eluted by continuous charging of a buffer mixture of triethylammonium phosphate in water and acetonitrile (2:3 v/v) in a continuous gradient.  
     
     
         12 . High-purity cardiodilatin fragments R 1 -ANP(105-121)-R 2  having a chain length of 17-37 amino acids in total, wherein R 1  represents an amino acid chain of sequence ANP(90-104) or fragments thereof having a chain length of 0-15 amino acids, and R 2  represents an amino acid chain of sequence ANP(122-126) or fragments thereof having a chain length of 0-5 amino acids, characterized in that they are substantially free of peptide impurities and exhibit a single migration peak in the purity analysis using capillary electrophoresis.  
     
     
         13 . The high-purity cardiodilatin fragments of  claim 12 , characterize in that R 1  represents an amino acid sequence selected from the group of ANP(95-104), ANP(99-104) and ANP(102-104).  
     
     
         14 . The high-purity cardiodilatin fragments of  claim 12  or  13 , characterized in that R 2  represents an amino acid sequence selected from the group of ANP(122-125) and ANP(122-126).  
     
     
         15 . The high-purity cardiodilatin fragments according to one of claims  12 - 14 , selected from the group of ANP(95-126), ANP(99-126), P(102-126), and ANP(103-126).  
     
     
         16 . Pharmaceutical formulations, containing the high-purity cardiodilatin fragment according to one of claims  12 - 15  in addition to physiologically acceptable adjuvants or additives.  
     
     
         17 . Peptide fragments having the amino acid sequence R 1 -ANP(105-108), wherein R 1  represents an amino acid chain of sequence ANP(90-104) or fragments thereof having a chain length of 0-15 amino acids, as well as their derivatives modified by protecting groups.  
     
     
         18 . Peptide fragment having the amino acid sequence ANP(109-120), as well as derivatives thereof modified by protecting groups.  
     
     
         19 . Peptide fragments having the amino acid sequence ANP(109-121)-R 2 , wherein R 2  represents an amino acid chain of sequence ANP(122-126) or fragments thereof having a chain length of 0-5 amino acids, as well as their derivatives modified by protecting groups.  
     
     
         20 . Peptide fragments having the amino acid sequence Cys 121 -R 2 , wherein R 2  represents an amino acid chain of sequence ANP(122-126) or fragments thereof having a chain length of 3-5 amino acids, as well as their derivatives modified by protecting groups.