Liquid phase peptide synthesis of KL-4 pulmonary surfactant

Abstract

The invention realtes to improved liquid phase processes for the preparation of the 21 residue protein component, (Lys-Leu 4 ) 4 -Lys, of the pulmonary surfactant KL-4. These process are amenable to large scale synthesis and one process employs a method of saponifying an ester which reduces the inherent racemization of the α-carbon.

1 . A process for producing the synthetic KL-4 pulmonary surfactant protein of the formula (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1) comprising the steps of: 
 a. reacting a 3-amino acid peptide residue of the formula H-Lys(Z)-Leu-Leu OH with a 5-amino acid peptide residue Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) to yield an 8-amino acid peptide residue, which is successively reacted with the 5-amino acid peptide to form an 18-amino acid peptide of the formula H-Leu-Leu (Lys(Z)-Leu 4 ) 3 Lys(Z)-OBzl (SEQ. ID No. 3);    b. reacting the 18 amino acid peptide residue with a 3-residue amino acid peptide of the formula: H-Leu-Leu-Lys(Z)-OBzl to form the protected 21-amino acid KL-4 protein; and    c. removing the protecting group of the 21-amino acid KL-4 protein by reaction a suitable acid to form the final KL-4-protein.    
   
   
       2 . A process for producing (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1) comprising the steps of: 
 a. reacting H-Leu-Leu-Lys(Z)-OBzl, Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2), and a peptide coupling agent in an inert organic solvent, to form: Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6);    b. reacting Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6) with a suitable acid with or without an inert organic solvent to form: H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6);    c. reacting H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6), Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) and a peptide coupling agent in an inert organic solvent to form: Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5);    d. reacting Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5) with a suitable acid, with or without an inert organic solvent to form: H-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5);    e. reacting, Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2), a peptide coupling agent, a saltand H-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5) in an inert solvent to form: Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3);    f. reacting Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3) with a suitable acid, with or without an inert organic solvent to form: H-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3);    g. reacting Z-Lys(Z)-Leu-Leu-OH, water, a peptide coupling agent and H-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3) in an inert organic solvent to form: Z-(Lys(Z)-Leu 4 ) 4 -Lys(Z)-OBzl (SEQ. ID No. 1);    h. reacting, acetic acid, water, a catalyst and Z-(Lys(Z)-Leu 4 ) 4 -Lys(Z)-OBzl (SEQ. ID No. 1) under an H 2  atmosphere of 2 to 2.2 bar to form: (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1).    
   
   
       3 . The process according to claim  2 (a), wherein the peptide coupling agent is HOOBT/HBTU and the inert organic solvent is DMF  
   
   
       4 . The process according to claim  2 (b), wherein the suitable acid is TFA and an organic solvent is absent.  
   
   
       5 . The process according to claim  2 (c), wherein the peptide coupling agent is HOOBT/HBTU and the inert organic solvent is DMF.  
   
   
       6 . The process according to claim  2 (d), wherein the inert organic solvent is ethyl acetate and the suitable acid is gaseous HCl.  
   
   
       7 . The process according to claim  2 (e), wherein the peptide coupling agent is HOOBT/HBTU, the salt is LiBF 4  and the inert organic solvent is DMF.  
   
   
       8 . The process according to claim  2 (f), wherein the inert solvent is EtOH and the suitable acid is trifluoroacetic acid.  
   
   
       9 . The process according to claim  2 (g), where the peptide coupling agent is HOOBT/HBTU, and the inert organic solvent is DMF.  
   
   
       10 . The process according to claim  2 (h), wherein the catalyst is 5% Pd/C.  
   
   
       11 . A process for producing (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1) comprising the steps of: 
 a. reacting a compound of the formula Boc-Lys(Z)Leu-Leu-OH and a peptide 
 coupling agent with a compound of the formula: 
 H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 2), a suitable organic solvent to form a compound of the formula: 
 Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4), wherein R is C 1-8 alkyl or phenylC 1-8 alkyl;  
 
 
   b. removing the carboxy protecting group by reacting a hydroxide, salt, water and Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) in an inert organic solvent, to form: 
 Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4);  
   c. reacting Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No.  4 ), H-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5), a salt, water and a peptide coupling agent in an inert organic solvent, to form: 
 Boc-(Lys(Z)-Leu 4 ) 4 -Lys(Z)-OBzl (SEQ. ID No. 1);  
 d. reacting trifluoroacetic acid, Boc-(Lys(Z)-Leu 4 ) 4 -Lys(Z)-OBzl (SEQ. ID No. 1), acetic acid, water and a catalyst under a H 2  atmosphere of 40 to 50 psi to form (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1).  
   
   
   
       12 . The process according to  claim 11  (a) wherein, the peptide coupling agent is HOOBT/HBTU, and the inert organic solvent is DMF  
   
   
       13 . The process according to claim  11 (b) wherein, the hydroxide is tetrabutylammonium hydroxide and the inert solvent is THF or DMF.  
   
   
       14 . The process according to  claim 11  (c) wherein, the peptide coupling agent is HOOBT/DIC, the salt is tetrabutylammonium chloride and the inert organic solvent is DMF.  
   
   
       15 . The process according to claim  11 (d) wherein, the catalyst is Pd/C.  
   
   
       16 . Intermediates useful in processes for the production of (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1) selected from the group consisting of: 
 H-Leu-Leu-Lys(Z)-OBzl, Boc-Lys(Z)-Leu-Leu-OR, Z-Lys(Z)-Leu-Leu-OR wherein R is C 1-8 alkyl or phenylC 1-8 alkyl and suitable acid salts thereof.    
   
   
       17 . Intermediates useful in processes for the production of (Lys-Leu 4 ) 4 Lys (SEQ. ID No. 1) selected from the group consisting of: 
 Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH, (SEQ. ID No. 2)    Boc-Leu-Leu-Lys(Z)-Leu 4 -Lys(Z)-OBzl (SEQ. ID No. 6),    H-Leu-Leu-Lys(Z)-LeU 4 -Lys(Z)-OBzl (SEQ. ID No. 6),    Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5)    H-Leu-Leu-(Lys(Z)-Leu 4 ) 2 -Lys(Z)-OBzl (SEQ. ID No. 5),    Boc-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3),    H-Leu-Leu-(Lys(Z)-Leu 4 ) 3 -Lys(Z)-OBzl (SEQ. ID No. 3),    -(Lys(Z)-Leu 4 ) 4 -Lys(Z)-OBzl, Boc-Leu-Leu-Lys(Z)-Leu 4 -Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4), wherein Z is benzyloxycarboxyl and suitable acid salts thereof.    
   
   
       18 . A method of saponifying an ester protected carboxy group of a peptide, which consists or reacting said protected peptide with a tetraalkylammonium hydroxide and water in an inert organic solvent at a low temperature to form the deprotected carboxy group.  
   
   
       19 . The method of  claim 18  wherein, the tetraalkylammonium hydroxide is tetrabutylammonium hydroxide, the inert organic solvent is DMF or THF and the temperature is about −20 to 0° C.  
   
   
       20 . A process for coupling polypeptide fragments having 8-25 amino acid residues by reacting said fragments with a suitable peptide coupling agent in the presence of a tetraalkylammonium halide as a solubilizing agent.  
   
   
       21 . A method of saponifying Ac-D-Nal-D-p-Cal-OMe which consists of treating this peptide with a tetraalkylammonium hydroxide and water in an inert organic solvent at a low temperature to form Ac-D-Nal-D-p-Cal-OH.  
   
   
       22 . The method of  claim 21  wherein, the tetraalkylammonium hydroxide is tetrabutylammonium hydroxide, the inert organic solvent is DMF or THF and the temperature is about −20 to 0° C.  
   
   
       23 . A method of saponifying Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OBzl (SEQ. ID No. 7) which consists of treating this peptide with a tetraalkylammonium hydroxide and water in an inert organic solvent at a low temperature to form Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OH (SEQ. ID No. 7).  
   
   
       24 . The method of  claim 23  wherein, the tetraalkylammonium hydroxide is tetrabutylammonium hydroxide, the inert organic solvent is DMF or THF and the temperature is about −20 to 0° C.