Process for the Synthesis of Therapeutic Peptides

Abstract

A process for the large-scale synthesis of a therapeutic peptide using solid-phase Fmoc-chemistry.

1 . A method for synthesizing a therapeutic peptide, the method comprising the steps of:
 (a) swelling Fmoc-Sieber resin comprising an Fmoc group in a dipolar aprotic solvent;   (b) de-protecting the Fmoc group of the resin with a solution of piperidine in a dipolar aprotic solvent;   (c) washing the resin after Fmoc de-protection with a dipolar aprotic solvent;   (d) activating an Fmoc-amino acid for coupling to the de-protected resin by dissolving the Fmoc-amino acid and 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) (HCTU) and/or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/1-hydroxybenzotriazole (HOBt) in a dipolar aprotic solvent, adding a base and stirring;   (e) contacting the activated Fmoc amino acid solution with the resin in a reactor to allow for coupling of the activated Fmoc-amino acid with the resin to form a coupled product;   (f) washing the coupled product;   (g) de-protecting the Fmoc group of the coupled product formed in step (e) or step (j) with a solution of piperidine in a dipolar aprotic solvent;   (h) washing the coupled product after Fmoc de-protection with a dipolar aprotic solvent;   (i) activating an Fmoc-amino acid for coupling to the de-protected coupled product by dissolving the Fmoc-amino acid and 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) (HCTU) and/or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/1-hydroxybenzotriazole (HOBt) in a dipolar aprotic solvent, adding a base and stirring;   (j) contacting the activated Fmoc-amino acid solution with the coupled product in a reactor to allow for coupling of the activated Fmoc-amino acid with the coupled product to form a new coupled product;   (k) washing the coupled product formed in step (j);   (l) repeating steps (g)-(k) until the peptide is formed;   (m) contacting the resin with a cleavage cocktail to cleave the peptide and simultaneously remove side chain protecting groups, resulting in a cleavage mixture and resin;   (n) filtering the cleavage mixture from the resin; and   (o) evaporating the cleavage mixture filtrate and precipitating the crude product from the concentrated solution with an organic solvent to yield a partially purified peptide;   
       wherein the peptide is a ghrelin analogue of formula I,
   R 1 -A 1 -A 2 -A 3 -A 4 -A 5 -R 2   (I),
 
 wherein: 
 A 1  is Apc or Inp; 
 A 2  is D-Bal, D-Bip, D-1Nal, or D-2Nal; 
 A 3  is D-Bal, D-1Nal, D-2Nal, or D-Trp; 
 A 4  is 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, or Thr(Bzl); 
 A 5  is Apc, Lys, or a bond; 
 R 1  is hydrogen; and 
 R 2  is OH or NH 2 ; 
 provided that, when A 5  is a bond, then:
 A 4  is Pff, Taz, or Thr(Bzl); or 
 A 1  is Apc and:
 A 2  is D-Bip or D-Bal; 
 A 3  is D-Bal; or 
 A 4  is 2Thi, 3Pal, or 4Pal. 
 
 
 
     
     
         2 . The method of  claim 1 , wherein the dipolar aprotic solvents used are each independently selected from the group consisting of: dimethylformamide (DMF); dimethylacetamine (DMA); N-methylpyrrolidone (NMP); and mixtures thereof. 
     
     
         3 . The method of  claim 1 , wherein the bases used are each independently selected from the group consisting of: N,N-diisopropylethylamine (DIEA); triethylamine (TEA); N-methylmorpholine (NMM); 2,4,6-trimethylpyrimidine (TMP); 2,3,5,6-tetramethylpyridine (TEMP); 2,6-di-tert-butyl-4-dimethylaminopyridine (DBDMAP); 4-dimethylaminopyridine (DHAP); and mixtures thereof. 
     
     
         4 . The method of  claim 1 , wherein the cleavage cocktail comprises a solution of up to 25% v/v trifluoroacetic acid (TFA), one or more scavengers, and dichloromethane (DCM), and wherein the scavengers are triisopropylsilane (TIPS), triethylsilane (TES), phenol, anisole, thioanisole, water, ethanedithiol (EDT), 1-dodecanethiol, dithiothreitol (DTT) and/or indole. 
     
     
         5 . The method of  claim 4 , wherein the scavengers are TIPS, TES, anisole and/or water. 
     
     
         6 . The method of  claim 1 , wherein the peptide comprises tert-butyloxycarbonyl (Boc) and/or tert-butyl ether (tBu) side chain protecting groups and the cleavage cocktail comprises:
 15 to 25% v/v TFA, and   2.5 to 12% v/v TIPS,   with the balance being DCM.   
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the solution in step (b) comprises piperidine and DMF and the concentration of piperidine is less than 20% (v/v). 
     
     
         9 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein A 1  is Inp. 
     
     
         17 . The method of  claim 16 , wherein the peptide is: 
       
         
           
                 
                 
               
                     
                   H-Inp-D-Bal-D-Trp-Phe-Apc-NH 2 ; 
                 
                     
                     
                 
                     
                   H-Inp-D-2-Nal-D-Trp-Phe-Apc-NH 2 ; 
                 
                     
                     
                 
                     
                   H-Inp-D-Bal-D-Trp-2-Thi-Apc-NH 2 ; 
                 
                     
                     
                 
                     
                   H-Inp-D-Bal-D-Trp-Taz-Apc-NH 2 ; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   H-Inp-D-Bal-D-Trp-Phe-Lys-NH 2 . 
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         18 . The method according to  claim 1 , wherein A 1  is Apc. 
     
     
         19 . The method according to  claim 18 , wherein the peptide is: 
       
         
           
                 
                 
               
                     
                   H-Apc-D-1-Nal-D-Trp-2-Thi-Apc-NH 2   
                 
                     
                   or 
                 
                     
                     
                 
                     
                   H-Apc-D-1-Nal-D-Trp-2-Thi-NH 2 . 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         20 . The method of  claim 1 , wherein, in step (f), the dipolar aprotic solvent is dimethylformamide (DMF).