Diketopiperazine Forming Dipeptidyl Linker

Abstract

The invention relates to a method for homogeneous solution phase peptide synthesis (HSPPS) of a N-terminal peptide fragment PEP-N and a C-terminal peptide fragment C-PEP, with C-PEP carrying a specific diketopiperazine (DKP) comprising C-terminal protecting group, which contains a handle group HG, with HG being connected to the C-terminus of the peptide fragmcnt; thereby this specific DKP comprising C-terminal protecting group can be selectively cleaved from the peptide as a conventionally used C-terminal protecting group. By the use of this DKP and HG comprising C-terminal protecting group, certain process steps in convergent peptide synthesis based on a combination of HSPPS and solid phase peptide synthesis (SPPS) can be avoided. The invention relates further to a method for the preparation of such specifically protected fragment C-PEP by SPPS by using a linker comprising a specific dipeptide and HG for connecting the growing peptide chain to the resin, which linker forms said DKP group, when the peptide fragment C-PEP is cleaved from the supporting resin; and further to the intermediates of the preparation method.

1 - 13 . (canceled) 
     
     
         14 . A method for the preparation of a DKP-PG forming linker DKP-L,
 wherein the DKP-PG forming linker DKP-L comprises a handle group HG, an optional spacer group SG, and a dipeptide DPR,   wherein DKP-PG is a protecting group comprising the handle group HG, the optional spacer group SG, and a diketopiperazine residue DKP;   wherein DKP is derived from the dipeptide DPR;   wherein SG is a spacer group conventionally used in peptide chemistry;   wherein DPR consists of a C-terminal residue Xaa1 and an N-terminal residue Xaa2, wherein Xaa2 has a side chain functional group FG;   wherein Xaa1 is selected from the group consisting of naturally occurring alpha amino acid residues, alpha-N-methylamino acid residues, L-Hpr residue, D-Hpr residue, DL-Hpr residue, 2-(C 1-5 -alkyl)-D-amino acid residues, 2-(C 1-5 -alkyl)-L-amino acid residues, 2-(C 1-5 -alkyl)-DL-amino acid residue and an HypX residue; wherein HypX is a compound of formula:   
       
         
           
           
               
               
           
         
         wherein
 X is O, S or C(R13)R14; 
 R5, R7, R12, R13 and R14 are identical or different and independently from each other selected from the group consisting of hydrogen, C 1-4  alkyl and O—R8; 
 R8 is a protecting group conventionally used for side chain protection in peptide chemistry, or a substituent of formula (Sub-R8); 
 
       
       
         
           
           
               
               
           
         
         wherein
 m8 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 R9 is C 1-4  alkyl; 
 
         wherein Xaa2 is selected from the group consisting of L-Lys residue, D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue, L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue, D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diamino butanoic acid residue, L-alpha,beta-diaminopropanoic acid residue, D-alpha,beta-diaminopropanoic acid residue, DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Ser residue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue, L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue, D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Asp residue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Glu residue; 
         wherein HG is either directly connected to a side chain functional group of Xaa2 (FG), or, if a SG is present, HG is connected to SG and SG is connected to FG; 
         wherein HG is a handle group selected from the group consisting of HGF-I, HGF-II, HGF-III, HGF-IV, HGF-V and HGF-VI, 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 (*) denotes the bond between HG and the carbonyl carbon or the side chain of the C-terminal amino acid of PEP-C, 
 (**) denotes the bond between HG and SG when a SG is present, or denotes the bond between HG and FG when no SG is present; 
 R1, R2, R3, R4, R10 and R11 are identical or different and independently from each other selected from the group consisting of hydrogen and O—C 1-4  alkyl, 
 s1-1, s2, s3, s4 and s6 are identical or different and independently from each other selected from the group consisting of 1, 2, 3 and 4, 
 s5-1 is 0, 1, 2, 3 or 4, 
 s1-2, s5-2 and s5-3 are identical or different and independently from each other 0 or 1, 
 T1-1 is O or NH, 
 T1-2 and T5-1 are O; 
 
         wherein said method comprises
 coupling Xaa2 to Xaa1; 
 optionally coupling SG to Xaa2, if SG is present; 
 coupling HG either to SG, if SG is present, or to Xaa2. 
 
       
     
     
         15 . The method according to  claim 14 , wherein
 HG is selected from the group consisting of HGF-I, HGF-IV and HGF-VI.   
     
     
         16 . The method according to  claim 14 , wherein
 HG is selected from the group consisting of HG-Ia, HG-Ib, HG-Ic, HG-Id, HG-II, HG-III, HG-IVa, HG-IVb, HG-Va, HG-Vb and HG-VI,   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 (*) and (**) are as defined in  claim 14 . 
 
       
     
     
         17 . The method according to  claim 14 , wherein
 SG is a spacer group selected from the group consisting of SG-I, SG-II, SG-III, SG-IV SG-V, SG-VI, and SG-VII;   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 m1, m5, m6, m7, m9, m10, mil and m12 are identical or different and independently from each other an integer of 1 to 500; 
 m2, m3 and m4 are identical or different and independently from each other 1, 2, 3 or 4, 
 (***) is the bond from SG to HG when a SG is present, 
 (****) is the bond between SG and Xaa2 when a SG is present, 
 
         wherein
 HG and Xaa2 are defined according to  claim 14 . 
 
       
     
     
         18 . The method according to  claim 14 , wherein
 Xaa1 is selected from the group consisting of L-N-methylglycine residue, D-N-methylglycine residue, DL-N-methylglycine residue, L-N-methylphenylalanine residue, D-N-methylphenylalanine residue, DL-N-methylphenylalanine residue, L-Pro residue, D-Pro residue, DL-Pro residue, side chain protected L-4Hyp residue, side chain protected D-4Hyp residue, side chain protected DL-4Hyp residue, L-4Hpr residue, D-Hpr residue and DL-Hpr residue.   
     
     
         19 . A method according to  claim 14 , wherein
 Xaa2 is selected from the group consisting of L-Lys residue, D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue, L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue, D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diaminobutanoic acid residue, L-alpha,beta-diaminopropanoic acid residue, D-alpha,beta-diaminopropanoic acid residue, DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Ser residue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue, L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue, D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Asp residue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Glu residue.   
     
     
         20 . A compound DKP-L as defined according to  claim 14 . 
     
     
         21 . A method for the preparation of a DKP-L-ResinA,
 wherein DKP-L-ResinA comprises a ResinA connected to a DKP-PG forming linker DKP-L,   wherein ResinA is a resin used conventionally as solid phase in SPPS,   wherein the DKP-PG forming linker DKP-L comprises a handle group HG, an optional spacer group SG, and a dipeptide DPR;   wherein DKP-PG is a protecting group comprising the handle group HG, the optional spacer group SG, and a diketopiperazine residue DKP;   wherein DKP is derived from the dipeptide DPR;   wherein DPR consists of a C-terminal residue Xaa1 and an N-terminal residue Xaa2, wherein Xaa2 has a side chain functional group FG;   wherein the carboxylic acid group of Xaa1 is connected to ResinA;   wherein SG is a spacer group conventionally used in peptide chemistry;   wherein Xaa1 is selected from the group consisting of naturally occurring alpha amino acid residues, alpha-N-methylamino acid residues, L-Hpr residue, D-Hpr residue, DL-Hpr residue, 2-(C 1-5 -alkyl)-D-amino acid residues, 2-(C 1-5 -alkyl)-L-amino acid residues, 2-(C 1-5 -alkyl)-DL-amino acid residue and an HypX residue; wherein HypX is a compound of formula:   
       
         
           
           
               
               
           
         
         wherein
 X is O, S or C(R13)R14; 
 R5, R7, R12, R13 and R14 are identical or different and independently from each other selected from the group consisting of hydrogen, C 1-4  alkyl and O—R8; 
 R8 is a protecting group conventionally used for side chain protection in peptide chemistry, or a substituent of formula (Sub-R8); 
 
       
       
         
           
           
               
               
           
         
         wherein
 m8 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 R9 is C 1-4  alkyl; 
 
         wherein Xaa2 is selected from the group consisting of L-Lys residue, D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue, L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue, D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diamino butanoic acid residue, L-alpha,beta-diaminopropanoic acid residue, D-alpha,beta-diaminopropanoic acid residue, DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Ser residue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue, L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue, D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Asp residue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Glu residue; 
         wherein HG is either directly connected to a side chain functional group of Xaa2 (FG), or, if a SG is present, HG is connected to SG and SG is connected to FG; 
         wherein HG is a handle group selected from the group consisting of HGF-I, HGF-II, HGF-III, HGF-IV, HGF-V and HGF-VI, 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 (*) denotes the bond between HG and the carbonyl carbon or the side chain of the C-terminal amino acid of PEP-C, 
 (**) denotes the bond between HG and SG when a SG is present, or denotes the bond between HG and FG when no SG is present; 
 R1, R2, R3, R4, R10 and R11 are identical or different and independently from each other selected from the group consisting of hydrogen and O—C 1-4  alkyl, 
 s1-1, s2, s3, s4 and s6 are identical or different and independently from each other selected from the group consisting of 1, 2, 3 and 4, 
 s5-1 is 0, 1, 2, 3 or 4, 
 s1-2, s5-2 and s5-3 are identical or different and independently from each other 0 or 1, 
 T1-1 is O or NH, 
 T1-2 and T5-1 are O; 
 
         wherein said method comprises method (X1) or method(X2); 
         wherein method(X1) comprises
 coupling the amino acid Xaa1 to ResinA; 
 coupling the amino acid Xaa2 to Xaa1; 
 optionally coupling SG to the side chain of Xaa2, if SG is present in DKP-L-ResinA; and 
 coupling HG either to SG, if SG is present in DKP-L-ResinA, or to Xaa2; and 
 
         wherein method(X2) comprises
 coupling DKP-PG forming linker DKP-L to ResinA. 
 
       
     
     
         22 . The method according to  claim 21 , wherein
 HG is selected from the group consisting of HGF-I, HGF-IV and HGF-VI.   
     
     
         23 . The method according to  claim 21 , wherein
 HG is selected from the group consisting of HG-Ia, HG-Ib, HG-Ic, HG-Id, HG-II, HG-III, HG-IVa, HG-IVb, HG-Va, HG-Vb and HG-VI,   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 (*) and (**) are as defined in  claim 21 . 
 
       
     
     
         24 . The method according to  claim 21 , wherein
 SG is a spacer group selected from the group consisting of SG-I, SG-II, SG-III, SG-IV SG-V, SG-VI, and SG-VII;   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 m1, m5, m6, m7, m9, m10, m11 and m12 are identical or different and independently from each other an integer of 1 to 500; 
 m2, m3 and m4 are identical or different and independently from each other 1, 2, 3 or 4, 
 (***) is the bond from SG to HG when a SG is present, 
 (****) is the bond between SG and Xaa2 when a SG is present, wherein 
 HG and Xaa2 are defined according to  claim 21 . 
 
       
     
     
         25 . The method according to  claim 21 , wherein
 Xaa1 is selected from the group consisting of L-N-methylglycine residue, D-N-methylglycine residue, DL-N-methylglycine residue, L-N-methylphenylalanine residue, D-N-methylphenylalanine residue, DL-N-methylphenylalanine residue, L-Pro residue, D-Pro residue, DL-Pro residue, side chain protected L-4Hyp residue, side chain protected D-4Hyp residue, side chain protected DL-4Hyp residue, L-4Hpr residue, D-Hpr residue and DL-Hpr residue.   
     
     
         26 . A method according to  claim 21 , wherein
 Xaa2 is selected from the group consisting of L-Lys residue, D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue, L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue, D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diaminobutanoic acid residue, L-alpha,beta-diaminopropanoic acid residue, D-alpha,beta-diaminopropanoic acid residue, DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Ser residue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue, L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue, D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Asp residue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Glu residue.   
     
     
         27 . The method according to  claim 21 , wherein
 ResinA is selected from the group consisting of hydroxymethylpolystyrene (HMPS) resins, polyethylene glycol (PEG) based resins, polystyrene resin, p-benzyloxybenzyl alcohol resins, chloromethyl polystyrene-divinylbenzene resins, and poly(vinyl alcohol)-graft-poly(ethylene glycol) (PVA-g-PEG) resins.   
     
     
         28 . A compound DKP-L-ResinA as defined according to  claim 21 .