Conjugation of Peptides

Abstract

Methods for the selective conjugation of peptides which comprises an enzymatic incorporation of a functional group at the C-terminal end of a peptide followed by reaction with a second compound comprising the moiety to be conjugated to the peptide, wherein said second compound comprises a functional group which selectively reacts with the incorporated functional group.

1 . A method for preparing a conjugated peptide, said method comprising the steps of
 i) reacting in one or more steps a peptide with a first compound bearing one or more functional groups, which are not accessible in any of the amino acids residues constituting said peptide, in the presence of an enzyme capable of catalysing the incorporation of said first compound into the C-terminal of said peptide to form a transacylated peptide, and   ii) reacting in one or more steps said transacylated peptide with a second compound comprising one or more functional groups, wherein said functional group(s) do not react with functional groups accessible in the amino acid residues constituting said peptide, and wherein said functional group(s) in said second compound is capable of reacting with said functional group(s) in said first compound so that one or more covalent bond between said transacylated peptide and said second compound is formed.   
   
   
       2 . A method according to  claim 1 , wherein a peptide P is reacted in one or more steps with a first compound, which is an α-amino acid amide represented by the formula 
     
       
         
         
             
             
         
       
       in the presence of carboxypeptidase to form a transacylated peptide of the formula 
     
     
       
         
         
             
             
         
       
       said transacylated peptide being further reacted in one or more steps with a second compound of the formula
   Y-E-Z 
 
       to form a conjugated peptide of the formula 
     
     
       
         
         
             
             
         
       
       wherein R represents a linker or a bond; 
       P′ represents a peptide formed by removing the C-terminal amino acid from the peptide P; 
       X represents a radical comprising one or more functional group not accessible in the amino acid residues constituting the peptide P′; 
       Y represents a radical comprising one or more functional group which functional group(s) do not react with functional groups accessible in the amino acid residues constituting the peptide P′, and which functional group(s) react with said functional group(s) present in X; 
       E represents a linker or a bond; 
       wherein A represents the moiety formed by the reaction between the functional groups comprised in X and Y; and 
       Z comprises the moiety to be conjugated to the peptide, wherein said moiety decrease the clearance of compounds of formula [a] in comparison with the clearance of P. 
     
   
   
       3 . The method according to  claim 2 , wherein A represents oxime, hydrazone, phenylhydrazone, semicarbazone or triazole moieties. 
   
   
       4 . The method according to  claim 2 , wherein the functional group present in X is selected from the group consisting of: keto-, aldehyde-, —NH—NH 2 , —O—C(O)—NH—NH 2 , —NH—C(O)—NH—NH 2 , —NH—C(S)—NH—NH 2 , —NHC(O)—NH—NH—C(O)—NH—NH 2 , —NH—NH—C(O)—NH—NH 2 , —NH—NH—C(S)—NH—NH 2 , —NH—C(O)—C 6 H 4 —NH—NH 2 , —C(O)—NH—NH 2 , —O—NH 2 , —C(O)—O—NH 2 , —NH—C(O)—O—NH 2 , —NH—C(S)—O—NH 2 , alkyne, nitril-oxide and azide. 
   
   
       5 . The method according to  claim 2 , wherein the functional group present in Y is selected from the group consisting of: keto-, aldehyde-, —NH—NH 2 , —O—C(O)—NH—NH 2 , —NH—C(O)—NH—NH 2 , —NH—C(S)—NH—NH 2 , —NHC(O)—NH—NH—C(O)—NH—NH 2 , —NH—NH—C(O)—NH—NH 2 , —NH—NH—C(S)—NH—NH 2 , —NH—C(O)—C 6 H 4 —NH—NH 2 , —C(O)—NH—NH 2 , —O—NH 2 , —C(O)—O—NH 2 , —NH—C(O)—O—NH 2 , —NH—C(S)—O—NH 2 , alkyne, nitril-oxide and azide. 
   
   
       6 . The method according to  claim 2 , wherein X is selected from the group consisting of keto- and aldehyde-derivatives, and Y is selected from the group consisting of —NH—NH 2 , —O—C(O)—NH—NH 2 , —NH—C(O)—NH—NH 2 , —NH—C(S)—NH—NH 2 , —NHC(O)—NH—NH—C(O)—NH—NH 2 , —NH—NH—C(O)—NH—NH 2 , —NH—NH—C(S)—NH—NH 2 , —NH—C(O)—C 6 H 4 —NH—NH 2 , —C(O)—NH—NH 2 , —O—NH 2 , —C(O)—O—NH 2 , —NH—C(O)—O—NH 2 , and —NH—C(S)—O—NH 2 . 
   
   
       7 . The method according to  claim 2 , wherein X represents alkyne, and Y represents azide or nitril-oxide. 
   
   
       8 . The method according to  claim 2 , wherein X represents azide or nitril-oxide, and Y represents azide. 
   
   
       9 . The method according to  claim 2 , wherein R and E each independently represents a bi-radical of straight, branched and/or cyclic C 1-10 alkane, C 2-10 alkene, C 2-10 alkyne, C 1-10 heteroalkane, C 2-10 heteroalkene, or C 2-10 heteroalkyne, wherein one or more homocyclic aromatic compound bi-radical or heterocyclic compound biradical may be inserted. 
   
   
       10 . The method according to  claim 2 , wherein said α-amino acid amide represents a compound selected from the group consisting of: 2-amino-3-oxo-butyramide, 2-amino-6-(4-oxo-pentanoylamino)-hexanoic acid amide, 2-amino-3-(2-oxo-2-phenyl-ethylsulfanyl)-propionamide, 2-amino-5-oxo-hexanoic acid amide, 2-amino-3-oxo-propionamide, 2-amino-6-(4-acetylbenzoylamino)hexanoic acid amide, 2-amino-3-oxopropionic acid amide, (2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide, (2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide, (2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide, (2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide, (2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid amide, 4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, (2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic acid amide, 3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, 2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, (2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide, and (S)-2-aminopent-4-ynoicacid amide and S-phenylacylcysteine amide. 
   
   
       11 . A method according to  claim 2 , wherein Z comprises one or more polyethylene glycol or methoxy polyethylene glycol radicals and amino derivatives thereof; straight, branched and/or cyclic C 1-22 alkyl, C 2-22 alkenyl, C 2-22 alkynyl, C 1-22 heteroalkyl, C 2-22 heteroalkenyl, C 2-22 heteroalkynyl, wherein one or more homocyclic aromatic compound biradical or heterocyclic compound biradical may be inserted, and wherein said C 1 -C 22  or C 2 -C 22  radicals may optionally be substituted with one or more substituents selected from hydroxyl, halogen, carboxyl and aryl, wherein said aryl may optionally be further substituted by one or more substituents selected from hydroxyl, halogen, and carboxyl; steroid radicals; lipid radicals; polysaccharide radicals; dextran radicals; polyamide radicals; polyamino acid radicals; PVP radicals; PVA radicals; poly(1-3-dioxalane) radicals; poly(1,3,6-trioxane) radicals; ethylene/maleic anhydride polymer radicals; Cibacron dye stuff radicals; and Cibacron Blue 3GA radical. 
   
   
       12 . A method according to  claim 11 , wherein Z comprises one or more polyethylene glycol or methoxy polyethylene glycol radical with a molecular weight of about 10, 20, 30 or 40 kDa. 
   
   
       13 . A method according to  claim 11 , wherein Z comprises one or more C 10-20 alkyl, optionally substituted with a carboxy group. 
   
   
       14 . A method according to  claim 11 , wherein Z represents a undecanoic acid radical. 
   
   
       15 . The method according to  claim 11 , wherein Z comprises one or more of C 15 alkyl, C 17 alkyl, Cibacron Blue 3GA or radical of the formula 
     
       
         
         
             
             
         
       
     
   
   
       16 . The method according to  claim 11 , wherein Z comprises one or more moieties that bind to albumin. 
   
   
       17 . The method according to  claim 1 , wherein the enzyme is Carboxypeptidase Y. 
   
   
       18 . The method according to  claim 2 , wherein P represents a peptide selected from the group consisting of: insulin, GLP-1, GLP-2, growth hormone, cytokines, TFF, melanocortin receptor modifiers and Factor VII. 
   
   
       19 . The method according to  claim 2 , which further comprises the step of formulating said conjugated peptide in a pharmaceutical composition. 
   
   
       20 . A conjugated peptide according to the formula 
     
       
         
         
             
             
         
       
       wherein P′, R, A, E and Z are as defined in  claim 2 , and wherein 
     
     
       
         
         
             
             
         
       
       is attached to P′ at the C-terminal end of P′ via a peptide bond. 
     
   
   
       21 . A peptide according to  claim 20  selected from the group consisting of: 
     Lys ε (4-((2-(1-(mPEGcarbonyl)piperidin-4yl)ethoxy)imino)pentanoyl) 192)hGH(1-192) amide, in which mPEG has a molecular weight of 20 kDa; 
     (Lys ε (4-((3-(palmitoylamino)propoxy)imino)pentanoyl)192)hGH(1-192) amide; 
     (Lys ε (4-((3-((2S)-2,6-mPEGcarbonylamino)hexanoylamino)propoxy)imino)pentanoyl)34)GLP-2(1-34) amide, in which mPEG has a molecular weight of 20 kDa; 
     (Lys ε (4-(1-(2-(3-(mPEG)propanoylamino)hydrazino)ethyl)benzoyl)192)hGH(1-92) amide, in which mPEG has a molecular weight of 10 kDa; 
     (S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu 3 ,Leu 10 ]GLP-2ylleucinylamino)propionic amide; 
     (S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu 3 ]GLP-2ylleucinylamino)propionic amide; 
     3-(3-(3-((4-((S)-2-Carbamoyl-3-([Glu 3 ,Leu 10 ]GLP-2ylleucinylamino)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic acid; 
     11-(4-(4-((2S)-2-Carbamoyl-2-(([Glu 3 ,Leu 10 ]GLP-2ylleucinylamino)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid; 
     11-(5-(4-((2S)-2-Carbamoyl-2-(([Glu 3 ,Leu 10 ]GLP-2ylleucinylamino)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid 1-(4-(4-((S)-2-carbamoyl-2-([Glu 3 ]GLP-2ylleucinylamino))phenoxymethyl)-1H-1,2,3-triazol-1-yl)undecanoic acid; 
     11-(5-(4-((S)-2-carbamoyl-2-([Glu 3 ]GLP-2ylleucinylamino))phenoxymethyl)-1H-1,2,3-triazol-1-yl)undecanoic acid; 
     2-([Glu 3 ]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decanyl)-1H-1,2,3-tetrazol-4-yl)methoxy)phenyl)propionamide; and 
     2-([Glu 3 ]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decanyl)-1H-1,2,3-tetrazol-5-yl)methoxy)phenyl)propionamide. 
   
   
       22 . A pharmaceutical composition comprising one or more peptides according to  claim 21 . 
   
   
       23 . A compound according for formula I 
     
       
         
         
             
             
         
       
       wherein A and E independently represent C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or arylene, all of which may optionally be substituted with one or more substituents selected from halogen, amino, cyano and nitro; 
       B and D represents —C(O)— or —NH— with the proviso that when B represents —C(O)— then D must represent —NH—, and when B represents —NH— then D must represent —C(O)—; 
       and F represents hydrogen or C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or arylene, all of which may optionally be substituted with one or more substituents selected from halogen, amino, cyano and nitro. 
     
   
   
       24 . A compound according to  claim 23  selected from the group consisting of: 
     (2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid amide, 
     4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, 
     (2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic acid amide, 
     3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, and 
     2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide. 
   
   
       25 . A compound according to formula II 
     
       
         
         
             
             
         
       
       wherein J and L independently represent C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or arylene, all of which may optionally be substituted with one or more substituents selected from halogen, amino, cyano and nitro; 
       and M represents hydrogen or C 1-6 alkyl. 
     
   
   
       26 . A compound according to formula 25 selected from the group consisting of: 
     (2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide, 
     (2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide, 
     (2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide, and 
     (2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide. 
   
   
       27 . A compound according to formula III 
     
       
         
         
             
             
         
       
       wherein Q represents represent C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or arylene, all of which may optionally be substituted with one or more substituents selected from halogen, amino, cyano and nitro; 
       and T represents hydrogen or C 1-6 alkyl. 
     
   
   
       28 . A compound according to formula IV 
     
       
         
         
             
             
         
       
       wherein J″ and L″ independently represent C 1-6 alkylene or arylene, all of which may optionally be substituted with one or more substituents selected from halogen amino, cyano and nitro. 
     
   
   
       29 . The compound according to  claim 28 , which is (S)-2-amino-3-(4-(propargyloxy)phenyl)propionyl amide. 
   
   
       30 . A conjugated peptide obtainable by a method according to  claim 2 . 
   
   
       31 . A method for conjugating a first peptide, wherein said peptide comprises an alkyne group, the method comprising reacting said first peptide with a second compound, wherein said second compound comprises a nitri-oxide to form a conjugated peptide, wherein said first peptide is bonded to said second compound via a isoxazoline moiety. 
   
   
       32 . A method for conjugating a first peptide, wherein said peptide comprises an nitril-oxide group, the method comprising reacting said first peptide with a second compound, wherein said second compound comprises an alkyne to form a conjugated peptide, wherein said first peptide is bonded to said second compound via a isoxazoline moiety.