Process of Preparing Guanylate Cyclase C Agonists

Abstract

The invention provides processes of preparing a peptide including a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249 described herein.

We claim: 
     
         1 . A process of preparing a peptide comprising a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249, wherein the GCC agonist sequence is n amino acid units in length, with the N-terminal unit at position 1 and the C-terminal unit at position n, the process comprising:
 providing a first fragment having a first sequence of amino acid units from position j through position k of the GCC agonist sequence, wherein j is an integer between 1 and n−1, k is an integer between 2 and n and is greater than j, and the first fragment is protected except for an amino group of the amino acid unit at position j, or alternatively, a carboxyl group of the amino acid unit at position k,   providing a second fragment having a second sequence of amino acid units from position h through position j−1 of the GCC agonist sequence or having a third sequence of amino acid units from position k+1 through position m of the GCC agonist sequence, wherein h is an integer between 1 and n−2 and is smaller than j, m is an integer between k+2 and n, and the second fragment is protected except for a carboxyl group of the amino acid unit at position j−1 or an amino group of the amino acid unit at position k+1, and   coupling the first and the second fragments via a solution-phase synthesis to yield a protected peptide having a sequence of amino acid units from position h through position k of the GCC agonist sequence or a protected peptide having a sequence of amino acid units from position j through position m of the GCC agonist sequence.   
     
     
         2 . The process of  claim 1 , wherein the GCC agonist sequence is selected from the group consisting of SEQ ID NOs: 1, 8, 9, 55, 56, 58, and 59. 
     
     
         3 . The process of  claim 1 , wherein the GCC agonist sequence is selected from the group consisting of SEQ ID NOs: 1 and 9. 
     
     
         4 . The process of  claim 3 , wherein at least one of the first and second fragments is provided via a solid-phase peptide synthesis. 
     
     
         5 . The process of  claim 4 , wherein the solid-phase peptide synthesis is a Fmoc solid-phase synthesis. 
     
     
         6 . The process of  claim 5 , wherein the Fmoc solid-phase synthesis is performed on 2-chlorotrityl resin. 
     
     
         7 . The process of  claim 3 , wherein h is 1, j is 7, and k is 16. 
     
     
         8 . The process of  claim 3 , wherein h is 7, j is 15, and k is 16. 
     
     
         9 . The process of  claim 8 , further comprising deprotecting an amino group of the amino acid unit at position 7 of the protected peptide having the sequence of amino acid units from position 7 through position 16 of the GCC agonist sequence to yield a position-7 reactive peptide. 
     
     
         10 . The process of  claim 9 , further comprising providing a third fragment having a sequence of amino acid units from position 1 through position 6 of the GCC agonist sequence, wherein the third fragment is protected except for a carboxyl group of the amino acid at position 6. 
     
     
         11 . The process of  claim 10 , wherein the third fragment is provided via a solid-phase peptide synthesis. 
     
     
         12 . The process of  claim 11 , wherein the solid-phase peptide synthesis is Fmoc solid-phase synthesis. 
     
     
         13 . The process of  claim 10 , further comprising coupling the third fragment and the position-7 reactive peptide via a solution-phase synthesis to yield a protected linear peptide having a sequence of amino acid units from position 1 through position 16 of the GCC agonist sequence. 
     
     
         14 . The process of  claim 13 , further comprising deprotecting the protected linear peptide to yield a deprotected linear peptide. 
     
     
         15 . The process of  claim 14 , further comprising oxidizing the deprotected linear peptide to yield the peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1 and 9. 
     
     
         16 . The process of  claim 1 , wherein at least one of the first and second fragments is provided via a solid-phase peptide synthesis. 
     
     
         17 . The process of  claim 16 , wherein the solid-phase peptide synthesis is Fmoc solid-phase synthesis. 
     
     
         18 . The process of  claim 1 , wherein each of the first and second fragments is not more than 10 amino acid units in length. 
     
     
         19 . The process of  claim 18 , wherein the second fragment has the second sequence of amino acid units from position h through position j−1 of the GCC agonist sequence and either one or both of the amino acid unit of the second fragment at position j−1 and the amino acid unit of the first fragment at position k is selected from the group consisting of glycine, proline, leucine, alanine, and arginine. 
     
     
         20 . The process of  claim 18 , wherein the second fragment has the second sequence of amino acid units from position h through position j−1 of the GCC agonist sequence and either one or both of the amino acid unit of the second fragment at position j−1 and the amino acid unit of the first fragment at position k is selected from the group consisting of glycine and proline. 
     
     
         21 . The process of  claim 18 , wherein the second fragment has the third sequence of amino acid units from position k+1 through position m of the GCC agonist sequence and either one or both of the amino acid unit of the second fragment at position m and the amino acid unit of the first fragment at position k is selected from the group consisting of glycine, proline, leucine, alanine, and arginine. 
     
     
         22 . The process of  claim 18 , wherein the second fragment has the third sequence of amino acid units from position k+1 through position m of the GCC agonist sequence and either one or both of the amino acid unit of the second fragment at position m and the amino acid unit of the first fragment at position k is selected from the group consisting of glycine and proline. 
     
     
         23 . The process of  claim 1 , wherein k is n, the second fragment has the second sequence of amino acid units from position h through position j−1 of the GCC agonist sequence, and the amino acid unit at positions j−1 is selected from the group consisting of glycine, proline, leucine, alanine, and arginine. 
     
     
         24 . The process of  claim 23 , further comprising deprotecting an amino group of the amino acid unit at position h of the protected peptide having a sequence of amino acid units from position h through position k of the GCC agonist sequence to yield a position-h reactive peptide. 
     
     
         25 . The process of  claim 24 , further comprising providing a fourth fragment having a sequence of amino acid units from position 1 through position h−1 of the GCC agonist sequence, wherein the fourth fragment is protected except for a carboxyl group of the amino acid unit at position h−1. 
     
     
         26 . The process of  claim 25 , wherein the fourth fragment is provided via a solid-phase peptide synthesis. 
     
     
         27 . The process of  claim 26 , wherein the solid-phase peptide synthesis is Fmoc solid-phase synthesis. 
     
     
         28 . The process of  claim 25 , wherein the fourth fragment is not more than 10 amino acid units in length. 
     
     
         29 . The process of  claim 25 , further comprising coupling the fourth fragment and the position-h reactive peptide via a solution-phase synthesis to yield a protected linear peptide having a sequence of amino acid units from position 1 through position n of the GCC agonist sequence. 
     
     
         30 . The process of  claim 29 , further comprising deprotecting the protected linear peptide to yield a deprotected linear peptide having a sequence of amino acid units from position 1 through position n of the GCC agonist sequence. 
     
     
         31 . The process of  claim 30 , further comprising oxidizing the deprotected linear peptide to yield the peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249. 
     
     
         32 . The process of  claim 31 , further comprising purifying the peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249, wherein the purification comprises adsorbing the peptide onto a polymeric adsorbent column, optionally rinsing the peptide with deionized water, eluting the optionally rinsed peptide off the polymeric adsorbent column with an alcohol aqueous solution to form a peptide solution, removing water and the alcohol from the peptide solution to precipitate the peptide, and optionally adding an ether to the dewatered peptide to facilitate precipitation of the peptide. 
     
     
         33 . The process of  claim 32 , wherein the alcohol aqueous solution comprises isopropanol. 
     
     
         34 . The process of  claim 32 , wherein the ether comprises diethyl ether. 
     
     
         35 . The process of  claim 32 , further comprising salt exchanging the peptide by washing the peptide with an aqueous solution comprising an ammonium salt before the purification. 
     
     
         36 . The process of  claim 35 , further comprising, lyophilizing the peptide after the salt exchanging step before the purification. 
     
     
         37 . A process of purifying the peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249, the process comprising adsorbing the peptide onto a polymeric adsorbent column, desalting the peptide, eluting the desalted peptide off the polymeric adsorbent column with an alcohol aqueous solution to form a peptide solution, removing water from the peptide solution, and adding an ether to the dewatered peptide to precipitate the peptide. 
     
     
         38 . The process of  claim 37 , wherein the alcohol aqueous solution comprises isopropanol. 
     
     
         39 . The process of  claim 37 , wherein the ether comprises diethyl ether. 
     
     
         40 . The process of  claim 37 , further comprising salt exchanging the peptide by washing the peptide with an aqueous solution comprising an ammonium salt before the purification. 
     
     
         41 . The process of  claim 40 , further comprising, lyophilizing the peptide after the salt exchanging step before the purification. 
     
     
         42 . A purified peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249, wherein the peptide has a bulk density of no less than 0.1 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5 g/mL. 
     
     
         43 . A purified peptide, wherein the peptide is purified by the process of any of  claims 37 - 41 . 
     
     
         44 . The purified peptide of  claim 42 , wherein the peptide is purified by the process of any of  claims 37 - 41 . 
     
     
         45 . The purified peptide of any of  claims 42 - 44 , wherein the peptide has a tap density of no less than 0.1 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, no less than 0.5 g/mL, or no less than 0.6 g/mL. 
     
     
         46 . The purified peptide of any of  claims 42 - 45 , wherein the peptide has a chromatographic purity of no less than 95%, no less than 97%, or no less than 98%. 
     
     
         47 . The purified peptide of any of  claims 42 - 46 , wherein peptide is substantially free of water. 
     
     
         48 . The purified peptide of any of  claims 42 - 47 , wherein peptide is substantially free of impurities selected from acetonitrile, acetamide, alcohols, ammonium, and TFA. 
     
     
         49 . The purified peptide of any of  claims 42 - 48 , wherein the peptide is substantially free of topoisomers. 
     
     
         50 . The purified peptide of any of  claims 42 - 49 , wherein the peptide comprises the GCC agonist sequence of SEQ ID NO: 1. 
     
     
         51 . The purified peptide of any of  claims 42 - 49 , wherein the peptide comprises the GCC agonist sequence of SEQ ID NO: 9.