Process of preparing guanylate cyclase C agonists
View Patent ↗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.
1. A process of preparing a peptide consisting of the GCC agonist sequence of SEQ ID NO: 1, 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, wherein at least one of the first and second fragments is provided via a solid-phase peptide synthesis; 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, and wherein the peptide is purified by a process comprising 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 isopropanol aqueous solution to form a peptide solution, removing water and the isopropanol from the peptide solution to precipitate the peptide, and optionally adding an ether to the dewatered peptide to facilitate precipitation of the peptide.
2. The process of claim 1 , wherein h is 7, j is 15, and k is 16.
3. The process of claim 2 , 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.
4. The process of claim 3 , 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.
5. The process of claim 4 , 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.
6. The process of claim 5 , further comprising deprotecting the protected linear peptide to yield a deprotected linear peptide and oxidizing the deprotected linear peptide to yield the peptide consisting of SEQ ID NO:1.
7. The process of claim 1 , wherein each of the first and second fragments is not more than 10 amino acid units in length.
8. The process of claim 7 , 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, leucine, and alanine, or 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, leucine, and alanine.
9. 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, leucine, and alanine.
10. The process of claim 9 , 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.
11. The process of claim 10 , 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, and
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.
12. The process of claim 11 , 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 and oxidizing the deprotected linear peptide to yield the peptide consisting of SEQ ID NO: 1.