Boc and fmoc solid phase peptide synthesis

Abstract

A solid phase method for synthesizing a peptide containing three or more amino acid residues utilizing both Boc and Fmoc protected amino acids and a chloromethylated polystyrene resin.

1 . A method for preparing a peptide comprising three or more amino acid residues having an N-terminal amino acid, a next to last amino acid adjacent to the N-terminal amino acid and a C-terminal amino acid, wherein said method comprises the steps of:
 (a) attaching a first amino acid to a solid support resin through an ester bond to form a first-coupled-product, which comprises (i) reacting an aqueous solution of cesium carbonate with an alcohol solution of the first amino acid to form a cesium salt of the first amino acid, (ii) obtaining a solvent free cesium salt of the first amino acid, (iii) reacting the solid support resin with the cesium salt of the first amino acid in a dry polar aprotic solvent to form a first-coupled product;   wherein the first amino acid corresponds to the C-terminal amino acid of the peptide, the first amino acid's non-side chain amino group is blocked by a Boc and the first amino acid does not have a side chain functionality requiring protection, and the solid support resin is a chloromethylated polystyrene resin;   (b) deblocking the Boc from the first-coupled-product with an acid to form a first-deblocked-coupled-product;   (c) optionally coupling a next-amino-acid to the first-deblocked-coupled-product, which comprises reacting the next-amino-acid with the first-deblocked-coupled-product in an organic solvent comprising a peptide coupling reagent to form a next-blocked-coupled-product, wherein the next-amino-acid has a non-side chain amino group blocked by Boc and if the next-amino-acid has one or more side chain functionalities then the side chain functionalities do not require protection or the side chain functionalities have protecting groups that are stable to acid and base reagents used to deblock Boc and Fmoc, respectively;   (d) deblocking the Boc from the next-blocked-coupled-product which comprises reacting the next-blocked-coupled-product with a an acid to yield a next-deblocked-coupled-product;   (e) optionally repeating steps (c) and (d), each cycle forming an (X+1)-next-deblocked-coupled-product where X is the number of desired cycle repetitions;   (f) coupling a next-amino-acid to the first-deblocked-coupled-product from (b), or optionally to the (X+1)-next-deblocked-coupled-product from (e), which comprises reacting the next-amino-acid with said first-deblocked-coupled-product or said (X+1)-next-deblocked-coupled-product in an organic solvent comprising a peptide coupling reagent to form a next-blocked-coupled-product, wherein the next-amino-acid has a non-side chain amino group blocked by Fmoc, provided that if the next-amino-acid has one or more side chain functionalities then the side chain functionalities do not require protection or the side chain functionalities have protecting groups that are stable to base reagents used to deblock Fmoc;   (g) deblocking the Fmoc from the next-blocked-coupled-product which comprises reacting the next-blocked-coupled-product with a primary or secondary amine to yield a next-deblocked-coupled-product;   (h) optionally repeating steps (1) and (g), each cycle forming an (X+1)-next-deblocked-coupled-product where X is the desired number of cycle repetitions, until the next to last amino acid of the peptide is coupled and deblocked;   (i) coupling an N-terminal amino acid to the (X+1)-next-deblocked-coupled-product, which comprises reacting the N-terminal amino acid with the (X+1)-next-deblocked-coupled-product in an organic solvent comprising a peptide coupling reagent to form a completed-blocked-coupled-product, wherein the N-terminal-amino-acid has a non-side chain amino group blocked by Boc or Fmoc;   (j) deblocking the Boc or Fmoc group from the completed-blocked-coupled-product, which comprises reacting the completed-blocked-coupled-product with an acid in the case of Boc, or a base in the case Fmoc, to form a completed-peptide-resin-product;   (k) if side-chain functionalities are present on the completed-peptide-resin-product then optionally deprotecting the side-chain functionalities of the completed-peptide-resin-product, which comprises reacting the completed-peptide-resin-product with the appropriate deprotecting reagents to form a deprotected-completed-peptide-resin-product; and   (l) cleaving the peptide from the solid support resin of the completed-peptide-resin-product or the deprotected-completed-peptide-resin-product to yield the peptide, which comprises reacting the completed-peptide-resin-product or the deprotected-completed-peptide-resin-product with ammonia, a primary amine or a secondary amine until the cleavage of the peptide from the resin is substantially complete;   provided that steps (f) and (g) must be carried out at least once in the synthesis of the peptide.   
     
     
         2 . A method according to  claim 1 , wherein the ammonia,
 primary amine or secondary amine of step (1) is in a solvent comprising an alcohol, and optionally, an aprotic polar solvent.   
     
     
         3 . A method according to  claim 1 , wherein step (1) further comprises the steps of:
 (i) precipitating the cleaved peptide from the solvent;   (ii) filtering the solid support resin and the precipitated peptide; and   (iii) extracting the peptide in an acid solution to isolate the peptide.   
     
     
         4 . A method according to  claim 1 , wherein the first amino acid is Boc-L-Thr. 
     
     
         5 . A method according to  claim 4 , wherein the first amino acid is Boc-L-Thr- cesium salt yielding Boc-L-Thr-resin as the first-coupled-product and H-L-Thr-resin is the first-deblocked-coupled-product. 
     
     
         6 . A method according to  claim 5 , wherein the acid used to deblock the Boc group in step (j) is TFA. 
     
     
         7 . A method according to  claim 6 , wherein the organic solvent is methylene chloride, chloroform, or dimethylformamide and the peptide coupling reagent is diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl-N′-(3-dimethyl-aminopropyl) carbodiimide. 
     
     
         8 . A method according to  claim 7 , which comprises carrying out steps (f) and (g) six times after the formation of the first-deblocked-coupled-product of the formula H-L-Thr-resin wherein the following amino acids are coupled in the order of Fmoc-L-Cys(Acm), Fmoc-L-Val, Fmoc-L-Lys(Boc), Fmoc-D-Trp, Fmoc-L-Tyr(O-t-Bu) and Fmoc-L-Cys-(Acm) to form H-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin. 
     
     
         9 . A method according to  claim 8 , which comprises coupling Boc-D-β-Nal to H-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin according to step (i) to form Boc-D-β-Nal-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin. 
     
     
         10 . A method according to  claim 9 , which comprises simultaneously deblocking the Boc group blocking D-β-Nal, the O-t-Bu group protecting Tyr and the Boc group protecting Lys of Boc-D-β-Nal-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin according to step (j) to yield the completed-peptide-resin-product of the formula H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-resin. 
     
     
         11 . A method according to  claim 10 , which comprises cleaving the peptide, H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr, from the solid resin by reacting H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cyx(Acm)Thr-resin with ammonia in a solvent comprising an alcohol, and optionally, an aprotic polar solvent until the cleavage is substantially complete to yield H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2 . 
     
     
         12 . A method according to  claim 11 , wherein the alcohol is methanol and the polar aprotic solvent is dimethylformamide. 
     
     
         13 . A method according to  claim 12 , which comprises simultaneously deprotecting the Acm groups protecting Cys and cyclizing the resulting deprotected Cys residues of the completed-peptide-resin-product of the formula H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2  by reacting H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2  with a solution of iodine in an alcohol until the deprotecting and cyclizing is substantially complete to yield H-D-β-Nal-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 . 
     
     
         14 . A method according to  claim 1 , wherein the peptide is H-D-β-Nal-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 . 
     
     
         15 . A method according to  claim 1 , wherein the peptide is a somatostatin analog. 
     
     
         16 . A method according to  claim 1  for preparing a peptide comprising three or more amino acid residues having an N-terminal amino acid, a next to last amino acid adjacent to the N-terminal amino acid and a C-terminal amino acid substantially as hereinbefore described and exemplified.