Solid phase peptide syntheses

Abstract

An improved method of deprotection in solid phase peptide synthesis is disclosed. In particular the deprotecting composition is added in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle. Thereafter, the ambient pressure in the vessel is reduced with a vacuum pull to remove the deprotecting composition without any draining step and without otherwise adversely affecting the remaining materials in the vessel or causing problems in subsequent steps in the SPPS cycle.

The invention claimed is: 
     
       1. A method of deprotection in batch solid phase peptide synthesis conducted in a batch reaction vessel in which the improvement comprises:
 adding an organic base deprotection composition neat to a mixture of a growing peptide chain, a solid phase support, and any excess activated amino acid from a preceding coupling cycle in the batch reaction vessel; 
 wherein the organic base is added to the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid from a preceding coupling cycle in the batch reaction vessel in a volume of 20% or less of the volume of the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid from a preceding coupling cycle in the batch reaction vessel; and 
 without any draining step of the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid from a preceding coupling cycle in the batch reaction vessel between the coupling step of any preceding coupling cycle and the addition of a deprotection composition for any successive cycle. 
 
     
     
       2. A method according to  claim 1  further comprising adding a successive amino acid to the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid from a preceding coupling cycle following the step of adding the deprotection composition. 
     
     
       3. A method according to  claim 1  comprising adding an organic base selected from the group consisting of piperidine, pyrrolidine, and 4-methyl piperidine. 
     
     
       4. A method according to  claim 1  wherein the organic base is a liquid added neat to the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid from a preceding coupling cycle in a ratio of between about 1:20 and 1:3 based upon the volume of the mixture. 
     
     
       5. A method according to  claim 3  comprising adding an organic base selected from the group consisting of piperidine, pyrrolidine, and 4-methyl piperidine in a volume ratio of about 1:5 based upon the volume of the mixture. 
     
     
       6. A method according to  claim 1  wherein the volume of the deprotection composition is less than 2 mL. 
     
     
       7. A method according to  claim 1  wherein the volume of the deprotection composition is less than 1 mL. 
     
     
       8. A method according to  claim 1  wherein the volume of the deprotection composition is between about 0.4 and 1.0 ml added to between about 3.8 and 4.2 ml of the mixture of the growing peptide chain, the solid phase support, and any excess activated amino acid. 
     
     
       9. A method of deprotection in solid phase peptide synthesis in which the improvement comprises:
 deprotecting a protected amino acid by combining and heating the protected amino acid and a liquid organic base in a reaction vessel; and 
 during or after the deprotecting step, reducing the ambient pressure in the vessel with a vacuum pull to remove the liquid organic base without any intervening draining step while simultaneously heating the vessel contents; and 
 without otherwise adversely affecting the remaining materials in the vessel or causing problems in any subsequent steps in the SPPS cycle. 
 
     
     
       10. A method according to  claim 9  comprising:
 applying microwave radiation to heat the deprotection step; and 
 applying microwave radiation to accelerate the vacuum removal step. 
 
     
     
       11. A method according to  claim 9  comprising reducing the pressure in the vessel to less than one atmosphere. 
     
     
       12. A method according to  claim 9  comprising:
 heating the combined protected amino acid and liquid organic base to between about 81° C. and 99° C. to accelerate the deprotection step; and 
 heating the vessel contents to between about 90° C. and 110° C. to accelerate the removal step. 
 
     
     
       13. A method of deprotection in solid phase peptide synthesis (SPPS) in which the improvement comprises deprotecting in a reaction vessel a protected amino acid below atmospheric pressure at a temperature of at least about 60° C. while providing a path for evaporating base to leave the reaction vessel. 
     
     
       14. A method according to  claim 13  further comprising carrying out a maximum of one washing step between the deprotecting and coupling steps in the SPPS cycle.