Controlled synthesis of peptide mixtures using mixed resins

• US 5,182,366 A
• Filed: 05/15/1990
• Issued: 01/26/1993
• Est. Priority Date: 05/15/1990
• Status: Expired due to Term

• Current Assignee: Daniel V. Santi, Verena D. Huebner
• Original Assignee: Daniel V. Santi, Verena D. Huebner
• Inventors: Huebner, Verena D., Santi, Daniel V.
• Primary Examiner(s): Lee, Lester L.
• Assistant Examiner(s): PERKINS, SUSAN
• Application Number:
  US07/523,791
• Time in Patent Office: 987 Days
• Field of Search: 530/334, 530/300, 530/333, 514/2
• US Class Current: 530/334
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00283 Reactor vessels with top opening
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/00418 using pressure
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00493 by sparging or bubbling with gases
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/045 using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

A method of preparing a mixture of peptides having a known composition and containing a peptide of a desired amino acid sequence is disclosed. The method involves three essential steps. First a given amount of a mixture of amino acyl or peptide derivatized resin is divided into a number of pools with each pool containing an equal molar amount of the resin mixture. Second a different single amino acid is coupled to the resin mixture in each of the pools and the coupling reaction is driven to completion. The peptide mixtures in each of the pools are then mixed together to obtain a complex peptide mixture containing each peptide in retrievable and analyzable amounts. The steps can be repeated to lengthen the peptide chains and methods can be employed to retrieve the desired peptide from the mixture and carry out analyses such as the determination of the amino acid sequence.

Claims

1. A method of preparing a mixture of peptides having different amino acid sequences, which mixture contains retrievable and analyzable amounts of each peptide, comprising:

(a) dividing an amount of a mixture of amino acyl or peptide derivatized resins into a plurality of subamounts;

(b) coupling a different single amino acid with each of the subamounts of resin to obtain a plurality of different peptide resins the coupling being carried out under conditions such that it is driven to substantial completion with each subamount; and

(c) combining known amounts of the different peptide resins together to obtain the mixture of peptides;

which mixture contains retrievable and analyzable amounts of each of the peptide resins.

2. The method as claimed in claim 1, wherein the amount of mixture in step (a) includes at least 5 different amino acyl or peptide derivatized resins in substantially equal molar amounts.

3. The method as claimed in claim 2, wherein the amount of mixture in step (a) is divided into at least 5 subamounts and wherein each of the subamounts is coupled in step (b) with a different single amino acid to obtain at least 5 different peptides in each subamount which are then combined in step (c) to obtain at least 25 different peptides, each present in retrievable and analyzable amounts.

4. The method as claimed in claim 1, wherein the amount of mixture in step (a) includes at least 10 different amino acyl or peptide derivatized resins which amount is divided into at least 10 subamounts.

5. The method as claimed in claim 4, wherein the 10 subamounts are each reacted with a different single amino acid in order to obtain at least 10 different peptides in each subamount and adding together the peptides from each subamount in step (c) in order to obtain at least 100 peptides, each present in retrievable and analyzable amounts.

6. The method as claimed in claim 1, wherein the amount of mixture in step (a) includes 20 amino acyl or peptide derivatized resins and the amount is divided into 20 subamounts with each subamount being reacted with a different single amino acid in step (b) in order to obtain 20 peptides in each of the subamounts which are then combined in step (c) to obtain a mixture of 400 peptides, each present in retrievable and analyzable amounts.

7. The method of preparing a mixture of peptides as claimed in claim 1, further comprising:

monitoring the coupling (b) by an analytical method to determine the degree of completion of the coupling.

8. The method of preparing a mixture of peptides as claimed in claim 7, wherein the analytical method includes the use of triketohydrine hydrate.

9. The method of preparing a mixture of peptides as claimed in claim 1, further comprising:

repeating the steps (a), (b) and (c) to increase the size of the peptides and the number of different peptides in the mixture.

10. The method of preparing a mixture of peptides as claimed in claim 9, further comprising:

retrieving at least one peptide from the mixture having a selected target property.

11. The method of preparing a mixture of peptides as claimed in claim 10, further comprising:

analyzing the peptide having the selected target property in order to determine its amino acid sequence.

12. The method of preparing a mixture of peptides as claimed in claim 9 wherein the combining is carried out using a mixture of acceptor amino acids or peptides derivatized to a solid support through their carboxy terminus.

13. The method as claimed in claim 10, wherein the target property is binding specifically to a peptide or protein.

14. The method as claimed in claim 10, wherein the protein is selected from an immunoglobin, a receptor, an enzyme, an antigen, and a receptor-binding ligand.

15. The method as claimed in claim 10, wherein the target property is binding specifically to DNA or RNA.

16. The method as claimed in claim 10, wherein the target property is binding to a carbohydrate or glycoprotein.

17. The method as claimed in claim 10, wherein the target property is transport or passage through a membrane.

18. The method as claimed in claim 10, wherein the target property is retention by a membrane.

19. The method as claimed in claim 10, wherein the target property is utilization as a substrate or inhibitor for a designated enzyme.

20. The method as claimed in claim 10, wherein the target property is a specified set of physical characteristics.

21. The method as claimed in claim 20, wherein the set of physical characteristics includes one or more characteristics selected from freezing point, molecular size, and solubility profile.

22. A method of preparing a mixture of peptides which mixture contains substantially equal molar amounts of a plurality of different known peptides, comprising:

(a) providing a plurality of pools each containing a mixture of amino acyl or peptide derivatized resins, each of which is present in substantially equal molar amounts;

(b) adding a different amino acid to each of the pools, the amino acid being added in an amount and under conditions so as to provide a substantially complete coupling reaction with all the resins in each pool to obtain different peptides in substantially equal molar amounts in each pool; and

(c) combining substantially equal molar ( amounts of different peptides together to obtain a mixture of peptides which mixture contains substantially equal molar amounts of the different peptides.

23. The method of preparing a mixture of peptides as claimed in claim 22, further comprising:

monitoring the coupling reaction in step (b) by an analytical method to determine the degree of completion of the coupling.

24. The method of preparing a mixture of peptides as claimed in claim 23, wherein the analytical method includes the use of triketohydrine hydrate.

25. The method of preparing a mixture of peptides as claimed in claim 22, further comprising:

repeating the steps (a), (b) and (c) to increase the size of the peptides and the number of different peptides in the mixture.