Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques

• US 5,049,656 A
• Filed: 12/21/1988
• Issued: 09/17/1991
• Est. Priority Date: 12/21/1988
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Nebraska
• Original Assignee: Board of Regents of the University of Nebraska
• Inventors: Van Heeke, Gino, Schuster, Sheldon M., Lewis, William, Wagner, Fred W., Stout, Jay, Wylie, Dwane E.
• Primary Examiner(s): Schain, Howard E.
• Assistant Examiner(s): Perkins, Susan M.
• Application Number:
  US07/288,009
• Time in Patent Office: 1,000 Days
• Field of Search: 435/91, 530/334, 530/333, 536/27, 536/28, 536/29
• US Class Current: 530/334
• CPC Class Codes: C07K 1/006 of peptides containing derivatised side chain amino acids
• CPC Class Codes: C07K 1/04 on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

Abstract

The invention is directed to a method for purifying sequentially synthesized peptides and oligonucleotides by immunoaffinity techniques. Selected products are lapped with an antigenic capping agent and are conjugated with antibodies that are specific for the capping agent.

Claims

1. A method for the synthesis of a polypeptide which comprises:

(a) preparing a C-terminus blocked peptide of at least one amino acid in length;

(b) reacting an N-blocked, C-activated amino acid with the C-terminus blocked peptide to produce a mixture of an extended peptide and an unreacted peptide;

(c) adding an antigenic N-terminus capping agent to the mixture to antigenically cap the unreacted peptide, wherien the antigenic N-terminus capping agent is an aromatic acylating capping agent that reacts with amine groups;

(d) combining the mixture of antigenically capped unreacted peptide and extended peptide with antibodies that are selective for the antigenic cap; and

(e) isolating the extended peptide thereby producing the polypeptide.

2. A method according to claim 1, wherein the C-terminus blocked peptide, chain extended peptide, unreacted peptide and antigenically capped unreacted peptide are immobilized on a solid support and the solid support is removed just prior to step (d).

3. a method according to claim 1, or 2, further comprising:

repeating steps (a) through (c) one or more times;

removing the N-block on the extended peptide after each repetition of step (c) to form a new C-terminus blocked peptide for each repetition of step (a); and

,

reacting the same or a different N-blocked, C-activated

4. A method according to claim 3, wherein the aromatic acylating agent is fluorescamine and its derivatives or a substituted or unsubstituted phthalic anhydride, benzoyl halide or naphthoyl halide, the substituents being selected from the group consisting of mono-, di- or tri-nitro;

mono-, di- or tri-methoxy;

mono-, di- or tri-cyano; and

mono-, di- or tri-carboxy; and

the halide being fluoro, chloro, bromo and iodo.

12. A method according to claim 1, 5, 7, 9, 10 or 11 wherein the antibodies are polyclonal and selectively immunoreact with the antigenic cap.

13. A method according to claim 1, 5, 7, 9, 10 or 11 wherein the antibodies are monoclonal and selectively immunoreact with the antigenic cap.

14. A method according to claim 1, 5, 7, 9, 10 or 11 wherein the antibodies are immobilized upon a solid support.

5. A method for the synthesis of a polypeptide which comprises:

(a) preparing a C-terminus immobilized peptide of at least one amino acid in length, said C-terminus immobilized peptide being immobilized upon a support;

(b) reacting an N-blocked, C-activated amino acid with the C-terminus immobilized peptide to produce a mixture of immobilized extended peptide and immobilized unreacted peptide;

(c) adding an antigenic N-terminus capping agent to the mixture to antigenically cap the immobilized unreacted peptide, wherein the antigenic N-terminus capping agent is an aromatic acylating capping agent that reacts with amine groups;

(d) removing the N-block from the immobilized extended peptide to produce a new C-terminus immobilized peptide;

(e) repeating steps (a) throughout (d) with the new C-terminus immobilized peptide and the same or different N-blocked, C-activated amino acid until the desired polypeptide sequence in immobilized form is produced;

(f) removing the desired polypeptide and the antigenically capped unreacted peptides from the support;

(g) combining the desired polypeptide and antigenically capped unreacted peptides with antibodies that are selective for the antigenic cap to form a conjugated mixture; and

(h) removing the desired polypeptide from the conjugated mixture.

6. A method according to claim 5 wherein blocking groups for pendant functional moieties of the amino acid residues of the peptides are also removed in step (f).

7. A method for the synthesis of a polypeptide which comprises:

conducting a series of reactions which couple together the amino acids of the polypeptide;

at the completion of each coupling reaction, antigenically capping the N-terminus of any unreacted peptide side product that did not undergo the coupling reaction, the capping being conducted with an antigenic aromatic acylating capping agent that reacts with amine groups, thereby producing antigenically capped side products; and

removing the antigenically capped side products by their conjugation with antibodies that are immunospecific for the antigenic cap.

8. A method according to claim 7, wherein the removing step is performed at the end of the series of coupling steps.

9. A method for separating a mixture of a synthesized N-blocked polypeptide and N-unblocked unreacted peptide side products which comprises:

antigenically capping the N-terminus of the N-unblocked unreacted peptide side products with an N-terminus antigenic aromatic acylating capping agent to form antigenically capped side products, and conjugating the antigenically capped side products with antibodies that are immunospecific for the antigenic cap.

10. A method for separating a mixture of a synthesized N-blocked polypeptide, and N-unblocked unreacted peptide side products which comprises:

capping the N-terminus of the N-blocked unreacted peptide side products with an N-terminus capping agent, employing SULFmoc, Fmoc or an acid stable, base labile acylating group as the N-block of the sequentially synthesized N-blocked polypeptide and conjugating the resulting mixture with antibodies that are immunospecific for the SULFmoc, Fmoc or acid stable, base labile acylating group.

11. A method according to claim 10, further comprising employing an antigenic capping agent to cap the unreacted peptide side products, wherein the antigenic capping agent is an aromatic acylating capping agent that reacts with amine groups, and, in addition to conjugating with the antibodies that are immunospecific for the SULFmoc, Fmoc or acid stable, base labile acylating group, further conjugating the resulting mixture with antibodies that are immunospecific for the N-terminus antigenic cap.

15. A kit for antigenically capping peptides, which comprises;

a solution of antigenic capping agent in inert solvent wherein the antigenic capping agent is an aromatic acylating capping agent suitable for reaction with amino groups; and

antibodies in a buffered medium wherein the antibodies are immunoselective for the antigenic cap.