Peptide nucleic acid precursors and methods of preparing same

• US 6,433,134 B1
• Filed: 01/05/2000
• Issued: 08/13/2002
• Est. Priority Date: 07/09/1998
• Status: Expired due to Fees

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Patron, Andrew P., Pervin, Azra
• Primary Examiner(s): Shah, Mukund J.
• Assistant Examiner(s): TRUONG, TAMTHOM NGO
• Application Number:
  US09/479,320
• Time in Patent Office: 951 Days
• Field of Search: 530/333, 530/300, 536/22.1, 536/26.7, 536/26.8, 536/25.3, 544/264, 544/265, 544/310, 544/296
• US Class Current: 530/300
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)

Abstract

Novel and efficient syntheses create novel piperazinone intermediates which facilitate the production and use of PNAs. Such syntheses and the products enhance the feasibility of a system which permits the rapid identification of PNA oligomers useful as therapeutics, diagnostics and/or gene characterization tools. A first component of the system is a universal PNA library that most preferably incorporates one or more universal nucleotide bases into carefully selected positions within each oligomer species thereby providing the library with the screening ability of a much larger library. The second component of the system is a high throughput screening system that includes a number of assays designed to provide information on the binding activities of the different PNAs to a target nucleotide sequence (generally, a DNA or RNA sequence). The third component is a software system especially designed to provide rapid analysis of the data collected from the high throughput screening system and to determine therefrom the sequence base identities and sequence lengths of PNA oligomers most likely to bind to and appropriately affect the target molecule.

Claims

1. A protected precursor which readily provides a peptide nucleic acid (PNA), said precursor consisting essentially of a cyclic substituted piperazinone having a nucleotide base covalently linked directly to one nitrogen in said cyclic piperazinone ring and having a first protecting group covalently linked to the other nitrogen in said piperazinone ring, said substituted piperazinone constituting an intermediate, which precursor may be readily hydrolyzed to a peptide nucleic acid monomer or reacted with a nucleophile to form a PNA derivative or an oligomer.

2. The precursor according to claim 1 wherein said nucleotide base is selected from the group consisting of thymine, cytosine, guanine, adenine, and universal bases which are capable of binding to any of the four natural nucleotide bases.

3. The precursor according to claim 1 wherein said linkage of said nucleotide base to said piperazinone includes an amide bond between an acetyl group substituted onto a nitrogen atom in a purine or a pyrimidine ring in said nucleotide base and said one nitrogen.

4. The precursor according to claim 3 wherein either an amino group linked to or an amido group that is a part of an aromatic ring of said nucleotide base has attached thereto at least one amino- or amido-protecting group as a second protecting group.

5. The precursor according to claim 4 wherein said second protecting group attached to said nucleotide base is one that is not normally removed under conditions which remove said first protecting group.

6. The precursor according to claim 5 wherein said first protecting group is Boc.

7. A method for preparing a protected precursor, which method comprises the steps of:

a) mixing ethylene diamine and a haloacetic acid equivalent having the formula Y—

CH₂CO—

X where X is hydrogen, halogen or OR, with R being hydrogen or lower alkyl, and Y is a leaving group, in solution;

b) heating said mixture of step (a) to form a cyclic piperazinone;

c) covalently coupling a nucleotide base to said cyclic piperazinone to create a base-substituted piperazinone; and

d) providing protection for an amido moiety of the product of step (c) to form an activated intermediate which serves as a precursor that may be readily hydrolyzed to a peptide nucleic (PNA) acid monomer or reacted with a nucleophile to form a PNA derivative or an oligomer.

8. The method according to claim 7 wherein said nucleotide base is selected from the group consisting of thymine, cytosine, guanine, adenine, and universal bases which are capable of binding to any of the four natural nucleotide bases.

9. The method according to claim 7 wherein said haloacetic acid equivalent is provided in at least a stoichiometric amount to said ethylene diamine and where X is H, Br, Cl or OR with R being H or lower alkyl.

10. The method according to claim 9 wherein said nucleotide base is added by initially treating said cyclic piperazinone with a haloacetylating agent to form an amide bond and create a haloacetyl-piperazinone which is subsequently reacted with said nucleotide base.

11. The method according to claim 10 wherein said leaving group is Cl or Br and said haloacetylating agent is chloroacetic anhydride or chloroacetylchloride.

12. The method according to claim 7 wherein said base-substituted piperazinone is further reacted to add a covalent amido-protecting group.

13. The method according to claim 12 wherein said further reaction is with di-t-butyl dicarbonate.

14. The method according to claim 7 wherein said nucleotide base is added to the secondary amino moiety in said piperazinone by reacting said cyclic piperazinone with a nucleotide base acetic acid to form said nucleotide base-piperazinone product.

15. The method according to claim 14 wherein said nucleotide base-piperazinone product is then reacted using a reagent selected from the group consisting of di-t-butyl dicarbonate, benzyl chloroformate and 2-nitrobenzenesulfonyl chloride, to protect the amido moiety in said piperazinone ring.

16. A method for preparing a protected precursor, which method comprises the steps of:

a) carrying out a reductive amination reaction between the α

-amino group of an α

-amino acid or an ester thereof and the carboxyl group of either an N-protected amino aldehyde or an N-protected amino ketone;

b) then deprotecting the primary amino group of the reaction product of a step (a);

c) heating said deprotected reaction product of step (b) to form a cyclic piperazinone;

d) covalently coupling a nucleotide base to said cyclic piperazinone to create a base-substituted piperazinone; and

e) providing protection for an amido moiety of the product of step (d) to form an intermediate which serves as a precursor that may be readily hydrolyzed to a peptide nucleic acid (PNA) monomer or reacted with a nucleophile to form a PNA derivative or an oligomner.

17. The method according to claim 16 wherein said amino aldehyde has the formula P^gHN—

CHR₁—

CHO, said amino ketone has the formula P^gHN—

CHR₁—

C(═

O)—

R₂ and said α

-amino acid or ester has the formula H₂N—

CHR₄—

COOR₃ wherein P^g is an amino-protecting group, R₁, R₃ and R₄ are H, lower alkyl, substituted lower alkyl or aromatic, and R₂ is lower alkyl, substituted lower alkyl, or aromatic.

18. The method according to claim 16 wherein said nucleotide base is linked to the secondary amino group in said piperazinone ring through a linkage which includes an acetyl residue which is substituted onto a nitrogen atom in a purine or a pyrimidine ring in said nucleotide base and which forms an amide bond with said secondary amino group.