Divalent nucleobase compounds and uses therefor

Patent metadata

Field	Value
Publication number	US-10370415-B2
Application number	US-201414782471-A
Country	US
Kind code	B2
Filing date	Apr 11, 2014
Priority date	Apr 11, 2013
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

Abstract

Described herein are novel divalent nucleobases that each bind two nucleic acid strands, matched or mismatched when incorporated into a nucleic acid or nucleic acid analog backbone (a genetic recognition reagent, or genetic recognition reagent). In one embodiment, the genetic recognition reagent is a peptide nucleic acid (PNA) or gamma PNA (?PNA) oligomer. Uses of the divalent nucleobases and monomers and genetic recognition reagents containing the divalent nucleobases also are provided.

First claim

We claim:

1. A genetic recognition reagent comprising a plurality of nucleobase residues attached to a nucleic acid or nucleic acid analog backbone, in which at least one nucleobase residue is a divalent nucleobase chosen from the following:

in which each instance of R 1 is, independently, a protecting group or H and X is CH or N.

2. The genetic recognition reagent of claim 1 , in which each instance of R 1 is, independently, H or a protecting group chosen from: methyl, formyl, ethyl, acetyl, anisyl, benzyl, benzoyl, carbamate, trifluoroacetyl, diphenylmethyl, triphenylmethyl, N-hydroxysuccinimide, benzyloxymethyl, benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl), 4-methylbenzyl, 4-nitrophenyl, 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 2,4,5-trichlorophenyl, thioanizyl, thiocresyl, cbz (carbobenzyloxy), p-methoxybenzyl carbonyl, 9-fluorenylmethyloxycarbonyl, pentafluorophenyl, p-methoxybenzyl, 3,4-dimethozybenzyl, p-methoxyphenyl, 4-toluenesulfonyl, p-nitrobenzenesulfonates, 9-fluorenylmethyloxycarbonyl, 2-nitrophenylsulfenyl, 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, and p-bromobenzenesulfonyl.

3. The genetic recognition reagent of claim 1 , in which the backbone is chosen from one of a DNA, RNA, peptide nucleic acid (PNA), phosphorothioate, locked nucleic acid, unlocked nucleic acid, 2′-O-methyl-substituted RNA, morpholino nucleic acid, threose nucleic acid, or glycol nucleic acid backbone, or any combination thereof.

4. The genetic recognition reagent of claim 1 , in which the backbone is a peptide nucleic acid (PNA) backbone.

5. The genetic recognition reagent of claim 1 , in which the backbone is a gamma peptide nucleic acid (γPNA) backbone.

6. The genetic recognition reagent of claim 5 , in which the backbone is PEGylated, with one or more PEG moieties of two to fifty (—O—CH 2 —CH 2 —) residues.

7. The genetic recognition reagent of claim 1 , in which the backbone is a γPNA backbone in which a backbone monomer residue of the γPNA backbone is

wherein R is a nucleobase residue, R 1 of the backbone monomer residue, R 2 and R 3 are, independently, H, amino acid side chains, linear or branched (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 1 -C 8 )hydroxyalkyl, (C 3 -C 8 )aryl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )aryl(C 1 -C 6 )alkylene, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkylene, PEGylated moieties of the preceding comprising from 1 to 50 (—O—CH 2 —CH 2 —) residues, —CH 2 —(OCH 2 —CH 2 ) q OP 1 , —CH 2 —(OCH 2 —CH 2 ) q —NHP 1 , —CH 2 —(OCH 2 —CH 2 ) q —SP 1 and —CH 2 —(SCH 2 —CH 2 ) q —SP 1 , —CH 2 —(OCH 2 —CH 2 ) r —OH, —CH 2 —(OCH 2 —CH 2 ) r —NH 2 , —CH 2 —(OCH 2 —CH 2 ) r —NHC(NH)NH 2 , or —CH 2 —(OCH 2 —CH 2 ) r —S—S[CH 2 CH 2 ] s NHC(NH)NH 2 , where P 1 is selected from the group consisting of H, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 3 -C 8 )aryl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )aryl(C 1 -C 6 )alkylene and (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkylene; q is an integer from 0 to 10, inclusive; r and s are each independently integers from 1 to 50, inclusive; where R 1 of the backbone monomer residue and R 2 are different and one of R 1 of the backbone monomer residue or R 2 is H.

8. The genetic recognition reagent of claim 7 , in which R 2 is H, R 1 of the backbone monomer residue is an amino acid side chain that is optionally PEGylated, with one or more PEG moieties of one to twelve (—O—CH 2 —CH 2 —) residues.

9. The genetic recognition reagent of claim 1 , in which the nucleobase residues are arranged in a sequence complementary to a target sequence of a nucleic acid.

10. The genetic recognition reagent of claim 1 , having from 3 to 25 nucleobase residues.

11. The genetic recognition reagent of claim 1 , having the structure:

where each instance of R 4 is a backbone monomer residue and each instance of R is one of the plurality of nucleobase residues, where at least one instance of R the divalent nucleobase, E are independently end groups, and “n” is zero or a positive integer ranging from 1 to 48, wherein the plurality of nucleobase residues forms a sequence that is complementary to a target sequence of a nucleic acid.

12. The genetic recognition reagent of claim 1 , in which the divalent nucleobase is chosen from JB1, JB2, JB3, or JB4.

13. The genetic recognition reagent of claim 1 , in which all instances of R 1 are H.

14. A monomer for production of a genetic recognition reagent comprising a backbone monomer for a genetic recognition reagent covalently attached to a divalent nucleobase chosen from the following:

in which each instance of R 1 is, independently, a protecting group or H and X is CH or N.

15. The monomer of claim 14 , in which the nucleobase is one of JB1, JB2, JB3 or JB4.

16. The monomer of claim 14 , in which R 1 is a protecting group.

17. The monomer of claim 16 , in which the protecting group is chosen from one or more of: methyl, formyl, ethyl, acetyl, anisyl, benzyl, benzoyl, carbamate, trifluoroacetyl, diphenylmethyl, triphenylmethyl, N-hydroxysuccinimide, benzyloxymethyl, benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl), 4-methylbenzyl, 4-nitrophenyl, 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 2,4,5-trichlorophenyl, thioanizyl, thiocresyl, cbz (carbobenzyloxy), p-methoxybenzyl carbonyl, 9-fluorenylmethyloxycarbonyl, pentafluorophenyl, p-methoxybenzyl, 3,4-dimethozybenzyl, p-methoxyphenyl, 4-toluenesulfonyl, p-nitrobenzenesulfonates, 9-fluorenylmethyloxycarbonyl, 2-nitrophenylsulfenyl, 2,2,5,7,8-pentamethyl-chroman-6- sulfonyl, and p-bromobenzenesulfonyl.

18. The monomer of claim 14 , in which the monomer is a peptide nucleic acid (PNA) monomer.

19. The monomer of claim 14 , in which the monomer is a gamma peptide nucleic acid (γPNA) backbone monomer.

20. The monomer of claim 14 , in which the backbone monomer is PEGylated, with one or more PEG moieties of two to fifty (—O—CH 2 —CH 2 —) residues.

21. The monomer of claim 14 , in which the monomer is a γPNA monomer having the structure:

wherein R is a nucleobase residue, R 1 of the γPNA monomer, R 2 and R 4 are, independently, H, amino acid side chains, linear or branched (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 1 -C 8 )hydroxyalkyl, (C 3 -C 8 )aryl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )aryl(C 1 -C 6 )alkylene, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkylene, PEGylated moieties of the preceding comprising from 1 to 50 (—O—CH 2 —CH 2 —) residues, —CH 2 —(OCH 2 —CH 2 ) q OP 1 , —CH 2 —(OCH 2 —CH 2 ) q —NHP 1 , —CH 2 —(OCH 2 —CH 2 ) q —SP 1 and —CH 2 —(SCH 2 —CH 2 ) q —SP 1 , —CH 2 —(OCH 2 —CH 2 ) r —OH, —CH 2 —(OCH 2 —CH 2 ) r —NH 2 , —

Assignees

• Univ Carnegie Mellon

Inventors

• Ly Danith H
• Gopalsamy Suresh Kumar
• Manna Arunava

Classifications

• C07H21/04 Primary

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C07D239/54

  as doubly bound oxygen atoms or as unsubstituted hydroxy radicals · CPC title

• C07D471/04

  Ortho-condensed systems · CPC title

• C07D475/08

  with a nitrogen atom directly attached in position 2 · CPC title