# US 10,370,415 B2 · Divalent nucleobase compounds and uses therefor

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- Number: US 10,370,415 B2
- Document ID: US10370415B2
- Status: Granted
- Kind: B2
- Application number: 14/782,471
- Filed: 2014-04-11
- Published: 2016-03-24
- Granted: 2019-08-06
- Earliest claimed: 2013-04-11
- Assignees: Carnegie Mellon University
- Applicants: Carnegie Mellon University
- Inventors: Danith H. Ly; Suresh Kumar Gopalsamy; Arunava Manna
- Web page: https://patentgrep.com/patent/US10370415B2

## CPC

- C07K 14/003: {Peptide-nucleic acids (PNAs)}
- C07D 239/47: One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
- C07D 239/54: as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
- C07D 251/16: to only one ring carbon atom
- C07D 471/04: Ortho-condensed systems
- C07D 475/08: with a nitrogen atom directly attached in position 2
- C07D 487/04: Ortho-condensed systems
- C07D 487/16: Peri-condensed systems
- C07H 1/06: Separation; Purification
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### 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 R1 is, independently, a protecting group or H and X is CH or N.

### Claim 2 (depends on 1)

The genetic recognition reagent of claim 1, in which each instance of R1 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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

The genetic recognition reagent of claim 5, in which the backbone is PEGylated, with one or more PEG moieties of two to fifty (—O—CH2—CH2—) residues.

### Claim 7 (depends on 1)

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, R1 of the backbone monomer residue, R2and R3 are, independently, H, amino acid side chains, linear or branched (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C1-C8)hydroxyalkyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene, (C3-C8)cycloalkyl(C1-C6)alkylene, PEGylated moieties of the preceding comprising from 1 to 50 (—O—CH2—CH2—) residues, —CH2—(OCH2—CH2)qOP1, —CH2—(OCH2—CH2)q—NHP1, —CH2—(OCH2—CH2)q—SP1 and —CH2—(SCH2—CH2)q—SP1, —CH2—(OCH2—CH2)r—OH, —CH2—(OCH2—CH2)r—NH2, —CH2—(OCH2—CH2)r—NHC(NH)NH2, or —CH2—(OCH2—CH2)r—S—S[CH2CH2]sNHC(NH)NH2, where P1 is selected from the group consisting of H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene and (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 10, inclusive; r and s are each independently integers from 1 to 50, inclusive; where R1 of the backbone monomer residue and R2 are different and one of R1 of the backbone monomer residue or R2is H.

### Claim 8 (depends on 7)

The genetic recognition reagent of claim 7, in which R2 is H, R1 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—CH2—CH2—) residues.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

The genetic recognition reagent of claim 1, having the structure:
where each instance of R4 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.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

The genetic recognition reagent of claim 1, in which all instances of R1 are H.

### Claim 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 R1 is, independently, a protecting group or H and X is CH or N.

### Claim 15 (depends on 14)

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

### Claim 16 (depends on 14)

The monomer of claim 14, in which R1 is a protecting group.

### Claim 17 (depends on 16)

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.

### Claim 18 (depends on 14)

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

### Claim 19 (depends on 14)

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

### Claim 20 (depends on 14)

The monomer of claim 14, in which the backbone monomer is PEGylated, with one or more PEG moieties of two to fifty (—O—CH2—CH2—) residues.

### Claim 21 (depends on 14)

The monomer of claim 14, in which the monomer is a γPNA monomer having the structure:
wherein R is a nucleobase residue, R1 of the γPNA monomer, R2 and R4 are, independently, H, amino acid side chains, linear or branched (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C1-C8)hydroxyalkyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene, (C3-C8)cycloalkyl(C1-C6)alkylene, PEGylated moieties of the preceding comprising from 1 to 50 (—O—CH2—CH2—) residues, —CH2—(OCH2—CH2)qOP1, —CH2—(OCH2—CH2)q—NHP1, —CH2—(OCH2—CH2)q—SP1 and —CH2—(SCH2—CH2)q—SP1, —CH2—(OCH2—CH2)r—OH, —CH2—(OCH2—CH2)r—NH2, —CH2—(OCH2—CH2)r—NHC(NH)NH2, or —CH2—(OCH2—CH2)r—S—S[CH2CH2]sNHC(NH)NH2, where P1 is selected from the group consisting of H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene and (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 10, inclusive; r and s are each independently integers from 1 to 50, inclusive; where R1 of the γPNA monomer and R2 are different, and one of R1 of the γPNA monomer or R2 is H, and R3 is H or a protecting group.

### Claim 22 (depends on 21)

The monomer of claim 21, in which R2 is H, R1 of the γPNA monomer is an amino acid side chain that is optionally PEGylated, with one or more PEG moieties of one to twelve (—O—CH2—CH2—) residues, and R3 is a protecting group.

### Claim 23

A nucleobase chosen from one the following:
in which each instance of R1 is, independently a protecting group or H, R is a reactive group and X is CH or N.

### Claim 24 (depends on 23)

The nucleobase of claim 23, chosen from one of the following:
in which each instance of R1 is, independently a protecting group or H.

### Claim 25 (depends on 14)

A kit comprising a monomer of claim 14 in a vessel.

### Claim 26 (depends on 25)

The kit of claim 25, further comprising monomers comprising each of JB1, JB2, JB3, JB4,
where R1 is a protecting group, or H,
JB7, JB8, JB9, JB9b, JB10, JB11, JB12, JB13, JB14, JB15, and JB16 nucleobases in separate vessels.

### Claim 27 (depends on 1)

An array comprising the genetic recognition reagent of claim 1.

### Claim 28 (depends on 1)

A method of detection of a target sequence in a nucleic acid, comprising contacting the genetic recognition reagent of claim 1 with a sample comprising nucleic acid and detecting binding of the genetic recognition reagent with the nucleic acid.

### Claim 29 (depends on 1)

A method of isolation and purification of a nucleic acid containing a target sequence, comprising, contacting a nucleic acid sample with the genetic recognition reagent of claim 1, separating the nucleic acid sample from the genetic recognition reagent, leaving any nucleic acid bound to the genetic recognition reagent bound to the genetic recognition reagent, and separating the genetic recognition reagent from any nucleic acid bound to the genetic recognition reagent.

### Claim 30 (depends on 29)

The method of claim 29 in which the genetic recognition reagent is immobilized on a substrate, comprising contacting the nucleic acid with the substrate, washing the substrate to remove unbound nucleic acid from the substrate, but leaving bound nucleic acid bound to the substrate, and eluting the bound nucleic acid from the substrate.

### Claim 31 (depends on 1)

The genetic recognition reagent of claim 1, wherein at least one nucleobase is JB1.

### Claim 32 (depends on 1)

The genetic recognition reagent of claim 1, wherein at least one nucleobase is JB2.

### Claim 33 (depends on 1)

The genetic recognition reagent of claim 1, wherein at least one nucleobase is JB3.

### Claim 34 (depends on 1)

The genetic recognition reagent of claim 1, wherein at least one nucleobase is JB4.

### Claim 35

A genetic recognition reagent comprising a plurality of nucleobase residues attached to a peptide nucleic acid backbone, in which at least one nucleobase is
where R1 is a protecting group, or H.

### Claim 36 (depends on 35)

The genetic recognition reagent of claim 35, wherein the backbone is a gamma peptide nucleic acid (γPNA) backbone.

### Claim 37 (depends on 14)

The monomer of claim 14, wherein the divalent nucleobase is JB1.

### Claim 38 (depends on 14)

The monomer of claim 14, wherein the divalent nucleobase is JB2.

### Claim 39 (depends on 14)

The monomer of claim 14, wherein the divalent nucleobase is JB3.

### Claim 40 (depends on 14)

The monomer of claim 14, wherein the divalent nucleobase is JB4.

### Claim 41 (depends on 23)

The nucleobase of claim 23, having the structure of JB1.

### Claim 42 (depends on 23)

The nucleobase of claim 23, having the structure of JB2.

### Claim 43 (depends on 23)

The nucleobase of claim 23, having the structure of JB3.

### Claim 44 (depends on 23)

The nucleobase of claim 23, having the structure of JB4.

### Claim 45 (depends on 23)

The nucleobase of claim 23, wherein R is carboxyl.

### Claim 46 (depends on 25)

The kit of claim 25, wherein the monomer comprises a peptide nucleic acid backbone monomer.

### Claim 47 (depends on 25)

The kit of claim 25, wherein the monomer comprises a γ-peptide nucleic acid backbone monomer.