US6660845B1
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US
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Non-aggregating, non-quenching oligomers comprising nucleotide analogues; methods of synthesis and use thereof
Assignee: DRUG ROYALTY TRUST 9
•
Filing: 1999-11-23
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Granted: 2003-12-09
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Expires: 2019-11-23
Abstract
Abstract The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.
Inventors
- 👤 Alexander A. Gall
- 👤 Igor V. Kutyavin
- 👤 Nicolaas M. J. Vermeulen
- 👤 Robert O. Dempcy
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 17:23
CPC Classifications
C; C07; C07D; C07D487/00; C07D487/02; C07D487/04; A; A61; A61K; A61K31/00; A61K31/33; A61K31/395; A61K31/495; A61K31/505; A61K31/519; C07H; C07H21/00; C07K; C07K14/00; C07K14/001; C07K14/003; C07K5/00; C07K5/04; C07K5/06; C07K5/06008; C07K5/06017; C07K5/06026; C07K5/06139; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; C12Q1/6816; C12Q1/6818
Claims Analysis
Total Claims: 57
Claim 1
Independent Claim
145 words
An oligomer comprising one or more peptide nucleic acid (PNA) residues, wherein one or more of said PNA residues comprises a base having the formula wherein R 1 and R 2 are independently —H, —OH, —OR 6 , —SH, NH 2 , or —NHR 7 ; R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; R 6 is —C 6 H 5 (phenyl) or a diphenylcarbamoyl (DPC) group; R 7 is a protecting group selected from the group consisting of dimethylamino(methylene), benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyl, acetyl and anisoyl (An) groups; and X is ═CH— or ═N—; provided that when R 1 is —NH 2 and R 2 is H, then X is N.
Claim 2
Dependent Claim
145 words
An oligomer comprising one or more peptide nucleic acid (PNA) residues, wherein one or more of said PNA residues comprises a base having the formula wherein R 1 and R 2 are independently —H, —OH, —OR 6 , —SH, NH 2 , or —NHR 7 ; R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; R 6 is —C 6 H 5 (phenyl) or a diphenylcarbamoyl (DPC) group; R 7 is a protecting group selected from the group consisting of dimethylamino(methylene), benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyl, acetyl and anisoyl (An) groups; and X is ═CH— or ═N—; provided that when R 1 is —NH 2 and R 2 is H, then X is N.
Claim 3
Dependent Claim
16 words
An oligomer according to claim 1 , wherein the oligomer comprises one or more DNA nucleotides.
Claim 4
Dependent Claim
15 words
An oligomer according to claim 1 , wherein the oligomer is a peptide nucleic acid.
Claim 5
Dependent Claim
14 words
An oligomer according to claim 1 , wherein the oligomer is a PNA/DNA chimera.
Claim 6
Dependent Claim
13 words
An oligomer according to claim 1 , further comprising a minor groove binder.
Claim 7
Dependent Claim
11 words
An oligomer according to claim 1 , further comprising a fluorophore.
Claim 8
Dependent Claim
12 words
An oligomer according to claim 6 , further comprising a fluorescence quencher.
Claim 9
Dependent Claim
13 words
An oligomer according to claim 7 , further comprising a minor groove binder.
Claim 10
Dependent Claim
11 words
An oligomer according to claim 4 , further comprising a fluorophore.
Claim 11
Dependent Claim
12 words
An oligomer according to claim 9 , further comprising a fluorescence quencher.
Claim 12
Dependent Claim
13 words
An oligomer according to claim 10 , further comprising a minor groove binder.
Claim 13
Dependent Claim
13 words
An oligomer according to claim 4 , further comprising a minor groove binder.
Claim 14
Dependent Claim
11 words
An oligomer according to claim 12 , further comprising a fluorophore.
Claim 15
Dependent Claim
168 words
A conjugate comprising: (a) a polymer comprising a plurality of monomer units; and (b) a fluorophore covalently attached thereto; wherein one or more of the monomer units comprises a base analogue, wherein each of the base analogues is independently selected from the group consisting of pyrazolopyrimidines and pyrrolopyrimidines having the structure wherein R 1 and R 2 are independently —H, —OH, —SH, or —NH 2 ; R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; and X is ═CH— or ═N—; provided that when R 1 is —NH 2 and R 2 is H, then X is ═N—, such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and lead to reduced quenching of the fluorophore, reduced self-association of the polymer or both reduced quenching of the fluorophore and reduced self-association of the polymer.
Claim 16
Dependent Claim
168 words
A conjugate comprising: (a) a polymer comprising a plurality of monomer units; and (b) a fluorophore covalently attached thereto; wherein one or more of the monomer units comprises a base analogue, wherein each of the base analogues is independently selected from the group consisting of pyrazolopyrimidines and pyrrolopyrimidines having the structure wherein R 1 and R 2 are independently —H, —OH, —SH, or —NH 2 ; R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; and X is ═CH— or ═N—; provided that when R 1 is —NH 2 and R 2 is H, then X is ═N—, such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and lead to reduced quenching of the fluorophore, reduced self-association of the polymer or both reduced quenching of the fluorophore and reduced self-association of the polymer.
Claim 17
Dependent Claim
37 words
The conjugate of claim 14 , having at least four consecutive purine residues in the polymer, wherein at least one of the at least four consecutive purine residues in the polymer is substituted by a base analogue.
Claim 18
Dependent Claim
11 words
The conjugate of claim 15 , further comprising a fluorescence quencher.
Claim 19
Dependent Claim
10 words
The conjugate of claim 15 wherein the polymer comprises DNA.
Claim 20
Dependent Claim
13 words
The conjugate of claim 15 wherein the polymer comprises peptide nucleic acid (PNA).
Claim 21
Dependent Claim
12 words
The conjugate of claim 15 wherein the polymer comprises a PNA/DNA chimera.
Claim 22
Dependent Claim
19 words
The conjugate of claim 19 , wherein the base analogue is present in the DNA portion of the polymer.
Claim 23
Dependent Claim
19 words
The conjugate of claim 19 , wherein the base analogue is present in the PNA portion of the polymer.
Claim 24
Dependent Claim
10 words
The conjugate of claim 14 , wherein X is ═N—.
Claim 25
Dependent Claim
24 words
The conjugate of claim 22 , wherein the base analogue is selected from the group consisting of pyrazolopyrimidinyladenine (PPA), pyrazolopyrimidinylguanine (PPG) and pyrazolopyrimidinylhypoxanthine (PPI).
Claim 26
Dependent Claim
10 words
The conjugate of claim 14 , wherein X is ═CH—.
Claim 27
Dependent Claim
21 words
The conjugate of claim 24 , wherein the base analogue is selected from the group consisting of 7-deazaadenine, 7-deazahypoxanthine and 7-deazaguanine.
Claim 28
Dependent Claim
15 words
The conjugate of claim 16 , wherein the fluorophore emits between 400 and 800 nm.
Claim 29
Dependent Claim
15 words
The conjugate of claim 26 , wherein the quencher absorbs between 400 and 800 nm.
Claim 30
Dependent Claim
12 words
The conjugate of claim 15 , further comprising a minor groove binder.
Claim 31
Dependent Claim
13 words
An oligomer microarray, comprising one or more oligomers according to claim 1 .
Claim 32
Dependent Claim
13 words
An oligomer microarray comprising one or more oligomers according to claim 15 .
Claim 33
Dependent Claim
24 words
The conjugate of claim 15 , wherein the base analogue is selected from the group consisting of pyrazolopyrimidinyladenine (PPA), pyrazolopyrimidinylguanine (PPG) and pyrazolopyrimidinylhypoxanthine (PPI).
Claim 34
Dependent Claim
13 words
The conjugate of claim 31 , wherein the base analogue is pyrazolopyrimidinylguanine (PPG).
Claim 35
Dependent Claim
12 words
The conjugate of claim 15 , wherein the purine residue is guanine.
Claim 36
Dependent Claim
24 words
The conjugate of claim 33 , wherein the base analogue is selected from the group consisting of pyrazolopyrimidinyladenine (PPA), pyrazolopyrimidinylguanine (PPG) and pyrazolopyrimidinylhypoxanthine (PPI).
Claim 37
Dependent Claim
13 words
The conjugate of claim 34 , wherein the base analogue is pyrazolopyrimidinylguanine (PPG).
Claim 38
Dependent Claim
43 words
An oligomer according to claim 1 , wherein R 3 is —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH.
Claim 39
Dependent Claim
16 words
An oligomer according to claim 36 , wherein the oligomer comprises one or more DNA nucleotides.
Claim 40
Dependent Claim
15 words
An oligomer according to claim 36 , wherein the oligomer is a peptide nucleic acid.
Claim 41
Dependent Claim
14 words
An oligomer according to claim 36 , wherein the oligomer is a PNA/DNA chimera.
Claim 42
Dependent Claim
11 words
An oligomer according to claim 39 , further comprising a fluorophore.
Claim 43
Dependent Claim
12 words
An oligomer according to claim 40 , further comprising a fluorescence quencher.
Claim 44
Dependent Claim
13 words
An oligomer according to claim 41 , further comprising a minor groove binder.
Claim 45
Dependent Claim
13 words
An oligomer according to claim 39 , further comprising a minor groove binder.
Claim 46
Dependent Claim
11 words
An oligomer according to claim 43 , further comprising a fluorophore.
Claim 47
Dependent Claim
13 words
An oligomer according to claim 36 , further comprising a minor groove binder.
Claim 48
Dependent Claim
11 words
An oligomer according to claim 36 , further comprising a fluorophore.
Claim 49
Dependent Claim
12 words
An oligomer according to claim 46 , further comprising a fluorescence quencher.
Claim 50
Dependent Claim
13 words
An oligomer according to claim 47 , further comprising a minor groove binder.
Claim 51
Dependent Claim
13 words
An oligomer microarray, comprising one or more oligomers according to claim 36 .
Claim 52
Dependent Claim
23 words
The conjugate of claim 15 , wherein three or fewer consecutive purine residues remain in the polymer once the substitution has been made.
Claim 53
Dependent Claim
12 words
The conjugate of claim 50 , wherein the purine residue is guanine.
Claim 54
Dependent Claim
23 words
The conjugate of claim 15 , wherein two or fewer consecutive purine residues remain in the polymer once the substitution has been made.
Claim 55
Dependent Claim
12 words
The conjugate of claim 52 , wherein the purine residue is guanine.
Claim 56
Dependent Claim
25 words
The conjugate of claim 33 having from 2-9 consecutive guanine residues, wherein the 2-9 guanine residues are substituted by an equal number of pyrazolopyrimidinylguanines (PPGs).
Claim 57
Dependent Claim
47 words
A conjugate according to claim 14 , wherein R 3 is —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; and X is ═CH—.
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