Polynucleotide crosslinking agents

Details Polynucleotide crosslinking agents Polynucleotide crosslinking agents

1998-11-10

2001-10-16

Riley, Jezia (Department: 1656)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C435S006120, C435S091100, C536S022100, C536S023100, C536S025300, C536S024300, C549S467000, C548S203000, C548S495000, C558S254000

Reexamination Certificate

active

06303799

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to photoactive compounds that can be incorporated into synthetic oligonucleotides to crosslink nucleic acid strands.
DESCRIPTION OF RELATED ART
The use of crosslinkable probes in nucleic acid hybridization assays to crosslink to target sequences is demonstrated in U.S. Pat. No. 4,826,967 by R. Glass the crosslinking compounds are based on furocoumarin (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications. However, the crosslinking group
ucleoside adduct is difficult to synthesize, particularly in large quantities.
In U.S. Pat. No. 5,082,934, Saba et al. describe a photoactivatible nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety. The resulting nucleoside analogue is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays. However, the sugar moiety limits the conformational flexibility of the crosslinking moiety.
In PCT Publication Number WO96/28438, Wood et al. describe photoactivatible non-nucleosidic coumarin derivatives wherein the coumarin moiety is joined within the backbone of an oligonucleotide probe via moieties other than deoxyribose or ribose. The stability and reactivity of these compounds are particularly useful for nucleic acid diagnostic assays and therapeutics because of the rapid crosslinking reaction between the coumarin moiety and the natural bases found in the opposite strand.
Nevertheless, there remain applications for crosslinkable probes in which reaction with a natural base is not desirable, but instead reaction between two non-natural reactants is the desired mode.
SUMMARY OF THE INVENTION
The current invention provides non-nucleosidic, stable, photoactive compounds that can be used as photo-crosslinking reagents in nucleic acid hybridization assays and therapeutic applications, as well as probes incorporating the compounds.
The compounds comprise aryl olefin derivatives prepared by linking the aryl moiety to a functionalized saturated or unsaturated hydrocarbon molecule, such as glycerol. The functionalized hydrocarbon moiety of the resulting compound is equivalent to the sugar of a nucleoside, while the aryl olefin moiety occupies the position of a base. Accordingly, the compounds can be inserted into growing polynucleotide chains using automated or manual techniques of polynucleotide synthesis. The double bond of the aryl olefin is a photoactive group that covalently crosslinks to suitable reactants in the complementary strand when an oligonucleotide containing this crosslinkable non-nucleoside analogue (the “probe”) is used in a hybridization assay and/or therapeutic application.
The photoactive compound has the formula
Y is H, F, lower alkyl, lower alkoxy, or lower fluoroalkyl,
Z is O or S;
R
1
is H, R′, —OR′, —SR′, or —NR′R″ in which R′ and R″ independently represent H, lower alkyl, haloalkyl, alkoxy, alkylcarbonyl, or aryl, heteroaryl, or polycyclic aryl, each optionally substituted with W;
R
2
is aryl, heteroaryl or polycyclic aryl, each optionally substituted with W;
B represents (1) a linear, branched, or cyclic hydrocarbon group containing from 2 to 10 carbon atoms and, if cyclic, containing a 5- or 6-membered ring or (2) a heterocyclic aromatic ring system containing a 5- or 6-membered ring, said B(1) or B(2) being substituted with 1, 2, or 3 groups of the formula R
3
, wherein R
3
independently represents H, OH, NH
2
, or COOH, or a protecting or coupling group capable of protecting or coupling a functional group during synthesis of a polynucleotide, or R
3
represents a nucleotide or a polynucleotide and wherein one to three carbon atoms of the hydrocarbon group can be replaced by an oxygen, nitrogen, or sulfur atom; except that B is not ribose or deoxyribose;
X represents (1) a bond, (2) a linear, branched, or cyclic hydrocarbon group containing 1 to 10 carbon atoms, in which optionally one to three carbon atoms of the hydrocarbon group are replaced by an oxygen, sulfur, or nitrogen atom wherein X is optionally substituted with 1-3 substituents selected from the group consisting of hydroxy, halogen, amino, amido, azido, carboxy, carbonyl, perfluoromethyl, and cyano functional groups; and wherein X is attached to the phenyl ring of said formula directly or through W;
n is 0, 1, 2, or 3;
each W independently represents a hydroxy, halogen, amino, amido, azido, nitro, thio, carboxy, carbonyl, perfluoromethyl, or cyano functional group; an unsubstituted hydrocarbyl group of 10 or fewer carbon atoms; or said hydrocarbyl group substituted with 1-3 of said functional groups or in which one carbon atoms replaced by an oxygen, sulfur, or nitrogen atom and wherein two Ws together can represent a ring fused to the phenyl ring of said formula;
with the provisos that (1) when X or W is a substituted hydrocarbon, the total number of substituents in X or W is less than the total number of carbon atoms in said X or W and no more than one substituent or heteroatom is attached to a given carbon, unless said substituents are halogen atoms on said given carbon, and (2) the total carbon atoms in all W substituents is 15 or fewer.


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patent: 3804243 A1 (1989-08-01), None
patent: 0 130 523 A2 (1985-01-01), None
patent: 0 324 616 A3 (1989-07-01), None
patent: WO 94/24120 (1994-10-01), None
patent: WO 96/28438 (1996-09-01), None
patent: WO 96/34984 (1996-11-01), None
patent: WO98/39280 (1998-09-01), None
Misiura et al., “Biotinyul and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides,”Nucleic Acids Research, 18:4345-4354 (1990).
Zehnder et al., “Cross-linking hybridization assay for direct detection of factor V Leiden mutation,”Clinical Chemistry, 43:1703-1708 (1997).

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