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US 2004/0175726 A1Published application
Compounds for protecting hydroxyls and methods for their use
AssigneeQuiatech AB, a Swedish corporation
InventorMarek Kwiatkowski
Life of this patent
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Abstract
A hydrocarbyldithiomethyl-modified compound of the Formula: R1—O—CH2—S—S—R2 or a salt thereof wherein R1 is an organic molecule and R2 is a hydrocarbyl is useful for protecting and/or blocking hydroxyl groups in organic molecules such as nucleotides. The hydrocarbyldithiomethyl-modified compounds can also be used for chemically synthesizing oligonucleotides and for sequencing nucleic acid compounds.
Claims57
A hydrocarbyldithiomethyl-modified compound comprising the Formula:
- R1—O—CH2—S—S—R2
- or a salt thereof, wherein
- R1 is an organic molecule;
- and
- R2 is a hydrocarbyl.
2 more elements
A hydrocarbyldithiomethyl-modified compound comprising the formula:
- 16
- or a salt thereof, wherein
- R1 is H, a protecting group, phosphate, diphosphate, triphosphate, or residue of a nucleic acid;
- R2 is a nucleobase;
- R3 is H, OH, or a protected form of OH;
- and
- R4, R5 and R6 are together or separately H, hydrocarbyl, or a residue of a solid support.
4 more elements
A compound comprising the Formula:
- 17
- or a salt thereof, wherein
- R1 is H, a protecting group, a phosphate, diphosphate, or a triphosphate, or a residue of a nucleic acid;
- R2is a nucleobase;
- R3 is H or OH, or a protected form of OH;
- and
- R4 is H or hydrocarbyl.
4 more elements
A method for modifying a nucleoside comprising the steps of:
- a) contacting a nucleoside having at least one hallogenomethyl-modified hydroxyl group with a thiosulfonate compound thereby forming a thiosulfonated nucleoside;
- and
- b) contacting said thiosulfonated nucleoside with a hydrocarbylthiol compound thereby forming a hydrocarbyldithiomethyl-modified nucleoside.
A method for sequencing a nucleic acid comprising the steps of:
- a) contacting a target nucleic acid with a primer under conditions wherein said primer anneals to said target nucleic acid in a sequence specific manner and wherein at least a portion of said primer is complementary to a portion of said target nucleic acid;
- b) incorporating a hydrocarbyldithiomethyl-modified nucleotide into said primer;
- and
- c) detecting incorporation of said hydrocarbyldithiomethyl-modified nucleotide, wherein said hydrocarbyldithiomethyl-modified nucleotide is complementary to said target nucleic acid at said hydrocarbyldithiomethyl-modified nucleotide's site of incorporation thereby identifying the sequence of one nucleobase of said target nucleic acid.
1 more element
A method for sequencing a nucleic acid comprising the steps of:
- a) contacting a target nucleic acid with a primer under conditions wherein said primer anneals to said target nucleic acid in a sequence specific manner and wherein at least a portion of said primer is complementary to a portion of said target nucleic acid;
- b) incorporating a first 3′-hydrocarbyldithiomethyl-modified nucleotide into said primer;
- c) detecting said incorporation of said first 3′-hydrocarbyldithiomethyl-modified nucleotide thereby identifying the sequence of a nucleobase of said target nucleic acid;
- d) removing said hydrocarbyldithiomethyl group from said first incorporated hydrocarbyldithiomethyl-modified nucleotide to form a first elongated primer having a free hydroxyl group;
- e) incorporating a second 3′-hydrocarbyldithiomethyl-modified nucleotide into said first elongated primer;
- and
- f) detecting said second hydrocarbyldithiomethyl-modified nucleotide thereby identifying the sequence of another nucleobase of said target nucleic acid, wherein said first 3′-hydrocarbyldithiomethyl-modified nucleotide and said second 3′-hydrocarbyldithiomethyl-modified nucleotide are complementary to said target nucleic acid at each said nucleotide's site of incorporation.
4 more elements
A compound comprising the Formula:
- 18
- wherein
- R1 is a H, a protecting group, a phosphate, diphosphate, or a triphosphate, or a residue of a nucleic acid;
- R2 is a nucleobase;
- R4, R5 and R6 are together or separately H or hydrocarbyl;
- and
- R7 is H, H-phosphonate or phosphoramidite.
4 more elements
An oligonucleotide synthesis support comprising the formula:
- 19
- wherein
- R1 is H, phosphate, diphosphate, triphosphate, or a protecting group,
- R2 is a nucleobase,
- R3 is H, OH, or a protected form of OH, and
- Z is a group effective for covalent attachment to a solid support, said solid support being effective for covalently bonding an oligonucleotide during oligonucleotide synthesis.
3 more elements
A method for synthesizing an oligonucleotide comprising the steps of:
- a) providing a 5′ protected first nucleoside covalently bonded to a solid support through a linker;
- b) deprotecting said first nucleoside at its 5′ position;
- c) covalently bonding another 5′ protected nucleoside to said first nucleotide at the 5′ position of said first nucleoside;
- d) deprotecting said another nucleoside at its 5′ position;
- and
- e) repeating steps c) and d) for adding additional protected nucleosides, said linker securing said first nucleotide to said solid support via a hydrocarbyldithiomethyl bond.
3 more elements
A method for synthesizing an oligoribonucleotide comprising the steps of:
- a) providing a first protected ribonucleoside covalently bonded to a solid support;
- b) covalently linking at least one 2′-hydrocarbyldithiomethyl-modified ribonucleoside to said first ribonucleoside to form an oligoribonucleotide;
- c) partially de-protecting said oligoribonucleotide under acidic or basic conditions;
- and
- d) contacting said oligoribonucleotide with a reducing agent under neutral conditions thereby completely de-protecting said oligoribonucleotide, wherein said method is effective for preventing cleavage or migration of intemucleotide phosphate bonds, and wherein said hydrocarbyldithiomethyl-modified ribonucleoside comprises a hydrocarbyldithiomethyl group bound at the 2′ position of said hydrocarbyldithiomethyl-modified ribonucleoside.
2 more elements
A method for sequencing a nucleic acid comprising the steps of:
- a) providing a primer array comprising a plurality of sequencing primers;
- b) contacting a target nucleic acid with said primer array under conditions wherein said sequencing primers anneal to said target nucleic acid in a sequence specific manner thereby forming target-primer complexes between complementary portions of said sequencing primers and said target nucleic acid;
- c) incorporating a first 3′ -hydrocarbyldithiomethyl-modified nucleotide into at least one sequencing primer portion of said target-primer complexes, said first 3′-hydrocarbyldithiomethyl-modified nucleotide being complementary to said target nucleic acid;
- and
- d) detecting said incorporation of said first 3′-hydrocarbyldithiomethyl-modified nucleotide, wherein said first 3′-hydrocarbyldithiomethyl-modified nucleotide is complementary to said target sequence at said first 3′-hydrocarbyldithiomethyl-modified nucleotide's site of incorporation.
2 more elements
A method for synthesizing an oligonucleotide comprising the steps of:
- a) providing a 5′ protected first nucleoside covalently bonded to a solid support through a hydrocarbyldithiomethyl containing linker;
- b) deprotecting said first nucleoside at its 5′ position;
- c) covalently bonding another 5′ protected nucleoside to said first nucleotide at the 5′ position of said first nucleoside;
- d) deprotecting said another nucleoside at its 5′ position;
- e) optionally repeating steps c) and d) for adding additional protected nucleosides thereby producing an oligonucleotide;
- f) optionally selectively cleaving a protecting group from said oligonucleotide thereby forming a partially deprotected oligonucleotide;
- g) selectively cleaving said hydrocarbyldithiomethyl containing linker;
- and
- h) isolating said partially deprotected oligonucleotide.
6 more elements
Dependencies are inferred from the claim text.