Organic compounds -- part of the class 532-570 series – Organic compounds – Fatty compounds having an acid moiety which contains the...
Reexamination Certificate
2001-09-12
2003-10-28
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Fatty compounds having an acid moiety which contains the...
C554S085000, C554S101000, C554S102000, C435S006120, C536S022100, C536S023100
Reexamination Certificate
active
06639088
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to biological chemistry in general. In particular, the invention relates to protecting hydroxyls in organic molecules.
BACKGROUND OF THE INVENTION
Temporary protection or blocking of chemically reactive functions in biological compounds is an important tool in the field of biological chemistry. To this end, researchers have developed a number of protecting groups. The vast majority of the known protecting groups, however, are acid or base labile and while there are also protecting groups that are labile under neutral conditions, most of these protecting groups are also somewhat acid and base labile. Greene, T W, “Protective Groups in Organic Synthesis”, publishers Wiley-Interscience (1981). Furthermore, many protecting groups suffer additional synthesis, side-reaction, and/or solubility problems. For example, only a few protecting groups applied as a part of a linking system between the solid phase and the oligonucleotide can withstand all the rigors of oligonucleotide synthesis and deprotection thereby facilitating the final purification of oligonucleotides free of truncated or depurinated fragments. See “Solid Phase Synthesis,” Kwaitkowski et al., PCT International Publication WO 98/08857 (1996). Selective post-synthetic derivatization of oligonucleotides also requires selectively cleavable protecting groups. See, e.g., Kahl & Greenberg, “Introducing Structural Diversity in Oligonucleotides via Photolabile, Convertible C5-substituted Nucleotides,”
J. Am. Chem. Soc.,
121(4), 597-604 (1999).
Protecting groups also should be removable. Ideally, the protecting group is removable under mild conditions, for example, without disturbing interactions between biomolecules. These types of protecting groups may be useful for deprotecting oligonucleotides without disturbing interactions between oligo/polynucleotide strands. For example, International Publication WO 96/23807 entitled “Novel Chain Terminators, The Use Thereof for Nucleic Acid Sequencing and Synthesis and a Method of their Preparation” discloses methods that use nucleotides that are reversibly blocked at the 3′ hydroxyl group. These reversibly blocked nucleotides can be used in sequencing methods where, unlike the well-known Sanger sequencing method that utilizes terminating dideoxynucleotides, the temporarily 3′-OH-protected intermediates can be converted into nucleotides having a free 3′-OH that may be further extended.
One such sequencing method that uses reversibly blocked nucleotides is known as Sequencing by Synthesis (SBS). SBS determines the DNA sequence by incorporating nucleotides and detecting the sequence one base at a time. To effectively sequence long stretches of a nucleic acid using SBS, it is advantageous to be able to perform multiple iterations of the single nucleotide incorporation. Accordingly, SBS-based methods require 3′-OH protecting groups that are removable under conditions that do not distrupt the primer and target DNA interactions. As such, there exists a need for nucleotide triphosphates that are reversibly blocked at the 3′ position and which are also effective substrates for DNA polymerases.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a hydrocarbyldithiomethyl-modified compound of the Formula:
R
1
—O—CH
2
—S—S—R
2
or a salt thereof, wherein R
1
is an organic molecule and R
2
is a hydrocarbyl. Before undergoing hydrocarbyldithiomethyl-modification, R
1
has at least one hydroxyl group, which after modification is in an ether linkage. In one embodiment, R
2
further includes a labeling group. The labeling group can be any type of labeling group including fluorescent labeling groups, which can be selected from the group consisting of bodipy, dansyl, fluorescein, rhodamin, Texas red, Cy 2, Cy 4, and Cy 6.
In another embodiment, R
1
—O represents modified or unmodified amino acids, peptides, proteins, carbohydrates, sterols, ribonucleosides, ribonucleotides, base- and/or sugar-modified ribonucleosides, base- and/or sugar-modified ribonucleotides, deoxyribonucleosides, deoxyribonucleotides, base- and/or sugar-modified deoxyribonucleosides, and base- and/or sugar-modified deoxyribonucleotides. R
1
can have more than one hydroxyl group and more than one of the hydroxyl groups can be modified with a hydrocarbyldithiomethyl moiety. For nucleotide embodiments, the hydrocarbyldithiomethyl modification can be at the 2′ and/or, 3′, and/or 5′ hydroxyl positions of the R
1
—O.
In another embodiment, R
2
includes a function that modifies the electron density of the dithio function, thereby modifying the stability of the dithiol. Such a function may be provided by a chemical group containing elements selected from the group consisting of oxygen, nitrogen, sulfur, and silicon.
In another aspect, the invention provides a hydrocarbyldithiomethyl-modified compound of the Formula:
or a salt thereof, wherein R
1
is H, a protecting group, phosphate, diphosphate, triphosphate, or residue of a nucleic acid, R
2
is a nucleobase, R
3
is H, OH, or a protected form of OH; and R
4
, R
5
and R
6
are together or separately H, hydrocarbyl, or a residue of a solid support. Suitable hydrocarbyls for R
4
, R
5
and R
6
include methyl, ethyl, isopropyl, and t-butyl. In one embodiment, R
4
, R
5
and R
6
together or separately further include a labeling group and/or an electron donating function or electron density modifying function. The electron density modifying function can be a heteroatom selected from the group consisting of oxygen, nitrogen, sulfur, and silicon.
In another aspect, the invention provides a compound of the Formula:
or a salt thereof, wherein R
1
is H, a protecting group, a phosphate, diphosphate, or a triphosphate, or a residue of a nucleic acid, R
2
is nucleobase, R
3
is H or OH, or a protected form of OH, and R
4
is H or hydrocarbyl. In one embodiment, R
4
is modified with a labeling group. In other embodiments, R
4
includes a derivatizable function, or R
4
includes nitrogen, or R
4
is covalently linked to a solid support.
In another aspect, the invention provides a method for modifying a nucleoside including the steps of: a) contacting a nucleoside having at least one hallogenomethyl-modified hydroxyl group with an thiosulfonate compound thereby forming a thiosulfonated nucleoside; and b) contacting the thiosulfonated nucleoside with a hydrocarbylthiol compound thereby forming a hydrocarbyldithiomethyl-modified nucleoside. Useful thiosulfonate compounds include alkylthiosulfonate and arylthiosulfonate.
In one embodiment, the method includes the step of labeling the hydrocarbyldithiomethyl-modified nucleoside.
In another aspect, the invention provides a method for sequencing a nucleic acid including the steps of: a) contacting a target nucleic acid with a primer wherein at least a portion of the primer is complementary to a portion of the target nucleic acid; b) incorporating a hydrocarbyldithiomethyl-modified nucleotide into the primer; and c) detecting incorporation of the hydrocarbyldithiomethyl-modified nucleotide, wherein the hydrocarbyldithiomethyl-modified nucleotide is complementary to the target nucleic acid at the hydrocarbyldithiomethyl-modified nucleotide's site of incorporation. In one embodiment, the incorporating step is catalyzed by a DNA polymerase. Useful sequencing methods that may use the method disclosed above include minisequencing and sequencing by synthesis whether performed in isolation or performed as a sequencing array.
In another aspect, the invention provides a method for sequencing a nucleic acid including the steps of: a) contacting a target nucleic acid with a primer wherein at least a portion of the primer is complementary to a portion of the target nucleic acid; b) incorporating a first hydrocarbyldithiomethyl-modified nucleotide into the primer; c) detecting the incorporation of the first hydrocarbyldithiomethyl-modified nucleotide; d) removing the hydrocarbyldithiomethyl group from the first incorporated hydrocarbyldithiomethyl-modif
Fish & Richardson P.C. P.A.
Quiatech AB
Riley Jezia
LandOfFree
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