Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-09-05
2004-06-08
Fredman, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S027100, C436S528000
Reexamination Certificate
active
06747142
ABSTRACT:
BACKGROUND OF THE INVENTION
Oligonucleotides bearing different functional groups and other functional entities have become a commonplace tool in many diagnostic and therapeutic applications. Accordingly, a large number of nucleosidic and non-nucleosidic phosphoramidite derivatives for functionalization of synthetic oligonucleotides have been reported. However, all these reagents, except for a few examples, allow introduction of only one functional moiety per phosphoramidite unit. This limitation makes synthesis of highly functionalized oligonucleotides somewhat problematic as a considerable number of incorporations may increase the overall yield of the functionalized oligonucleotide.
A precursor strategy is a system by which a single precursor is used to manufacture a variety of different products. The use of a precursor strategy is quite common in synthetic organic chemistry. As used herein, a precursor is a molecule capable of reacting with different compounds, such as modifiers, to produce a number of different products. A precursor molecule comprises a core and one or more reactive moieties. The “core” is the part of the compound that does not generally change and the part that often, but not necessarily, possesses some specific properties critical for the desired application. Thus, the core is generally untouched upon reaction with a modifier.
A “reactive moiety” is a group that reacts in a highly effective, preferably quantitative, and specific manner with a particular modifier to form a particular product or with a mixture of modifiers to form a pool of products. If a core part of a precursor contains some functionalities that are also capable of reacting with the modifier, during the reaction, these functionalities must be protected.
A precursor strategy will work successfully only if some demands are fulfilled. These demands include the following:
1. If a precursor is a complex molecule and is prepared by multi-step synthesis, the precursor reactive moiety or moieties must be stable in all conditions used during the synthesis after its introduction. However, this rule is not applicable if a reactive moiety is introduced at the very last step of the precursor synthesis.
2. It is highly desirable for the yield of the reaction between a precursor reactive moiety and a modifier to be close to quantitative. This is especially important when the precursor contains more than one reactive moiety.
3. The core part of a precursor must be stable in the conditions of transformation, that is, the conditions under which the precursor reacts with a modifier.
4. One or more modified sites, that is, parts of a product molecule that are formed after reaction between a precursor reactive moiety and a modifier, must tolerate the deprotection conditions if a deprotection step is necessary to prepare a desired product.
5. It is desirable for the transformation time to be relatively short.
Oligonucleotides bearing various functionalities have become common place tools in molecular biology and diagnostics. Goodchild, J., “Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of Their Synthesis and Properties,” Perspectives in Bioconjugate Chemistry, pp. 77-99 (1993). One of the most efficient routes to the synthesis of functionally modified oligonucleotides (FMOs) is the introduction of a precursor, that is, a nucleotide monomer bearing a reactive moiety, into the oligonucleotide. At the end of solid phase synthesis, the precursor reacts with a desired linker or modifier. This strategy enables one to synthesize a wide variety of FMOs from a single parent oligonucleotide.
MacMillan, A. and Verdine, G., “Engineering Tethered DNA Molecules by the Convertible Nucleoside Approach,” Tetrahedron, 47: 2603-2619 (1991), and Ferenz, A. and Verdine, G., “Aminolysis of 2′-Deoxyinosine Aryl Ethers: Nucleoside Model Studies for the Synthesis of Functionally Tethered Oligonucleotides,” Nucleosides & Nucleotides, 11: 1749-1763 (1992), have elaborated a convertible nucleoside strategy to prepare functionally tethered oligonucleotides (FTOs). This convergent strategy is based on the use of O-substituted deoxyuridine and deoxyinosine as convertible nucleosides. Upon treatment with aqueous amines, precursor oligonucleotides containing convertible nucleosides undergoes a transformation giving rise to a FTO.
Buhr et al., U.S. Pat. No. 5,466,786, described the incorporation of a 2′-deoxy-2′-O-(ethoxycarbonylmethyl)-cytidine into an oligonucleotide. After solid phase synthesis and deprotection, the ester group, which is a reactive moiety, can be hydrolyzed to a carboxy group by treatment with NaOH or derivatized to an amide or substituted amide by a reaction with NH
3
or a primary aliphatic amine.
Hebert et al., Tetrahedron Letters, 35: 9509-9512 (1994), reported the N-acylation of a DMT-hydroxymethylpyrrolidinol precursor with a number of carboxylic acids. N-substituted DMT-hydroxymethylpyrrolidinols were further phosphitilated and used for the preparation of phosphodiester oligomer combinatorial libraries.
U.S. Pat. No. 5,902,879 to Polouchine, the entire disclosure of which is hereby incorporated by reference, discloses the addition of individual precursor moieties to nucleosides and nucleotides and compounds synthesized therefrom. U.S. patent application Ser. No. 09/655,317, filed Sep. 5, 2000, the entire disclosure of which is hereby incorporated by reference, discloses the addition of individual precursor moieties to non-nucleosides and non-nucleotides and compounds synthesized therefrom.
SUMMARY OF THE INVENTION
The present invention provides for the synthesis of compounds containing multiple precursor groups, such as methoxyoxalamido (MOX), and allowing the efficient synthesis of highly functionalized oligonucleotides and oligomers. Also, the present invention provides a branching unit that helps to further increase the density of functional groups on synthetic oligonucleotides and oligomers. The present invention also provides for compounds containing multiple precursor groups, such as methoxyoxalamido (MOX) precursor groups.
The present invention provides a compound, or a salt thereof, having the formula (I):
A—X
n
(I)
wherein A represents an organic moiety, n is at least 2, each X is independently selected from the group consisting of: —NRCOCONu, —NHCOCR
2
CR
2
CONu, —NHCOCR═CRCONu, —NHCOSSCONu, —NRCOCOOCR
3
,
wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile.
The present invention also provides a method for forming a compound, comprising:
reacting, by nucleophilic addition, a first compound containing a first moiety selected from the group consisting of: —NRCOCOOCR
3
,
wherein each R independently represents H or a substituted or unsubstituted alkyl group, with a second compound (HNu) containing at least three primary or secondary amines to form a third compound containing a second moiety selected from the group consisting of: —NRCOCONu, —NHCOCR
2
CR
2
CONu, —NHCOCR═CRCONu, and —NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, wherein one of the at least three primary or secondary amines of the second compound acts as a nucleophile in the nucleophilic addition, leaving at least two unreacted primary or secondary amines in the second moiety;
reacting the third compound with at least two compounds, which may be the same or different, each containing a third moiety independently selected from the group consisting of: —COCOOCR
3
, —COCR
2
CR
2
CO—, —COCR═CRCO— and —COSSCO—, wherein each of the at least two compounds reacts with one of the at least two unreacted primary or secondary amines of the second moiety to form a compound containing at least two moieties independently selected from the group consisting of: —NRCOCOOCR
3
,
wherein each R independently represents H or a substituted or unsubstituted alkyl group.
REFERENCES:
patent: 5112962 (1992-05-01), Letsinger et al.
patent: 5241060 (1993-08-01), Engel
Chunduru Suryaprabha
Fidelity Systems, Inc.
Fredman Jeffrey
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