Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-04-13
2001-11-20
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S023100, C536S024300, C536S024330, C536S025330, C536S025340, C536S025600, C536S026100
Reexamination Certificate
active
06320041
ABSTRACT:
TECHNICAL FIELD
This invention relates to compositions utilized to chemically join two molecules of interest. More particularly, the compositions of the present invention are crosslinking reagents utilized to chemically join a desired molecule to an oligonucleotide.
BACKGROUND OF THE INVENTION
The preparation of oligonucleotide conjugates is generally accomplished through the use an oligonucleotide modified with a primary amine (Agrawal, S. (1994) Functionalization of oligonucleotides with amino groups and attachment of amino specific reporter groups.
Methods in Molecular Biology
26; Protocols for Oligonucleotide Conjugates. (S. Agarwal, Ed.) pp. 73-92, Humana Press, Totowa, N.J. (Review), Meyers, R. (1994) Incorporation of Modified Bases into Oligonucleotides.
Methods in Molecular Biology
26; Protocols for Oligonucleotide Conjugates. (S. Agarwal, Ed.) pp. 93-120, Humana Press, Totowa, N.J. (Review)). In most cases, amide or thiourea bonds are formed with conjugars containing an activated carboxyl or isothiocynate (ITC) functionality.
Although functionalization of many conjugars is routine, a number of conjugars have proved to be very difficult to transform into activated carboxyl or ITC derivatives either because of the complex synthesis involved or the inherent instability of the final compound. In an effort to circumvent these difficulties the coupling partners have been reversed placing the carboxylic acid function on the oligonucelotide, and the amine on the conjugar. The literature contains several examples of 5′ terminal oligonucleotide linkers that contain a carboxyl funtionality. Unfortunately these methods have several disadvantages. Kremsky et al. ((1987) Immobilization of DNA via oligonucleotides containing and aldehyde or carboxylic acid group at the 5′ terminus.
Nucleic Acids Research
15, 2891-2909), describe conjugation with a protected 5′ terminal oligonucleotide carboxyl group requiring cleavage of the methyl ester protecting group, followed by in situ activation with N-hydroxysuccinimide (“NHS”) and a coupling reagent to achieve conjugation. The disadvantage of this method is the number of steps required to achieve the desired conjugate.
In another approach, the protecting group is a benzyl ester, which can be directly coupled to an amine (Endo, M., Gaga, Y., and Komiyama, M., (1994) A novel phosphoramidite for the site-selective introduction of functional groups into oligonucleotides via versatile tethers.
Tetrahedron Letter
33, 3879-3882). However, this procedure calls for conditions that require treatment with a primary amine for 48 hours and is incompatible with base sensitive oligonucleotides, such as methylphosphonates (Hogrefe, R. I., Vaghefi, M. M., Reynolds, R. A., Young, K. M., and Arnold, L. J. Jr. (1993) Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.
Nucleic Acids Research
21, 2031-2038).
A third approach describes the formation of a phosphoramidate bond between a 3′ or 5′ phosphorylated oligonucleotide and an amino acid, followed by subsequent activation of the carboxyl moiety with carbodiimide (Gottikh, M., Asseline, U., and Thoung, N. T. (1990) Synthesis of oligonucleotides containing a carboxyl group at either their 5′ end or their 3′ end and their subsequent derivitization by an intercalating agent.
Tetrahedron Letters
31, 6657-6660). The number of steps required to prepare these conjugates makes these methods generally undesirable.
Consequently there is a need for a fast and effective means of forming conjugating oligonucleotides to a variety of molecules while requiring a minimal amount of post-synthetic chemistry. In particular linker compositions are needed that may be synthesized from primary amine containing compounds that are relatively easy to obtain; comprise a non-labor intensive and efficient means for attachment to an oligonucleotide such as a phosphoramidite reagent that can be coupled directly to the 5′ terminus; that are compatible with the conditions of oligonucleotide synthesis and that provide a highly reactive functionality, such as a succinimydyl or pentafluorophenyl ester. In addition, linker compositions that may be utilized in conjugations performed on support bound linker-modified oligonucleotides prior to deprotection and cleavage are particularly preferred.
SUMMARY OF THE INVENTION
The present invention relates generally to compositions of pre-activated linkers for the rapid modification of oligonucleotides.
In one aspect of the present invention a composition is provided of the formula,
wherein
R
1
is
R
3
is —CH
3
, —O—CH
2
—CH
2
—CN or —O—CH
3
;
R
4
is o-chlorophenyl, &bgr;-cyanoethyl;
X
1
is O, S or Se; and
n is 5 to 15.
In another aspect of the invention a composition is provided of the formula,
wherein
R
1
is
R
2
is —CH
3
, —O—CH
2
—CH
2
—CN or —O—CH
3
;
X is O, S or Se; and
n is 5 to 15.
In one embodiment of this aspect the compositions above further comprising a nucleic acid, preferably a nucleic acid from 1 to 1,000 nucleotides in length, a nucleotide bound to a support matrix or a solid support matrix.
In yet another aspect of the invention a composition is provided comprising a nucleoside from 1 to 1,000 nucleotides in length conjugated to a reporter group utilizing the compositions above, wherein said reporter group is selected from the group consisting of fluorescent, bioluminescent, chemiluminscent, colorimetric and radioactive agents. In particular, when the reporter group is a fluorescent reporter group it may be selected from the group consisting of fluorescein, pyrene, 7-methoxycoumarin, Cascade Blue™, 7-aminocoumarin (“AMCA-X”), dialkylaminocoumarin, Pacific Blue, Marina Blue, BODIPY 493/503™, BODIPY Fl-X™, (4,6-dichlorotriainyl)aminofluorescien) (“DTAF”), Oregon Green 500™, 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoate (“Dansyl-X”), 6-(fluorescein-6-carboxamido)hexylamine (“6-FAM”), Oregon Green 488™, Oregon Green 514™, Rhodamine Green-X™, Rhodol Green™, NBD, Tetrachlorofluorescein (“TET”), 2′, 4′, 5′, 7′tetrabromosulfonefluorescien, BODIPY-R6G™, BODIPY-Fl Br
2
™, BODIPY 530/550™, hexachlorofluorescein (“HEX”), BODIPY 558/568™, BODIPY-TMR-X™, 1-(3-carboxybenzyl)-4-(5-(4-methoxyphenyl)oxazol-2yl)pyridinium bromide (“PyMPO”), BODIPY 564/570™, 6-(tetramethylrhodamine-5(6)-carboxamido)hexylamine (“TAMRA”), Cy3™, Rhodamine Red-X™, BODIPY 576/589™, CarboxyXrhodamine™, BODIPY 581/591™, Texas Red-X, BODIPY-TR™, Cy5™, and naphthofluorescein.
In one embodiment of this aspect the nucleic acid may be a deoxynucleotide or a ribonucleotide or a combination of DNA and RNA. In another embodiment a first nucleic acid may be conjugated to a second nucleic acid wherein the first nucleic acid is from 1 to 1,000 nucleotides in length and the second nucleic acid is from 1 to 1,000 nucleotides utilizing the composition above.
In yet another embodiment of this invention a composition is provided comprising a nucleic acid from 1 to 1,000 nucleotides in length conjugated to a biomolecule or an organic molecule utilizing the composition.
In still another aspect of the invention the following compounds are provided:
Hogrefe Richard I.
Vaghefi Morteza M.
Riley Jezia
Trilink Biotechnologies, Inc.
Waller David B.
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