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Oligonucleotide labeling reactants and their use

a technology of oligonucleotide and reactant, applied in the field of oligonucleotide labeling reactants and their use, can solve the problems of reducing the purity of these analogues, and reducing the purity of the analogues, so as to improve the labeling of oligonucleotides

Inactive Publication Date: 2004-12-23
HOVINEN JARI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides improved labeling of oligonucleotides with desired numbers of lanthanide(III) chelates. The invention provides a versatile method for direct attachment of lanthanide(III) chelates to the oligonucleotide structure during chain assembly, without the need for purification procedures. The invention also provides a method for introducing lanthanide(III) chelates at the desired position on the oligonucleotide structure. The invention also provides a simplified method for preparing nucleoside building blocks with additional functional groups in their structure. Overall, the invention simplifies the process of labeling oligonucleotides and allows for the preparation of large-scale oligonucleotide conjugates with additional functional groups."

Problems solved by technology

All of these methods have their own drawbacks.
Since the double helix formation of DNA is based on hydrogen bonding between the complementary base residues, tethers attached to the base moieties often weaken these interactions.
Introduction of tethers to the phosphate backbone gives rise to new chiral centers and makes the purification of these analogues difficult.
Introduction of the tether arm to the carbohydrate moiety, in turn, often decreases the coupling efficiency of the phosphoramidite (steric hindrance).
Furthermore, synthesis of these blocks is commonly extremely laborious.
Although design of non-nucleosidic blocks may look attractive on paper, very often their syntheses suffer from complexity, low coupling yields and problems associated with the storage and handling of the phosphoramidites.
Since normally an excess of linker molecule and rather vigorous reaction conditions has to be used, laborious purification procedures cannot bc avoided.
However, the method involves rather laborious synthesis of a 5-halogeno or 5-mercuriochloro nucleoside.
Since in all the cases the labeling reaction is performed in aqueous solution with an excess of labeling reactants, laborious purification procedures cannot be avoided.
Especially when attachment of several labels is required the isolation and characterization of the desired conjugate is extremely difficult, and often practically impossible.
However, such labels and labeled biomolecules suffer from many commonly known drawbacks such as Raman scattering, other fluorescent impurities, low water solubility, concentration quenching etc.
Thus multilabeling of oligonucleotides with organic fluorophores may not enough enhance detection sensitivity needed in several applications.
However the synthetic strategy described allows only preparation of chelates where the nucleobase is conjugated to the chelate structure limiting the chelate stability and versatility.

Method used

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  • Oligonucleotide labeling reactants and their use
  • Oligonucleotide labeling reactants and their use
  • Oligonucleotide labeling reactants and their use

Examples

Experimental program
Comparison scheme
Effect test

example 2

[0108] The synthesis of 5'-O-(4,4'-dimethoxytrityl)-N3-(N6-trifluoroacetam-idohexyl)-thymidine (2)

[0109] The title compound was synthesized as described in example 1 for compound 1 by using 5'-O-(4,4'-dimethoxytrityl)thymidine as the starting material. The yield was 76%. .sup.1H NMR (DMSO-d.sub.6; 500 MHz): .delta. 9.35 (1H, br t, J 5.2, NH); 7.54 (1H, d, J 1.1, H-6); 7.38-7.23 (9H, DMT); 6.88 (4H, d, DMT); 6.22 (1H, t, J 6.6, H-1'); 5.31 (1H, d, J 4.6, 3'-OH); 4.31 (1H, m, H-3'); 3.89 (1H, m, H-4'); 3.77 (2H, m, NCH.sub.2); 3.72 (6H, s, 2.OCH.sub.3); 3.21 (1H, dd, J 5.8 and 10.6H-5'); 3.16 (1H, dd, J 3.0 and 10.6, H-5"); 3.15 (2H, m, CH.sub.2NH); 2.24 (1H, m, H-2"); 2.17 81H, m, H-2'); 1.49 (3H, d, J 1.1 5-CH.sub.3); 1.48 (2H, m, NCH.sub.2CH.sub.2); 1.45 (2H, m, CH.sub.2CH.sub.2NH); 1.26 (4H, m, 2.CH.sub.2). .sup.13C NMR (DMSO-d.sub.6) .delta.: 162.5 (C4), 158.1 (C.dbd.O), 156.1 (q, J.sub.C,F 35.9, CF.sub.3); 150.2 (C2); 144.7 (DMT); 134.3 (C6); 129.7, 127.8, 127.66, 126.7, 113.1 (...

example 3

[0110] The synthesis of 2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-N3-(N6-triflu-oroacetamidohexyl)uridine 3'-O-(2-cyanoetlyl N,N-diisopropyl)phosphoramidi-te (3)

[0111] Predried compound 1 and 2-cyanoethyl N,N,N',N'-tetraisopropylphosph-ordiamidite (1.5 eq) were dissolved in dry acetonitrile. 1H tetrazole (1 eq; 0.45 M in acetonitrile) was added, and the mixture was stirred for 30 min at room temperature before being poured into 5% NaHCO.sub.3 and extracted with dichloromethane and dried over Na.sub.2SO.sub.4. Precipitation from cold (-70.degree. C.) hexane yielded the title compound as a white powder. Compound 3: .sup.31P NMR (CDCl.sub.3): .delta. 148.6 (0.5 P), 148.4 (0.5 P).

example 4

[0112] The synthesis of 5'-O-(4,4'-dimethoxytrityl)-N3-(N6-trifluoroacetam-idohexyl)-thymidine 3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (4)

[0113] Phosphitylation of compound 2 as described in example 3 for compound 1 yielded the title compound as a white powder. Compound 4: .sup.31P NMR (CDCl.sub.3): .delta. 148.6 (0.5 P), 148.4 (0.5 P).

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Abstract

The invention relates to a novel labeling reactant of formula (I) suitable for labeling an oligonucleotide wherein: R is a temporary protecting group. A is either a phosphorylating moiety or a solid support tethered to a bridge point Z via a linker arm E. E' is a linker arm between G and Z. G is a bivalent aromatic structure, tethered to two iminodiacetic acid ester groups N(COOR''')2 or G is a structure selected from a group consisting of or G is a protected functional group. The invention further concerns a method for direct attachment of a conjugate group to an oligonucleotide structure enabling the attachment of a desired number of these groups during chain assembly. The method comprises a Mitsonobu alkylation.

Description

[0001] This invention relates to novel compounds and methods for labeling of oligonucleotides using machine assisted solid phase chemistry.[0002] The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.[0003] Synthetic oligonucleotides tethered to various ligands have been used as research tools in molecular biology [see e.g.: Goodchild, Bioconjugate Chem., 1990, 3, 166; Uhlman and Peyman, Chem. Rev., 1990, 90, 543; Sigman, et al. Chem. Rev., 1993, 93, 2295; O'Donnel and McLaughlin in Bioorganic Chemistry, Nucleic Acids, Hecht SM, ed. Oxford Univ. Press, 1996, p. 216]. They have been applied to genetic analysis, and to elucidate mechanism of gene function. Oligonucleotides carrying reporter groups have had widespread use for automated DNA sequencing, hybridization affinity chromatography and fluorescence microscopy. Oligonucleotide-bio...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07F9/58C07F9/6503C07F9/6558C07H19/10C07H19/20C07H21/00
CPCC07F9/582C07F9/65033C07F9/65583C07H19/10C07H19/20C07H21/00C07F9/58C07F9/65031Y02P20/55
Inventor HOVINEN, JARITAKALO, HARRI
Owner HOVINEN JARI
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