Oligonucleotide labeling reactants and their use

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...

AI Technical Summary

Benefits of technology

[0014] Another objective of the invention is to provide a highly simplified method for the preparation of nucleosidic building blocks that allow large-scale preparation of oligonucleotide conjugates containing additional functional groups in their structure.

[0015] The invention provides improved labeling reactants and a versatile method for direct attachment of a desired number of conjugate groups to the oligonucleotide structure during chain assembly. Hence solution phase labeling and laborious purification procedures can be avoided. The key reaction in the synthetic strategy towards nucleosidic oligonucleotide building blocks is a Mitsunobu alkylation which allows introduction of various labeling reactants to the nucleoside, and finally to the oligonucleotide structure. When oligonucleotides labeled with lanthanide(III) chelates are synthesized, initially precursors of lanthanide(III) chelates are introduced to the oligonucleotide structure during chain assembly, and they are converted to the corresponding lanthanide(III) chelates during deprotection steps.

[0072] (i) The nucleosidic protected functional group tethered building blocks can be synthesized in a few days using cheap reagents, equimolar reagent ratios, and simple purification procedures. The starting materials are commercially available and can also be prepared in a single step using standard well-documented textbook protocols [Gait, M. Oligonucleotide Synthesis, a Practical Approach, IRL Press, 1990]. The key reaction in the present invention is the Mitsunobu alkylation of the above mentioned 5'-O-protected nucleoside and the appropriate linker molecule i.e. a primary alcohol where additional functional groups are protected. Under the reaction conditions employed 3'-O-protection of the nucleoside is not required. These nucleosides are finally converted to the corresponding phosphoramidites in conventional manner, and they can be purified either by precipitation from cold hexanes, or by silica gel column chromatography. Since the products are solids, their storage and handling does not suffer from the problems associated with oily non-nucleosidic phosphoramidites.

[0080] (v) If a ligand structure / structures is / are incorporated to the oligonucleotide chain during chain assembly, it / they can be converted to the corresponding lanthanide(III) chelates during slightly modified deprotection steps. Hence laborious solution phase labeling as well as synthesis of the activated chelates and oligonucleotides tethered to functional groups can be avoided.

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.

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