Method for solution phase synthesis of oligonucleotides

Abstract

This invention discloses an improved method for the sequential solution phase synthesis of oligonucleotides. The method lends itself to automation and is ideally suited for large scale manufacture of oligonucleotides with high efficiency.

Description

FIELD OF THE INVENTION[0001] This invention relates to the field of nucleic acid chemistry. Specifically, this invention describes a novel method for preparing oligonucleotides. The method utilized herein for preparing said oligonucleotides is called PASS, an acronym for Product Anchored Sequential Synthesis.BACKGROUND OF THE INVENTION[0002] Until quite recently, the consideration of oligonucleotides in any capacity other than strictly informational was unheard of. Despite the fact that certain oligonucleotides were known to have interesting structural possibilities (e.g. t-RNAs) and other oligonucleotides were bound specifically by polypeptides in nature, very little attention had been focused on the non-informational capacities of oligonucleotides. For this reason, among others, little consideration had been given to using oligonucleotides as pharmaceutical compounds.[0003] There are currently at least three areas of exploration that have led to extensive studies regarding the use...

AI Technical Summary

Benefits of technology

[0020] The present invention is a method for the sequential solution phase synthesis of oligonucleotides that increases reaction yields and allows for scale-up possibilities. As opposed to traditional schemes in which the 3'-end of the growing oligonucleotide is bound to a solid support, the present invention is characterized by use of an anchor group attached to the 5'-end of the growing oligonucleotide that allows successfully coupled product to be separated from unreacted starting materials. In one embodiment, the anchor group also serves as the 5'-OH protecting group and the coupling reaction occurs in solution. The successfully reacted oligomer will contain the protecting group, while the unreacted oligomer will not, and the materials can be partitioned based on the presence of the anchor / protecting group. In a preferred embodiment, the anchor group reacts covalently with a derivatized solid support, such as a resin, membrane or polymer.

[0021] Specifically, the invention provides a method for the solution phase synthesis of a wide variety of oligonucleotides and modified oligonucleotides comprising reaction of a 5'-protected monomer unit with the 5'-end of a growing oligonucleotide chain in solution. In an additional aspect of the invention, following reaction between the 5'-protected monomer unit and the growing oligonucleotide, the unreacted monomer may be oxidized to form a charged species that may be easily partitioned from the remainder of the reaction medium. In the preferred embodiments of the invention the monomer units are phosphoramidites, which upon activation and oxidation are converted to phosphates. The charged phosphate species can be easily partitioned from the remainder of the reaction medium.

[0023] The method of this invention is not limited to phosphoramidite coupling chemistry, but is compatible with other coupling reactions such as H-phosphonate or phosphate triester coupling chemistry. This method also lends itself to automation and is ideally suited for the large scale manufacture of oligonucleotides with high efficiency.

Problems solved by technology

Although there has been a fair amount of activity in the development of modified oligonucleotides for use as pharmaceuticals, little attention has been paid to the preparation and isolation of these compounds on a scale that allows clinical development.

The conventional laboratory scale 1 .mu.mole automated oligonucleotide synthesis does not provide a sufficient amount of the compound of interest to enable clinical development.

At each step--and in the case of the initial reaction with the solid support--there are reactive sites that fail to react with the 5'-protected monomer, which results in oligonucleotides that have not been extended by one nucleotide monomer (failure sequences).

This increases the difficulty of purifying the crude product away from the failure sequences.

Additionally, even after high resolution purification has been achieved, it remains very difficult to verify the sequence and composition of the product, especially if it contains non-standard nucleotides.

It is, however, a highly inefficient process in terms of overall process yield based on input monomer.

It has been recognized that the automated solid phase synthesis approach does not readily lend itself to be scaled to a level that allows efficient manufacture of oligonucleotide pharmaceuticals.

The inefficiency of the solid phase synthesis is created to a large extent by the heterophase monomer coupling reaction and by the covalent attachment of both unreacted failure sequences and reaction product to the same support bead.

As a result, purification of crude synthetic oligonucleotides to a state acceptable for clinical studies is extremely cumbersome and inefficient.

This method, however, does not overcome the primary problem associated with solid phase synthesis, in that a considerable monomer excess is still required to minimize failure sequences.

Additionally, the method does not provide consistently satisfactory yields.

The weakness of this approach is the unacceptably low recovery of support bound oligonucleotide after each reaction step.

Additionally, this method does not address the problem of failure sequences that must be capped and carried through to the final product.

Again, this method does not address the problem of failure sequences bound to the resin.

Due to the comparatively low yield of phosphotriester coupling this approach has not been widely accepted.

In solution, deprotection of the 5'-DMT group is impaired by the reversibility of acid induced detritylation.

To date, trityl groups which allow anchoring of the product to a resin or membrane during oligonucleotide synthesis in solution have not been designed.

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