Method for solution phase synthesis of oligonucleotides

Details Method for solution phase synthesis of oligonucleotides Method for solution phase synthesis of oligonucleotides

1998-04-06

2001-07-17

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025330, C536S025340, C536S025400, C530S338000, C530S322000

Reexamination Certificate

active

06262251

ABSTRACT:

FIELD OF THE INVENTION
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
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.
There are currently at least three areas of exploration that have led to extensive studies regarding the use of oligonucleotides as pharmaceutical compounds. In the most advanced field, antisense oligonucleotides are used to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions. Additionally, the discovery of RNA species with catalytic functions—ribozymes—has led to the study of RNA species that serve to perform intracellular reactions that will achieve desired effects. And lastly, the discovery of the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk and Gold (1990) Science 249:505) has shown that oligonucleotides can be identified that will bind to almost any biologically interesting target.
SELEX is a method for identifying and producing nucleic acid ligands, termed “nucleic acid antibodies”, e.g., nucleic acids that interact with target molecules (Tuerk and Gold (1990) Science 249:505). The method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity.
The use of antisense oligonucleotides as a means for controlling gene expression and the potential for using oligonucleotides as possible pharmaceutical agents has prompted investigations into the introduction of a number of chemical modifications into oligonucleotides to increase their therapeutic activity and stability. Such modifications are designed to increase cell penetration of the oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA and to improve their pharmacokinetic properties.
Recent research has shown that RNA secondary and tertiary structures can have important biological functions (Tinoco et al. (1987) Cold Spring Harb. Symp. Quant. Biol. 52:135; Larson et al. (1987) Mol. Cell. Biochem. 74:5; Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364; Resnekov et al. (1989) J. Biol. Chem. 264:9953). PCT Patent Application Publication WO 91/14436, entitled “Reagents and Methods for Modulating Gene Expression Through RNA Mimicry,” describes oligonucleotides or oligonucleotide analogs which mimic a portion of RNA able to interact with one or more proteins. The oligonucleotides contain modified internucleoside linkages rendering them nuclease-resistant, have enhanced ability to penetrate cells, and are capable of binding target oligonucleotide sequences.
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 &mgr;mole automated oligonucleotide synthesis does not provide a sufficient amount of the compound of interest to enable clinical development. For clinical development oligonucleotides must be produced in gram-scale to multigram scale amounts at a minimum. Although there are reports of large-scale oligoribonucleotide syntheses in the literature, the term “large-scale” has been applied to the 1 to 10 &mgr;mole scale, rather than gram-scale or kilogram-scale amounts. (Iwai et al. (1990) Tetrahedron 46:6673-6688).
The current state of the art in oligonucleotide synthesis is automated solid phase synthesis of oligonucleotides by the phosphoramidite method, which is illustrated in Scheme 1. (Beaucage and Iyer (1992) Tetrahedron 48:2223-23 11; Zon and Geiser (1991) Anti-Cancer Drug Design 6:539-568. Matteucci and Caruthers (1981) J. Am. Chem. Soc. 103:3185-3191). Briefly the 3′-terminal nucleoside of the oligonucleotide to be synthesized is attached to a solid support and the oligonucleotide is synthesized by addition of one nucleotide at a time while remaining attached to the support. As depicted in Scheme 1 a nucleoside monomer is rotected (P
1
) and the phosphoramidite is prepared (1). The phosphoramidite (referred to as the 5′-protected monomer unit) is then covalently attached to the growing oligonucleotide chain (2), via a phosphite triester linkage, through the 5′-hydroxy group of the ribose ring of the growing oligonucleotide chain to yield the oligonucleotide product (3), in which the majority of the growing oligonucleotide chain has been extended by one nucleotide, but a significant percent of chains are not extended. The product (3) is then oxidized to yield the phosphate triester (4). Prior to the addition of the next base to the growing nucleotide chain, the 5′-hydroxyl group must be deprotected. As can be seen in Scheme 1 (compound 4), however, not all of the reactive sites on the solid support react with the 5′-protected monomer. These unreacted sites (referred to as failure sequences) must, therefore, be protected (referred to as capping) (5) prior to deprotection of the 5′-hydroxyl group (6). Subsequent monomers, which have also been protected and converted to the phosphoramidite, are then sequentially added by coupling the 5′-end of the growing oligomer to the 3′-end of the monomer. Each coupling reaction extends the oligonucleotide by one monomer via a phosphite triester linkage. 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). When the synthesis is complete the desired oligonucleotide (6 (n+1 sequence)) is deprotected and cleaved from the resin, together with all of the failure sequences (n, n−x).
The yield of conventional solid phase oligonucleotide synthesis decreases exponentially with the number of monomers coupled. 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.
Automated oligonucleotide synthesis on solid supports is very efficient for the preparation of small amounts, 0.001 to 0.01 mmol, of a variety of sequences in a minimum amount of time with reasonable yield. It is, however, a highly inefficient process in terms of overall process yield based on input monomer. Typically a 16 fold excess of phosphoramidite is necessary per monomer addition. 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. (Zon and Geiser (1991) Anti-Cancer Drug Design 6:539-568).
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

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