Large scale synthesis of oligonucleotides and their...

Details Large scale synthesis of oligonucleotides and their... Large scale synthesis of oligonucleotides and their...

1998-11-20

2001-10-09

Geist, Gary (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025340, C536S026700, C536S026800

Reexamination Certificate

active

06300486

ABSTRACT:

TABLE OF CONTENTS
1. Introduction
2. Background of the Invention
2.1. Synthetic Oligonucleotides and Their Utility
2.2. Preparative Methods
3. Summary of the Invention
4. Brief Description of the Figures
5. Detailed Description of the Invention
5.1. Functionalized Solid Support
5.2. Synthesis of Oligonucleotide H-Phosphonates
5.2.1. Suggested Modification of a Commercial Automated DNA Synthesizer
5.2.2 Conditioning of the Solid Support and Initiation of the Condensation Cycles
5.2.3. Choice of Appropriate Solvents
5.3. Recovery of Excess Reagent Monomer
6. Examples
6.1. General Procedures
6.2. Functionalization of Solid Support
6.3. Preparation of DNA H-Phosphonate
6.4. Oxidation of the Oligonucleotide H-Phosphonate to the Thiophosphate
6.5. Oxidation of the Oligonucleotide H-Phosphonate to the Phosphodiester and the Phosphoramidate Analog
6.6. Alternative Procedure For Oxidation of The H-Phosphonate to the Phosphoramidate
6.7. Cleavage of the Oligonucleotide from the Copolymer Support
6.8. Recovery of Excess Protected Nucleoside H-Phosphonate
6.9. HPLC Purification of Monomethoxytrityl Protected Oligonucleotide; Detritylation and Further Work Up
6.10. Preparation of Monomethoxytritylnucleoside H-Phosphonates
1. INTRODUCTION
The present invention describes methods for the efficient synthesis of oligonucleotides of variable length, as well as their associated analogs. The materials and methods disclosed herein provide a unique procedure by which a high degree of substitution on the solid support may be achieved, while observing high conversions of the reactants present and isolating high yields of the desired end-products. These characteristics, along with the surprising ability to recover valuable excess reagent monomers, combine to give a convenient and highly cost effective means for the production of nucleic acid fragments. In addition, novel monomethoxytritylated nucleoside H-phosphonate compounds are disclosed which are useful reagents for the high efficiency synthesis of polynucleotides.
2. BACKGROUND OF THE INVENTION
2.1. Synthetic Oligonucleotides and There Utility
Oligonucleotides and, in particluar, oligodeoxynucleotides (DNA) enjoy a special status among the tools used by the scientific biological community. In its pursuit of knowledge regarding the intimate workings and details of the body's mechanism for expressing particular traits and the development of certain abnormalities and mammalian disease, sequence-specific oligonucleotides have been used in recombinant host-vector systems and, in diagnostic assays, as intermediates in the preparation of labeled oligonucleotide probes. DNA fragments of a few to tens of bases in length are useful models for the study of the local interaction of DNA with foreign agents or known therapeutic compounds (See, for example, Lippard et al. in
Science
1985, 230, 412).
Recently, oligodeoxynucleotides, which are complementary to certain gene messager RNA or viral sequences, have been reported to inhibit the spread of disease related to viral and retroviral infectious agents (See, for example, Matsukura et al. in
Proc. Natl. Acad. Sci. USA
1987, 84, 7706, and references cited therein). It has also been reported that oligonucleotides can bind to duplex DNA via triple helix formation and, presumably, inhibit transcription and/or DNA synthesis (See, Moser and Dervan in
Science
1987, 238, 645).
These oligonucleotides are referred to as “antisense” compounds, and they, themselves, represent a whole class of therapeutic agents which exhibit antiviral activity and/or inhibit viral DNA synthesis or protein synthesis. Moreover, analogs of DNA having internucleotide phosphate linkages different from the phosphate diester groups of normal DNA have been found to possess their own unique characteristics which are desirable in certain applications. For example, methyl phosphonate analogs of DNA, perhaps being uncharged, demonstrate greater hydrophobicity and readily pass through cell membranes while inhibiting protein synthesis. The thiophosphate analogs are more resistant to degradation by nucleosides than their phosphate diester counterparts and are thus expected to have a higher persistence in vivo and greater potency. Phosphoramidate derivatives of oligonucleotides are known to bind to complementary polynucleotides and have the additional capability of accommodating covalently attached ligand species (See, for example, Froehler et al. in
Nucleic Acids Res.
1988, 16(11), 4831).
Thus, oligonucleotides and their associated analogs have a well-established utility in biological and chemical research, but their synthesis is invariably time consuming, tedious, and costly.
2.2. Preparative Methods
Since the pioneering work by Merrifield and co-workers in the area of solid phase-supported synthesis of polypeptides, the preparation of synthetic “biological” polymers under heterogeneous conditions has been pursued with keen interest by research workers in many laboratories. Of particular and present interest in the fields of molecular biology and biological chemistry is the development of a practical in vitro synthesis of DNA, RNA, or their associated analogs and derivatives. Among the earliest work in the area of solid phase-supported synthesis of oligonucleotides was that of Lestinger and co-workers (See, e.g.,
J. Am. Chem. Soc.
1966, 88(22), 5319) who described the use of “popcorn” copolymers prepared from styrene, p-vinylbenzoic acid, and p-divinylbenzene. These copolymers were found to be insoluble in water, alkaline solutions, and “all organic solvents examined” (Ibid, at page 5320, col. 1). The condensation chemistries described in these references involve multiple steps including the phosphorylation of polymer bound nucleoside followed by coupling of the phosphoryl derivative with an added nucleoside. The protocols associated with this, and similar techniques, required reaction times in the order of days but disclosed the use of a variety of sugar hydroxyl protecting groups including trityl, monomethoxytrityl, and dimethoxytrityl moieties (See, e.g.,
Biochemistry
1967, 6(5), 1379).
Thereafter, other derivatives of styrene polymers were developed as the insoluble polymer support. Of note were the tritylated polymers described by Melby and Strobach (See, e.g.,
J. Am. Chem. Soc.
1967, 89(2), 450). The solvent in popular use for the condensation reactions described in the foregoing references was pyridine, but certain solubility problems persisted, especially with particular acylated nucleosides as starting materials (See, e.g.,
J. Org. Chem.
1969, 34(2), 427). Although fairly high loading of initial nucleosides were possible, the degress of conversion remained very low and were, in fact, inversely proportional to the degree of loading. Other solvents, such as methylene chloride, dioxane, and dimethylformamide were described as either “unsuitable” or provided “diminished conversions” (See, e.g.,
J. Org. Chem.
1969, 34(2), on page 423).
More recently, Merrifield and co-workers described, in
J. Org. Chem.
1978, 43(14), 2845, yet another derivatized styrene polymer, an aminomethylated resin, useful in the preparation of a 4-(oxymethyl)phenylacetamido-methylpoly(styrene-co-divinylbenzene), the preferred solid support for polypeptide syntheses. Nevertheless, the drawbacks associated with organic polymeric solid supports, which have led research workers in the field to abandon them in favor of inorganic supports, are perhaps best summarized in U.S. Pat. No. 4,415,732, issued Nov. 15, 1983 to Caruthers and Beaucage which states, in part, that “prior art polymers used in such synthesis have proven inadequate for reasons such as: (1) slow diffusion rates of activated nucleotides into the support; (2) excessive swelling of various macroporous, low cross-linked support polymers; and (3) irreversible absorption of reagent onto the polymer” (col. 1, lines 34-40). Caruthers and Beaucage also describe in this patent the utility of phosphoramidite compounds as intermediates in the preparation of polynucleotides and prefer, as most researche

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