Solid phase synthesis of oligonucleotides using carbonate...

Details Solid phase synthesis of oligonucleotides using carbonate... Solid phase synthesis of oligonucleotides using carbonate...

1999-06-22

2001-04-24

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S022100, C536S023100, C536S024300, C536S025310, C536S025330, C536S025340

Reexamination Certificate

active

06222030

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to nucleic acid chemistry and to the chemical synthesis of oligonucleotides. More particularly, the invention relates to an improved method for synthesizing oligonucleotides wherein carbonates are used as hydroxyl-protecting groups and “alpha-effect” nucleophiles such as peroxides are used in the deprotection thereof. The invention has utility in the fields of biochemistry, molecular biology and pharmacology, and in medical diagnostic and screening technologies.
BACKGROUND
Solid phase chemical synthesis of DNA fragments is routinely performed using protected nucleoside phosphoramidites. S. L. Beaucage et al. (1981)
Tetrahedron Lett
. 22:1859. In this approach, the 3′-hydroxyl group of an initial 5′-protected nucleoside is first covalently attached to the polymer support. R. C. Pless et al. (1975)
Nucleic Acids Res
. 2:773 (1975). Synthesis of the oligonucleotide then proceeds by deprotection of the 5′-hydroxyl group of the attached nucleoside, followed by coupling of an incoming nucleoside-3′-phosphoramidite to the deprotected hydroxyl group. M. D. Matteucci et a. (1981)
J. Am. Chem. Soc
. 103:3185. The resulting phosphite triester is finally oxidized to a phosphorotriester to complete the intemucleotide bond. R. L. Letsinger et al. (1976)
J. Am. Chem. Soc
. 9:3655. The steps of deprotection, coupling and oxidation are repeated until an oligonucleotide of the desired length and sequence is obtained. This process is illustrated schematically in
FIG. 1
(wherein “B” represents a purine or pyrimidine base, “DMT” represents dimethoxytrityl and “iPR” represents isopropyl.
The chemical group conventionally used for the protection of nucleoside 5′-hydroxyls is dimethoxytrityl (“DMT”), which is removable with acid. H. G. Khorana (1968)
Pure Appl. Chem
. 17:349; M. Smith et al. (1962)
J. Am. Chem. Soc
. 84:430. This acid-labile protecting group provides a number of advantages for working with both nucleosides and oligonucleotides. For example, the DMT group can be introduced onto a nucleoside regioselectively and in high yield. E. I. Brown et al. (1979)
Methods in Enzymol
. 6:109. Also, the lipophilicity of the DMT group greatly increases the solubility of nucleosides in organic solvents, and the carbocation resulting from acidic deprotection gives a strong chromophore, which can be used to indirectly monitor coupling efficiency. M. D. Matteucci et al. (1980)
Tetrahedron Lett
. 21:719. In addition, the hydrophobicity of the group can be used to aid separation on reverse-phase HPLC. C. Becker et al. (1985)
J. Chromatogr
. 326:219.
However, use of DMT as a hydroxyl-protecting group in oligonucleotide synthesis is also problematic. The N-glycosidic linkages of oligodeoxyribonucleotides are susceptible to acid catalyzed cleavage (N. K. Kochetkov et al.,
Organic Chemistry of Nucleic Acids
(New York: Plenum Press, 1972)), and even when the protocol is optimized, recurrent removal of the DMT group with acid during oligonucleotide synthesis results in depurination. H. Shaller et al. (1963)
J. Am. Chem. Soc
. 8:3821. The N-6-benzoyl-protected deoxyadenosine nucleotide is especially susceptible to glycosidic cleavage, resulting in a substantially reduced yield of the final oligonucleotide. J. W. Efcavitch et al. (1985)
Nucleosides
&
Nucleotides
4:267. Attempts have been made to address the problem of acid-catalyzed depurination utilizing alternative mixtures of acids and various solvents; see, for example, E. Sonveaux (1986)
Bioorganic Chem
. 4:274. However, this approach has met with limited success. L. J. McBride et al. (1986)
J. Am. Chem. Soc
. 108:2040.
Conventional synthesis of oligonucleotides using DMT as a protecting group is problematic in other ways as well. For example, cleavage of the DMT group under acidic conditions gives rise to the resonance-stabilized and long-lived bis(p-anisyl)phenylmethyl carbocation. P. T. Gilham et al. (1959)
J. Am. Chem. Soc
. 81:4647. Protection and deprotection of hydroxyl groups with DMT are thus readily reversible reactions, resulting in side reactions during oligonucleotide synthesis and a lower yield than might otherwise be obtained. To circumvent such problems, large excesses of acid are used with DMT to achieve quantitative deprotection. As bed volume of the polymer is increased in larger scale synthesis, increasingly greater quantities of acid are required. The acid-catalyzed depurination which occurs during the synthesis of oligodeoxyribonucleotides is thus increased by the scale of synthesis. M. H. Caruthers et al., in
Genetic Engineering: Principles and Methods
, J. K. Setlow et al., Eds. (New York: Plenum Press, 1982).
Considerable effort has been directed to developing 5′-O-protecting groups which can be removed under non-acidic conditions. For example, R. L. Letsinger et al. (1967)
J. Am. Chem. Soc
. 89:7147, describe use of a hydrazine-labile benzoyl-propionyl group, and J. F. M. deRooij et al. (1979)
Real. Track. Chain. Pays
-
Bas
. 9:537, describe using the hydrazine-labile levulinyl ester for 5′-OH protection (see also S. Iwai et al. (1988)
Tetrahedron Lett
. 22:5383; and S. Iwai et al. (1988)
Nucleic Acids Res
. 16:9443). However, the cross-reactivity of hydrazine with pyrimidine nucleotides (as described in F. Baron et al. (1955)
J. Chem. Soc
. 2855 and in V. Habermann (1962)
Biochem. Biophys. Acta
55:999), the poor selectivity of levulinic anhydride and hydrazine cleavage of N-acyl protecting groups (R. L. Letsinger et al. (1968),
Tetrahedron Lett
. 22:2621) have made these approaches impractical. H. Seliger et al. (1985),
Nucleosides
&
Nucleotides
4:153, describes the 5′-O-phenyl-azophenyl carbonyl (“PAPco”) group, which is removed by a two-step procedure involving transesterification followed by &bgr;-elimination; however, unexpectedly low and non-reproducible yields resulted. Fukuda et al. (1988)
Nucleic Acids Res. Symposium Ser
.19, 13, and C. Lehmann et al. (1989)
Nucleic Acids Res
. 17:2389, describe application of the 9-fluorenylmethylcarbonate (“Fmoc”) group for 5′-protection. C. Lehmann et al. (1989) report reasonable yields for the synthesis of oligonucleotides up to 20 nucleotides in length. The basic conditions required for complete deprotection of the Fmoc group, however, lead to problems with protecting group compatibility. Similarly, R. L. Letsinger et al. (1967),
J. Am. Chem. Soc
. 32:296, describe using the p-nitrophenyloxycarbonyl group for 5′-hydroxyl protection. In all of the procedures described above utilizing base-labile 5′-O-protecting groups, the requirements of high basicity and long deprotection times have severely limited their application for routine synthesis of oligonucleotides.
Still an additional drawback associated with conventional oligonucleotide synthesis using DMT as a hydroxyl-protecting group is the necessity of multiple steps, particularly the post-synthetic deprotection step in which the DMT group is removed following oxidation of the intemucleotide phosphite triester linkage to a phosphorotriester. It would be desirable to work with a hydroxyl-protecting group that could be removed via oxidation, such that the final two steps involved in nucleotide addition, namely oxidation and deprotection, could be combined.
The problems associated with the use of DMT are exacerbated in solid phase oligonucleotide synthesis where “microscale” parallel reactions are taking place on a very dense, packed surface. Applications in the field of genomics and high throughput screening have fueled the demand for precise chemistry in such a context. Thus, increasingly stringent demands are placed on the chemical synthesis cycle as it was originally conceived, and the problems associated with conventional methods for synthesizing oligonucleotides are rising to unacceptable levels in these expanded applications.
The invention is thus addressed to the aforementioned deficiencies in the art, and provides a novel method for synthesizing oligonucleotides, wherein th

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