Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-30
2001-01-16
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025310, C536S025340, C536S025410
Reexamination Certificate
active
06175006
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the synthesis and purification of polynucleotides, and more particularly, to automated techniques for solid phase synthesis and purification of polynucleotides using phosphoramidite and/or hydrogen phosphonate chemistries.
A key factor in the recent advances in molecular biology has been the development of reliable and convenient methods for synthesizing polynucleotides, e.g. Itakura,
Science
, Vol. 209, pgs. 1401-1405 (1980); and Wallace et al, pgs. 631-663, in Scouten, ed.
Solid Phase Biochemistry
(John Wiley & Sons, New York, 1982). As the use of synthetic polynucleotides has increased, the demand for even greater convenience in the preparation of pure, ready-to-use polynucleotides has also increased. This demand has stimulated the development of many improvements in the basic procedures for solid phase synthesis, e.g. Sinha et al,
Nucleic Acids Research
, Vol. 12, pgs. 4539-4557 (1984)(beta-cyanoethyl in phosphoramidite chemistries); Froehler et al,
Tetrahedron Letters
, Vol. 27, pgs. 469-472 (1986)(H-phosphonate chemistry); Germann et al,
Anal. Biochem
., Vol. 165, pgs. 399-405 (1987); and Ikuta et al,
Anal. Chem
., Vol. 56, pgs. 2253-2256 (1984)(rapid purification of synthetic oligonuclectides by way of trityl moieties); Molko et al, European patent publication 241363 dated 3 Apr. 1987 (improved base-labile acyl protection groups for exocyclic amines), and the like.
In spite of such progress, difficulties are still encountered in current methods of polynucleotide synthesis and purification. For example, H-phosphonate and phosphoramidite monomers readily degrade in the presence of even trace amounts of water. This contributes greatly to their very short in-solution shelf lives, and the need to use large molar excesses of reactants to drive the coupling reactions to completion in reasonable times. The presence of water-degraded reactants leads to less pure final products and to more expensive syntheses. This problem is particularly acute for large scale (milligram and greater) syntheses, as well as for small scale (less than 1 umole) syntheses. In the case of the former, the cost of the monomeric reactants makes up the greatest portion of the overall cost of synthesis. Any reduction in the excess molar amount of reactants needed to adequately drive the coupling reaction would lead to substantial cost reductions in the synthesis of large amounts of polynucleotides, e.g. the milligram and gram quantities contemplated for therapeutic use. In the case of the latter, degradation of the reactants by trace amounts of water limits the efficiency that can be achieved on currently available commercial synthesizers, and increases the cost of synthesis because greater amounts of reactant must be used to counter losses due to water and oxygen present in trace amounts in the solvents, tubing, and vessels, or due to water and oxygen leaking into the system from faulty connections, valves, and the like.
As a further example, derivatized controlled pore glass (CPG), the current support of choice in most solid phase methodologies, can be responsible for spurious indications of coupling yields, e.g. Pon et al,
BioTechniques
, Vol. 6, pgs. 768-775 (1988). Moreover, CPG, like most glasses, lacks chemical stability in some of the highly corrosive deprotection reagents, such as concentrated ammonia and trichloroacetic acid, used in polynucleotide synthesis. As a consequence, the CPG support itself can be degraded in the deprotection steps and can be a source of contamination of the final product. This problem is exacerbated by the relative long reaction times required to remove currently used protection groups for exocyclic amines. An extended period of deprotection is required to remove these groups after the polynucleotide has been cleaved from the solid phase support. Thus, complete automation of synthesis and purification has been impractical. Another problem with CPG is that its surface supports chain growth at sites other than those associated with the 5′ terminus of an attached nucleoside. Such “extraneous” chain growth gives rise to a heterogeneous population of 5′-blocked (usually tritylated) polynucleotides. Typically, the “extraneous” tritylated products lack one or more 3′ nucleotides. This, of course, prevents one from successfully taking advantage of the relatively high hydrophobicity of the trityl group to purify “correct sequence” polynucleotides. Incorrect-sequence extraneous chains are also tritylated. Finally, the hydrophilic nature of CPG causes it to absorb water present in trace amounts in the solvents, which leads to the degradation of the highly water-sensitive monomeric reactants.
In view of the above, the field of solid phase polynucleotide synthesis could be significantly advanced by the availability of alternative support materials (i) which have the favorable mechanical properties of CPG, but which also possess greater chemical stability under the reaction conditions of polynucleotide synthesis, (ii) which provide less opportunity for extraneous chain growth during synthesis, and (iii) which would permit more efficient syntheses, particularly under conditions of reduced molar excess of reactants. The use of such materials coupled with improved exocyclic protection groups would not only allow practical automation of polynucleotide synthesis and purification in a single instrument, but also would permit more efficient and less expensive syntheses of polynucleotides and their derivatives.
SUMMARY OF THE INVENTION
The invention is directed to a system and method for solid phase polynucleotide synthesis on a nonswellable porous polystyrene support by H-phosphonate and/or phosphoramidite chemistries. The use of the polystyrene support (1) significantly reduces the number of failure sequences caused by extraneous initiation of polynucleotide chain growth on the support material, (2) permits deprotection of the polynucleotide product in the presence of the support material by virtue of its superior stability in the presence of deprotection reagents, and (3) permits more efficient syntheses, particularly under conditions of reduced molar excess of the monomeric reactants.
As used herein, “nonswellable” means that the porous polystyrene material remains substantially mechanically rigid, in particular does not increase in volume, when exposed to solvents, reactants, and products of the phosphoramidite and/or hydrogen phosphonate chemistries. Preferably, “nonswellable” means that the volume of the porous polystyrene support increases in volume no more than 5-10% over its dry volume when exposed to the solvents, reactants, and products of the phosphoramidite and/or hydrogen phosphonate chemistries. Mechanical rigidity is desirable for efficient and uniform transfer of reagents to the polynuclectide chain during synthesis.
An important feature of the invention is efficient monomer coupling to the growing polynucleotide chains under conditions of reduced molar excess of monomer over polynucleotide. It is believed that the increased efficiency results from the hydrophobic nature of the nonswellable porous polystyrene support, which unlike CPG does not accumulate moisture, even over many coupling cycles. In particular, with the use of the nonswellable porous polystyrene supports, much lower amounts of reactants are required for achieving equivalent effective reactant concentrations adjacent to the growing polynucleotide as those achieveable with CPG.
Preferably, the correct-sequence polynucleotides synthesized by the method of the invention are purified from the crude mixture cleaved from the reaction column by passage of the crude mixture over an adsorbent which preferentially adsorbs the 5′-blocking group of the correct-sequence polynucleotides. More preferably, whenever the 5′-blocking group is a trityl, the adsorbent is a nonswellable polystyrene solid.
REFERENCES:
patent: 4373071 (1983-02-01), Itakura
patent: 4401796 (1983-08-01), Itakura et al.
patent: 4415732 (1983-11-01)
Andrus William A.
McCollum Christie D.
Zon Gerald
Macevicz Stephen C.
Powers Vincent M.
The Perkin-Elmer Corporation
Wilson James O.
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