Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1998-06-19
2000-01-18
Crane, L. Eric
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 2533, 536 2534, C07H 2100, C07H 2102, C07H 2104
Patent
active
060158956
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a linker arm for solid support oligonucleotide synthesis and to a process for production thereof.
BACKGROUND ART
The art of organic chemistry on solid supports is generally known. A useful review article on this topic may be found in "Organic Chemistry on Solid Supports"by Fruchtel et al., Angew. Chem. Int. Ed. Engl., 1996, 35, pgs. 17-42, the contents of which are hereby incorporated by reference.
As discussed in Fruchtel et al., the art has developed automated solid-phase synthesis of polypeptides, oligonucleotides and oligosaccharides. Of particular interest here is solid-phase synthesis of oligonucleotides. The following are useful review articles/textbooks on this topic:
Beaucage et al., Tetrahedron, 1992, 48, pg. 2223;
Beaucage et al., Tetrahedron, 1993, 49, pgs. 6123-6194
Davis et al., Innovation and Perspectives in Solid Phase Synthesis (Ed.: R. Epton), Intercept, Andover, 1992, pg. 63; and
Montserra et al., Tetrahedron, 1994, 50, pg. 2617; the contents of each of which are hereby incorporated by reference.
In the solid-phase synthesis of oligonucleotides, it is known to synthesize the oligonucleotide on an inorganic solid support bearing a succinyl linker arm--see, for example, any of the following references:
Caruthers et al., Genetic Engineering, Plenum Press, New York (1982), Vol. 4, pgs. 1-17;
Letsinger et al., Genetic Engineering, Plenum Press, New York (1985), Vol. 5, pg. 191;
Froehler et al., Nucleic Acids Research, 14:5399-5407 (1986); and
Matteucci et al., Journal of American Chemical Society, 103:3185-3186 (1981); the contents of each of which are hereby incorporated by reference.
Typically, the succinyl linker arm has the following general formula: ##STR1## Thus, the succinyl group links the growing oligonucleotide from its terminal 3' hydroxyl group by an ester bond to a primary amine on the support, which may be, for example, conventional controlled pore glass (CPG) or silica, by an amide bond. Once the desired oligonucleotide has been synthesized, it is freed or cleaved from the succinyl linker arm hydrolyzing the ester carbonyl group. The hydrolysis agent is usually concentrated ammonium hydroxide. Typically, this reaction can take from 1-4 hours to complete. With improvements to current solid-phase oligonucleotide synthesizers, this cleavage step can represent 50% or more of the total time require to synthesize the desired oligonucleotide.
Thus, there have been various recent attempts in the art to develop improved linker arms for use in solid-phase oligonucleotide synthesis.
Of particular note is U.S. Pat. No. 5,112,962 [Letsinger et al. (Letsinger)], the contents of which are hereby incorporated by reference. Letsinger teaches a linker arm for solid support synthesis of oligonucleotides and oligonucleotide derivatives have the following formula: ##STR2## Thus, Letsinger teaches an oxalyl linker arm which purportedly release the synthesized oligonucleotide or oligonucleotide derivate in a period of 1-30 minutes in a manner that leaves the oligonucleotide fully protected. The oxalyl linker arm purportedly can be rapidly cleaved by 5% ammonium hydroxide in methanol, ammonium hydroxide, wet tertiary amine, triethylamine/alcohol, triethylamine/methanol, triethylamine/ethanol, aqueous trimethylamine and other bases. Unfortunately, the oxalyl linker arm of Letsinger suffers from its purported advantage. Specifically, the present inventors have discovered that the oxalyl linker arm of Letsinger is susceptible to significant spontaneous hydrolysis (e.g. spontaneous hydrolysis of approximately 10-40% per month) which renders it difficult it to use in commercial operations. This is illustrated in more detail hereinbelow. The oxalyl linker arm is also difficult to prepare because it requires using oxalyl chloride, which is highly reactive, toxic and dangerous.
Accordingly, the art is still in need of a linker arm capable of offering the advantages of the succinyl linker arm (ease of production/use) and the oxalyl linker arm (short cleavage
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Pon et al.(I), "Hydroquinon-O,O'-Diacetic Acid as a More Labile Replacement for Succinic Acid Linkers in Solid-Phase Oligonucleotide Synthesis," Tetrahedron Letters, 38(19), 3327-3330 (May 12, 1997).
Pon et al.(II), "Rapid Automated Derivatization of Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium or Phosphonium Coupling Reagants," Tetrahedron Letters, 38(19), 3331-3334 (May 12, 1997).
Pon et al.(III), "Hydroquinone-O,O'-Diacetic Acid (`Q-Linker`) as a Replacement for Succinyl and Oxalyl Linker Arms in Solid Phase Oligonucleotide Synthesis," Nucleic Acids Research, 25(18), 3629-3635 (1997).
Tetrahedron, (incl. Tetrahedron Reports), vol. 44, No. 14, Oxford GB, pp. 4331-4338 XP000673489 T. Tanaka et al.
Biotechniques, vol. 6, No. 8, Jan. 1, 1998, pp. 768-770, 773-775, XP000562920, Pon R.T et al.
Biophosphates and Their Analogues, Synthesis, Structure, Metabolism and Activity, vol. 3, 1987, pp. 3-21, XP000671967 M.H. Caruthers et al.
Pon Richard T.
Yu Shuyuan
Crane L. Eric
University Technologies International Inc.
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