Sulfurizing reagent: 3-aryl-1,2,4-dithiazoline-5-ones

Details Sulfurizing reagent: 3-aryl-1,2,4-dithiazoline-5-ones Sulfurizing reagent: 3-aryl-1,2,4-dithiazoline-5-ones

1997-07-16

2002-12-31

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025300, C558S095000, C548S129000, C548S130000

Reexamination Certificate

active

06500944

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to the synthesis of novel sulfur transfer reagents and to the sulfurization of the internucleotide linkages of oligonucleotides with the reagents.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. Oligonucleotide phosphorothioates are of considerable interest in nucleic acid research and are among the analogues tested as oligonucleotide therapeutics. Oligonucleotides phosphorothioates contain internucleotide linkages in which one of the nonbridging oxygen atoms of the phosphate group is replaced by a sulfur atom. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive, and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See eg.,
Methods in Molecular Biology, Vol
20:
Protocols for Oligonucleotides and Analogs,
pp. 165-189 (S. Agrawal, ed., Humana Press, 1993);
Oligonucleotides and Analogues: A Practical Approach,
pp. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman,
Chemical Reviews,
90: 543-584 (1990); Agrawal and Iyer,
Curr. Op. in Biotech.
6: 12 (1995); and
Antisense Research and Applications
(Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al.,
J. Molec. Biol.
72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese,
Tetrahedron Lett.
34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to automated synthesis. Beaucage and Carruthers,
Tetrahedron Lett.
22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
These latter approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild,
Tetrahedron Lett.
28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al.,
Biochemistry
23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al.,
Biochemistry
27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al.,
Proc. Natl. Acad. Sci. USA
85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3′-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Internucleoside linkages are formed between the 3′ functional group of the incoming nucleoside and the 5′ hydroxyl group of the 5′-most nucleoside of the nascent, support-bound oligonucleotide. In the phosphoramidite approach, the internucleoside linkage is a phosphite linkage, whereas in the H-phosphonate approach, it is an H-phosphonate internucleoside linkage. To create the sulfur-containing phosphorothioate internucleoside linkage, the phosphite or H-phosphonate linkage must be oxidized by an appropriate sulfur transfer reagent. In the H-phosphonate approach, this sulfurization is carried out on all of the H-phosphonate linkages in a single step following the completion of oligonucleotide chain assembly, typically using elemental sulfur in a mixed solvent, such as CS
2
/pyridine. In contrast, the phosphoramidite approach allows stepwise sulfurization to take place after each coupling, thereby providing the capability to control the state of each linkage in a site-specific manner. Based on superior coupling efficiency, as well as the capacity to control the state of each linkage in a site-specific manner, the phosphoramidite approach appears to offer advantages.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 &mgr;mol to 1 mmol and higher). See Padmapriya et al.,
Antisense Res. Dev.
4: 185 (1994). Several modifications of the standard phosphoramidite processes have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al.,
Tetrahedron
50: 9255 (1994); Theisen et al.,
Nucleosides & Nucleotides
12: 43 (1994); and Iyer et al.,
Nucleosides & Nucleotides
14: 1349 (1995)) and isolation (Kuijpers et al.,
Nucl. Acids Res.
18: 5197 (1990); and Reddy et al.,
Tetrahedron Lett.
35: 4311 (1994)) of oligonucleotides.
It is imperative that an efficient sulfur transfer reagent is used for the synthesis of oligonucleotide phosphorothioates via the phosphoroamidite approach. Elemental sulfur is not efficient due to poor solubility and slow sulfurization reaction. A number of more efficient sulfurizing reagents have been reported in recent years. These include phenylacetyl disulfide, (Kamer et al.,
Tetrahedron Lett.
30: 6757-6760 (1989)), H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent)(Iyer et al.,
J. Org. Chem.
55: 4693-4699 (1990)), tetraethylthiuram disulfide (TETD)(Vu et al.,
Tetrahedron Lett.
32: 3005-3008 (1991)), dibenzoyl tetrasulfide (Rao et al.,
Tetrahedron Lett.
33: 4839-4842 (1992)), bis(O,O-diisopropoxyphosphinothioyl) disulfide (S-Tetra)(Stec et al.,
Tetrahedron Lett.
33: 5317-5320 (1993)), benzyltriethyl-ammonium tetrathiomolybate (BTTM) (Rao et al.,
Tetrahedron Lett.
35: 6741-6744 (1994)), bis(p-toluenesulfonyl) disulfide (Effimov et al.,
Nucl. Acids Res.
23: 4029-4033 (1995)), 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH)(Xu et al.,
Nucleic Acid Res.
24:1602-1607 (1996)), and 1,2,4-dithiazolidine-3,5-dione (DtsNH)(Xu et al.,
Nucleic Acid Res.
24:1602-1607 (1996)). Both Beaucage reagent and TETD are commercially available. Beaucage reagent has been widely used, however, its synthesis and stability are not optimal. In addition, the by-product formed by Beaucage reagent during sulfurization, 3H-2,1-benzoxanthiolan-3-one-1-oxide, is a potential oxidizing agent that can lead to undesired phosphodiester linkages under certain conditions. Therefore, its application in large-scale synthesis of oligonucleotide phosphorothioates may not be particularly suitable. We report the novel preparation of 3-phenyl-1,2,4-dithiazoline-5-one as a potential alternative sulfurizing reagent.
There is, therefore, a continuing need to develop new sulfur transfer reagents and processes for sulfurizing oligonucleotides. Ideally, such sulfur transfer reagents should be inexpensive to make, stable in storage and in solution, and highly efficient in sulfurization.
BRIEF SUMMARY OF THE INVENTION
The invention provides new sulfur transfer reagents, 3-aryl-1,2,4-dithiazoline-5-ones, for use in sulfurizing oligonucleotides. The sulfur transfer reagents according to the invention are inexpensive to make, stable in storage and in solution for thirty days, and highly efficient in sulfurization.
In a first aspect, the invention provides novel sulfur transfer reagents having the general structure (1)
wherein R is
or any substituted heterocyclic or substituted aromatic group. The sulfur transfer reagent 3-phenyl-1,2,4-dithiazoline-5-one is a particularly preferred embodiment of the invention and has the structure (2)
A second aspect provides for the syn

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