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New Product: 0.5M CSO for non-aqueous oxidation in DNA synthesis

Iodine-based oxidizers have been the standard for DNA and RNA synthesis since the advent of automated synthesizers.1 They are fast and efficient oxidizers, typically requiring less than 30 seconds for complete oxidation of phosphite triesters to phosphate triesters. However, while iodine-based oxidizers work well for most applications, there are some circumstances where non-aqueous oxidizers may be advantageous, especially where the bases or linkages being produced are sensitive to the presence of water and/or iodine during synthesis. For example, using low water oxidizers has been shown to improve the synthesis of oligos containing methyl phosphonates.2,3 Non-aqueous oxidizers, typically peroxides, including tert-butyl hydroperoxide, cumene hydroperoxide, hydrogen peroxide, and bis-trimethylsilyl peroxide, among others, have also been employed in DNA synthesis.5,6 These peroxides tend to be unstable, requiring that they be freshly formulated just prior to use, and so are difficult to use in routine automated synthesis.7

In 1996, we investigated the use of (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO)4 as a non-aqueous oxidizer in DNA synthesis.2 We found that a 0.5M solution of CSO in acetonitrile worked well as an oxidizer for the synthesis of oligos containing multiple incorporations of 7-deaza-dG, compared with iodine oxidation which caused substantial degradation.

More recently, CSO has been used for synthesizing oligos that incorporate the phosphonoacetate modification.8 A solution of 0.1M CSO is recommended for the oxidation of PACE modifications as the phosphonite internucleotide linkage is more easily oxidized than the phosphite internucleotide linkage. When synthesizing DNA-phosphonoacetate chimeric oligos, a 0.5M CSO solution is recommended. CSO has also been used as an oxidizer in 2’-O-DMAOE modified siRNA oligo synthesis.9

CSO Structure

CSO
CSO

Comparison of CSO with Iodine

We prepared the following oligo using 0.5M CSO in acetonitrile and compared it to the same oligo synthesized using standard 0.02M Iodine in THF/Water/Pyridine.

5’-TTA TTG TTC TTA TTG TTC TT-3’

We found the products to be virtually identical on RP HPLC, as shown in Figure 1.

A second pair of oligos was synthesized incorporating NHS-carboxy-dT (10-1535) to compare the performance of 0.5M CSO in acetonitrile to the standard 0.02M iodine oxidizer in an oligo containing a very sensitive modified base.

5’-TTT TTT TTT TTT TTT XTT TTT TTT T – 3’ (where X = NHS-Carboxy-dT)

The resulting oligonucleotides, shown in Figure 2, were of comparable purity by RP HPLC.

We can conclude that CSO is an effective, stable, non-aqueous oxidizer. Despite the demonstrated effectiveness of CSO as a non-aqueous oxidizer, the cost and the quality of the CSO has been prohibitive for use in formulating CSO as an oxidizer for routine use. We are now pleased to offer 0.5M CSO in acetonitrile as an effective, stable, non-aqueous oxidizer for DNA synthesis. We also offer 0.1M CSO for PACE chemistry at a more affordable price.

Non-aqueous oxidizers may prove beneficial in applications where it is desirable to avoid exposure to iodine or water. This might include on-chip or chamber-based synthesizers, and with phosphoramidites that are sensitive to the presence of iodine or moisture.

Figure 1: Mixed Base oligo using CSO or I2

Figure 2: Sensitive oligo using CSO or I2

FIG 1
FIG 2

References:

1. M.H. Caruthers, et al., Gene Amplif Anal, 1983, 3, 1-26.

2. M.A. Reynolds, et al., Nucleic Acids Res, 1996, 24, 4584-4591.

3. The Glen Report, 1996, 9, 8-9.

4. I. Ugi, et al., Nucleosides and Nucleotides, 1988, 7, 605 - 608.

5. S.L. Beaucage, and M.H. Caruthers, Current Protocols in Nucleic Acid Chemistry, 2000, 1, 3.3.1 - 3.3.20.

6. Y. Hayakawa, M. Uchiyama, and R. Noyori, Tetrahedron Letters, 1986, 27, 4191-4194.

7. N.D. Sinha, D.P. Michaud, S.K. Roy, and R.A. Casale, Nucleic Acids Res, 1994, 22, 3119-3123.

8. D.J. Dellinger, D.M. Sheehan, N.K. Christensen, J.G. Lindberg, and M.H. Caruthers, J Amer Chem Soc, 2003, 125, 940-950.

9. T.P. Prakash, J.F. Johnston, M.J. Graham, T.P. Condon, and M. Manoharan, Nucleic Acids Res, 2004, 32, 828-33.

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