Glen Report 10.17: More Novel Monomers

4-Thio-dU, 5'-Amino-dT, 2'-F-Pyrimidines, 5,6-Dihydro-Pyrimidines, 2'-Phosphoramidites

4-Thio-dU

Demand for sulfur modified bases continues to expand for investigations of oligonucleotide structure, but primarily for cross-linking purposes. We are happy to broaden our line of sulfur modified nucleoside phosphoramidites with the addition of 4-thio-dU (1). We have protected this monomer as the S-cyanoethyl ether1, (2) which is stable during synthesis and readily removed by ammonium hydroxide.

It is critical to add 50mM sodium hydrosulfide (NaSH) to the ammonium hydroxide used for deprotection. Especially if room temperature deprotection is carried out, this technique radically reduces the level of ammonolysis which would lead to undesired dC. Moreover, in critical applications, it is also desirable (3) to remove the cyanoethyl protecting group (1M DBU in acetonitrile, 3h/RT) prior to the ammonium hydroxide cleavage and deprotection.

Further reactions of oligonucleotides specifically at the sulfur residue have been described (3), allowing the incorporation of a wide variety of functional groups at these positions.

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5'-Amino-dT

Applications requiring the use of peptide nucleic acids (PNA) continue to grow in popularity and the need for PNA/DNA chimeras has, consequently, become more significant. We already have offered 5'-Amino-dT where the amino group is protected as the trifluoroacetate. This was designed to be easily removed by ammonium hydroxide during the cleavage and deprotection. However, it has been brought to our attention that the free 5'-amine is capable of reacting with the thymine base during deprotection, with substantial loss of amine reactivity. We have therefore discontinued this product and now offer 5'-Amino-dT (2) protected with an MMT group. The MMT group should remain on the oligonucleotide during cleavage and deprotection steps. It should be removed in aqueous acid after removal of the ammonia. The MMT group can, of course, also be used in reverse phase purification techniques. Alternatively, the MMT group can be removed on the synthesizer to allow amine-specific reactions to be carried out in the synthesis column.

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2'-F-Pyrimidines

2'-Deoxy-2'-fluoro-nucleosides adopt an RNA-type sugar conformation, presumably due to the high electronegativity of fluorine. Because of this sugar conformation, RNA duplexes (A-form) are generally more thermodynamically stable than DNA duplexes (B-form). As expected, the addition of 2'-F-RNA residues to oligodeoxynucleotides progressively increases the thermal stability of their duplexes with RNA. The stabilization is additive at approximately 2° per residue. This compares favorably with 2'-OMe-RNA at around 1.5° and RNA at 1.1° per residue. In the meantime, base pair specificity remains intact.(4)

2'-F-RNA phosphodiester linkages are not nuclease resistant, although the corresponding phosphorothioate linkages are highly resistant.

Researchers usually design antisense oligonucleotides to form duplexes with RNA which are substrates for RNase H. Uniformly modified 2'-F-RNA/RNA duplexes are not substrates for RNase H. However, it is straightforward to prepare chimeric 2'-F-RNA/DNA phosphorothioate oligonucleotides which exhibit enhanced binding to the RNA target, are substrates for RNase H, and are highly nuclease resistant.

Following many requests from our customers, we are now introducing 2'-F-C (3) and 2'-F-U (4) phosphoramidite monomers. If we are able to supply the corresponding A and G monomers at a reasonable price, we will introduce them later.

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2’-F-RNA Phosphoramidites

5,6-Dihydro-dU and dT

Cellular DNA is constantly being damaged by oxidation and alkylation, by free radicals, and by ultraviolet and ionizing radiation. The body has therefore evolved a number of repair enzyme systems to excise and repair these lesions. The study of the resulting lesions and the enzymes responsible for their repair is currently a major area of scientific research.(5) One group of compounds of particular interest are the 5,6-dihydro pyrimidines. They are a naturally occurring class of compounds that are structural components of alanine transfer RNA. Dihydrouracil is also a major base damage product formed by exposure of cytosine in DNA to ionizing radiation under anoxic conditions.

A major difficulty in the study of damaged nucleobases is their inherent instability. This has made it difficult to incorporate them into synthetic oligonucleotides for model studies since they must be stable to the conditions used for oligonucleotide synthesis, cleavage and deprotection. This makes it necessary to do most experiments using preparations of DNA which are then exposed to various agents followed by isolation and characterization of the damaged products. However, the use of our UltraMILD monomers allows strongly basic hydrolytic conditions to be avoided during cleavage and deprotection, opening the possibility that oligos incorporating some damaged bases might be synthesized. Oligonucleotides synthesized using 5,6-dihydro-dU (5) or 5,6-dihydro-dT (6) and UltraMILD monomers can be cleaved using either concentrated ammonium hydroxide or 50 mM potassium carbonate in anhydrous methanol. Complete cleavage and deprotection can be accomplished at room temperature in 2-4 hours without damaging either the dihydro-dU or dihydro-dT bases.

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2'-5' Linked Oligonucleotides

Cellular DNA and RNA are made up of ribo- and 2'-deoxyribonucleic acids linked together via 3'-5' phosphodiester linkages and by far comprise the bulk of polynucleic acids found in cells. Much less common are oligonucleotides which have 2'-5' linkages. However, a unique feature of 2'-5' linked oligonucleotides is their ability to bind selectively to complementary RNA.(6, 7) These features suggest a number of interesting uses for 2'-5' linked oligos such as their use as RNA specific probes or in antisense oligos.

Recently, chimeric oligos have been synthesized using 3'-deoxy-2'-phosphoramidites and 2'-deoxy-3'-phosphoramidites. (3) Using these amidites the authors synthesized phosphorothioate oligos with 2'-5' linkages and chimeras with 2'-5' linked ends and 3'-5' linked central regions. They found that 2'-5' phosphorothioate oligos: 1) bind selectively to complementary RNA with the same affinity as phosphodiester oligos; 2) exhibit much nonspecific binding to cellular proteins; 3) do not activate RNase H. In experiments with Chinese hamster ovary cells transfected with human 5a-reductase-II (5aR-II), chimeric antisense oligos complementary to the 5' untranslated region of 5aR-II, containing seven 3'-5' linkages in the center, were effective in inhibiting 5aR-II protein in a dose dependent manner. The same oligos with 2'-5' linkages only were ineffective in inhibiting 5aR -II protein synthesis.

Glen Research now offers all of the 3'-deoxy-2'-phosphoramidites (7-10) for use in synthesizing 2'-5' oligonucleotides.

Product Information

2'-5' Linked Oligonucleotide Phosphoramidites and Supports

References

  1. T.T. Nikifirov and B.A. Connolly, Tetrahedron Lett., 1991, 32, 3851-3854.
  2. T.T. Nikiforov and B.A. Connolly, Nucleic Acids Res., 1992, 20, 1209-1214
  3. R.S. Coleman and E.A. Kesicki, J. Amer. Chem. Soc., 1994, 116, 11636-11642.
  4. A.M. Kawasaki, et al., J. Med. Chem., 1993, 36, 831-841.
  5. L. Augeri, Y.M. Lee, A.B. Barton, and P.W. Doetsch, Biochemistry, 1997, 36, 721-729.
  6. P.A. Giannaris and M.J. Damha, Nucleic Acids Research, 1993, 21, 4742-4749.
  7. P. Bhan, A. Bhan, M.K. Hong, J.G. Hartwell, J.M. Saunders, and G.D. Hoke, Nucleic Acids Res, 1997, 25, 3310-3317.