Diastereomer Separation of Azobenzene-Tethered Oligodeoxyribonucleotides and Determination of Their Absolute Configurations by Enzymatic Digestion

Abstract

Two diastereomers were produced by the introduction of azobenzene-tethering prochiral linker (2,2-bis(hydroxymethyl)propionic acid) in the modified ODN, which had been used for the photoregulation of DNA functions. We found that this modified ODN with sequence 5′-…pNpXpN…-3′ (p = phosphate; N = nucleoside; X = azobenzene residue) could be digested to pX (the phosphate at the 5′ side of X was left) by an over excess of Phosphodiesterase I. By comparing the retention time of pX from the separated diastereomer with that of authentic R- or S-pX on chiral HPLC, absolute configuration could be easily determined.

Keywords:

• Diastereomer separation
• modified oligodeoxynucleotide
• absolute configuration
• enzymatic digestion
• Phosphodiesterase

This work was financially supported by Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST). The Mitsubishi Foundation (for H.A.), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan are also acknowledged.

Supporting Information Available. Supplemental Figure 1 (Molecular structures of the modified polysaccharide used for coating silica-gel in chiral columns), Supplemental Figure 2 (HPLC patterns of the products obtained from the digestion of β−AXT9nt by Phosphodiesterase I), Supplemental Figure 3 (HPLC patterns of pX obtained from the digestion of AXG7nt and CXA9nt by Phosphodiesterase I), Supplemental Figure 4 (HPLC patterns on CHIRALCEL AD-RH column of the diastereomers of 5′-TpX-3′ obtained from the digestion of T₆XT by Phosphodiesterase I), Supplemental Figure 5 (HPLC patterns of the products obtained from the digestion of α-TXT₆ and T₇X by Phosphodiesterase I), and Supplemental Scheme (Synthetic route of pX) are available.

Notes

^a HPLC conditions: Merck LiChrospher 100 RP-18(e) column; linear gradient of 7.5–17.5% acetonitrile/water (50 mM ammonium formate (pH7.0)) in 40 minutes; 0.5 mL/min.

^b See Scheme 1 for the sequences.

^a The gradient program: 15–18.5% acetonitrile/water in 30 minutes; then 18.5–50% acetonitrile/water in 10 minutes; at last washing with 50% acetonitrile/water for 10 minutes.

^b The gradient program: 14.5–20% acetonitrile/water in 30 minutes; then 20–50% acetonitrile/water in 10 minutes; at last washing with 50% acetonitrile/water for 20 minutes.

^cOnly in the case of T₆XT, the pX obtained from α-diastereomer has longer retention than that from β-diastereomer.

1. Yamana, K.; Mitsui, T.; Isuno, T.; Nakano, H. Incorporation of two anthraquinonylmethyl groups into the 2′-O-positions of oligonucleotides: increased affinity and sequence specificity of anthraquinone-modified oligonucleotides in hybrid formation with DNA and RNA. Bioconj. Chem. 1996, 7, 715–720.

2. Yamana, K.; Iwase, R.; Furutani, S.; Tsuchida, H.; Zako, H.; Yamaoka, T.; Murakami, A. 2′-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA. Nucleic Acids Res. 1999, 27, 2387–2392.

3. Santoro, S.W.; Joyce, G.F.; Sakthivel, K.; Gramatikova, S.; Baras, C.F. RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 2000, 122, 2433–2439.

4. Okamoto, A.; Kanatani, K.; Saito, I. Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNA typing. J. Am. Chem. Soc. 2004, 126, 4820–4827.

5. Christensene, U.B.; Pedersen, E.B. Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilization of dsDNA and discrimination of DNA over RNA. Nucleic Acids Res. 2002, 30, 4918–4925.

6. Kool, E.T. Replacing the nucleobases in DNA with designer molecules. Accounts Chem. Res. 2002, 35, 936–943.

7. Fukui, K.; Morimoto, M.; Segawa, H.; Tanaka, K.; Shimidzu, T. Synthesis and properties of an oligonucleotide modified with an acridine derivative at the artificial abasic site. Bioconj. Chem. 1996, 7, 349–355.

8. Endo, M.; Azuma, Y.; Saga, Y.; Kuzuya, A.; Kawai, G.; Komiyama, M. Molecular design for a pinpoint RNA scission. Interposition of oligoamines between two DNA oligomers. J. Org. Chem. 1997, 62, 846–852

9. Asanuma, H.; Ito, T.; Komiyama, M. Photo-responsive oligonucleotides carrying azobenzene in the side-chains. Tetrahedron Lett. 1998, 39, 9015–9018.

10. Asanuma, H.; Liang, X.G.; Komiyama, M. meta-Aminoazobenzene as a thermo-insensitive photo-regulator of DNA-duplex formation. Tetrahedron Lett. 2000, 41, 1055–1058.

11. Asanuma, H.; Takarada, T.; Yoshida, T.; Tamaru, D.; Liang, X.G.; Komiyama, M. Enantioselective incorporation of azobenzenes into oligodeoxyribonucleotide for effective photoregulation of duplex formation. Angew Chem. Int. Ed. Eng. 2001, 40, 2671–2673.

12. Asanuma, H.; Ito, T.; Yoshida, T.; Liang, X.G.; Komiyama, M. Photoregulation of the formation and dissociation of a DNA duplex by using the cis-trans isomerization of azobenzene. Angew Chem. Int. Ed. Eng. 1999, 38, 2393–2395.

13. Asanuma, H.; Liang, X.G.; Yoshida, T.; Yamazawa, A.; Komiyama, M. Photocontrol of triple-helix formation by using azobenzene-bearing oligo(thymidine). Angew Chem. Int. Ed. Eng. 2000, 39, 1316–1319.

14. Asanuma, H.; Liang, X.G.; Yoshida, T.; Komiyama, M. Photocontrol of DNA duplex formation by using azobenzene-bearing oligonucleotides. Chem. Bio. Chem. 2001, 2, 39–44.

15. Liang, X.G.; Asanuma, H.; Komiyama, M. Photoregulation of DNA triplex formation by azobenzene. J. Am. Chem. Soc. 2002, 124, 1877–1883.

16. Yamazawa, A.; Liang, X.G.; Asanuma, H.; Komiyama, M. Photoregulation of the DNA polymerase reaction by oligonucleotides bearing an azobenzene. Angew Chem. Int. Ed. Eng. 2000, 39, 2356–2358.

17. Asanuma, H.; Tamaru, D.; Yamazawa, A.; Liu, M.Z.; Komiyama, M. Photoregulation of the transcription reaction of T7 RNA polymerase by tethering an azobenzene to the promoter. Chem. Bio. Chem. 2002, 3, 786–789.

18. Liang, X.G.; Asanuma, H.; Kashida, H.; Takasu, A.; Sakamoto, T.; Kawai, G. NMR study on the photoresponsive DNA tethering an azobenzene. Assignment of the absolute configuration of two diastereomers and structural determination of their duplex in the trans-form. J. Am. Chem. Soc. 2003, 125, 16408–16415.

19. Home page of Daicel Chemical Industries: http://www.daicel.co.jp/chiral/e/product/instruction.

20. For 5′-CAAATTAATACGXACTCACTATAGGG-3′, the retention time of the two diastereomers is 22.2 and 25.8 minutes, respectively (a linear gradient 6.5–13.5% acetonitrile/water in 40 minutes); For 5′-ApXpA-3′, the retention time of the two diastereomers is 74.7 and 78.8 minutes, respectively (a linear gradient 12.5–17.5% acetonitrile/water in 120 minutes).

21. Laskowski, M., Sr. Venom Exonuclease. in The Enzymes 3rd Ed., Vol,. 4, ed. Boyer, P.D., Academic Press: New York, 1971, 4, 313–328.

22. Razzell, W. Phosphodiesterases. in Methods in Enzymology 6th ed. Colowick, S.; Kaplan, N., Academic Press: New York, 1963, 236–258.

23. The fractions at 12.3, 18.5, and 35.7 minutes could be assigned from ESI-MS as pApX (Obsd. 703.0, Calcd. for [pApX-H⁺] 703.2), pX (Obsd. 392.4, Calcd. for [pX-H⁺] 392.1), and ApX (Obsd. 625.6, Calcd. for [ApX-H⁺] 625.2), respectively.

24. Pure 5′-ApX-3′ could be obtained from the treatment of 5′-pApX-3′ with alkaline phosphatase. Further treatment of 5′-ApX-3′ with Phosphodiesterase I did not produce pX at all.

25. Isomers of TpX could not be separated on CHIRALCEL OJ-R column or ODS column under all the conditions we tried.

26. Liang, X.G.; Asanuma, H.; Komiyama, M. Phenylazonaphthalene as a superb photo-regulator for DNA-triplex formation. Tetrahedron Lett. 2001, 42, 6723–6725.

27. Khorana, G.H. Phosphodiesterases. in The Enzymes 2nd Ed. Vol. 5. Boyer P.D.; Lardy, H.; Myrbäck, K., Eds., Academic Press: New York, 1961, 5, 79–94.

28. Matsubara, H.; Hasegawa, S.; Fujimura, S.; Shima, T.; Sugimura, T. Studies on poly(adenosine diphosphate ribose) V. Mechanism of hydrolysis of poly(adenosine diphosphate ribose) by snake venom phosphodiesterase. J. Biol. Chem. 1970, 245, 3606–3611.

29. Razzell, W.E.; Khorana, H.G. Studies on polynucleotides IV. Enzymatic degradation. The stepwise action of venom phosphodiesterase deoxyribo-oligonucleotides. J. Biol. Chem. 1959, 234, 2114–2117.

30. Felix, F.; Potter, J.L.; Laskowski, M. Action of phosphodiesterase on deoxyribooligonucleotides carrying a monoesterified phosphate on carbon 3′. J. Biol. Chem. 1960, 235, 1150–1154.

31. Nossal, N.G.; Singer, M.F. The processive degradation of individual polyribonucleotide chains. J. Biol. Chem. 1968, 243, 913–922.

32. Melgar, E.; Goldthwait, D.A. Deoxyribonucleic acid nucleases I. The use of a new method to observe the kinetics of deoxyribonucleic acid degradation by deoxyribonuclease I, deoxyribonuclease II, and Escherichia Coli endonuclease I. J. Biol. Chem. 1968, 243, 4401–4408.

33. Zhang, L.; Peritz, A.E.; Meggers, E. A simple glycol nucleic acid. J. Am. Chem. Soc. 2005, 127, 4174–4175.

34. Zhang, L.; Peritz, A.E.; Meggers, E. Synthesis of glycol nucleic acids. Synthesis 2006, 127, 645–653.

35. Schlegel, M.K.; Peritz, A.E.; Kittigowittana, K.; Zhang, L.; Meggers, E. Duplex formation of the simplified nucleic acid GNA. Chem. Bio. Chem. 2007, 8, 927–932.