HETEROCYCLES

Communication | Regular issue | Vol. 87, No. 5, 2013, pp. 1023-1028
Received, 9th March, 2013, Accepted, 4th April, 2013, Published online, 5th April, 2013.
DOI: 10.3987/COM-13-12697

■ Post-Modification of Triazole-Linked Analogues of DNA for Positively Charged Variants

Tomoko Fujino, Yusuke Miyauchi, Nobuhide Tsunaka, Koudai Okada, and Hiroyuki Isobe*

Department of Chemistry and Advanced Institute for Materials Research (WPI-AIMR), Tohoku University, Aoba-ku, Sendai 980-8578, Japan

Abstract

We report a concise post-modification method to convert an electroneutral triazole-linked DNA (TLDNA) to a positively charged triazolium-linked analogue, TLDNA+. A one-step methylation of oligothymine TLDNA successfully afforded TLDNA+ with a dramatic improvement in the solubility. The pentameric oligothymine TLDNA+ formed a stable triple helix with natural oligoadenine DNA as well as a mercury-mediated self-duplex.

One of the most common problems accompanied with non-phosphorus, electroneutral DNA analogues is the low solubility of the oligomers. Despite the chemical and biological stability benefitted from the absence of the negatively charged phosphate linkages, the electroneutral strand of oligonucleotides lacks electrostatic supports for the solubility. This solubility issue also arose, when we prepared long oligomers of triazole-linked DNA (TLDNA).1 Specifically, unique functions of the oligothymine TLDNA congeners such as electron-transporting materials2 or lure substrates for reverse-transcriptase3 prompted us to explore further applications with longer oligomers, but the solubility problem became severer as the oligomers lengthened. We herein report a concise post-modification method for the preparation of positively charged variants, i.e., triazolium-linked analogue of DNA (TLDNA+). A one-step methylation of oligothymine TLDNA successfully afforded TLDNA+ with an improved solubility, and the pentameric oligothymine TLDNA+ formed a triple helix with natural oligoadenine DNA in addition to a mercury-mediated self-duplex.

We found that TLDNA can be charged positively via a one-step post-modification of oligothymine congeners. Thus, we first examined dimer 1a with a single triazole unit for the post-modification with methylation reaction.4 The reaction took place effectively with 1a and iodomethane at reflux temperature in acetonitrile and, after washing the crude material with chloroform, the corresponding TLDNA+ 2a was obtained in 96% yield as an analytically pure material without chromatographic purification (Scheme 1). We also found that protective groups for 5'-acetylene or 3'-hydroxyl group are unnecessary for this methylation protocol. Tetramer 1b was thus converted to TLDNA+ 2b with iodomethane in DMF in 71% yield.5 The same conditions were also applicable to longer 1c to afford the corresponding pentamer TLDNA+ 2c in 99% yield. The post-modification with methylation was concise enough to be exerted in a large quantity.

The positively charged congener, TLDNA+, showed an improved and excellent solubility in aqueous media. As a representative example, the solubilities of pentamers were compared. A saturated aqueous solution of the pentamer (TLDNA 1c or TLDNA+ 2c) was prepared in SSPE buffer [10 mM sodium phosphate (pH 7.0), 100 mM sodium chloride and 0.10 mM ethylenediamine tetraacetic acid],6 and the concentration was determined by measuring the absorbance at 260 nm of an appropriately diluted solution. We determined the saturated concentration in SSPE buffer as 14.5 µM for the neutral TLDNA 1c and 11.8 mM for the positively charged TLDNA+ 2c. The results showed 814-fold improvement in the water solubility via methylation and demonstrated the effectiveness of the post-modification method for the preparation of soluble congeners.

Helix formation of TLDNA+ with natural complementary DNA was examined to reveal the affinity improvement of the positively charged variant. In order to obtain reproducible and reliable data for the melting point analysis, we optimized several conditions and found that the sigmoid melting curves were reproducibly obtained with oligoadenine (dA)20 as the complementary strand and hexafluorophosphate (PF6–) as the counter anion of TLDNA+ 2c.7 During this initial screening, a 2:1 stoichiometry of thymine base (T): adenine base (A) was also indicated for pentamer 2c and (dA)20 (i.e., 8:1 molar ratio of 2c:(dA)20), and we confirmed this optimal ratio by Job plot analysis. As shown in Figure 1a, the Job plot at 0 °C showed the minimal change of Δ%absorbance at 0.7 of the T/(T+A) molar ratio. The result showed the 2:1 stoichiometry of T:A for the optimal complexation and suggested the formation of a triplex with oligothymine 2c and oligoadenine (dA)20.8 The subsequent analysis of melting curve also confirmed the triplex formation: As can be seen in the representative melting curve for 2c and (dA)20 in Figure 1b, two-phase transitions in the melting curve were detected.9 The sigmoid curve showed two melting points (Tm) at 6 °C and 17 °C, respectively.10 Considering the Job plot data, we ascribed the first transition to the triplex-to-duplex transition and the second transition to the duplex-to-single strand transition.11 These Tm values are very high for pentameric oligonucleotides, as the Tm of a natural duplex with (dT)5 can be expected around –33 °C.12 The neutral TLDNA 1c did not show the melting curve at accessible temperatures, indicating that Tm of neutral 1c may be below the freezing point of the solvent.13 We also confirmed the helical arrangement of the three strands with the CD spectrum (Figure 1c).

Finally, we examined the mercury-mediated self-duplex formation of TLDNA+ 2c. We previously found that two molecules of the neutral oligothymine TLDNA form a duplex with an electron transporting ability on a solid surface through N-Hg-N bond formation with thymine bases.2 Similarly, upon mixing with Hg(ClO4)2•nH2O in water/acetonitrile solution, the positively charged oligothymine TLDNA+ 2c formed the mercury-mediated duplex 3 spontaneously. The duplex 3 was detected as the corresponding hydrated species by ESI MS spectrum, and the helical arrangement was confirmed by CD spectrum (Figure 2, S5, S6).2

In summary, we developed a post-modification method of TLDNA for the positively charged variants, TLDNA+. The method is concise enough to be exerted with various triazole derivatives from the click chemistry.14 The positive charges of the new DNA analogue successfully improved the water solubility and, moreover, binding affinity toward negatively charged natural DNA. The triplex formation with natural DNA may also lead to the development of new functional congeners.11,15 A mix sequence of TLDNA+ and a chimeric combination of TLDNA/TLDNA+, for instance, through solid-phase and convergent synthesis are another interesting substrates for biological or materials applications in future.

ACKNOWLEDGEMENTS
We thank Prof. N. Teramae (Tohoku University) for the CD instrument. This work was partly supported by KAKENHI (Nos. 20108015, 23550041 and 24241036).

References

1. (a) H. Isobe, T. Fujino, N. Yamazaki, M. Guillot-Nieckowski, and E. Nakamura, Org. Lett., 2008, 10, 3729; CrossRef (b) T. Fujino, N. Yamazaki, and H. Isobe, Tetrahedron Lett., 2009, 50, 4101; CrossRef (c) T. Fujino, N. Tsunaka, M. Guillot-Nieckowski, W. Nakanishi, T. Iwamoto, E. Nakamura, and H. Isobe, Tetrahedron Lett., 2010, 51, 2036; CrossRef (d) T. Fujino, N. Yamazaki, A. Hasome, K. Endo, and H. Isobe, Tetrahedron Lett., 2012, 53, 868. CrossRef
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5. The yield of tetramer 2b was lower than other cases, because the compound was amphiphilic and therefore dissolved slightly in organic solvents used for precipitation.
6. The saturated solution was prepared as follows: The compound was immersed in SSPE buffer at 50 °C and stirred for 3 h. After gradually cooling the solution to ambient temperature, precipitates were removed by filtration with membrane filter (pore size 200 nm) to afford the saturated solution.
7. We do not understand the detailed mechanism of the condition-dependency of the melting curves at this stage. However, similar effects were observed with cationic guanidium oligonucleotides, which may suggest that the stronger electrostatic interactions with natural strands may affect the helix formation. See for instance: (a) R. O. Dempcy, K. A. Browne, and T. C. Bruice, Proc. Natl. Acad. Sci. USA, 1995, 92, 6097; CrossRef (b) A. Blaskó, R. O. Dempcy, E. E. Minyat, and T. C. Bruice, J. Am. Chem. Soc., 1996, 118, 7892. CrossRef
8. No triplex formation has been detected with electroneutral TLDNA under identical conditions. The electronic complementarity between natural DNA and TLDNA+ may have facilitated the triplex formation. Similar effects were observed in the neutral thiourea-linked oligonucleotide and cationic S-methylthiourea-linked oligonucleotide. See: D. P. Arya and T. C. Bruice, Bioorg. Med. Chem. Lett., 2000, 10, 691. CrossRef
9. The hyperchromicity of TLDNA upon melting tends to be smaller than that of natural DNA, which has been also observed with the duplex of electroneutral congeners (ref. 1).
10. The Tm values were obtained by calculation of inflection points of the melting curve using a melting program (VWTP-780) from JASCO.
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12. We calculated the theoretical Tm value of (dT)5•(dA)5 duplex with the following equation: ΔH/{ΔS + 1.987 ln(Ct/4)} – 273.15 + 12.5 log[Na+] where Ct is the total strand concentration. See: J. SantaLucia, Jr., H. T. Allawi, and P. A. Seneviratne, Biochemistry, 1996, 35, 3555. CrossRef
13. Electroneutral 10-mer TLDNA recorded the Tm value of 61.1 °C (ref. 1).
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