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Bio-organic Chemistry Laboratory, Depart

Technology Guwahati, Guwahati-781039, A

in; Fax: +91-361-258-2349

† Dedicated to Professor Isao Saito on the

‡ Electronic supplementary information (data, macromodel study. See DOI: 10.103

Cite this: DOI: 10.1039/c3ra44120b

Received 2nd August 2013Accepted 23rd August 2013

DOI: 10.1039/c3ra44120b

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Unnatural triazolyl nucleoside stabilizes an abasic sitecontaining DNA duplex equally as the stabilization of anatural A–T pair†‡

Subhendu Sekhar Bag,* Rajen Kundu and Sangita Talukdar

A better than reported abasic duplex stabilization was achievedwith

the novel triazolylphenanthrene nucleoside TPhenBDo. The large

surface area, polarizability and strong stacking propensity of tri-

azolylphenanthrene play a major role in the thermal stabilization of

the TPhenBDo–F duplex comparable to that of a natural A–T pair via a

strong intercalative stacking interaction.

An abasic site in genomic DNA is among the most commonforms of DNA lesions.1 Due to a lack of coding information, ifle unrepaired by the base excision repair (BER) machinery, anabasic site (Ap) can lead to deleterious mutations that aredangerous to cellular survival and replication.1 Generally, an Ap(F) site destabilizes DNA locally, thereby providing a specialaffinity for repair enzymes to repair it.1,2 Therefore, stabilizingan abasic site is important for the design of new diagnostics andchemotherapeutics. The design of molecules that stabilizeabasic sites is therefore a growing research area.

In an effort to unfold the mechanism by which DNA repairenzymes recognize and distinguish abasic DNA from normalDNA, several research efforts have been put forth to recognise/stabilize abasic DNA via the design of small molecule inter-callators,3 oligonucleotide (ODN) probes containing non-nucleosidic building blocks4 and/or modied nucleosides5 asnucleobase surrogates. While all the reported non-nucleosidebase surrogates4 led to a thermal stabilization of 0–8 �C, probescontaining modied nucleosides5 were found to stabilize anabasic site to a much greater extent than those of the corre-sponding natural duplexes with an adenine–abasic pair.However, these nucleoside/non-nucleoside base surrogatesstabilize abasic duplexes but are less stable than the natural A–T

ment of Chemistry, Indian Institute of

ssam, India. E-mail: ssbag75@iitg.ernet.

occasion of his 72nd birthday.

ESI) available: Synthesis, spectroscopic9/c3ra44120b

Chemistry 2013

pair.4,5 As an example, ODNs containing pyrenyl deoxyribosideoffer the highest reported thermal stability of an abasic DNA,the stability is slightly lower (by 1.6–2.2 �C) than a natural A–Tpair.5a,g

As a part of our ongoing research efforts towards the designof unnatural nucleosides with tuned photophysical properties,we have recently observed that the click chemistry generatedtriazolyl donor/acceptor aromatic chromospheres containingunnatural nucleoside bases (TPhenBDo and TNBBAc) produce aself-pair and a hetero-pair with a promising stability that iscomparable to the stability of a control A–T pair.6 The higheststability in self-pairing is most likely a result of the large size oftriazolylphenanthrene which helps in the slipping past of oneanother leading to a strong stacking interaction.

Inspired by the large surface area (248 A2 against natural A–Tpair surface area 273 A2, calculated from macromodel),7 highpolarizability, high stacking propensity (higher than adeni-ne)5a,8 and our recent observation on the strong self-pair/hetero-pair stabilization,6 we thought that it would be worthwhile tostudy abasic DNA stabilization using our unnatural tri-azolylphenanthrene nucleoside TPhenBDo (1, Fig. 1a). Therefore,we have synthesized TPhenBDo nucleoside following a modiedprocedure of our recently published protocol.6 Previously, wehave used TMS-N3 which produced bis-toluolyl protectedb-azidonucleoside 2 in a very low yield starting from Hoffer's

Fig. 1 (a) Structure of the unnatural triazolyl nucleoside (TB ¼ TPhenBDo) andmodel abasic site (F). (b) Schematic presentation of possible intercalative p-stacking interaction at the abasic site.

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Scheme 1 Synthesis of TPhenBDo nucleoside and the oligonucleotides. Reagentsand conditions: (a) CsN3, DMSO; (b) column separation; (c) 9-phenanthreneacetylene, CuSO4, Na-ascorbate, DIPEA, 80 �C, 12 h; (d) NaOMe–MeOH, r.t. 12 h;(e) DMTrCl, DMAP, pyridine, 12 h; (f) (i-Pr2N)2PO(CH2)2CN, DIEA, DCM, rt, 1.5 h; (g)DNA synthesis.

Table 2 Tm values and thermodynamic parameters for several duplexesa

ODNs X:Y Tm DTm �DG037 �DH0 �DS0

1$2 TPhenBDo:F 52.2 +1.0 13.4 129.1 373.19$2 A:F 37.2 �14.0 7.5 111.7 336.310$2 G:F 37.5 �13.7 7.7 97.5 289.711$2 C:F 35.1 �16.1 6.9 90.5 269.612$2 T:F 35.8 �15.4 7.4 101.6 303.98$7 F:TPhenBDo 49.0 �2.2 12.0 125.8 367.38$3 F:A 35.6 �15.6 7.3 81.9 240.98$4 F:G 35.7 �15.5 7.2 87.3 258.58$5 F:C 33.4 �17.8 6.8 63.4 182.68$6 F:T 33.5 �17.7 6.7 75.9 223.39$6 A:T 51.2 — 12.9 117.4 337.11$7 TPhenBDo:

TPhenBDo 53.6 +2.4 12.8 107.4 305.0

a All samples contained 2.5 mM of each strand of DNA in 50 mM sodiumphosphate, 100 mM sodium chloride, 0.1 mM EDTA, at pH 7.0, roomtemperature. Units of DG and DH are in kcal mol�1, while DS is in calK�1 mol�1. The error in Tm is estimated at �0.3 �C and in free energy�3%. DTm ¼ difference in Tm compared to the corresponding naturalA:T pair.

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chlorosugar (21%).6 However, in this present protocol9 CsN3wasused to react with Hoffer's chlorosugar 3 to afford a 1 : 10mixture of a- and b-anomers from which the b-anomer 2 wasisolated in pure form by column chromatography in 90% yield(Scheme 1). b-Azide 2, upon the click reaction with 9-phe-nanthryl acetylene followed by deprotection, produced thedesired triazolylphenanthrene nucleoside TPhenBDo(1) in excel-lent yield (Scheme 1). The nucleoside 1 was then incorporatedinto short oligonucleotide sequences (ODN 1, 7) followingphosphoramidite chemistry via an automated DNA/RNAsynthesizer (ESI,‡ Section 1).

Two complementary 13-mer oligonucleotides (ODN 1, 7)containing TPhenBDo at the central position of the strands weresynthesized. The abasic site containing ODNs (2, 8) and allpossible natural ODNs (3–6; 9–12) were purchased. The twosequence context was chosen to study the effects of the ankingbases (T base for X and A base for Y) on the abasic duplexstability (Table 1). The stabilities in aqueous buffer of all theduplexes containing the TBs–F pairs were then evaluated withthermal denaturation studies (Table 2). To compare duplexstability and pairing selectivity, ODN 1–2, 7–8 were also pairedagainst the four natural bases, and the thermal stability of thenatural A–T pair in the same central position (ODN 9$6) was alsoexamined.

From the thermalmelting temperature it is evident that all theduplexes containing unnatural nucleoside (TB:F or F:TB) pairedwith an abasic site aremore stable than the duplexes between anyof the natural bases and the abasic site X (¼ A, G, C, T):F orF:Y(¼ A, G, C, T) in both the sequence contexts. The natural A–T pairduplex (ODN 9$6), has a Tm of 51.2 �C. A strong destabilization of14.0 �C in Tm is the result when A is paired against an abasic site(9$2, A:F) which is possibly due to the disruption of continuousstacking in the helix at the abasic site.5a However, the

Table 1 Oligonucleotide sequences containing a TPhenBDo nucleoside/abasic site (

ODNs Sequences

1 50-CGCAAT TPhenBDo TAACGC-30

2 50-GCGTTA F ATTGCG-30

3 50-GCGTTA A A TTGCG-30

4 50-GCGTTA G A TTGCG-30

5 50-GCGTTA C A TTGCG-30

6 50-GCGTTA T A TTGCG-30

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triazolylphenanthrene nucleobase stabilizes the abasic site wellleading to thermal stabilization of the duplexes (TB:F orF:TB) by13.4–15 �C higher than that of the duplexes A:F and/or F:A(Table 2). Furthermore, when the triazolylphenanthrene nucleo-side 1 is paired opposite the abasic site, the duplex formed (1$2,TPhenBDo:F) is found to be equally as stable as the control A–Tpair (DTm ¼ +1.0 �C and DG ¼ +0.5 kcal mol�1).

It is also evident that TPhenBDo shows signicant selectivity(by 0.7–1.3 kcal mol�1 and 0.8–3.7 �C in Tm higher stability) forthe abasic site over all four natural bases (Table 2 and ESI,‡Section 10). The model abasic nucleoside 2 also shows a strongpreference for pairing with TPhenBDo over natural bases (by a14.7–17.1 �C increase in Tm and a gain of 5.7–6.5 kcal mol�1 instability). Interestingly, the self-pair duplex formed by TPhenBDo

(1$7, TPhenBDo:TPhenBDo) is found to be 2.4 �C more stable than a

control A–T pair. Moreover, TPhenBDo offers better thermalstabilization (by �2.6–3.2 �C) of the abasic duplex than thehighest reported stabilization provided by unnatural nucleosidebase surrogates.5a,g To the best of our knowledge this is thestrongest stabilization of an abasic duplex achieved by ourdesigned nucleoside, TPhenBDo.

An examination of the role of anking bases on the stabili-zation of an abasic site reveals that the anking –A– bases lead

F) and their natural complements

ODNs Sequences

7 50-GCGTTA TPhenBDo ATTGCG-30

8 50-CGCAAT F TAACGC-30

9 50-CGCAAT A TAACGC-30

10 50-CGCAAT G TAACGC-30

11 50-CGCAAT C TAACGC-30

12 50-CGCAAT T TAACGC-30

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Fig. 2 (a) UV-visible spectra of single strand ODN 1 containing TPhenBDo and theabasic duplex TPhenBDo:F (ODN 1$2). (b) CD spectra of the shown abasicduplexes.

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to higher duplex stability compared to anking –T– bases irre-spective of the natural or unnatural bases opposite the abasicsite (Table 2). This is probably because of the fact that the largersurface area possessed by the purine neighbor –A– tends toclose the gap in the abasic site by stacking more than that of thepyrimidine neighbor.10 On the other hand, the nucleosideTPhenBDo in general, when anked between –T– bases shows ahigher duplex stability compared to the stability of the duplexeswhen anked between –A– bases in the case of mis-pairing withany natural bases (ESI,‡ Section 10).6 Therefore a cooperativeeffect of anking bases is reected in the higher stabilization ofthe duplex TPhenBDo:F (by 3.2 �C) than the F:TPhenBDo duplexwhich is only slightly less stable (by 2.2 �C) compared to acontrol A–T pair (Table 2). However, in the reverse sequence(F:TPhenBDo) the stabilization of the abasic DNA is also high butcomparable to that offered by reported pyrene nucleosides.5a,g

These observations are remarkable especially considering thefact that the large surface area of triazolylphenanthrene rendersit able to offer both the inter- and intra-strand stacking inter-actions within the nearby natural bases in the duplex at theabasic site; thus covering the full space leading to a higherabasic duplex stabilization than any other reported bases.4,5

The van't Hoff analyses of the thermal denaturation curvesfor all the duplexes reveal that the abasic site stabilization by theunnatural TPhenBDo nucleoside (TPhenBDo:F and F:TPhenBDo) isdriven by a more favourable enthalpy (higher DDH by 8.4–11.7kcal mol�1) change compared to a natural A–T pair (Table 2, andESI,‡ Section 9). Also the process of coil to helix formation isaccompanied by a slightly higher and comparable change infree energy for the TPhenBDo:F and F:TPhenBDo pairs. This isprobably because of the comparable surface area that leads to aproper geometric t and stacking with the neighboring bases atthe abasic site.

These results show that the intercalative stacking interactionwith DNA bases plays a dominant role in stabilizing a duplexcontaining an abasic site paired with the hydrophobic TPhenBDo

unnatural base. The strong pairing selectivity between TPhenBDo

and the abasic nucleoside (compared to the natural bases) andequal stabilization of the TPhenBDo:F duplex to that of a controlA–T pair can be rationalized through a best geometric t, highpolarizability, large size and strong stacking ability of the tri-azolylphenanthrene moiety of the TPhenBDo nucleoside.5a,g,8

The UV-visible spectra of both the duplexes clearly show abathochromic shi (of�9–10 nm) along with a 27–28% increasein hypochromicity in comparison to the corresponding singlestranded ODNs containing TPhenBDo in both the sequencecontexts (Fig. 2a and ESI,‡ Sections 5 and 6) indicating a strongintercalative stacking interaction between the triazolylphenan-threne and DNA base pairs at the abasic site.11 The uorescencelife time experiments show the single exponential decay of theTPhenBDo nucleoside in both the abasic duplexes. A higher decaytime is observed in the duplexes compared to their singlestranded form. This result suggests there is only one mode ofinteraction, possibly intercalation, of TPhenBDo with the neigh-boring base pairs at the abasic site. Steady state uorescenceanisotropy of triazolylphenanthrene in the duplexes ODN 1$2(TPhenBDo:F) and ODN 8$7 (F:TPhenBDo) shows anisotropy values

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three times and two times higher, respectively, compared totheir corresponding single strands (ODN 1 or ODN 7) indicatingthat the triazolylphenanthrene is more strongly stacked,possibly via the intercalation between the base pairs in theduplexes (ESI,‡ Sections 7 and 8). This result is also consistentwith the observations from the UV-visible spectral analyses.12

CD spectral analysis of the duplexes (TPhenBDo:F or TPhenB-

Do:F) reveals the presence of negative induced CD signals forboth the duplexes at �307 nm corresponding to the chromo-phores' absorption band supporting a possible intercalativestacking interaction operative in the duplex containing theabasic site paired with the unnatural nucleoside, TPhenBDo

(Fig. 2b).4c,5g,12c The slight difference in the relative intensity ofthese bands in the two sequence context is indicative of a subtlechange in the intercalation site or a shi of the chromophore atthe abasic site that is also supported by a macromodel study.12c

The Amber* minimized structures of the abasic duplexespaired against TPhenBDo show an intrahelical conformation ofboth the TPhenBDo and abasic site wherein the TPhenBDo reachesdeeper into the interior of the helix at the abasic site enablingan optimal positioning for stacking interactions.5d,7 Boththe inter-/intra-strand stacking occurs more in the duplexTPhenBDo:F compared to its reverse sequence resulting in acomparatively smaller gap which is reected in a space llingmodel indicating a more mobile nature of the reverse sequence(F:TPhenBDo) which is probably the cause of less stabilitycompared to the normal sequence (ESI‡).

In summary, the high stabilization of the TPhenBDo:F duplex,which is comparable to that of a natural A–T pair, represents aremarkable improvement in the stability and selectivity overpreviously reported non-hydrogen-bonded base pairs. The largesurface area, polarizability and strong stacking propensity play amajor role in offering high duplex stabilization via a strongintercalative stacking interaction which is evident from highduplex stability, UV-visible spectroscopy, increased uorescenceanisotropy and negative induced CD signal analysis. Currently, weare incorporating multiples of such nucleosides decorated withdonor/acceptor chromophores to make helical arrays which maynd applications in the study of DNA-mediated charge transfer.

This work was nancially supported by the Department ofScience and Technology [DST: SR/SI/OC-69/2008], Govt.of India.

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Notes and references

1 (a) G. Vesnaver, C.-N. Chang, M. Eisenberg, A. P. Grollmanand K. J. Breslauer, Proc. Natl. Acad. Sci. U. S. A., 1989, 86,3614–3618; (b) O. D. Scharer, Angew. Chem., Int. Ed., 2003,42, 2946.

2 (a) S. T. Hoehn, C. J. Turner and J. Stubbe, Nucleic Acids Res.,2001, 29, 3413; (b) J. Chen, F.-Y. Dupradeau, D. A. Case,C. J. Turner and J. Stubbe, Nucleic Acids Res., 2008, 36, 253.

3 (a) N. Berthet, J. F. Constant, M. Demeunynck, P. Michonand J. Lhomme, J. Med. Chem., 1997, 40, 3346; (b)K. Yoshimoto, S. Nishizawa, M. Minagawa and N. Teramae,J. Am. Chem. Soc., 2003, 125, 8982.

4 (a) S. M. Langenegger and R. Haner, ChemBioChem, 2005, 6,848; (b) S. M. Langenegger and R. Haner, Chem. Biodiversity,2004, 1, 259; (c) V. L. Malinovskii, D. Wenger and R. Haner,Chem. Soc. Rev., 2010, 39, 410.

5 (a) T. J. Matray and E. T. Kool, J. Am. Chem. Soc., 1998, 120,6191; (b) E. T. Kool, J. C. Morales and K. M. Guckian, Angew.Chem., Int. Ed., 2000, 39, 990; (c) S. Smirnov, T. J. Matray,E. T. Kool and C. los Santos, Nucleic Acids Res., 2002, 30,5561; (d) C. Brotschi, G. Mathis and C. J. Leumann, Chem.–Eur. J., 2005, 11, 1911; (e) C. Verhagen, T. Bryld,M. Raunkær, S. Vogel, K. Buchalova and J. Wengel, Eur. J.Org. Chem., 2006, 2538; (f) S. Nakano, Y. Uotani, K. Uenishi,M. Fujii and N. Sugimoto, Nucleic Acids Res., 2005, 33, 7111;(g) F. Wojciechowski, J. Lietard and C. J. Leumann, Org.

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Lett., 2012, 14, 5176; (h) S. Pitsch, S. Wendeborn, B. Jaunand A. Eschenmoser, Helv. Chim. Acta, 1993, 76, 2161.

6 S. S. Bag, S. Talukdar, K. Matsumoto and R. Kundu, J. Org.Chem., 2013, 78, 278.

7 Maestro, version 9.0, Schrodinger, LLC, New York, NY, 2009.8 K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheils,P. L. Paris, D. C. Tahmassebi and E. T. Kool, J. Am. Chem.Soc., 1996, 118, 8182.

9 (a) D.-W. Chen, A. E. Beuscher, IV, R. C. Stevens,P. Wirsching, R. A. Lerner and K. D. Janda, J. Org. Chem.,2001, 66, 1725; (b) A. Stimac and J. Kobe, Carbohydr. Res.,2000, 329, 317; (c) N. A. Kolganova, V. L. Florentiev,A. V. Chudinov, A. S. Zasedatelev and E. N. Timofeev,Chem. Biodiversity, 2011, 8, 568.

10 (a) L. Ayadi, C. Coulombeau and R. Lavery, Biophys. J., 1999,77, 3218; (b) J. Sagi, A. B. Guliaev and B. Singer, Biochemistry,2001, 40, 3859.

11 (a) G. Dougherty and J. R. Pilbrow, Int. J. Biochem., 1984, 16,1179; (b) M. Nakamura, Y. Fukunaga, K. Sasa, Y. Ohtoshi,K. Kanaori, H. Hayashi, H. Nakano and K. Yamana, NucleicAcids Res., 2005, 33, 5887.

12 (a) A. Murakami, M. Nakaura, Y. Nakatsuji, S. Nagahara,Q. Tran-Cong and K. Makino, Nucleic Acids Res., 1991, 19,4097; (b) M. Nakamura, Y. Fukunaga, K. Sasa, Y. Ohtoshi,K. Kanaori, H. Hayashi, H. Nakano and K. Yamana, NucleicAcids Res., 2005, 33, 5887; (c) R. Lyng, A. Rodger andB. Norden, Biopolymers, 1991, 31, 1709.

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