A coumarin-modified pyrimidine nucleoside (1) has been synthesized using Cu(I)-catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross-links are produced upon irradiation of ODN containing 1 at 350 nm. Cross-linking occurs via [2+2] cycloaddition reaction with the opposing thymidine, 2′-deoxycytidine, or 2′-deoxyadenosine. A much higher reactivity was observed with dT than dC or dA. Irradiation of the dT-1 and dC-1 cross-linked products at 254 nm leads to a reversible ring-opening reaction, while such phenomenon was not observed with dA-1 adducts. The reversible reaction is ultrafast and complete within 50 s – 90 s. Consistent photoswitching behavior was observed over 6 cycles of irradiation at 350 nm and 254 nm. To the best of our knowledge, this is the first example of photoswitchable interstrand cross-linking formation induced by a modified pyrimidine nucleoside. Compound 1 is a novel tool for developing reversible DNA photoswitches which will lead to new applications in chemistry, biology, and nanotechnology.

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A coumarin-modified pyrimidine nucleoside (1) has been synthesized using a Cu I -catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross-links are produced upon irradiation of ODNs containing 1 at 350 nm. Cross-linking occurs through a [2+2] cycloaddition reaction with the opposing thymidine, 2'-deoxycytidine, or 2'-deoxyadenosine. A much higher reactivity was observed with dT than dC or dA. Irradiation of the dT-1 and dC-1 cross-linked products at 254 nm leads to a reversible ringopening reaction, while such phenomena were not observed with dA-1 adducts. The reversible reaction is ultrafast and complete within 50-90 s. Consistent photoswitching behavior was observed over 6 cycles of irradiation at 350 nm and 254 nm. To the best of our knowledge, this is the first example of photoswitchable interstrand cross-linking formation induced by a modified pyrimidine nucleoside. Figure 4. Reversibility of the DNA interstrand photo-cross-linking by a coumarin-modified ODN duplex 10 over six cycles of 50 min UV irradiation at 350 nm and 6 min at 254 nm.

Previous reports showed that consecutive incorporations of triazole-modified 2′-deoxyuridines stabilized DNA•DNA and DNA•RNA duplexes due to additional πstacking between triazole moieties. In this work, several triazole-modified thymidines (1−3) have been synthesized via Cu-catalyzed azide−alkyne cycloaddition reaction and incorporated into oligodeoxyribonucleotides (ODNs). Compounds 1−3 decreased the stability of DNA•DNA and DNA• RNA duplexes. Among these triazole-modified thymidines, 3 with a coumarin-modified triazole moiety showed the most destabilizing effect on ODN duplexes while 1 with an unsubstituted triazole moiety showed the least destabilizing effect. These data suggested that a bulky substituent at the 4 position of triazole ring further destabilizes DNA duplex possibly due to steric hindrance interfering Watson−Crick base pairing. The destabilizing effect per modified nucleoside is smaller for two or three consecutive incorporations than single incorporation. Density functional theory computation suggested the presence of π-stacking interactions among the modified triazole moieties at the C5-methyl group of thymine, which may slightly stabilize the DNA duplex. However, due to the noncoplanar conformation between the modified triazole groups and thymine moieties, the destabilizing effect caused by the steric hindrance is larger than the stabilizing effect arisen from π-stacking. Although the triazole moieties slightly alter the pK a values of thymidine, they did not affect the pH dependence of DNA duplex stability. Similar to native ODNs, the stability of the DNA duplexes containing 1−3 decreased in the following order: pH 7.0/ pH 8.0 > pH 6.0 > pH 9.0 > pH 5.0. Our study provides additional insights into and a novel guide for duplexes' thermal stability caused by base modifications.