Most photoinduced DNA cross-link formation by a bifunctional aryl derivative is through a bisquinone methide. DNA cross-linking via a bisarylcarbocation remains a less explored area. We designed and synthesized a series of naphthalene boronates that produce DNA interstrand cross-links via a carbocation upon UV irradiation. A free radical was generated from the naphthalene boronates with 350 nm irradiation and further converted to a carbocation by electron transfer. The activation mechanism was determined using the orthogonal traps, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and methoxyamine that react with either the free radical or the carbocation but not both. This represents a novel example of photoinduced DNA cross-link formation via carbocations generated from a bisaryl derivative. This work provides information useful for the design of novel photoactivated DNA cross-linking agents.

Three novel H₂O₂-activated aromatic nitrogen mustard prodrugs (6–8) are reported. These compounds contain a DNA alkylating agent connected to a H₂O₂-responsive trigger by different electron-withdrawing linkers so that they are inactive towards DNA but can be triggered by H₂O₂ to release active species. The activity and selectivity of these compounds towards DNA were investigated by measuring DNA interstrand cross-link (ICL) formation in the presence or absence of H₂O₂. An electron-withdrawing linker unit, such as a quaternary ammonia salt (6), a carboxyamide (7), and a carbonate group (8), is sufficient to deactivate the aromatic nitrogen mustard resulting in less than 1.5 % cross-linking formation. However, H₂O₂ can restore the activity of the effectors by converting a withdrawing group to a donating group, therefore increasing the cross-linking efficiency (>20 %). The stability and reaction sites of the ICL products were determined, which revealed that alkylation induced by 7 and 8 not only occurred at the purine sites but also at the pyrimidine site. For the first time, we isolated and characterized the monomer adducts formed between the canonical nucleosides and the aromatic nitrogen mustard (15) which supported that nitrogen mustards reacted with dG, dA, and dC. The activation mechanism was studied by NMR spectroscopic analysis. An in vitro cytotoxicity assay demonstrated that compound 7 with a carboxyamide linker dramatically inhibited the growth of various cancer cells with a GI₅₀ of less than 1 μM, whereas compound 6 with a charged linker did not show any obvious toxicity in all cell lines tested. These data indicated that a neutral carboxyamide linker is preferable for developing nitrogen mustard prodrugs. Our results showed that 7 is a potent anticancer prodrug that can serve as a model compound for further development. We believe these novel aromatic nitrogen mustards will inspire further and effective applications.

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 ring-opening 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.

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 ring-opening 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.

A series of arylboronic esters containing different aromatic substituents and various benzylic leaving groups (Br or N⁺Me₃Br⁻) have been synthesized. The substituent effects on their reactivity with H₂O₂ and formation of quinone methide (QM) have been investigated. NMR spectroscopy and ethyl vinyl ether (EVE) trapping experiments were used to determine the reaction mechanism and QM formation, respectively. QMs were not generated during oxidative cleavage of the boronic esters but by subsequent transformation of the phenol products under physiological conditions. The oxidative deboronation is facilitated by electron-withdrawing substituents, such as aromatic F, NO₂, or benzylic N⁺Me₃Br⁻, whereas electron-donating substituents or a better leaving group favor QM generation. Compounds containing an aromatic CH₃ or OMe group, or a good leaving group (Br), efficiently generate QMs under physiological conditions. Finally, a quantitative relationship between the structure and activity has been established for the arylboronic esters by using a Hammett plot. The reactivity of the arylboronic acids/esters and the inhibition or facilitation of QM formation can now be predictably adjusted. This adjustment is important as some applications may benefit and others may be limited by QM generation.

A highly efficient chemical ligation was developed for quantitative conjugation of PNA with DNA (PNA or peptide) by using the copper-catalyzed azide–alkyne cycloaddition reaction. Whereas PNAs with an alkyne at the C-terminus and an azide at the N-terminus have been used, an efficient click–click reaction occurs. The PNA click ligation is sequence specific and capable of single nucleotide discrimination.