Singlet Oxygen DOI: 10.1002/anie.201003612 Reversible pH-Regulated Control of Photosensitized Singlet Oxygen Production Using a DNA i-Motif** Thomas Tørring, Rasmus Toftegaard, Jacob Arnbjerg, Peter R. Ogilby,* and Kurt V. Gothelf* Singlet molecular oxygen ( 1 O 2 ) is a reactive intermediate that is important in fields that range from materials science to medicine. [1] Singlet oxygen has a characteristic chemistry in which molecules are oxygenated that sets it apart from the ground triplet state of oxygen ( 3 O 2 ). These oxygenation reactions can be important in processes that include polymer degradation and cell death. Singlet oxygen is commonly and conveniently produced by photosensitization. [2] In this process, light is used to create an excited state of a given molecule, the photosensitizer, which in turn transfers its energy of excitation to 3 O 2 to generate 1 O 2 . The judicious use of light and sensitizers facilitates a great deal of control in the production of 1 O 2 , [3] which can then be applied, for example, in the selective killing of undesired cells (e.g., the medical procedure of photo- dynamic therapy, PDT, whereby cancerous tissue can be removed [4] ). Even though significant progress has been made in the development and use of biologically relevant 1 O 2 sensitizers, the available systems are still often indiscriminate, which in turn limits their usefulness. Thus, the development of sensitizer systems that provide more control and selectivity over the photosensitized production of 1 O 2 has received increasing attention. [1, 3, 5] In particular, systems that allow reversible switching of the photosensitizer between “on” and “off” configurations have gained interest. A convenient method to control the ability of a photo- sensitizer to produce 1 O 2 is to alter the efficiency with which energy can be transferred from the excited state of the sensitizer to 3 O 2 . This principle has been demonstrated by selectively placing the 1 O 2 sensitizer close to a molecule that can quench the excited state of the sensitizer by using a positioning system that can then be manipulated to change the distance between the sensitizer and the quencher. Use of positioning systems based on cleavable peptides [6, 7] or dynamic DNA structures [8, 9] has resulted in appreciable control in the production of 1 O 2 . Moreover, this approach has been successfully applied in vivo. [9] In 2005, OShea and co-workers reported a related approach by which a change in pH could be used to control photosensitized 1 O 2 production with a sensitizer closely linked to a pH-responsive amine. [10] At high pH values, the excited state of the sensitizer was quenched by electron transfer from the amine, whereas at low pH values, protonation of the lone pair of electrons on the amine precluded quenching by electron-transfer, thereby allowing the photosensitized pro- duction of 1 O 2 . The amount of 1 O 2 produced varied by a factor of 10.6 upon changing from high to low pH values. [10] We report herein a new approach to reversibly control photosensitized 1 O 2 production by using a DNA i-motif as a pH-sensitive regulator. The i-motif is a four-stranded DNA structure formed from sequences that contain stretches of cytosine (C) residues. [11] The structure is stable at slightly acidic pH values, where the Cs are partially protonated and form a quadruplex structure of interdigitated C–CH(+) base pairs. Under alkaline conditions, however, the Cs are fully deprotonated and the i-motif is no longer stable, thus leading to denaturation and stretching of the DNA sequence. The i-motifs have been explored intensively as nanoscale switch- ing devices. [12] In our current design, a 1 O 2 sensitizer and a quencher of the sensitizer are covalently linked to each end of a DNA sequence that contains an i-motif. The principle of the pH- regulated 1 O 2 sensitizer/quencher/i-motif (SQI) is illustrated in Figure 1 a. Under acidic conditions (pH < 5) the i-motif quadruplex is stable and holds the sensitizer and the quencher in close proximity. Upon irradiation, the fluorescent state of the sensitizer is efficiently deactivated by the adjacent quencher, thereby precluding the ultimate formation of the triplet state of the sensitizer which is the immediate precursor to 1 O 2 . As the pH is raised above 5, the C residues are deprotonated and the i-motif is no longer stable. In turn, the increased distance between the sensitizer and the quencher has the consequence that the sensitizer is no longer immedi- ately deactivated and 1 O 2 production can take place. To more efficiently separate the sensitizer and the quencher in the open state, a complementary oligonucleotide strand can be added to form a rigid DNA helix between the two moieties (Figure 1a). At low pH values, the formation of the i-motif will efficiently compete with the formation of the DNA hybrid, thus providing the basis for an effective pH-respon- sive switch. The SQI conjugate was generated by synthesizing a phosphoramidite analogue of the 1 O 2 sensitizer pyropheo- [*] T. Tørring, Prof. K.V. Gothelf Danish National Research Foundation: Center for DNA Nanotechnology, Department of Chemistry and iNANO Langelandsgade 140, 8000 Aarhus C (Denmark) E-mail: kvg@chem.au.dk Homepage: http://www.cdna.dk R. Toftegaard, Dr. J. Arnbjerg, Prof. P. R. Ogilby Danish National Research Foundation: Center for Oxygen Microscopy and Imaging, Department of Chemistry Aarhus University, Langelandsgade 140, 8000 Aarhus C (Denmark) E-mail: progilby@chem.au.dk Homepage: http://www.chem.au.dk/ ~ comi/ [**] The work was supported by the Danish National Research Foundation. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201003612. A ngewandte Chemi e 7923 Angew. Chem. Int. Ed. 2010, 49, 7923 –7925  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim