Nucleobase-caged peptide nucleic acids: PNA/ PNA duplex destabilization and light-triggered PNA/PNA recognition Samit Guha, Julia Graf, Björn Göricke and Ulf Diederichsen* The 2-(o-nitrophenyl)-propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N-(2-aminoethyl)glycine peptide nucleic acids (aeg-PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc-a NPP -aeg-OH and Fmoc-c NPP -aeg-OH were synthesized and incorporated into PNA sequences by Fmoc solid phase synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc-cleavage, coupling, capping, and resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV-LED irradiation at 365 nm. The nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both strands. PNA/PNA recognition can be completely restored by UV light-triggered release of the photolabile protecting group. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: caging group; double strand recognition; nucleobase pairing; peptide nucleic acid (PNA); photolabile protecting group Introduction Caging with photolabile protecting groups involves the modification of biologically active molecules to temporarily block and inactivate functionalities [1,2]. The activity of biomolecules is masked by intro- duction of caging groups at key positions allowing to restore the function externally triggered by irradiation [3]. Various caging groups with high selectivity and orthogonality are addressed providing cleavage under mild conditions [4]. The caging group strategy can be applied to various biomolecules such as ATP [1], amino acids [5], proteins [6], nucleic acids [7,8], sugars [9], lipids [10] and as a temporary photolabile N-terminal protecting group for solid phase synthesis for N-(2-aminoethyl)glycine peptide nucleic acid (aeg-PNA) [11]. An azobenzene photoswitch was further incorpo- rated in aeg-PNA in order to gain photocontrol over DNA and RNA hybridization [12]. However, nucleobase caging groups are not applied so far for the modulation of aeg-PNA double strand stabilities. Caging of aeg-PNA is of high relevance because aeg-PNA is well-established as DNA and RNA structural mimic nicely resembling oligonucleotide pairing complexes with high duplex stabilities and resistance toward enzymatic degradation [13,14]. Despite numerous applications known for the use of aeg-PNA [15] that will benefit from the availability of caged PNA, our need for a light-triggered modulation of PNA/PNA duplex formation was derived from a recently reported application of aeg-PNA as recog- nition unit in artificial SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein-like peptide-PNA hybrids for membrane fusion [16]. The approximation of lipid vesicles is initiated by PNA/PNA duplex formation, thereby, starting the membrane fusion process. In order to test the hypothesis for SNARE-mediated membrane fusion following a zipper-like recogni- tion process resulting in a tetrameric helical peptide bundle, aeg-PNA oligomers were envisioned introducing sterical hindrance for nucleobase recognition at one end of the forming duplex. The synthesis of adenine and cytosine-caged nucleo amino acids Fmoc-a NPP -aeg-OH (1) and Fmoc-c NPP -aeg-OH (2) is reported using the 2-(o-nitrophenyl)-propyl (NPP) caging group as introduced by Pfleiderer [17]. Incorporation of the caged nucleo amino acids in PNA oligomers by spss provided the opportunity to study the modulation of PNA/PNA double strand stability based on number and positioning of caging groups. A stepwise reduction of stability by ΔT m =5–3  C for each caging group up to complete inhibition of duplex formation was obtained. Complete recovery of PNA/PNA duplex formation was induced by photocleavage. 2-(o-Nitrophenyl)-propyl was chosen as caging group because it is compatible with the cleavage conditions in solid phase pep- tide synthesis and it can be cleaved by irradiation at 365 nm yielding a-methyl nitrostyrene as by-product that is less harmful as, for example, nitrosoaldehyde resulting from ortho-nitrobenzyl caging groups and respective derivatives [17–20]. With respect to positioning, the NPP group at the nucleobase, Heckel reported NPP-caged thymidine at the O 4 -position [21] and the O 6 -caged guanosine [22]. Furthermore, he introduced the NPP caging group in nucleotides at the N 6 -position of adenine and the N 4 -position of cytosine [23]. In a first attempt, we decided to prepare the N 6 -adenine and N 4 -cytosine-caged nucleo amino acids Fmoc- a NPP -aeg-OH (1) and Fmoc-c NPP -aeg-OH (2). Caging groups at the exocyclic amino groups are expected to influence the nucleobase pairing for sterical reasons but still allow complete hydrogen bond- ing at the Watson–Crick site. Therefore, N 6 -adenine and N 4 -cytosine * Correspondence to: Ulf Diederichsen, Institut für Organische und Biomolekulare Chemie, Universität Göttingen, Tammannstraße 2, 37077 Göttingen, Germany. E-mail: udieder@gwdg.de Institut für Organische und Biomolekulare Chemie, Universität Göttingen, Tammannstraße 2, 37077 Göttingen, Germany J. Pept. Sci. 2013 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 21 February 2013 Revised: 22 March 2013 Accepted: 24 March 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/psc.2514