1‐Ethynylpyrene as a Tunable and Versatile Molecular Beacon for DNA

Abstract

Fluorescent or luminescent probes that are sensitive to the local environment within DNA duplexes represent important tools for DNA hybridisation [1] and conformational changes caused by DNA±protein interactions, [1] or for the detection of physiologically important DNA base mismatches or lesions on DNA chips or microarrays.[2] As a consequence, there is a continuously increasing demand for new fluorophores that have a clear and specific range of spectral characteristics which are tunable to distinct excitation or emission wavelengths. One suitable and important way to create new emission properties is to attach chromophores covalently to natural DNA bases. Recently, we applied this modification strategy to the preparation of photoexcitable charge donors, which have been used for the investigation of DNA-mediated electron transport. [3,4] Herein, we report the properties of DNA duplexes bearing the 1-ethynylpyrene moiety (PyÀCC) covalently attached to the bases dX = dA, dC, dG, or dU. Three structural features of these PyÀCCÀdX-modified DNA duplexes are important: i) a clear steric separation of the pyrene moiety from the DNA base stack due to the rigid ethynyl group, ii) a strong electronic coupling between the pyrene and the base moiety provided by the acetylene bridge and iii) a partial stacking of the base moiety as part of the delocalised PyÀCCÀdX chromophore. Moreover, the incorporation of the PyÀCCÀdX moiety could influence only the local conformation, but should not perturb the overall B-DNA duplex conformation.The PyÀCCÀdX-modified oligonucleotides were synthesised by a semiautomated synthetic strategy with solid-phase Sonogashira-type cross-coupling conditions (Scheme 1). [4,5] It is important to note that a time-consuming synthesis of PyÀCCÀ modified phosphoramidites [6] can be avoided because this modification protocol is based on commercially available DNA building blocks. First, the oligonucleotide was synthesised by following standard protocols on a DNA synthesiser up to the position of the PyÀCCÀdX unit. At this position, either 8-bromo-2'-deoxyadenosine, 2'-deoxy-5-iodocytidine, 8-bromo-2'-deoxyguanosine, or 2'-deoxy-5-iodouridine was inserted automatically without the final deprotection of the terminal 5'-OH group. Subsequently, the CPG vials were removed from the synthesiser and a Sonogashira-coupling reagent mixture containing Pd(PPh 3 ) 4 (60 mm), 1-ethynylpyrene (120 mm) and CuI (60 mm) in DMF/Et 3 N (3.5:1.5) was added to the CPG vials under dry conditions with syringes. After a coupling time of 3 h at room temperature, the CPGs were washed with different solvents, dried and attached to the DNA synthesiser to finish the synthesis automatically. Modification of the standard procedures for deprotection and cleavage of the oligonucleotides from the solid phase, or during workup was not necessary. The PyÀCCÀdX-modified oligonucleotides were purified by semipreparative HPLC and identified by MALDI-TOF mass spectrometry. HPLC analysis of the unpurified oligonucleotides showed excellent coupling ...

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

“…It was found in studies of 1-ethynylpyrene-modified uracil incorporated into DNA that the strength of the 405 nm pyrene absorption band can be mainly correlated to the strength of the base-stacking interaction with the neighbouring bases. [43,46] Compared to the DNA systems presented in this reference, the effects monitored in our study are relatively small. A reason for this might be found in the generally lower stability of RNA, which leads to weaker stacking interactions.…”

“…[42] The central emission wavelength of the singly modified duplexes is red-shifted by roughly 35 nm (444-467 nm) with respect to the chromophore, and the fluorescence spectrum is in good accordance with previous studies on similar systems. [32,43,46] These strong changes have been attributed to strong coupling of the dye to the base; this results in an intramolecular exciplex with an at least partial charge transfer from the pyrene to the adenine. [15,[47][48][49][50] The strength of this exciplex coupling is strongly dependent on the attachment site of the pyrene to the base.…”

Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs

“…This is in contrast to the emission of the related 5-pyrenylethynyldeoxycytidine which resembles more closely an isolated pyrene, as previously noted. 47 The benefit to variation of the 6-substituent is shown by improvement of fluorophore brightness, primarily by increasing the extinction coefficient at the longest wavelength absorption band used for excitation. As well, variation of the nature of the heteroaromatic substituent at position 6 produces compounds with varying sensitivity to the polarity of the environment, as judged by the emission Stokes shift and emission intensity.…”

“…47 Spectral characterization of a duplex containing a single 5-pyrenylethynyldeoxycytidine insert was described as pyrene-like exhibiting vibronic structure in the emission (λ emiss. = 408, 430 nm).…”

Structure‐Sensitive and Self‐Assembled Helical Pyrene Array Based on DNA Architecture

“…[20] One suitable and important way to fulfil these requirements is to attach chromophores covalently to natural DNA bases. [4,21] Recently, we introduced 5-(pyren-1-yl)-2'-deoxyuridine (PydU), [22][23][24] 1-ethynylpyrene, [25,26] and other chromophores [27] as fluorescent probes and photoinducable electron donors in DNA. Herein, we describe the properties of DNA duplexes that have been functionalized by a helical stack consisting of five adjacent PydU moieties.…”