A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA Heidi-Kristin Walter1, Bettina Olshausen2, Ute Schepers2 and Hans-Achim Wagenknecht*1 Full Research Paper Address: 1Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany and 2Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), H.-v.-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany Open Access Beilstein J. Org. Chem. 2017, 13, 127–137. doi:10.3762/bjoc.13.16 Received: 17 October 2016 Accepted: 03 January 2017 Published: 20 January 2017 Email: Hans-Achim Wagenknecht* - This article is part of the Thematic Series "Chemical biology". * Corresponding author Guest Editor: H. B. Bode Keywords: dyes; fluorescence; nucleic acid; oligonucleotide © 2017 Walter et al.; licensee Beilstein-Institut. License and terms: see end of document. Abstract The arabino-configured analog of uridine with a propargyl group at the 2’-position was synthesized and incorporated into DNA by solid-phase chemistry. The fluorescence quantum yields of DNA strands that were postsynthetically modified by blue and green emitting cyanine-styryl dyes were improved due to the arabino-configured anchor. These oligonucleotides were used as energy transfer donors in hybrids with oligonucleotides modified with acceptor dyes that emit in the yellow-red range. These combinations give energy transfer pairs with blue–yellow, blue–red and green–red emission color changes. All combinations of arabino- and riboconfigured donor strands with arabino- and ribo-configured acceptor strands were evaluated. This array of doubly modified hybrids was screened by their emission color contrast and fluorescence quantum yield. Especially mixed combinations, that means donor dyes with arabino-configured anchor with acceptor dyes with ribo-configured anchor, and vice versa, showed significantly improved fluorescence properties. Those were successfully applied for fluorescent imaging of DNA after transport into living cells. Introduction The “click”-type reactions [1], in particular the 1,3-dipolar cycloaddition between alkynes and azides (CuAAC) is a broadly applied strategy for postsynthetic oligonucleotide modification since both reactive groups are not present in nucleic acids [2-5]. Although Huisgen described the uncatalyzed reaction yielding 1,2,3-triazoles already in the 1960s [6], the bioorthogonality with respect to proteins and nucleic acids emerged after Sharpless [7] and Meldal [8] had reported that catalysis by Cu(I) enhances not only reaction rates but improves also regioselectivity. The formation of oligonucleotide oxidation side products by Cu(I) is avoided by the use of chelating Cu(I) ligands, in particular tris[(1-benzyl-1H-1,2,3-triazol-4- 127 Beilstein J. Org. Chem. 2017, 13, 127–137. yl)methyl]amine (TBTA) and better water-soluble derivatives [9,10]. The CuAAC cannot only be applied for conventional postsynthetic oligonucleotide modification in solution but also on solid phase [11] and for the introduction of multiple postsynthetic modifications [12]. The azide groups for CuAAC are typically placed onto the fluorescent dyes since azides are not compatible with phosphoramidite chemistry. The alkyne groups as reactive precursors are attached to the oligonucleotide [13], especially at the 5-position of pyrimidines [13], the 7-position of 7-deazapurines [14], and the 2’-position of ribofuranosides [11,15]. These positions were chosen since they are typically accepted by DNA polymerases in primer extension experiments and PCR [4,16]. To develop fluorescently labelled oligonucleotides that undergo energy transfer reactions [17] we recently applied 2’-propargylmodified uridine 1 as DNA building block (Scheme 1) [15,18,19]. A simple look on the three-dimensional structure of double-helical DNA elucidates that the positioning of the fluorophores in the major groove may be improved by inversion of the configuration at the 2’-position of the anchor nucleoside sugar. In fact, arabino nucleic acids are an important class of antisense oligonucleotides [20] since their first report [21]. The orientation of the 2’-OH group in the arabino configuration towards the major groove yields hybrids with RNA that show a slightly lower thermal stability compared to DNA/RNA hybrids. In order to evaluate this structural influence for our fluorescently labelled oligonucleotides, we developed and synthesized the 2’-propargyl-modified arabino-configured uridine analog 2, incorporated it into DNA by automated phosphoramidite chemistry, “clicked” it to a variety of our recently established, photostable cyanine-styryl dyes and probed the fluorescence and energy transfer properties by determination of quantum yields and emission color contrasts. Scheme 1: 2’-Propargylated nucleosides as “clickable” DNA/RNA building blocks with ribo (1) and arabino (2) configuration. Results and Discussion The synthesis of the phosphoramidite 7 (Scheme 2) was straightforward and includes mainly protecting group chemistry since it starts with the commercially available arabino- configured uridine analog 3. The 3’- and 5’-hydroxy functions of nucleoside 3 were selectively protected by the Markiewicz silyl ether [22]. The central step of the whole synthetic procedure was the alkylation of the 2’-OH function of nucleoside 4 by propargylic bromide which worked in 65% yield in the presence of NaH as base. After removal of the silyl protecting group from nucleoside 5, the 5’-position of nucleoside 2 was again protected by 4,4’-dimethoxytrityl chloride (DMTr-Cl) and, finally, the 3’-position of nucleoside 6 was phosphitylated. Remarkably, the overall yield of phosphoramidite 7 with the optimized conditions over the described five steps is 54%. Automated DNA synthesis with 7 as building block required a slightly extended coupling time of 10 min. The phosphoramidite for the “clickable” nucleoside 1 is commercially available. After preparation, the detritylated oligonucleotides DNA1a (“a” = arabino) and DNA1r (“r” = ribo) were cleaved from the resin and deprotected with conc. NH4OH at 45 °C for 16 h. The lyophilized oligonucleotides were reacted with the azide-modified dyes D1–D4 in the presence of Cu(I) and TBTA, as mentioned above. The reaction was performed in H2O/DMSO/t-BuOH 3:3:1 and was completed after 1.5 h at 60 °C. The modified oligonucleotides were purified by ethanol precipitation in the presence of EDTA to remove copper ions and subsequently by semi-preparative HPLC. Finally, the modified oligonucleotides were identified by MALDI–TOF mass spectrometry (see Supporting Information File 1) and annealed with the corresponding unmodified counterstrand. The four fluorophores D1 [23], a blue emitter excitable at 389 nm, D2 [24], D3 [19], and D4 [24], all green emitters excitable at 450–460 nm, that were “clicked” to the oli (...truncated)