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Letter Cite This: Org. Lett. 2018, 20, 3962−3965

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Flexible Alkyne-Linked Thymidine Phosphoramidites and Triphosphates for Chemical or Polymerase Synthesis and Fast Postsynthetic DNA Functionalization through Copper-Catalyzed Alkyne−Azide 1,3-Dipolar Cycloaddition Alessandro Panattoni,†,‡ Radek Pohl,† and Michal Hocek*,†,‡

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Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, CZ-16610 Prague 6, Czech Republic ‡ Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic S Supporting Information *

ABSTRACT: Two alternative flexible alkyne-linked thymine nucleosides (propargyl-diethylene glycol- or undecyn-linked 5hydroxymethyluracil derivatives), as well as their phosphoramidites and triphosphates, were designed and synthesized. The nucleoside 3′-O-phosphoramidites were successfully incorporated into oligonucleotides on a solid support, whereas the nucleoside triphosphates served as good substrates for polymerase synthesis of modified DNA, which underwent fast and efficient copper-catalyzed alkyne−azide 1,3-dipolar cycloaddition (CuAAC) reactions.

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for PCR reactions with long templates. However, because of steric hindrance of the DNA, the directly linked ethynyl group in EdU-modifed DNA is rather inefficient in CuAAC reactions (recently, we determined that, under mild conditions in the presence of lower amounts of Cu(I) to prevent DNA damage, the average conversion in densely modified DNA is ca. 36% only).13 Moreover, partial hydration of the alkyne during the phosphoramidite synthesis has been observed, and therefore a silyl-protected EdU phosphoramidite has to be used.14 To circumvent the lower reactivity of EdU in DNA, the extended octadiyne-linked phosphoramidite5 and dNTP (dUoTP)8 have been developed and are now used as the gold standard for synthesis of clickable DNA (but are not suitable for metabolic labeling). The CuAAC reactivity of the octadiynyl group in DNA is much higher, and the approach has been used for highdensity DNA modification through click reaction.8 However, because of the hydrophobic nature of the octadiyne, the substrate activity of the dUoTP is not optimal (especially in PCR) and hydrophobically modified DNA may tend to aggregate in water. Another drawback is that all dUe and dUo building blocks are synthesized through the Sonogashira reaction from the expensive 5-iodo-2′-deoxyuridine.4,5,7,8 Therefore, we designed and synthesized alternative clickable deoxyuridine building blocks bearing longer and more flexible linkers between the alkyne and the pyrimidine and to test one hydrophilic (oligoethylene glycol) and one hydrophobic tether

ostsynthetic modifications and bioconjugations of DNA find a wide range of applications in chemical biology and diagnostics.1 The copper-catalyzed alkyne−azide 1,3-dipolar cycloaddition (CuAAC)2 plays a prominent role in DNA modifications3 because of its bioorthogonality and efficiency. Terminal alkynes and azides are biocompatible and mostly inert within living systems, and the triazoles resulting from the CuAAC reactions are chemically stable in physiological media and are considered to be nontoxic. Many alkyne-containing nucleoside phosphoramidite building blocks4−6 for solid-phase synthesis of clickable oligonucleotides (ONs), as well as alkyne-7−9 or azide-linked9,10 2′-deoxyribonucleoside triphosphates (dNTPs) for polymerase synthesis of clickable DNA have been reported. In most building blocks, the reactive terminal alkyne is either linked directly to the nucleobase at position 5 of pyrimidines or at position 7 of 7-deazapurines4,7 or connected as an extended and more flexible octa-1,7-diynyl group.5,8 Because of relatively easy accessibility, the most common clickable nucleotide building blocks are 2′-deoxyuridine derivatives: 5-ethynyl-dU (EdU, dUe , 1a) and 5-(1,7octadiynyl)-dU (dUo, 1b) and their 3′-phosphoramidites4,5 for chemical and 5′-O-triphosphates7,8 for enzymatic synthesis. EdU (1a) is used11 for metabolic labeling of DNA synthesis because it penetrates the cell membranes and is intracellularly phosphorylated to triphosphate (dUeTP), which serves as a substrate for incorporation of EdU into genomic DNA, where it can be detected by fluorescent staining through CuAAC reactions. The dUeTP is a very good substrate for DNA polymerases7 (comparable to natural TTP)12 and works even © 2018 American Chemical Society

Received: May 15, 2018 Published: June 13, 2018 3962

DOI: 10.1021/acs.orglett.8b01533 Org. Lett. 2018, 20, 3962−3965

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Scheme 2. PEX Synthesis of Modified DNA and (a) SteadyState Fluorescent Measurements of Products of CuAAC Reaction of ON_1UR with TAMRA-N3; (b) PAGE Shift Analysis of CuAAC of DNA_1UR with PEG-N3; (c) Observed Reaction Rate Constants of CuAAC of DNA_1UR with PEG-N3, Determined through Gel Shift Analysis

Scheme 1. Structures and Scheme of Synthesis of the AlkyneModified Phosphoramidites and dURTPs

The synthesis started from 3′,5′-di-O-acetyl protected thymidine (3, Scheme 1). The radical bromination by treatment with NBS in the presence of AIBN formed in situ the reactive 5-bromomethyluracil derivative which was, without isolation, treated with alkyne-linked alcohol 4c or 4d to furnish the desired protected alkyne-functionalized nucleosides 5c and 5d in good yields (52% and 57%, respectively). Subsequent treatment with NH3 in 1,4-dioxane afforded the nucleosides dUpeg and dUun in almost-quantitative yields. Deprotection, 5′O-DMTritylation, and treatment with O-(cyanoethyl)-diisopropylamino-chlorophosphite according to the literature protocol15 provided the phosphoramidite building blocks 7c (51% yield) and 7d (66% yield). Also the 5′-O-triphosphorylation of unprotected nucleosides 1c and 1d was performed following a well-established procedure16 consisting of reaction with POCl3, followed by tributylammonium pyrophosphate, and finally treatment with triethylammonium bicarbonate (TEAB) and provided the desired triphosphates dUpegTP (2c) and dUunTP (2d) in 22% and 48% yields, respectively. The phosphoramidite building blocks 7c and 7d were successfully used for synthesis of oligonucleotides ON_Upeg and ON_Uun using standard phosphoramidite synthesis on solid support using an automated DNA synthesizer. Therefore, we performed a CuAAC reaction on the obtained oligonucleotides clicking onto them the commercially available 5carboxyltetramethylrhodamine-azide (TAMRA-N3, Scheme 2). The formation of the clicked product was proven by MALDITOF MS (for spectra, see Figures S27−S30 in the Supporting Information).

The new dNTPs, dUpegTP (2c) and dUunTP (2d), were then tested as substrates for KOD XL DNA polymerase.17 In primer extension (PEX) experiments, we used either a 19-mer template (for sequences, see Table S3) encoding for incorporation of one modified dUR nucleotide, or a 31-mer template for incorporation of 4 dUR nucleotides. Polyacrylamide gel electrophoreses (Figure 1a−d) and MALDI TOF MS spectrometry (spectra in Figures S17−S20) confirmed the formation of the desired full-length products in all cases. We also tested other DNA polymerases, i.e., BST large fragment, vent(exo-), and PWO, which also gave full-length products in PEX using the 19-mer template (Figure S4). PCR amplifications were performed with either a shorter 98 bp or a longer 235 bp DNA templates using KOD XL DNA polymerase. In addition to the new dNTPs (dUpegTP and dUunTP), we also tested the standard clickable dNTPs (dUeTP and dUoTP) for comparison. Figure 1e shows that in the PCR with shorter template, we observed formation of a strong modified amplicon with dUeTP and with the PEG-linked 3963

DOI: 10.1021/acs.orglett.8b01533 Org. Lett. 2018, 20, 3962−3965

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the CuAAC. Thus, the PEX products (DNA_1UR) obtained by the incorporation of dURTPs using PrimPEX-FAM and Temp1PEX were, after silica-membrane-based purification, reacted with 5 equiv of PEG-N3 in the presence of CuBr. The large molecular weight of the PEG-substituent leads to a significant decrease in the mobility of PEG-clicked DNA on polyacrylamide gel, allowing a good separation of the spots of the starting alkyne-linked DNA and the CuAAC product. Quantification of the intensity of spots on PAGE allowed us to determine the conversions of CuAAC. Reaction conversions were measured at different times and are shown in Scheme 2B, confirming again an almost 2-fold higher conversion for the peg-linked DNA (DNA_1Upeg, 63% conversion) compared to standard octadiynyl-modified DNA (DNA_1Uo, 33% conversion) and somewhat better than the undecynyl-modified DNA (DNA_1Uun, 44% conversion). On the other hand, ethynyl-linked DNA showed a much lower reactivity with only 18% conversion after 1 h and 50% conversion after 5 h. Simplified kinetic studies were performed employing a similar assay, and the reaction of alkyne-DNA with PEGazide was quenched at several time intervals employing a solution of EDTA, which is useful to strongly chelate Cu(II), and propiolic acid, which quickly reacts with the excess of the azide, removing it from the reaction mixture. Reaction conversions at every time interval were plotted against time using OriginPro 2016. Fitting with the exponential curve Exp1dec (y = A1e−k + y0) provided the observed reaction rate constants (kobs), which are reported in Scheme 2. The obtained values clearly show approximately twice faster reaction of PEGlinked DNA_1Upeg compared to octadiynyl- and undecynyllinked DNA (Scheme 2). Furthermore, we investigated the click reaction on the 98 bp PCR product. In these DNA duplexes, all the thymidines (except for those in primers) are modified, resulting in a high density of alkyne modifications. The CuAAC can give a wide dispersion of labeled products. Because of this fact, agarose gel analysis are very difficult, and we opted, then, for the use of the fluorescent TAMRA-N3 as the click partner. In this way, we could measure the total fluorescence of the final product, thus having a cumulative outcome which evaluates the efficiency of both the PCR and CuAAC as a unique process. As previously discussed, dUeTP is very well incorporated by the KOD XL polymerase, and the PCR provides a larger amount of product with respect to the other terminal alkynyl- substrates. In fact, when the reaction with the azide is carried out for long times, e.g. 1 h, the ethynyl-DNA gives the higher total fluorescence intensity (see the Figure S35). However, when reactions are carried out for a short time, the influence of the rate of CuAAC is predominant; in fact, after incubation for 15 min, the PEG− alkyne linker was confirmed to be the best choice, leading to the higher total fluorescence of the product (Figure S34). In summary, we designed and prepared the two new clickable thymidine derivatives, bearing alkyne linked to pyrimidine through long flexible linkers. The dUpeg contains a hydrophilic linker, whereas dUun has a hydrophobic tether. The synthesis starts from inexpensive natural thymidine and is straightforward and efficient. The nucleosides were converted to phosphoramidites 7c and 7d which are useful for solid-phase synthesis of ONs in an automatic synthesizer. We also synthesized the corresponding dNTPs (dUpegTP and dUunTP), which were good substrates for KOD XL polymerase in PEX. In PCR amplification, the peg-linked dUpegTP was a better substrate compared to standard octadiynyl-linked dUoTP, while dUunTP

Figure 1. PAGE analysis of single-modified PEX products after incorporation of (a) dUpegTPs or (b) dUunTPs and PEX products with 4 modifications after incorporation of (c) dUpegTPs or (d) dUunTPs. Agarose gel analysis of (e) 98 bp PCR products and (f) 235 bp PCR products.

dUpegTP, whereas the hydrophobic dNTPs (dUoTP and dUunTP) gave only very weak products of PCR. It should be noted that we had to carefully optimize the conditions and use 10 mM of MgSO4 to facilitate incorporation of the dUpeg nucleotide (see Table S1−S2). Similarly, using the longer 235 bp template, we observed strong modified amplicons when using dUeTP and dUpegTP, while the standard dUoTP gave a weak amplification and the dUunTP did not show formation of any PCR product (Figure 1f). To study and compare the use of the four clickable dURTP building blocks for DNA postsynthetic functionalization, we synthesized modified ONs containing each one dUR nucleotide by PEX followed by magnetoseparation and reacted the ON_1UR with fluorescent TAMRA-N3 (Scheme 2) at 37 °C for 15 min or 1 h in the presence of CuBr. In this simple and fast experiment, it was possible to qualitatively follow the progress of CuAAC reaction through steady-state fluorescence measurements (Scheme 2A). To our delight, at the short time (15 min), the ON_1Upeg appeared to be the most reactive giving the strongest fluorescence of the clicked conjugate (ON_1Upeg‑TAMRA). This azide was also efficiently clicked on the octadiynyl- and the undecynyl-modified oligonucleotides, while the CuAAC was significantly slower on ethynyl-PEX products. The same reaction was repeated at 37 °C for 45 min, and the products were purified and analyzed by MALDI TOF MS spectrometry, showing that, at this reaction time, ethynyland octadiynyl- PEX products were still present in the reaction mixtures, while in the case of PEG-alkynyl- or undecynylmodified DNA, the CuAAC was quantitative. However, the fluorescence spectroscopy analysis, though fast and facile, is not suitable for accurate quantification of the kinetics of CuAAC reaction of alkyne-linked DNA. Therefore, we designed a more laborious but fully quantitative experiment involving the use of a FAM-labeled primer in the PEX experiment and use of long nonfluorescent azide (PEG-N3) for 3964

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provided PCR products only when the shorter template was used. The flexibility and length of the new linkers resulted in a significant advantage for the reactivity in CuAAC. The ONs and DNA modified with dUpeg reacted approximately twice faster compared to standard octadiynyl-linked DNA. Therefore, the triphosphate dUpegTP and phosphoramidite 7c building blocks have good potential for applications in DNA modifications through CuAAC chemistry, in particular in biological applications, where the fast rate of the click reaction is crucial.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01533. Complete experimental section, characterization of compounds and ONs (including copies of MALDI TOF MS and NMR spectra), additional gels, fluorescence measurements, PCR conditions and ONs sequences (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Michal Hocek: 0000-0002-1113-2047 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Academy of Sciences of the Czech Republic (Praemium Academiae to M.H.), the Czech Science Foundation (18-03305S to M.H.), by Marie Sklodowska-Curie Innovative Training Network (ITN) Click Gene (H2020-MSCA-ITN-2014-642023 to A.P.), and by the European Regional Development Fund; OP RDE (No. CZ.02.1.01/0.0/0.0/16_019/0000729 to M.H.).



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DOI: 10.1021/acs.orglett.8b01533 Org. Lett. 2018, 20, 3962−3965