Synlett 2024; 35(06): 691-697
DOI: 10.1055/a-2201-3756
cluster
Special Issue to Celebrate the Centenary Year of Prof. Har Gobind Khorana

Fluorescent 2′-Deoxyuridine (dU) Analogue: Tropolonyl triazolyl-dU (tt-dU) Exhibits Solvatochromism/HeLa Cell Internalization and Its Triphosphate (tt-dUTP) Is Incorporated into DNA Enzymatically

Sagarika Meher
a   School of Chemical Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar, Jatani-752050, Odisha, India
b   Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai, 400094, India
,
Chandrasekhar Reddy Gade
a   School of Chemical Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar, Jatani-752050, Odisha, India
b   Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai, 400094, India
,
a   School of Chemical Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar, Jatani-752050, Odisha, India
b   Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai, 400094, India
› Author Affiliations

This project has been supported by SERB-New Delhi core research grant (grant number: CRG/2020/001028).
 


Abstract

This era has witnessed the development and extensive application of modified nucleosides, including fluorescent nucleosides that clinically served humankind. Most fluorescent nucleoside analogues are derived from benzenoid aromatic scaffolds. However, the non-benzenoid aromatic moiety, tropolone, which exhibits unique hydrogen bonding and metal chelating properties, also occurs in nature. Recently, we introduced the tropolone unit at deoxyuridine through an ethyne linker and prepared its DNA analogues, which are fluorescent. This report describes the synthesis of a new troponyl triazolyl-dU (tt-dU) analogue, possessing a triazolyl linker, through click chemistry. tt-dU exhibits fluorescence with solvatochromism and enters into Hela cells without any cytotoxicity. Its triphosphate (tt-dUTP) was also synthesized and incorporated enzymatically into DNA, as shown in primer extension experiments. The unique photophysical properties and metal-chelating ability of the tropolone group make tt-dU a promising modified nucleoside.


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Zoom Image
Figure 1 Previously reported nucleosides having (a–d) a triazolyl linkage, and (e) a troponyl nucleoside. The current troponyl nucleosides (tt-dU, tt-dUTP) are shown in the box.

Nucleic acids (DNA/RNA) are genetic materials of living systems that regulate various biological processes such as metabolic control, catalysis, and energy transmission.[1] Structurally, DNA/RNA molecules are biopolymers of nucleosides connected through phosphodiester bonds. Nucleosides are composed of a pentose sugar ring (deoxyribose/ribose) and heterocyclic nucleobases such as purine (A/G) or pyrimidine (T/C/U).[2] Various modified nucleosides and their nucleic acids have been synthesized and studied to understand the DNA/RNA-based biochemical process, which has led to the development of gene-based therapeutic drugs.[3] Radio- and fluorescence-labeled DNA/RNA analogues have also been synthesized to investigate the role of such structures inside cells in real time.[4] [5] Fluorescent nucleic acids (FNA) are more economical and environmentally friendly than their radiolabeled analogues; however, native nucleic acids are not fluorescent. Thus, FNAs have provided powerful tools for monitoring biochemical phenomena in real-time through the use of high-resolution fluorescence microscopy.[6,7] FNA nucleosides are classified as isomorphic, enlarged, extended, and chromophoric base analogues, depending on their chemical structure and their relationship to the natural nucleobases.[8] Isomorphic nucleoside analogues differ from natural nucleosides by the presence of small substituents or by the number and position of heteroatoms in the heterocyclic core. The expanded nucleosides have additional aromatic rings fused to the purine or pyrimidine core. The extended nucleoside analogues contain fluorophore-linked nucleobases.[8] Chromophoric nucleoside analogues comprise a bulky aromatic chromophore instead of the nucleobase residue.[9] Environment-sensitive fluorophores (ESF), another class of fluorophores, exhibit fluorescence changes that arise from secondary structure formation or intramolecular interactions induced by changes in the microenvironment (polarity/viscosity/pH). These fluorescence responses are manifested in valuable photophysical features such as changes in absorption or emission wavelengths (solvatochromism), fluorescence lifetime, quantum yield, or color.[10] Microenvironment-sensitive fluorescent molecules are ubiquitous research tools for sensing biomolecules and studying inter-biomolecular interactions inside a cell.[11] Synthetic nucleoside mimics are also explored as therapeutic drug candidates for antiviral, antimicrobial, antitumor, and anticancer agents. In the search for new biologically active nucleoside analogues, the native structure of nucleosides can be modified with different synthetic methodologies.[12] [13] The metal-mediated cross-coupling reactions (Sonogashira, Stille, Suzuki–Miyaura, Heck, etc.) are perhaps the most successful and widely used chemistry today for synthesizing novel nucleosides.[14] [15] However, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC or click reaction), one of the most important bioorthogonal reactions, has also been broadly utilized to modify oligonucleotides (ONs) and DNA (Figure [1a–d]).[16] [17] [18] The copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides with terminal alkynes has high specificity and efficiency in connecting two different molecular entities. In 2001, Sharpless and Meldal independently introduced this to provide 1,2,3-triazole moieties regioselectively.[19] Click chemistry bagged the Nobel Prize in 2022 and undoubtedly plays a vital role in medicinal chemistry and drug discovery. Click reactions are very appealing for biological applications since they are, by definition, fast, stereospecific (although not necessarily enantioselective), and can be conducted in aqueous solutions with high yield.[17] [20] [21] These properties facilitate biomolecular conjugation in vitro or, if the reaction is non-toxic, even in cells or in vivo where the concentration of target biomolecules is typically low.[22] Recently, many triazole-modified nucleosides have been reported and reviewed by several groups, such as Brown and co-workers, and Rentmeister and co-workers. These modified nucleosides have been used by various groups as photoswitchable DNA interstrand crosslinking agents for thermal stability studies and DNA fluorescence mismatch sensing.[23] [24] Nielsen and co-workers reported the effect of triazole-modified 2-deoxyuridines on the stability of DNA:DNA and DNA:RNA duplexes.[25] [26] Hocek and co-workers introduced azidophenyl to label DNA using click chemistry for electrochemical detection of DNA–protein interactions.[27] Fujimoto and co-workers synthesized sensitive DNA probes for photochemical ligation.[18] However, the majority of the alterations in nucleosides are based on benzenoid and heterocyclic aromatic scaffolds. In the repertoire of functional DNA synthesis, DNA analogues conjugated to non-benzenoid moieties are not well explored. Tropolone is a non-benzenoid aromatic scaffold whose derivatives constitute troponoid natural products.[28] Tropolone has unique intramolecular hydrogen bonding, metal chelating, and therapeutic properties.[29] [30] Recently, tropolone has been employed as a novel scaffold for tuning the structural and functional properties of peptides and DNA. The conjugation of tropolone on a nucleobase was achieved by a Pd-catalyzed Sonogashira coupling reaction.[31] The tropolonyl deoxyuridine (tr-dU) nucleoside analogue exhibits pH-dependent fluorescence (Figure [1e]).[32] Herein, we have conjugated an azido tropolone unit at a modified nucleoside, ethynyl-dU, in the presence of Cu(I) ions through click chemistry and undertaken a series of biochemical evaluations.

5-Iodo-2′-deoxyuridine was modified to give 5-ethynyl-2′-deoxyuridine (2) by following a reported procedure (Scheme [1]).[31] Tropolone was derivatized to give the azide functionalized tropolone 5-azido-tropolonyl benzoate (3) in five steps, as described in the Supporting Information, with all but the last step being well documented.[33] This azide derivative 3 was coupled with 5-ethynyl-2′-deoxyuridine (2) through a Cu-catalyzed [3+2] cycloaddition reaction (Click chemistry), which produced the desired Bz- protected thymidine nucleoside, tt-bzdU (4) (Scheme [1]). The triphosphate analogue, tt-dUTP (6), was synthesized from nucleoside 4 by reaction with POCl3 followed by reaction with pyrophosphate. The synthesis of the triphosphate is an extensive procedure that requires great care. After phosphorylation, the Bz group was deprotected with ammonium hydroxide solution. We obtained tropolonyl deoxyuridinyl triphosphate (tt-dUTP, 6) in considerable yield after HPLC purification. Nucleoside tt-dU (5) was synthesized for fluorescence and biochemical studies from compound 4 by deprotecting the benzoyl group. All the compounds were characterized by NMR and ESI-HRMS analysis (Supporting Information, Figure S1–S12).

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Scheme 1 Synthesis of tropolonyl-dU (tt-dU, 5) and its triphosphate analogue (tt-dUTP, 6)

Our previous work showed that tropolonyl nucleoside exhibited solvent-dependent fluorescence behavior.[31] Tropolone exhibits the π-π* and n-π* electronic transitions, and intramolecular charge transfer,[34] all of which contribute to the photophysical properties of the nucleoside. Here, we performed photophysical studies on tt-dU (5) to examine the effect of the triazole and tropolone rings on its absorption and emission properties. The absorption and emission spectra of the modified nucleoside tt-dU (5) were recorded in a range of solvents (MeOH, EtOH, DMSO, DMF, EtOAc, Dioxane, ACN, THF, DCM, CHCl3, benzene, and toluene), and the spectral properties are summarized in Figure [2] (full data are provided in Table S1 of the Supporting Information). The absorption peaks of tt-dU (5) appeared at shorter wavelengths in non-hydrogen-bonding solvents than in hydrogen-bonding solvents. We observed two absorption maxima (λabs= 305, 445 nm) when nonpolar solvents were used (Supporting Information, Table S1, entries 1–7), whereas a ca. 2–5 nm bathochromic shift was observed with polar or protic solvents (Figure [2]A). In the fluorescence spectra, the emission maxima range from 432 to 464 nm (Figure [2]B/2C). The nucleoside, tt-dU (5), exhibited solvatochromism, but the trend was not continuous from nonpolar to polar solvents. We extracted the quantum yields of tt-dU (5) in different solvent systems, taking aqueous quinine sulfate in H2SO4 as a reference, as summarized in Figure [2]D. Importantly, tt-dU (5) exhibited the highest fluorescence quantum yield (ca. 1.3%) in aromatic solvents (toluene and benzene), DMF, and DMSO, whereas lower quantum yields were observed in the protic polar solvents (MeOH, EtOH), and the lowest quantum yield was observed in non-hydrogen bonding polar solvent ACN (0.3%). When the photophysical properties of tt- dU (having a triazolyl linkage) and tr-dU (having an alkyne linkage) were compared, two major points were noted: (1) the quantum yield of tt-dU (5) was slightly lower than the previously reported tr-dU (Figure [1]) analogue, and (2) the trend was not completely continuous for quantum yields in different solvent polarity or for the plot for Reichard’s solvent polarity parameter E T(30) vs. quantum yield (see the Supporting Information, Figure S13). This result probably stems from the flexibility gained by the introduction of the triazolyl linkage in tt-dU (5), in contrast to the alkyne linkage in the case of tr-dU that does not allow bond rotation. In DMSO and DMF solvents there may be restriction in rotation of tt-dU due to the viscous nature of the solvent. However, we did not observe any sensitivity towards solvent viscosity in the case of tr-dU.[31] We assume that π-π interactions of tt-dU (5) with aromatic solvent lowered the HOMO-LUMO energy gap in comparison to other given solvents and enhanced the fluorescence. The hydrogen-bond acceptor solvents DMF/DMSO encapsulate the tt-dU (5), possibly lowering the HOMO-LUMO energy gap and enhancing the fluorescence significantly. The protic solvents could disrupt the intramolecular hydrogen bonding between the tropolone residue and tt-dU and decrease the quantum yield.

Zoom Image
Figure 2 Absorption (A), emission (B), normalized emission (C) spectra, quantum yield (D) of tt-dU (5) in various solvents (20 μM), and tautomeric structure of tt-dU in solvent (E).
Zoom Image
Figure 3 HOMO-LUMO of tt-dU (5)
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Figure 4 Confocal microscopic images for tt-dU (5) localized in HeLa cells. Cells were treated with tt-dU (100 μM), incubated for 12 h, also stained with DAPI for co-localization. Images in the (A) DAPI (blue channel), (B) TRITC (red), and (C) FITC (green) channels. (D) Expanded image of FITC. Merged images of (E)DAPI and TRITC, (F)DAPI and FITC.

We then calculated the optimized structure of tt-dU at the DFT (B3LYP) level and extracted the HUMO-LUMO diagram and energy difference (Figure [3]). Our results show the coplanar organization of the triazole uracil, with the triazole-CH pointing toward O4 (of C4 carbonyl in the pyrimidine ring) of the uracil probably through a C–H···O hydrogen bonding interaction and the lone pair repulsion of the N/O. This structural preference is well documented in the literature.[16] From the above studies, it is evident that tt-dU (5) is an environment-sensitive fluorophore, although it does not exhibit a large quantum yield. It can be used as a probe for further biochemical evaluations.

We executed MTS assays to evaluate the cytotoxicity of nucleoside tt-dU (5) for both normal (HEK239T) and malignant (HeLa) cell lines (Supporting Information, Figure S15). The concentration-dependent cell viability showed no substantial cytotoxicity with tt-dU in either cell line; thus, this nucleoside can be utilized for further studies. Nucleosides and similar compounds are prodrugs for a wide range of diseases, including antiviral, anticancer, and antibiotic drugs. Fluorescent nucleoside analogues have recently demonstrated better cell permeability and are being used for labeling biomolecules in vitro and in vivo.[35] [36] Thus, the cell permeability of fluorescent tt-dU (5) into HeLa cell lines was investigated. HeLa cells were incubated with tt-dU (5) for 12/24 h and stained with DAPI by following the standard protocol and observed under a confocal microscope. Images were captured in bright light and various channels, i.e., DAPI (blue channel, λex 358 nm) (Figure [4]A), FITC (green channel, λex 490 nm) (Figure [4]C/5D), and TRITC (red channel, λex 570 nm) (Figure [4]B). The tt-dU (5) was primarily identified in the nucleus region of the cell. DAPI-stained, tt-dU-treated cells (Figure [4]A) reveal DAPI localization at the cellular nucleus. For this study, no transfecting reagents were used. Colocalization tests of tt-dU (5) with DAPI were carried out in both channels (green/red). Pearson’s coefficient values (r) for the red and green channels are 0.76 and 0.75, respectively (for 12 h incubation time) (Figure [4]E/4F). These values were computed by using Fiji: ImageJ with the JACoP plugin.[37] The results were the same for both 12 h and 24 h incubation times. Thus tt-dU (5) nucleoside entered into the cell nucleus without any transfecting agent.

We also investigated the enzymatic incorporation of tt-dUTP (6) into DNA primer (P1: 5′-TGTAAAACGACGGCCAGT-3′) guided by template DNA (T1: 3′-ACATTTTGCTGCCGGTCA AGTCGAGGCAT 5′) with a high fidelity DNA polymerase (Therminator), through a primer extension experiment (Figure [5]A). The reaction mixture was analyzed by LCMS (Figure [5]B and Figure S12). Pleasingly, we obtained the expected mass peak at m/z 1515.32, which was nearly identical to the calculated mass [M+3NH4]4– 1515.75, while M = 6009.05. This mass data confirms the incorporation of tt-dU into the primer (P1).

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Figure 5 (A) Primer extension reactions of tt-dUTP (6) with DNA polymerase (Therminator); (B) ESI-Mass spectra of primer extended product (P1*).

In conclusion, we have accomplished the synthesis of tropolonyl triazolyl deoxyuridine (tt-dU, 5) nucleoside, its triphosphate (tt-dUTP, 6), and the corresponding DNA.[38] [39] Our photophysical studies strongly support the conclusion that the tt-dU nucleoside exhibits fluorescence (Φ f ~1.3% in toluene). Its fluorescence characteristics also depend upon the solvent polarity; i.e., it is higher in nonpolar solvents and lower in polar/protic solvents. This nucleoside (tt-dU, 5) was permeable in cell lines (HeLa cells) and exhibited fluorescence. It was mainly localized at the cellular nucleus. It had no significant cytotoxicity against HEK293T or HeLa cell lines. Importantly, its triphosphate analogue (tt-dUTP, 6) was incorporated into DNA enzymatically through a primer extension reaction with DNA polymerase (Therminator). Hence, tt-dU (5) is a promising fluorescent nucleoside analogue that could be applicable for the design of DNA-based fluorescence probes. It may bind with metal ions (Cu2+) and regulate the metal-dependent biochemical processes owing to the metal-chelating properties of the tropolone residue.


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We thank Dr. Supriya Kumari and Dr. Manjusha Dixit (SBS, NISER-Bhubaneswar) for providing the facility to record confocal images.

Supporting Information


Corresponding Author

Nagendra K. Sharma
School of Chemical Sciences, National Institute of Science Education and Research (NISER)
Bhubaneswar, Jatani-752050, Odisha
India   

Publication History

Received: 27 July 2023

Accepted after revision: 30 October 2023

Accepted Manuscript online:
30 October 2023

Article published online:
27 November 2023

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Zoom Image
Figure 1 Previously reported nucleosides having (a–d) a triazolyl linkage, and (e) a troponyl nucleoside. The current troponyl nucleosides (tt-dU, tt-dUTP) are shown in the box.
Zoom Image
Scheme 1 Synthesis of tropolonyl-dU (tt-dU, 5) and its triphosphate analogue (tt-dUTP, 6)
Zoom Image
Figure 2 Absorption (A), emission (B), normalized emission (C) spectra, quantum yield (D) of tt-dU (5) in various solvents (20 μM), and tautomeric structure of tt-dU in solvent (E).
Zoom Image
Figure 3 HOMO-LUMO of tt-dU (5)
Zoom Image
Figure 4 Confocal microscopic images for tt-dU (5) localized in HeLa cells. Cells were treated with tt-dU (100 μM), incubated for 12 h, also stained with DAPI for co-localization. Images in the (A) DAPI (blue channel), (B) TRITC (red), and (C) FITC (green) channels. (D) Expanded image of FITC. Merged images of (E)DAPI and TRITC, (F)DAPI and FITC.
Zoom Image
Figure 5 (A) Primer extension reactions of tt-dUTP (6) with DNA polymerase (Therminator); (B) ESI-Mass spectra of primer extended product (P1*).