Replacement of the phosphodiester backbone between canonical nucleosides with a dirhenium carbonyl “click” linker—a new class of luminescent organometallic dinucleoside phosphate mimics

Abstract

The first-in-class luminescent dinucleoside phosphate analogs with a [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] “click” linker as a replacement for the natural phosphate group are reported together with the synthesis of luminescent adenosine and thymidine derivatives having the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] entity attached to positions 5′ and 3′, respectively. These compounds were synthesized by applying inverse-electron-demand Diels–Alder and copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition reactions in three or four steps. The obtained compounds exhibited orange emission (λ_PL ≈ 600 nm, Φ_PL ≈ 0.10, and τ = 0.33–0.61 μs) and no toxicity (except for one nucleoside) to human HeLa cervical epithelioid and Ishikawa endometrial adenocarcinoma cancer cells in vitro. Furthermore, the compounds’ ability to inhibit the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacterial strains was moderate and only observed at a high concentration of 100 μM. Confocal microscopy imaging revealed that the “dirhenium carbonyl” dinucleosides and nucleosides localized mainly in the membranous structures of HeLa cells and uniformly inside S. aureus and E. coli bacterial cells. An interesting finding was that some of the tested compounds were also found in the nuclei of HeLa cells.

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Introduction

Chemical modifications of nucleic acids and their building blocks such as nucleosides and nucleotides have grown as an important field of academic and industrial studies. Many modified nucleosides and nucleotides have been marketed as anticancer and antiviral drugs.^1–3 Furthermore, the discovery of novel chemically modified nucleosides and nucleotides has been beneficial for the development of RNA/DNA-targeting technologies such as antisense and short interfering RNA as well as the increasingly popular CRISPR-Cas9 gene editing technique.^4–8 Structural alterations of canonical nucleic acid components pertains to the modification of the nucleobase, the (deoxy)ribose and the phosphodiester backbone.^1,2,9–11 In the case of the phosphodiester backbone, three general classes of modifications can be distinguished. The first class comprises the substitution of the nonbonding oxygen atom in the phosphodiester backbone. The second deals with the replacement of the bonding oxygen atom, while the third involves the complete replacement of the phosphodiester backbone with a nonphosphodiester linker. There have been many examples of the phosphodiester backbone modifications reported in the literature. Relevant examples include phosphorothioate,^12,13 phosphoroamidate,¹⁴ boranophosphate,^15,16 amide,^5,6,8,17,18 formacetal,¹⁹ amine,²⁰ guanidinium,^21,22 and 1,2,3-triazole^23–27 internucleotide linkers.

Alternatively to these organic modifications, there is growing interest in nucleic acids modified with coordination or organometallic metal complexes.^28–41 The most notable examples of such modifications resulted in metallo-DNA nanostructures and nanowires.^29,33 Furthermore, metal-containing oligonucleotides and nucleosides were shown to exhibit useful redox,^38,42 radioactive,⁴³ and luminescence⁴⁴ properties. Literature data indicate that at least 13 metals, namely Cr, Mn, W, Tc, Fe, Re, Co, Os, Ir, Rh, Hg, Pd, and Sn, have been used for the organometallic derivatization of nucleosides.³⁰ Four classes of organometallic nucleosides have been postulated based on the position of the metal-containing group within the nucleoside/nucleotide scaffold.³⁰ The most common class comprises compounds in which a metal-containing entity is attached to the nucleobase part of a given nucleoside or nucleotide. On the other hand, the group of canonical dinucleoside phosphates with organometallic modifications in the phosphodiester backbone is the least prevalent.^45–48 Dinucleoside phosphates A and B (Fig. 1) are examples of this least abundant class of compounds.^45,46

Fig. 1 Structures of canonical DNA dinucleoside phosphate, metal–containing dinucleoside phosphates A and B and compounds 4, 5 and 6 studied in this work. Ac = acetyl; Bz = benzoyl; DMTr = 4,4′-dimethoxytrityl; and Lev = levulinyl.

To the best of our knowledge, there are no literature reports on dinucleoside phosphates in which the entire phosphodiester backbone has been replaced by the luminescent organometallic linker. Therefore, we attempted to synthesize this new class of compounds as part of our research on the chemistry and biology of organometallic nucleic acid components.^49,50

This paper describes the synthesis, photophysical properties, anticancer/antibacterial activity, and results of the imaging studies of the first-in-class dinucleosides 4, 5, and 6 (Fig. 1 and Scheme 1) bearing a luminescent dirhenium carbonyl [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] 1,2,3-triazolyl (“click”) linker in place of the canonical phosphate backbone. Furthermore, in this work, we report on luminescent dirhenium carbonyl “click” thymidine and adenosine conjugates 10 and 11 (Scheme 2). This work emphasizes on how the complex functional (luminescent) artificial “nucleosides” can be obtained by using simple cycloaddition reactions in a few synthetic steps. Furthermore we showed that the fate of these nucleosides could be followed in living cells by the luminescence confocal microscopy.

Scheme 1 Synthetic pathway to compounds 4, 5, and 6. Reagents and conditions: (i) DCM, ambient temperature, and 18 h; (ii) toluene, reflux, and 30 min; (iii) CuSO₄·5H₂O, Na ascorbate, tert-BuOH, H₂O, ambient temperature, and 2 h; (iv, v, vi) CuSO₄·5H₂O, Na ascorbate, tert-BuOH, H₂O, ambient temperature, and 3 h. DCM = dichloromethane.

Scheme 2 Synthetic pathway to compounds 10 and 11. Reagents and conditions: (i) DCM, ambient temperature, and 18 h; (ii) toluene, reflux, and 2 h; (iii) DCC, DMAP, DCM, ambient temperature, and 1.5 h; (iv, v) CuSO₄·5H₂O, Na ascorbate, tert-BuOH, H₂O, ambient temperature, and 3 h. DCM = dichloromethane; DMAP = 4-(dimethyl)aminopyridine; and DCC = N,N′-dicyclohexylcarbodiimide.

The 1,2,3-triazole scaffold was chosen as the primary component of the internucleotide linker because it can be easily synthesized via the copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC, “click”) reaction which is being widely utilized in nucleic acid chemistry.^11,49,51 Re(I)-carbonyl, on the other hand, was chosen as the metal-containing motif due to its inherent photoluminescence properties.^52,53 Luminescent Re(I) conjugates of biologically relevant compounds such as sugars, drugs and so forth have been used as imaging probes and phototoxic agents.^54–58 Furthermore Re(I)-carbonyl complexes have been used as IR probes⁵⁹ and as “cold” isostructural counterparts to the ^99mTc radionuclide used in positron emission tomography.⁶⁰ In this study, we used dinuclear [Re₂(μ-Cl)₂(CO)₆(-μ-1,2-diazine)] rhenium derivatives. Dinuclear rhenium 1,2-diazine complexes have generally received significantly less attention than mononuclear Re(I)-carbonyl complexes.^61–67 Like the majority of their mononuclear Re(I)-carbonyl counterparts, they exhibit strong phosphorescence in the range of ca. 550–620 nm. This emission is assigned to the lowest triplet state of the metal-to-ligand charge transfer (³MLCT) character resulting from a d(Re₂(μ-Cl)₂(CO)₆) → π*(pyridazine) electronic transition.^65,67 Luminescent [Re₂(μ-Cl)₂(CO)₆(μ-1,2-pyridazine)] conjugates with estradiol,⁶⁵ glycodendrons,^63,64 and peptide nucleic acids (PNA)^61,62 have been reported in the literature e.g., as probes for cell imaging which encouraged us to use them as confocal microscopy-detectable labels in the course of the studies reported here.

Results and discussion

Synthesis

Dinucleoside 4 was synthesized in four steps, while the synthesis of dinucleosides 5 and 6 required three synthetic steps each (Scheme 1). The key for all procedures 1,2,4,5-tetrazine (C) was obtained according to the literature.⁶⁸ The first step of the synthetic sequence was an inverse-electron-demand Diels–Alder (IEDDA) [4 + 2]-cycloaddition reaction^69,70 of 1,2,4,5-tetrazine (C) with tripropargylamine (D) which afforded ligand 1 in 44% yield. Refluxing of 1 with Re(CO)₅Cl in a toluene solution resulted in the [Re₂(μ-Cl)₂(CO)₆] complex 2 in 61% yield. In the subsequent step, complex 2 was reacted with 5′-azido-2′,3′-O-isopropylidene adenosine⁷¹ (F) under CuAAC reaction conditions to afford an intermediate 3 in 21% yield. In the last step, the CuAAC reaction of 3 with 3′-azido-3′-deoxythymidine (G) resulted in “click” dinucleoside 4 in 40% yield. Compounds 5 and 6 were synthesized in 38 and 42% yields via a CuAAC reaction of complex 2 and (F) or (G) in 38 and 42% yield, respectively.

Nucleosides 11 and 10 were synthesized in four steps according to Scheme 2. In the first step, an IEDDA reaction of 1,2,4,5-tetrazine (C) with 6-heptynoic acid (H) afforded 5-(pyridazine-4-yl)pentanoic acid 7 in 31% yield. In the second step, the rhenium complex E was reacted with ligand 7 which afforded acid 8 in 87% yield. In the following step, acid 8 was transformed into the corresponding amide 9 (87% yield) by conjugation with propargylamine (I) under Steglich reaction conditions.⁷² The last step involved a CuAAC reaction of amide 9 with azidonucleoside (F) or (G) which afforded target conjugates 10 and 11 in 47 and 45% yield, respectively.

At this point it should be emphasised that the synthetic approach for 4–6, 10 and 11 compounds vividly shows how different types of cycloaddition reactions together with simple coordination and amide bond formation reactions might be utilized for the engineering of structurally complex and functional (in this case luminescent) artificial nucleic acid components.

With the exception of 3, all compounds were characterized by ¹H NMR, ¹³C NMR and FTIR spectroscopy, high-resolution mass spectrometry, and elemental analysis. The resulting analytical data confirmed the proposed structures. The ¹H-NMR spectra are shown in Fig. S1–S10 (ESI†). Also, the structures of compounds 2 and 8 in the solid state were determined by single-crystal X-ray structural analysis. All compounds were found to be stable in the solid state and could be stored as such for months without decomposition. On the other hand, synthetic intermediate 3 decomposes in organic solvents. We also noticed gradual decomposition of other complexes in DMSO solution when left overnight at room temperature. This did not, however, interfere with their structural assignment and spectroscopic characterization.

X-ray structure determination. Crystals of compounds 2 and 8 suitable for a single-crystal X-ray diffraction analysis (XRD) were obtained by slow convectional diffusion of n-heptane or n-hexane into a solution of the respective complex in chloroform at room temperature. Both complexes crystallized in the monoclinic space group P2₁/n with one molecule of the given compound in the asymmetric part of the unit cell. Fig. 2 and 3 show the molecular diagrams of the examined compounds with the atom-labeling scheme together with selected geometrical parameters. Table S1 (ESI†) summarizes the crystal and structural refinement data. The full list of bond lengths (Å), valences and torsion angles (°) can be found in Tables S2–S7 (ESI†).

Fig. 2 Molecular diagram of 2 with atomic displacement ellipsoids at the 50% probability level. The H-atoms are shown as small spheres of arbitrary radius. Selected bond lengths, distances [Å], and angles [°]: Re1⋯Re2, 3.5459(3); Re1–Cl1, 2.4830(12); Re1–Cl2, 2.4978(12); Re2–Cl1, 2.4925(13); Re2–Cl2, 2.5034(12); Re1–N1, 2.204(5); Re2–N2, 2.204(4); Re1–C12, 1.918(6); Re1–C13, 1.942(7); Re1–C14, 1.899(6); Re2–C15, 1.905(6); Re2–C16, 1.914(6); Re2–C17, 1.907(6); N1–N2, 1.354(6); C7–C8, 1.196(10); C10–C11, 1.180(10); Re1–N1–N2, 120.1(3); Re2–N2–N1, 119.5(3); Re1⋯Re2–C16, 115.88(19); Re2⋯Re1–C13, 115.04(18).

Fig. 3 Molecular diagram of 8 with atomic displacement ellipsoids at the 50% probability level. The H-atoms are shown as small spheres of arbitrary radius. Selected bond lengths, distances [Å], and angles [°]: Re1⋯Re2, 3.5564(6); Re1–Cl1, 2.5033(19); Re1–Cl2, 2.503(2); Re2–Cl1, 2.4921(19); Re2–Cl2, 2.4883(19); Re1–N1, 2.204(7); Re2–N2, 2.182(7); Re1–C10, 1.905(9); Re1–C11, 1.935(9); Re1–C12, 1.892(10); Re2–C13, 1.889(10); Re2–C14, 1.922(7); Re2–C15, 1.897(9); N1–N2, 1.356(10); C9–O1, 1.306(11); C9–O2, 1.227(11); Re1–N1–N2, 119.4(5); Re2–N2–N1, 120.7(5); Re1⋯Re2–C14, 115.6(3); Re2⋯Re1–C11, 118.4(3).

Similar to the literature data,⁶⁷ the X-ray analysis revealed that the coordination sphere around the Re(I) atoms of the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] moiety in complexes 2 and 8 was distorted octahedral. The Re⋯Re distance in these two complexes was 3.5459(3) and 3.5564(6) Å, respectively. Within the coordination spheres of Re1 and Re2, the planes defined by equatorial C-atoms and bridging Cl atoms were inclined to themselves by 135.92(11) (2) and 134.90(16)° (8) (Fig. S11 [ESI†]). In both compounds, the pyridazine moiety was planar, with deviation from planarity of 0.008 (2) and 0.007 Å (8). In compound 2, the triple bond lengths were 1.180(10) and 1.196(10) Å for C10–C11 and C7–C8, respectively. In the crystal lattice of 8, the adjacent molecules of the complex formed dimers, where individual molecules were held together by O–H⋯O hydrogen bonds arranged in an R²₂(8) motif (Fig. S12 [ESI†]) (d(D⋯A) = 2.607(10) Å; <(D–H⋯A) = 172(5)°). In turn, in the crystal lattice of 2, there were only weak intermolecular hydrogen bonds found.

Photophysical characterization and DFT calculations

The absorption and emission properties of compounds 2, 4–6, and 8–11 were almost identical and dominated by the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] group (Table 1).

Table 1 Photophysical data for compounds 2, 5 and 6, and 8–11 in air-equilibrated DCM and DMSO (*) at ambient temperature

Compound	λ abs [nm] (ε [×10^3 M^−1 cm^−1])	λ PL [nm]	λ abs * [nm]	λ PL * [nm]	Φ PL(±10%)	τ [μs]	τ* [μs]
* data in air-equilibrated DMSO at ambient temperature.
2	370 (10.0)	612	335	630	0.08	0.53	0.04
4	370 (8.0)	600	340	614	0.06	0.49	0.31
5	365 (8.9)	596	342	625	0.07	0.33	0.29
6	371 (6.3)	605	347	617	0.08	0.53	0.30
8	363 (8.5)	589	330	640	0.11	0.61	0.41
9	359 (5.9)	590	332	650	0.11	0.60	0.41
10	360 (7.4)	588	340	616	0.08	0.61	0.24
11	363 (6.9)	589	335	620	0.12	0.60	0.40

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The UV/Vis absorption spectra of the studied complexes were characterized by the presence of a broad unstructured band at ca. 370 nm owing to metal-to-ligand (dπ(Re)–π*(pyridiazine)) charge transfer states (³MLCT).^61,65 At higher energies (ca. 260 nm) an intense band assigned to the pyridazine intraligand π–π* transitions was present. All compounds display luminescence with a broad emission band in a range of 588–612 nm in air-equilibrated DCM at ambient temperature (Table 1, and Fig. 4). In DMSO solution, a slight red shift of the emission was observed (Fig. 4 and S13†). This broad, unstructured emission was assigned to phosphorescence from the lowest ³MLCT.^61,65,66 The long decay times typical of triplet emissions further substantiate this assignment.

Fig. 4 Electronic absorption (red line) and luminescence (black line) spectra of 2 in DCM (left) and 5 in DMSO (right).

For a deeper understanding of the electronic structure of the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] emitting part common to all the examined compounds, DFT and TD-DFT computations were conducted on the model complex 2. According to the TD-DFT calculations, the four first excited states S₁, S₂, and T₁, T₂ of 2 involve the two highest occupied molecular orbitals (HOMO and HOMO−1) and one lowest unoccupied molecular orbital (LUMO). The orbital localization analysis showed that the HOMO, HOMO−1, and LUMO were localized on the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] moiety (Fig. 5). The HOMO and HOMO−1 had major contribution from metal centers, whereas the LUMO was mainly localized on pyridazine. Thus the calculated first excited states were characterized by dominant MLCT transitions.

Fig. 5 DFT-calculated iso-surface contour plots (iso-value = 0.035) of the lowest unoccupied molecular orbital (LUMO) and the highest and second highest occupied molecular orbitals (HOMO and HOMO−1) of the model compound 2.

Imaging of cells with luminescent probes or compounds has several limitations. One of these is a cell's ability to show autofluorescence which occurs in the range of 350 to 550 nm and is related to the presence of aromatic cellular components (e.g., aromatic amino acids, NADPH, and riboflavin). Autofluorescence is a short-lived process typically characterized by emission decay times of several nanoseconds. The photophysical characteristics of compounds 2, 4–6, and 8–11, which include their ability to emit in the region lacking cell autofluorescence and exhibiting long emission decay times, support the use of these rhenium complexes in luminescence confocal microscopy imaging of live cells.

Biological activity and confocal microscopy. Compounds 4–6, 10, and 11 represent a novel class of luminescent organometallic nucleoside derivatives with unknown biological properties and pharmacological activities. Therefore, their biological potential was first investigated by anticancer and antibacterial activity screening. Next, the localization of these compounds in model eukaryotic and prokaryotic cells was examined using luminescence confocal microscopy. The anticancer activity of dinucleosides 4–6, nucleosides 10 and 11, and nonnucleoside rhenium complexes 2, 8, and 9 was screened in vitro against human cervical epithelioid carcinoma HeLa and endometrial adenocarcinoma Ishikawa cells using the MTT assay. Additionally, the toxicity of the compounds was also examined against nontumorigenic L929 cells. Table 2 presents the IC₅₀ values determined from the experiments.

Table 2 Effects of compounds 2, 4–6, and 8–11 on the viability of HeLa, Ishikawa, and L929 cells, after 24 h of treatment. The results are expressed as IC₅₀ (μM) ± SEM from two independent experiments. Cisplatin (CisPt) was used as the reference anticancer drug

	IC50 (μM)
	2	4	5	6	8	9	10	11	CisPt
a IC50 value was higher than the maximal compound concentration tested.
L929	7.3 ± 0.3	>100^a	>100	>100	76.2 ± 13	18.7 ± 0.9	56.8 ± 11.4	>100	24.4 ± 2.0
HeLa	6.4 ± 0.6	>100	>100	>100	48.0 ± 3.4	9.2 ± 1.0	42.7 ± 4.4	>100	4.5 ± 0.3
Ishikawa	10.6 ± 0.7	>100	>100	>100	57.3 ± 2.9	21.1 ± 1.6	42.6 ± 2.6	>100	10.4 ± 1.6

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In the MTT assay, dinucleosides 4, 5 and 6 did not cause any cytotoxicity in the tested cell lines (IC₅₀ > 100 μM). However, their precursor, i.e., complex 2, showed the highest cytotoxicity against all the examined cell lines. The activity of complex 2 against HeLa and Ishikawa cells was comparable to that of cisplatin (CisPt). Unfortunately, complex 2 displayed higher toxicity than CisPt against nontumorigenic L929 cells which likely decreased its value from the standpoint of real anticancer applications. In the case of nucleosides 10 and 11, only the former compound displayed a moderate level of activity, with IC₅₀ values of 42.7 ± 4.4 and 42.6 ± 2.6 μM against HeLa and Ishikawa cells, respectively. The activity of compound 10 was comparable to that of carboxylic acid 8. In contrast, amide 9, a propargyl amine derivative of 8, exhibited strong cytotoxicity toward HeLa and Ishikawa cancer cells as well as unfavorable activity against nontumorigenic L929 cells. The two most effective anticancer complexes 2 and 9 were intermediates in the synthesis of target nucleoside compounds. Also both these intermediates showed highly undesirable toxicity against nontumorigenic L929 cells. Driven by these reasons, more in-depth anticancer activity studies of 2 and 9 were not carried out. Because some anticancer nucleosides may show their activity after a longer treatment time,⁷³ the MTT experiment was also carried out after 72 h of exposure; however, no changes in the IC₅₀ values were noticed (data not shown). In summary, the MTT assay showed that nucleosides 4–6, 11 and 10 are appropriate for imaging in live HeLa cells with luminescence confocal microscopy due to their low cytotoxic activity. In the following stage of biological studies we used the broth microdilution technique to test the antibacterial activity of dinucleosides 4–6, 10, and 11 against Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Escherichia coli NCTC 8196 bacterial strains. The 24 h treatment period and the same concentration range as for the MTT assay were employed. The minimal inhibitory concentration (MIC) values for the widely used antibiotics ciprofloxacin (CIP) and streptomycin (STR) were determined in order to compare the activities of the tested compounds. For CIP, the MIC values were 0.04 and 0.8 μM for E. coli and S. aureus, respectively, while in the case of STR the value was 1.4 μM for both bacterial strains. The antibacterial activity of rhenium compounds was so weak that under the applied conditions their MIC values could not be determined. However, moderate suppression of bacterial growth was seen at a high concentration of 100 μM (Fig. 6).

Fig. 6 Growth inhibition of E. coli NCTC 8196 and S. aureus ATCC 6538 cells after 24 h treatment with compounds 4–6, 10, and 11 at a concentration of 100 μM. Percentage of bacterial growth was calculated in comparison with untreated control (100% of growth), and the results are presented as mean ± SEM from two independent experiments.

Dinucleoside 6 (69% growth inhibition) and nucleoside 11 (51% growth inhibition) caused the highest growth inhibition of E. coli cells. The remaining compounds were significantly less effective in inhibiting the growth of E. coli. Notably, 6 and 11 are both derivatives of 3′-azido-3′-deoxythymidine, which may have an influence on their antibacterial activity. On the other hand, compounds 6 and 11 as well as other examined compounds caused less than 50% growth inhibition of S. aureus cells. This finding is interesting because it contradicts the stronger luminescence stemming from the interiors of S. aureus cells stained with the studied compounds in comparison with the E. coli cells (described in the Confocal luminescence microscopy section). It can be speculated that the higher antibacterial activity and lower luminescence intensity of compounds observed in E. coli cells were caused by the degradation of these compounds accompanied by the production of toxic degradation products. Studies on antibacterial activity revealed that the examined compounds 4–6, 10, and 11 are largely nontoxic to bacteria, which is a valuable finding to proceed with imaging by luminescence confocal microscopy.

Confocal luminescence microscopy. To study the cellular uptake and localization of compounds 4–6, 10, and 11 in HeLa cells, confocal luminescence microscopy was used. After 30 minutes of treatment, a luminescence signal from the interiors of the cells was unambiguously observed supporting the uptake of the compounds (Fig. 7 and Fig. S14†). Rhenium dinucleosides 4 and 5 featured less intense emissions than did the rest of the series. This weaker emission was intensified slightly when a 2-hour incubation time was applied. Further extension of the treatment time to over 2 hours did not result in a brighter emission.

Fig. 7 Confocal luminescence images of viable HeLa cells stained with dinucleosides 4–6 and nucleosides 10 and 11. The images were taken after 30 min treatment time.

The staining pattern of HeLa cells was very similar for compounds 6, 10, and 11. The example photographs in Fig. 8 show details of dinucleoside 6 localization in the viable HeLa cell.

Fig. 8 Cellular distribution of dinucleoside 6 in viable HeLa cells. The images were taken after 30 min treatment time. Left column—luminescence image, right column—merged images of luminescence and transmitted light. Lysosomes/vesicles are denoted by inverted arrows; plasmalemma and nuclear envelope are denoted by hollow arrows; filopodia are denoted by the loop; mitochondria are denoted by full-tip arrows. The compound in the nucleus is denoted by asterisk.

Localization of compound 6 in the plasmalemma, nuclear envelope (hollow arrows in Fig. 8), and filopodia (loop in Fig. 8) was unambiguously confirmed. In addition, a granularly diffused pattern of emission was observed in the area of the cytoplasm which surrounds the nucleus. Within this space, bright circular structures were seen which are interpreted as lysosomes or vesicles (inverted arrows in Fig. 8). Furthermore, compound 6 was localized in the elongated structures scattered throughout the cell (arrows in Fig. 8) which were classified as mitochondria. Due to its DNA-protective role, it is difficult for many xenobiotics to cross the nuclear envelope. However, dinucleoside 6 crossed the nuclear envelope barrier and was localized in the nucleus (asterisk in Fig. 8). It is worth noting that the nuclear localization of compounds 10 and 11 was also unambiguously confirmed. In the case of dinucleosides 4 and 5, a more granular and less diffuse staining pattern of the cytoplasm was observed. Compounds 4 and 5 were not found in the nuclei of HeLa cells. Overall, the examined compounds showed a non-selective pattern of staining. They were mainly localized in lipidic membrane compartments and organelles of the cells. In comparison with luminescence imaging of human HeLa cells, the imaging of prokaryotic bacterial cells is a more challenging task. This is because it is difficult to penetrate the bacterial cell wall combined with efflux proteins which represent an evolutionary design to protect bacteria from external toxins. The low antibacterial activity of 4–6, 10, and 11 against E. coli NCTC 8196 and S. aureus ATCC 6538 strains prompted us to conduct luminescence confocal microscopy imaging in these bacteria (Fig. 9).

Fig. 9 Confocal luminescence images of E. coli and S. aureus bacteria stained with dinucleosides 4–6 and nucleosides 10 and 11. The images were taken after 1 hour treatment time. Left column—luminescence images, right column—merged images of luminescence and transmitted light.

After one hour of incubation, each of the tested compounds penetrated into E. coli and S. aureus bacterial cells (Fig. 9). However, it should be emphasized that less intense luminescence was observed for Gram-negative E. coli than for Gram-positive S. aureus cells. This feature can likely be explained by the weakened penetration of the assayed compounds through the E. coli bacterial envelope. Luminescence confocal microscopy enabled recognition of the characteristic stick-like morphology of E. coli cells as well as the sphere-shaped S. aureus cells. However, due to the small sizes of the bacteria and technical limitations further intracellular localization studies were not performed.

Conclusions

In this account, first-in-class luminescent organometallic analogs of canonical dinucleoside phosphates are reported. These contain the [Re₂(μ-Cl)₂(CO)₆(μ-pyridazine)] “click” linker in place of the phosphate linker present in canonical dinucleosides. The paper also reports the synthesis of adenosine and thymidine derivatives with a dirhenium carbonyl “click” entity attached at positions 5′ and 3′. The dinucleosides were synthesized in three- (5, 6) or four-step (4) procedures by applying IEDDA and CuAAC “click” reactions as key synthetic approaches. Dirhenium nucleosides and their synthetic precursors displayed orange phosphorescence with Φ_PL ≈ 0.10 and τ = 0.33–0.61 μs. With the exception of nucleoside 10, the synthesized nucleosides did not exhibit any toxic effect against human cancer HeLa and Ishikawa cells, as confirmed by biological activity assays. Additionally, the compounds were not toxic to S. aureus and E. coli bacterial cells. Favorable photophysical properties together with the low toxicity of 4–6, 10 and 11 enabled their confocal luminescence microscopy imaging in HeLa cells (as the eukaryotic model) as well as in S. aureus and E. coli (as the prokaryotic model) cells. Confocal imaging results showed that the studied compounds localized mainly in the membranous structures and compartments of HeLa cells. Additionally, compounds 6, 10, and 11 were detected in the nuclear region which indicates some interactions with nuclear components. The tested bacterial cells were more uniformly stained. In conclusion, a novel class of artificial nucleic acid components with luminescence properties were synthesized and biologically characterized. These compounds may serve as attractive building blocks for the synthesis of luminescent nucleic acid analogs for biological and other applications.

Materials and methods

Reagents and general procedures

All preparations were carried out using standard Schlenk techniques. Chromatographic separations involved the use of silica gel 60 (Merck, 230–400 mesh ASTM). Toluene was distilled and purged with argon prior to use. Other solvents were of reagent grade and used without prior purification. Tripropargylamine, propargylamine, 4-(dimethyl)aminopyridine, N,N′-dicyclohexylcarbodiimide, Re(CO)₅Cl, 3′-azido-3′-deoxythymidine, 6-heptynoic acid and Na ascorbate were purchased from a commercial supplier and used without further purification. ¹H NMR (600 MHz) and ¹³C{H} NMR (150 MHz) spectra were recorded on a Bruker Avance III 600 spectrometer operating at 298 K in Fourier transform mode. Chemical shifts are reported in δ units (ppm) using the residual signal of DMSO (¹H δ 2.50 ppm, ¹³C δ 39.5) or CD₃OD (¹³C δ 49.00) as reference. All the mass spectra were recorded using a Synapt G2-Si mass spectrometer (Waters) equipped with an ESI source and quadrupole-TOF mass analyzer. The mass spectrometer was operated in negative ion detection mode. The measurements were performed with the capillary voltage set to 2.7 kV and sampling cone voltage to 20 V. The source temperature was 110 °C. To ensure the accuracy of mass measurements, data were collected in centroid mode and mass was corrected during acquisition using leucine enkephalin solution as an external reference (Lock-Spray). The results of the measurements were processed using MassLynx 4.1 software (Waters) incorporated with the instrument. IR spectra (KBr pellets) were measured on a FTIR Nexus Nicolet apparatus. Microanalyses were carried out by the Analytical Services of the Polish Academy of the Sciences (Łódź). HPLC separations were performed with a Shimadzu Prominence system equipped with a PDA detector, using an analytical Phenomenex Lux 5 μm Silica (2) 100 A (150 × 21.2 mm) column. Detection was accomplished at λ = 254 nm and a flow rate of 8 mL min⁻¹ was applied. Pyridazine = pydz, adenine = ade, thymine = thym, triazole = triaz, and isoprop = isopropylidene.

Synthesis

Synthesis of 1. To a stirred solution of 1,2,4,5-tetrazine (500 mg, 6.1 mmol) in dichloromethane (5 mL) at ambient temperature, triethynylamine (863 μL, 6.1 mmol) was added dropwise. During and after addition, evolution of the dinitrogen was observed. The resulting reaction mixture was stirred at ambient temperature for 18 h. Then, all volatiles were evaporated under reduced pressure. The resulting material was subjected to column chromatography on SiO₂ (dichloromethane/methanol 50 : 3 v/v). Compound 1 was obtained as a pale yellow solid in 44% (500 mg) yield.

(1) ¹H NMR (600 MHz, DMSO-d₆): δ 9.17–9.16 (bs, 2H, H-pydz), 7.59 (q, J_H,H = 4.8, 2.4 Hz, 1H, H-pydz), 3.71 (s, 2H, CH₂N), 3.38 (d, J_H,H = 2.4 Hz, 4H, CH₂C≡C), 3.26 (t, J_H,H = 2.4 Hz, 2H, C≡CH) ppm. ¹³C{¹H} NMR (150 MHz, DMSO-d₆): δ = 152.0, 151.3, 137.8, 126.1, 78.6, 76.3, 52.9, 41.6. MS (TOF ESI+): m/z = 186.1038 (M + H⁺) (calcd for C₁₁H₁₂N₃: 186.1031). FTIR (KBr ν [cm⁻¹]): 3190, 2823, 2112, 2105, 1591, 1437, 1119, 927, 670. Anal. calcd for C₁₁H₁₁N₃: C, 71.33; H, 5.99; N, 22.69%. Found: C, 71.15; H, 5.82; N, 22.54%.

Synthesis of 2. To a stirred solution of Re(CO)₅Cl (796 mg, 2.2 mmol) in toluene (10 mL) at ambient temperature, ligand 1 (200 mg, 1.1 mmol) was added dropwise. The resulting reaction mixture was refluxed for 30 min. Then, all volatiles were evaporated under reduced pressure. The resulting material was subjected to column chromatography on Al₂O₃ (diethyl ether). Chromatography afforded the pure complex 2 as a yellow solid in 61% (528 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 10.06 (d, J_H,H = 6.0 Hz, 1H, H-pydz), 10.04 (s, 1H, H-pydz), 8.20 (d, J_H,H = 4.2 Hz, 1H, H-pydz), 4.00 (s, 2H, CH₂N), 3.53 (s, 4H, CH₂C≡C), 3.27 (s, 2H, C≡CH). ¹³C NMR (150 MHz, CD₃OD): δ = 195.6, 195.5, 191.7, 191.6, 164.2, 163.3, 148.8, 133.2, 79.1, 75.5, 54.3, 43.6. MS (TOF ESI−): m/z = 795.9045 (M − H⁺) (calcd for C₁₇H₁₀N₃O₆Cl₂Re₂: 795.9062). FTIR (KBr ν [cm⁻¹]): 3307, 3103, 2922, 2129, 2110, 2036, 1942, 1925, 1905, 1597, 1042. Anal. calcd for C₁₇H₁₁N₃O₆Cl₂Re₂: C, 25.63; H, 1.39; N, 5.27%. Found: C, 25.55; H, 1.64; N, 5.07%.

Synthesis of 3. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a light-protected solution of 2 (50 mg, 0.06 mmol) and 5′-azido-2′,3′-O-isopropylidene adenosine (20 mg, 0.06 mmol) in 8 mL of water/tert-butanol (2/6 v/v) (degassed with argon). The resulting reaction mixture was protected against light and stirred at ambient temperature for 1.5 h. Then, then reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/2 v/v). Chromatography afforded 3 as a yellow solid in 21% (15 mg) yield. MS (TOF ESI−): m/z = 1128.0 (M − H⁺). FTIR (KBr ν [cm⁻¹]): 3299, 2925, 2854, 2049, 2030, 1939, 1911, 1721, 1709, 1633, 1595, 1454, 1213, 1100.

Synthesis of 4. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a light-protected solution of 3 (50 mg, 0.04 mmol) and 3′-azido-3′-deoxythymidine (11 mg, 0.04 mmol) in 8 mL of water/tert-butanol (2/6 v/v). The resulting reaction mixture was protected against light and stirred at ambient temperature for 3 h. Then, the reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting crude residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/2 v/v). Subsequently, the analytically pure 4 was obtained as a yellow solid in 40% (25 mg) yield by preparative HPLC (t = 9.07 min, dichloromethane/methanol 50/2 v/v).

¹H NMR (600 MHz, DMSO-d₆): ¹H NMR (600 MHz, DMSO-d₆): δ = 11.32 (s, 2H, NH thym), 10.04 (d, J_H,H = 6.0 Hz, 1H, H-pydz), 10.01 (bs, 1H, H-pydz), 8.28 (s, 1.5H, 1,2,3-triaz and ½H-pydz), 8.27 (bs, 0.5 H, ½H-pydz), 8.20 (s, 1H, 1,2,3-triaz), 8.19 (s, 1H, ade), 7.94 (s, 1H, ade), 7.81 (d, J_H,H = 0.6 Hz, 1H, thym), 7.32 (s, 2H, NH₂ ade), 6.42 (t, J_H,H = 6.6 Hz, 1H, H1′ deoxyribose), 6.23 (d, J_H,H = 1.8 Hz, 1H, H1′ ribose), 5.46 (dd, J_H,H = 6.3, 1.8 Hz, 1H, H2′ ribose), 5.33 (dt, J_H,H = 8.4, 5.4 Hz, 1H, H3′ deoxyribose), 5.27 (t, J_H,H = 5.4 Hz, 1H, OH), 5.15 (dd, J_H,H = 6.3, 3.6 Hz, 1H, H3′ ribose), 4.76 (dd, J_H,H = 14.4, 5.4 Hz, 1H, H5′ ribose), 4.66 (dd, J_H,H = 14.4, 7.8 Hz, 1H, H5′ ribose), 4.55 (m, 1H, H4′ ribose), 4.19 (app q, J_H,H = 3.6 Hz, 1H, H4′ deoxyribose), 3.96 (s, 2H, CH₂), 3.80 (s, 2H, CH₂), 3.78 (s, 2H, CH₂), 3.70 (m, 1H, H5′ deoxyribose), 3.62 (m, 1H, H5′ deoxyribose), 2.71 (m, 1H, H2′ deoxyribose), 2.64 (m, 1H, H2′ deoxyribose), 1.81 (d, J_H,H = 0.6 Hz, 3H, CH₃ thym), 1.51 (s, 3H, isoprop), 1.30 (s, 3H, isoprop).

¹³C NMR (150 MHz, DMSO-d₆): δ = 191.8, 191.7, 166.3, 157.4, 154.1, 152.3, 150.1, 149.7, 145.0, 142.1, 138.2, 133.0, 130.9, 126.4, 125.0, 120.7, 115.9, 108.1, 91.4, 86.7, 86.6, 86.3, 85.2, 83.1, 62.2, 61.2, 54.9, 52.7, 50.0, 49.9, 49.5, 39.0, 27.4, 25.5, 12.4. MS (TOF ESI−): m/z = 1395.1367 (M − H⁺) (calcd for C₄₀H₃₉N₁₆O₁₃Cl₂Re₂: 1395.1375). FTIR (KBr ν [cm⁻¹]): 3396, 2955, 2923, 2048, 2029, 1939, 1911, 1694, 1642, 1476, 1212, 855. Anal. calcd for C₄₀H₄₀N₁₆O₁₃Cl₂Re₂: C, 34.41; H, 2.89; N, 16.05%. Found: C, 34.58; H, 3.04; N, 15.87%.

Synthesis of 5. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a solution of 2 (50 mg, 0.06 mmol) and 5′-azido-2′,3′-O-isopropylidene adenosine (40 mg, 0.12 mmol) in 8 mL of water/tert-butanol (2/6 v/v). The resulting reaction mixture was protected against light and stirred at ambient temperature for 3 h. Then, the reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/2 v/v). Chromatography afforded the analytically pure compound 5 as a yellow solid in 38% (35 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 10.02 (dd, J_H,H = 5.7, 0.6 Hz, 1H, H-pydz), 9.98 (bd, J_H,H = 1.2 Hz, 1H, H-pydz), 8.27 (s, 2H, 1,2,3-triaz), 8.24 (dd, J_H,H = 5.7, 2.4 Hz, 1H, H-pydz), 8.18 (s, 2H, ade), 7.91 (s, 2H, ade), 7.32 (bs, 4H, NH₂ ade), 6.22 (d, J_H,H = 2.4 Hz, 1H, H1′ ribose), 5.45 (dd, J_H,H = 6.3, 2.4 Hz, 2H, H2′ ribose), 5.14 (dd, J_H,H = 6.3, 3.0 Hz, 2H, H3′ ribose), 4.74 (dd, J_H,H = 14.0, 5.4 Hz, 2H, H5′ ribose), 4.65 (dd, J_H,H = 14.0, 7.8 Hz, 2H, H5′ ribose), 4.53 (m, 2H, H4′ ribose), 3.89 (s, 2H, CH₂), 3.73 (app d, J_H,H = 1.8 Hz, 4H, CH₂), 1.50 (s, 6H, isoprop), 1.29 (s, 6H, isoprop). ¹³C NMR (150 MHz, CD₃OD): δ = 195.6, 195.4, 191.7, 157.3, 154.1, 150.1, 149.6, 144.9, 142.0, 133.0, 126.2, 120.7, 115.9, 91.4, 86.6, 85.2, 83.1, 54.8, 52.7, 49.8, 27.4, 25.5. MS (TOF ESI−): m/z = 1460.1731 (M − H⁺) (calcd for C₄₃H₄₂N₁₉O₁₂Cl₂Re₂: 1460.1752). FTIR (KBr ν [cm⁻¹]): 3334, 2955, 2925, 2048, 2030, 1942, 1912, 1723, 1640, 1597, 1474, 1236, 870, 647. Anal. Calcd for C₄₃H₄₃N₁₉O₁₂Cl₂Re₂: C, 35.34; H, 2.97; N, 18.21%. Found: C, 35.34; H, 3.17; N, 18.00%.

Synthesis of 6. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a solution of 2 (50 mg, 0.06 mmol) and 3′-azido-3′-deoxythymidine (32 mg, 0.12 mmol) in 8 mL of water/tert-butanol (2/6 v/v). The resulting reaction mixture was protected against light and stirred at ambient temperature for 3 h. Then, the reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/5 v/v). Chromatography afforded the analytically pure compound 6 as a yellow solid in 42% (35 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 11.36 (s, 2H, NH thym), 10.07 (d, J_H,H = 6.0 Hz, 1H, H-pydz), 10.05 (bs, 1H, H-pydz), 8.32 (dd, J_H,H = 6.0, 1.8 Hz, 1H, H-pydz), 8.26 (s, 2H, 1,2,3-triaz), 7.82 (d, J_H,H = 0.6 Hz, 2H, thym), 6.43 (t, J_H,H = 6.6 Hz, 2H, H1′ deoxyribose), 5.35 (dt, J_H,H = 9.0, 4.8 Hz, 2H, H3′ deoxyribose), 5.30 (t, J_H,H = 5.4 Hz, 2H, OH), 4.20 (app q, J_H,H = 3.6 Hz, 2H, H4′ deoxyribose), 4.01 (s, 2H, CH₂), 3.85 (s, 4H, CH₂), 3.70 (m, 2H, H5′ deoxyribose), 3.62 (m, 2H, H5′ deoxyribose), 2.71 (m, 2H, H2′ deoxyribose), 2.63 (m, 2H, H2′ deoxyribose), 1.81 (s, 6H, CH₃ thym). ¹³C NMR (150 MHz, CD₃OD): δ = 195.5, 195.4, 191.9, 191.7, 166.3, 164.1, 163.1, 152.3, 149.9, 145.3, 138.1, 133.0, 130.6, 125.1, 111.7, 86.7, 86.4, 62.3, 61.3, 55.0, 50.1, 39.0, 12.4. MS (TOF ESI−): m/z = 1330.1001 (M − H⁺) (calcd for C₃₇H₃₆N₁₃O₁₄Cl₂Re₂: 1330.0997). FTIR (KBr ν [cm⁻¹]): 3434, 3068, 2930, 2048, 2030, 1938, 1913, 1693, 1469, 1273, 1103, 1051, 771. Anal. calcd for C₃₇H₃₇N₁₃O₁₄Cl₂Re₂ and 1.333 n-hexane: C, 37.38; H, 3.88; N, 12.59%. Found: C, 37.54; H, 3.64; N, 11.77%.

Synthesis of 7. To a stirred solution of 1,2,4,5-tetrazine (500 mg, 6.10 mmol) in dichloromethane (5 mL) at ambient temperature, 6-heptynoic acid (770 μL, 6.10 mmol) was added dropwise. Evolution of the dinitrogen gas was observed. The resulting reaction mixture was stirred at ambient temperature for 18 h. Then, all volatiles were evaporated under reduced pressure affording a yellowish solid residue. The residue was subjected to crystallization from a mixture of dichloromethane/n-hexane to afford analytically pure 5-(pyridazine-4-yl)pentanoic acid 7 as a pale yellow crystalline solid in 31% (342 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 12.04 (s, 1H, COOH), 9.12 (bs, 1H, H-pydz), 9.08 (dd, J_H,H = 5.4, 1.2 Hz, 1H, H-pydz), 7.53 (dd, J_H,H = 4.8, 2.4 Hz, 1H, H-pydz), 2.62 (t, J_H,H = 7.8 Hz, 2H, CH₂), 2.24 (t, J_H,H = 7.8 Hz, 2H, CH₂), 1.60 (qui, J_H,H = 7.8 Hz, 2H, CH₂), 1.49 (qui, J_H,H = 7.8 Hz, 2H, CH₂). ¹³C{¹H} NMR (150 MHz, DMSO-d₆): δ = 174.2, 152.7, 151.0, 141.4, 126.1, 33.2, 31.1, 28.5, 23.8. MS (TOF ESI+): m/z = 181.0973 (M + H⁺) (calcd for C₉H₁₃N₂O₂: 181.0977). FTIR (KBr ν [cm⁻¹]): 3073, 2930, 2503, 1934, 1708, 1590, 1032, 673, 492. Anal. calcd for C₉H₁₂N₂O₂: C, 59.99; H, 6.71; N, 15.55%. Found: C, 59.98; H, 6.45; N, 15.46%.

Synthesis of 8. To a stirred solution of Re(CO)₅Cl (1201 mg, 3.32 mmol) in toluene (20 mL) at ambient temperature, ligand 7 (300 mg, 1.66 mmol) was added. The resulting reaction mixture was refluxed for 2 h. Then, all volatiles were evaporated under reduced pressure. The solid residue obtained after evaporation was crystallized from a mixture of dichloromethane/n-hexane to afford the analytically pure complex 8 as a yellow crystalline solid in 87% (1200 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ 12.05 (s, 1H, COOH), 9.12 (s, 1H, H-pydz), 9.08 (d, J_H,H = 4.8 Hz, 1H, H-pydz), 7.53 (dd, J_H,H = 4.8, 1.8 Hz, 1H, H-pydz), 2.62 (t, J_H,H = 7.2 Hz, 2H, CH₂), 2.24 (t, J_H,H = 7.2 Hz, 2H, CH₂), 1.61 (qui, J_H,H = 7.2 Hz, 2H, CH₂), 1.49 (qui, J_H,H = 7.2 Hz, 2H, CH₂). ¹³C NMR (150 MHz, CD₃OD): δ = 195.7, 195.6, 191.8, 191.7, 177.0, 164.9, 163.0, 151.7, 133.4, 34.2, 33.0, 297, 25.4. MS (TOF ESI−): m/z = 790.8998 (M − H⁺) (calcd for C₁₅H₁₁N₂O₈Cl₂Re₂: 790.9007) FTIR (KBr ν [cm⁻¹]): 2049 (CO), 2028 (CO), 1930 (CO), 1900 (CO), 1701 (C=O). Anal. calcd for C₁₅H₁₂N₂O₈Cl₂Re₂: C, 22.76; H, 1.53; N, 3.54%. Found: C, 22.65; H, 1.68; N, 3.69%.

Synthesis of 9. To a stirred solution of 8 (50 mg, 0.06 mmol) in dichloromethane (10 mL) at ambient temperature, propargylamine (3 μL, 3 mg), 4-(dimethyl)aminopyridine (1 mg, 0.01 mmol) and N,N′-dicyclohexylcarbodiimide (31 mg, 0.15 mmol) were added. The resulting reaction mixture was stirred at ambient temperature for 1.5 h. Then, the solution was filtered and all volatiles were evaporated under reduced pressure. The resulting material was subjected to column chromatography on SiO₂ (dichloromethane/methanol 50/1 v/v). After evaporation of the eluting mixture the solid residue was crystallized from dichloromethane/n-hexane to afford the analytically pure compound 9 as a yellow solid in 87% (45 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 10.16 (d, J_H,H = 1.2 Hz, 1H, H-pydz), 10.05 (dd, J_H,H = 6.0, 1.2 Hz, 1H, H-pydz), 8.27 (t, J_H,H = 5.4 Hz, 1H, NH), 8.19 (q, J_H,H = 6.0, 1.8 Hz, 1H, H-pydz), 3.84 (dd, J_H,H = 5.4, 2.4 Hz, 2H, CH₂C≡C), 3.08 (t, J_H,H = 2.4 Hz, 1H, C≡CH), 2.91 (t, J_H,H = 7.2 Hz, 2H, CH₂), 2.15 (t, J_H,H = 7.2 Hz, 2H, CH₂), 1.66 (qui, J_H,H = 7.2 Hz, 2H, CH₂), 1.59 (qui, J_H,H = 7.2 Hz, 2H, CH₂). ¹³C NMR (150 MHz, DMSO-d₆): δ = 195.6, 195.5, 191.8, 191.7, 175.1, 164.9, 163.0, 151.6, 133.4, 80.6, 72.0, 36.0, 34.7, 32.9, 29.8, 26.0. MS (TOF ESI−): m/z = 827.9303 (M − H⁺) (calcd for C₁₈H₁₄N₃O₇Cl₂Re₂: 827.9324). FTIR (KBr ν [cm⁻¹]): 3301, 3069, 2933, 2121, 2048, 2028, 1904, 1659, 1523, 1456. Anal. calcd for C₁₈H₁₅N₃O₇Cl₂Re₂: C, 26.09; H, 1.82; N, 5.07%. Found: C, 26.01; H, 2.08; N, 4.98%.

Synthesis of 10. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a solution of 9 (50 mg, 0.06 mmol) and 5′-azido-2′,3′-O-isopropylidene adenosine (20 mg, 0.06 mmol) in 8 mL of water/tert-butanol (2/6 v/v). The resulting reaction mixture was protected against light and stirred at ambient temperature for 3 h. Then, the reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/3 v/v). Chromatography afforded the analytically pure compound 10 as a yellow solid in 47% (33 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 10.16 (d, J_H,H = 1.8 Hz, 1H, H-pydz), 10.04 (dd, J_H,H = 6.0, 0.6 Hz, 1H, H-pydz), 8.28 (s, 1H, 1,2,3-triaz), 8.27 (t, J_H,H = 5.4 Hz, 1H, CONH), 8.19 (d, J_H,H = 2.4 Hz, 1H, H-pydz), 8.18 (s, 1H, ade), 7.77 (s, 1H, ade), 7.38 (bs, 2H, NH₂ ade), 6.22 (d, J_H,H = 2.4 Hz, 1H, H1′ ribose), 5.45 (dd, J_H,H = 6.0, 2.4 Hz, 1H, H2′ ribose), 5.13 (dd, J_H,H = 6.0, 3.0 Hz, 1H, H3′ ribose), 4.73 (dd, J_H,H = 14.1, 5.4 Hz, 1H, H5′ ribose), 4.65 (dd, J_H,H = 14.1, 7.8 Hz, 1H, H5′ ribose), 4.53 (m, 1H, H4′ ribose), 4.23 (d, J_H,H = 5.4 Hz, 2H, NHCH₂), 2.89 (t, J_H,H = 7.2 Hz, 2H, CH₂), 2.14 (t, J_H,H = 7.2 Hz, 2H, CH₂), 1.65 (qui, J_H,H = 6.6 Hz, 2H, CH₂), 1.58 (qui, J_H,H = 7.2 Hz, 2H, CH₂), 1.52 (s, 3H, isoprop), 1.31 (s, 3H, isoprop). ¹³C NMR (150 MHz, CD₃OD): δ = 195.6, 195.5, 191.7, 164.9, 163.1, 151.6, 133.4, 115.9, 91.5, 86.4, 85.2, 83.1, 54.7, 52.7, 49.5, 36.1, 35.5, 32.9, 32.7, 29.7, 27.4, 26.0, 25.5, 23.6, 14.3. MS (TOF ESI−): m/z = 1160.0671 (M − H⁺) (calcd for C₃₁H₃₀N₁₁O₁₀Cl₂Re₂: 1160.0669). FTIR (KBr ν [cm⁻¹]): 3326, 2954, 2924, 2048, 2029, 1909, 1640, 1597, 1457, 1376, 1212, 1081, 854, 479. Anal. calcd for C₃₁H₃₁N₁₁O₁₀Cl₂Re₂: C, 32.07; H, 2.69; N, 13.27%. Found: C, 32.21; H, 2.90; N, 13.32%.

Synthesis of 11. Sodium ascorbate (10 mg, 0.05 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) were added to a solution of 9 (50 mg, 0.06 mmol) and 3′-azido-3′-deoxythymidine (16 mg, 0.06 mmol) in 8 mL of water/tert-butanol (2/6 v/v). The resulting reaction mixture was protected against light and stirred at ambient temperature for 3 h. Then, the reaction mixture was poured into water and extracted with chloroform. The organic layer was dried over anhydrous Na₂SO₄, filtered through a Schott funnel and all volatiles were evaporated under reduced pressure. The resulting residue was subjected to preparative TLC on SiO₂ (dichloromethane/methanol 50/3 v/v). Chromatography afforded the analytically pure compound 11 as a yellow solid in 45% (30 mg) yield.

¹H NMR (600 MHz, DMSO-d₆): δ = 11.35 (s, 1H, NH thym), 10.16 (d, J_H,H = 1.2 Hz, 1H, H-pydz), 10.04 (dd, J_H,H = 5.4, 1.2 Hz, 1H, H-pydz), 8.34 (t, J_H,H = 6.0 Hz, 1H, CONH), 8.20 (dd, J_H,H = 6.0, 1.8 Hz, 1H, H-pydz), 8.10 (s, 1H, 1,2,3-triaz), 7.80 (d, J_H,H = 1.2 Hz, 1H, thym), 6.40 (t, J_H,H = 6.6 Hz, 1H, H1′ deoxyribose), 5.33 (dt, J_H,H = 8.4, 5.4 Hz, 1H, H3′ deoxyribose), 5.27 (t, J_H,H = 5.4 Hz, 1H, OH), 4.30 (d, J_H,H = 6.0 Hz, 2H, NHCH₂), 4.18 (app q, J_H,H = 3.6 Hz, 1H, H4′ deoxyribose), 3.68 (m, 1H, H5′ deoxyribose), 3.59 (m, 1H, H5′ deoxyribose), 2.91 (t, J_H,H = 7.2 Hz, 2H, CH₂), 2.68 (m, 1H, H2′ deoxyribose), 2.63 (m, 1H, H2′ deoxyribose), 2.18 (t, J_H,H = 7.2 Hz, 2H, CH₂), 1.80 (d, J_H,H = 0.6 Hz, 3H, CH₃ thym), 1.68 (qui, J_H,H = 7.2 Hz, 2H, CH₂), 1.62 (qui, J_H,H = 7.2 Hz, 2H, CH₂) ppm. ¹³C NMR (150 MHz, CD₃OD): δ = 191.7, 175.5, 163.1, 152.3, 151.6, 146.5, 138.1, 133.4, 123.8, 111.7, 86.7, 86.3, 62.2, 61.1, 39.0, 36.1, 35.5, 32.9, 32.7, 29.7, 26.0, 12.4 ppm. MS (TOF ESI−): m/z = 1095.0292 (M − H⁺) (calcd for C₂₈H₂₇N₈O₁₁Cl₂Re₂: 1095.0291). FTIR (KBr ν [cm⁻¹]): 3331, 3067, 2956, 2927, 2047, 2028, 1909, 1688, 1529, 1458, 1272, 1101, 774, 520. Anal. calcd for C₂₈H₂₈N₈O₁₁Cl₂Re₂: C, 30.69; H, 2.58; N, 10.23%. Found: C, 30.82; H, 2.86; N, 10.17%.

X-ray structure determination

Good quality single-crystals of 2 and 8 were selected for the X-ray diffraction experiments at T = 100(2) K. Diffraction data were collected on an Agilent Technologies SuperNova Dual Source diffractometer with CuKα radiation (λ = 1.54184 Å) using CrysAlis Pro software.⁷⁴ The analytical numerical absorption correction using a multifaceted crystal model based on expressions derived by R.C. Clark & J.S. Reid (2) and numerical absorption correction based on Gaussian integration over a multifaceted crystal model (8) were applied.⁷⁵ The structural determination procedure was carried out using the SHELX package.⁷⁶ The structures were solved with the intrinsic phasing method and then successive least-square refinement was carried out based on the full-matrix least-squares method on F² using the SHELXL program.⁷⁶ The hydroxyl H-atom in the case of 8 was located on the Fourier difference electron density map and refined with the O–H bond length restrained to 0.82 Å, with U_iso(H) = 1.5U_eq(O). The remaining H-atoms were positioned geometrically with the C–H bond length equal to 0.93 and 0.97 Å for the aromatic and methylene H-atoms, respectively, and constrained to ride on their parent atoms with U_iso(H) = 1.2U_eq(C). The figures for this publication were prepared using the Olex2 program.⁷⁷ CCDC 2224012 (2) and 2224011 (8) contain the supplementary crystallographic data for this paper.†

Photophysical characterization and DFT calculations

Electronic absorption spectra were measured with a Shimadzu UV 2700 spectrophotometer. Stationary emission was obtained using a Fluorolog-3 spectrofluorometer (model FL3-22, Horiba Jobin Yvon). The spectra were corrected for the spectral sensitivity of the instrument. Photoluminescence curves, recorded as a function of wavelength (λ), were subsequently multiplied by λ² in order to convert counts per wavelength into counts per wavenumber. The quantum yield of luminescence, Φ, was determined by a reference method, using 0.05 mol dm⁻³ quinine sulfate in sulfuric acid (Φ_f = 0.51) as the standard.⁷⁸ Photoluminescence decay lifetimes in a nanosecond range were determined by time-resolved single photon counting (SPC) on an Edinburgh FL 900 CDT spectrofluorometer (excitation by an IBH Nanoled emitting at 370 nm, pulse width <1.5 ns was used). Photoluminescence decays were analyzed using FAST Advanced Analysis of Fluorescence Kinetics software (Edinburgh Instruments, version 3). The DFT calculations were performed with Gaussian 16 (C.01)⁷⁹ quantum-chemical software packages at the M11L^80,81/def2-SVP⁸² theory level. The C-PCM⁸³ polarizable continuum model was applied with the solvent parameters of dichloromethane. Ground-state (S₀) and first excited triplet state (T₁) geometries were found to possess no imaginary frequencies. The reported computational results refer to the optimized T₁ state geometry, as it is most relevant in terms of the discussed photophysics.

Cell viability studies

The effect of compounds 2, 4, 5, 6, 8, 9, 10, 11 and cisplatin (CisPt) on the viability of mammalian cells was determined using the MTT reduction assay. The MTT assay enables one to assess the number of metabolically active (viable) cells in a given cell population. The MTT assay protocol is based on the conversion of water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble formazan product by NAD(P)H-dependent oxidoreductase enzymes, present in viable cells. Human cervical cancer HeLa cells (ATTC®—CCL-2™), endometrial adenocarcinoma Ishikawa cells (ECACC 99040201) and murine fibroblast L929 cells (ATTC®—CCL-1) were plated in 96-well microplates with a density of 1 × 10⁴ cells per well, according to the international standard ISO 10993–5:2009(E) (American National Standard 2009), in Dulbecco's modified Eagle's medium F12 (DMEM-F12, Biowest), supplemented with 10% fetal bovine serum (FBS, Biowest) and 100 units per ml of penicillin G with 100 μg ml⁻¹ of streptomycin (Biowest). After overnight incubation of the cells, at 37 °C, under a humidified atmosphere containing 5% CO₂, the culture medium was removed and replaced with a freshly prepared solution of the compounds in culture medium or medium alone (control). Stock solutions (10 mM) of each compound were prepared in DMSO and diluted with growth medium, supplemented with 10% FBS, to obtain concentrations ranging from 1.56 to 100 μM. Cisplatin (Sigma-Aldrich P4394) was used as the control anticancer drug. The final content of DMSO in the solutions did not exceed 1% and an additional control group with 1% DMSO was included to rule out solvent effects. Cells were incubated with the compounds at 37 °C in 5% CO₂, for 24 h. After incubation, the medium was removed and MTT was added at a concentration of 50 μg per well. The plates were incubated for the next 2 h at 37 °C in 5% CO₂. The formazan crystals were then solubilized in 150 μl of DMSO and quantified by spectrophotometric measurements at 570 nm using the SpectraMax i3 Multi-Mode Platform (Molecular Devices). Each concentration was tested in triplicate with two independent experiments and the results were calculated as a percentage of viable cells of the control group. The IC₅₀ values (compound concentration that inhibits cell viability by 50%) were determined using a nonlinear regression, from the plot of % viability against the log dose of the compounds, using GraphPad Prism 5.01 software.

Antibacterial activity

The antibacterial activity of dinucleosides 4, 5, and 6 and nucleosides 10 and 11 was evaluated using E. coli NCTC 8196 and S. aureus ATCC 6538 strains. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the compound preventing the visible growth of the microorganism using the microdilution method according to EUCAST guidelines [ISO 20776-1 (2006)]. Each compound was pipetted into wells of 96-well microplates as a series of two-fold dilutions in Mueller–Hinton broth in the concentration range from 1.56 to 100 μM. All compounds were dissolved in DMSO. The final concentration of DMSO in the medium was 1% which had no influence on the growth of the microorganisms. Ciprofloxacin hydrochloride and streptomycin sulfate salt were purchased from a commercial supplier and were used as reference antibacterial agents. Then bacteria were added with an inoculum of approximately 5 × 10⁵ CFU mL⁻¹. The plates were incubated at 37 °C for 24 h and the optical density at 600 nm was measured using the SpectraMax i3 Multi-Mode Platform (Molecular Devices). Due to the weak activity of the tested compounds, MIC determination was impossible and the percentage of bacterial growth was calculated for each concentration, and the untreated control was taken as 100% of bacterial growth.

Confocal fluorescence microscopy

For confocal microscopy analysis E. coli NCTC 8196 and S. aureus ATCC 6538 strains were first grown on an L-agar plate to obtain single colonies. The day before microscopic observation, single colonies of each strain were transferred to Mueller–Hinton broth and were cultured for 24 h at 37 °C using an automated shaking incubator (140 rpm). After incubation, the tested dinucleosides (4, 5, and 6) and nucleosides (10, 11) were added to bacteria at a concentration of 10 μM and treated for 1 hour. Then 10 μl of each sample was put on microscopic slides and immediately imaged using a confocal fluorescence microscope. In order to compare the uptake of the tested compounds by Gram-negative and Gram-positive microorganisms, the excitation and fluorescence detection parameters were initially unified, but due to the significant differences in fluorescence intensity between species observed during the experiment, the UV diode laser (405 nm) power for E. coli was set at 5% and for S. aureus at 2% of the nominal potency. The detection parameters remained the same for both species. The emission was collected by the PMT detector in the range of 500–700 nm (gain: 750, offset: 0, scan acquisition speed: 600 Hz, and line average – 6×).

HeLa cells (ATCC Cat. no CCL-2™, human epithelial cells) were cultivated in Dulbecco's modified Eagle's medium F12 (DMEM-F12, Biowest), free of fluorescent markers supplemented with 10% fetal bovine serum (FBS, Biowest) at 37 °C under a humidified atmosphere with 5% CO₂. Twenty-four hours before the experiment, HeLa cells at a density of 1 × 10⁵ mL⁻¹ were plated in a chamber culture slide with a glass bottom and cultured under the same conditions. After that, the cells were incubated for 30 min or 2 h with a freshly prepared 10 μM solution of tested dinucleosides (4, 5, and 6), nucleosides (10 and 11) and their precursors (2 and 9), then rinsed with phosphate buffered saline (PBS) (3×) and immediately imaged under a fluorescence confocal microscope. Similarly, as for prokaryotic cells in the experiment on live HeLa cells to compare the fluorescence intensities of all tested nucleosides and dinucleosides and their precursors as well as the control, the excitation and fluorescence detection parameters were unified. The compounds were excited with a UV diode laser of 405 nm wavelength and 5% power. The emission was collected by the PMT detector in the range of 500–700 nm (gain: 750, offset: 0, scan acquisition speed: 400 Hz, and line average – 3×). In the experiment demonstrating the intracellular localization of the compounds, the analyses were performed after 30 min and 2 hours of incubation with tested compounds. In order to best visualize the cells, the laser power (2–5%) and detection parameters were adjusted to the signal intensity.

All imaging was performed using a DMI 600 SC inverted microscope (HC PL APO CS2 63×/1.40 oil immersion objective or 100×/1.40 oil immersion objective) and a scanning confocal system TCS SP8 (Leica Microsystem, Wetzlar, Germany) operated using LAS 2.0.2.15022 software.

Statistical analysis

All data in the text and figures are provided as mean ± SEM from two independent experiments. The results were analyzed using GraphPad Prism version 5.01, 2007. P < 0.05 was considered as the level of statistical significance.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

K. K. thanks the National Science Center in Cracow, Poland (grant OPUS UMO-2018/29/B/ST5/00055) for financial support. The X-Ray diffractometric measurements were performed at the Core Facility for Crystallographic and Biophysical Research to support the development of medicinal products sponsored by the Foundation for Polish Science (FNP).

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Footnote

† Electronic supplementary information (ESI) available. CCDC 2224012 and 2224011. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d2dt03995h

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