N⁴-Benzoyl-N³-benzyl-2′-deoxycytidine

Abstract

We have recently demonstrated that the pendant tert-butyldiphenylsilyl (TBDPS) groups in the short oligonucleotides TBDPS-5′-CG-3′-3′-GC-5′-TBDPS and TBDPS-5′-CGG-3′-3′-GGC-5′-TBDPS promoted the formation of new lipophilic and stable tetramolecular G-quadruplexes (GQs). Encouraged by these findings, we sought to investigate the effect of alternative lipophilic substituents at the flanking cytidine residues on GQ formation and properties. Herein, we reported on the synthesis and spectroscopic characterization of the new N⁴-benzoyl-N³-benzyl-2′-deoxycytidine, which was obtained during our attempts to synthesize N⁴-benzoyl-5′-O-benzyl-2′-deoxycytidine.

1. Introduction

G-Quadruplexes (GQs) are DNA secondary structures formed by guanine-rich sequences that assemble into stacked G-tetrads through Hoogsteen hydrogen bonding [1]. Their topology and stability are strongly influenced by the nucleotide sequence and the presence of monovalent cations, particularly K⁺ and Na⁺ [2]. Beyond their well-established biological relevance as therapeutic targets [3] in cancer [4,5,6,7] and neurodegenerative diseases [8,9,10], GQs have also emerged as versatile building blocks in supramolecular chemistry and nanotechnology [11,12,13], where their predictable self-assembly, structural polymorphism, and cation-responsive behavior have been exploited for the development of functional materials [14,15], molecular devices [16,17], and biosensing platforms [18,19].

As part of our ongoing efforts to develop modified oligonucleotides for GQ-based supramolecular systems, we recently demonstrated that the introduction of two tert-butyldiphenylsilyl (TBDPS) groups at the 5′-positions of the terminal 2′-deoxycytidine residues in G-rich oligonucleotides containing a 3′–3′ inversion of polarity (1 and 2, Figure 1) promoted the formation of stable GQ constructs (3 and 4) and the G-wire assembly [20].

The lipophilicity of the TBDPS groups has been already investigated by us and other groups to generate GQ aptamers capable of preventing gp120-mediated binding of HIV-1 to CD4 T-cell receptors [21,22].

To investigate the effect of alternative lipophilic protecting groups at the flanking cytidine 5′-positions on GQ assembly and properties, firstly we selected the benzyl group for the introduction at the 5′-position of 2′-deoxycytidine. Herein, we report on the synthesis and spectroscopic characterization of the new N⁴-benzoyl-N³-benzyl-2′-deoxycytidine (5), obtained during our attempts to obtain N⁴-benzoyl-5′-O-benzyl-2′-deoxycytidine (6).

2. Results and Discussion

N⁴-Benzoyl-2′-deoxycytidine (7a, Scheme 1) is a commercially available synthetic intermediate widely used in the preparation of modified nucleosides, nucleotides [23,24,25], and oligonucleotides [26,27,28].

Herein, we investigated whether a selective 5′-O-benzylation of nucleoside 7a could be achieved using one equivalent each of both NaH and BnBr.

The reaction proceeded in 30 min, affording a single product on thin layer chromatography (TLC) with a higher R_f value than the starting material. After chromatographic purification, the isolated compound was characterized by 1D and 2D NMR spectroscopy (Figures S1–S6, Supplementary Materials) and ESI MS (Figure S7). The spectroscopic data were consistent with the structure of compound 5, rather than the 5′-O-benzyl isomer 6. In detail, the resonance of the benzylic carbon at 46.5 ppm provided preliminary evidence that it was bonded to a nitrogen atom rather than to an oxygen atom. The ¹H–¹³C HMBC experiment confirmed that the benzylic carbon was attached to the N³-atom, since heteronuclear correlations were observed between the benzylic methylene protons, centered at approximately 5.34 ppm, and the pyrimidine C2- and C4-carbons, resonating at 150.8 and 156.4 ppm, respectively (Figure S5). Unfortunately, the geometry around the C=N bond of the N⁴-benzoyl imine moiety could not be determined by NOESY spectroscopy (mixing time = 0.3 s) (Figure S6), since the observed NOE correlations could not be unambiguously assigned.

N³-Alkylation reactions of compounds 7a and 2′-deoxycytidine (8a) have some precedents in the literature [29,30,31]. In particular, the direct alkylation at the N³-position of both compounds has been reported even in the absence of an added base, although considerably longer reaction times were required. This behavior could be rationalized by invoking the participation of their minor N⁴-imino tautomeric forms 7b and 8b, respectively. For completeness, all the possible minor tautomeric forms of 7a are summarized in Scheme 1 (7a–d).

In our case, the treatment of compound 7a with benzyl bromide in the absence of a base did not result in alkylation reaction, and only the starting material was recovered. Therefore, the formation of compound 5 may be rationalized through the base-mediated pathway proposed in Scheme 1. Accordingly, treatment of 7a with NaH generated the resonance-stabilized anion 10a–d, in which the negative charge can be delocalized over the N³-C4-N⁴-benzoyl and N³-C2-O² frameworks. Subsequent S_N2 reaction of the N³-centered resonance form (10b) with benzyl bromide would afford compound 5. The exclusive isolation of the N³-benzylated product 5, together with the absence of detectable O²-, 5′-O-, 3′-O- and N⁴-benzylated derivatives, is consistent with a preferential N³-alkylation under the employed reaction conditions. However, the experimental data do not allow the origin of the observed regioselectivity to be established. Likewise, they do not allow the relative acidities of the sugar hydroxyl groups and proton involved in the nucleobase tautomerism to be assessed.

3. Materials and Methods

All the reagents and solvents were commercially available and used without further purification. ¹H and ¹³C NMR spectra were acquired on the Bruker Avance 700 MHz spectrometer (Bruker-Biospin, Billerica, MA, USA) using DMSO-d₆ as solvent. NMR chemical shifts are reported in parts per million (δ) relative to residual solvents signals: (CD₂H)(CD₃)SO 3.54 for ¹H NMR and (CD₃)₂SO 40.4, for ¹³C NMR. The ¹H and ¹³C NMR chemical shifts were assigned through 2D NMR experiments. The NMR spectra were processed with the MestReNova (Mestrelab Research, Santiago de Campostela, Spain) suite. The ESI MS spectrum was acquired on the Applied Biosystem mass spectrometer equipped with a triple quadrupole mass analyzer (ThermoFisher, Waltham, MA, USA). Column chromatography was carried out on silica gel-60 (Merck, 0.063–0.200 mm, Darmstatd, Germany). TLC analyses were carried out on F₂₅₄ silica gel plates (0.2 mm thick, Merck). TLC spots were detected under UV light (254 nm).

N⁴-Benzoyl-N³-benzyl-2′-deoxycytidine (5)

A stirred solution of the compound 7 (0.10 g, 0.30 mmol) in DMF (2.0 mL) was cooled to 0 °C and, under N₂ atmosphere, NaH (7.2 mg, 0.30 mmol) was added in one portion. After 10 min BnBr (36 μL, 0.30 mmol) was added, and the reaction was warmed to r.t. After 30 min (TLC monitoring: AcOEt/CH₃OH, 99:1, R_f = 0.48), the reaction was quenched with saturated NH₄Cl (aq.), diluted with AcOEt (20 mL) and washed with Brine (20 mL). The organic layer was separated, dried over Na₂SO₄, filtered, and concentrated under rotary evaporation. Purification of the crude over a silica gel column eluted with increasing amounts of AcOEt in CH₃OH (up to 2%) afforded pure 5 (80% yield) as an amorphous white solid. ¹H NMR (700 MHz, DMSO-d₆) δ 7.93 (d, J = 8.2 Hz, 1H, 6-H), 7.81–7.87 (m, 2H, orho-H Bz), 7.59–7.55 (m, 1H, para-H Bz), 7.46–7.42 (m, 2H, arom., meta-H Bz), 7.41–7.36 (complex signal, 4H, orho-H and meta-H Bn), 7.35–7.29 (m, 1H, arom., para-H Bn), 6.47 (d, J = 8.1 Hz, 1H, 5-H), 6.26 (apparent t, J = 6.6 Hz, 1H, 1′-H), 5.49–5.23 (complex signal, 3H, CH₂Bn and 3′-OH), 5.07 (t, J = 5.2 Hz, 1H, 5′-OH), 4.32–4.24 (m, 1H, 3′-H), 3.88–3.85 (m, 1H, 4′-H), 3.68–3.54 (m, 2H, 2 × 5′-H), 2.28–2.17 (m, 2H, 2 × 2′-H). ¹³C NMR (APT, 176 MHz, DMSO-d₆) δ 176.7 (C=O), 156.4 (C4), 150.8 (C2), 138.8 (C6), 137.7 (ipso-C Bn), 136.8 (ipso-C Bz), 133.3 (para-C, Bz), 130.1 (ortho-C, Bz), 129.3 (meta-C, Bn), 129.2 (meta-C, Bz), 128.3 (ortho-C, Bn), 128.0 (para-C, Bn), 98.2 (C5), 88.6 (C4′), 86.5 (C1′), 70.9 (C3′), 61.9 (C5′), 46.5 (CH₂Bn), 40.8 (C2′). ESI MS m/z 422, ([M + H]⁺ calcd. for C₂₃H₂₄N₃O₅ 422).

4. Conclusions

Lipophilic oligonucleotides [32,33,34,35,36] represent a class of biomolecules with potential applications in chemical biology and DNA-based biomaterials. During our studies aimed at introducing alternative lipophilic substituents at the 5′-positions of the flanking cytidine residues of GQ-forming oligonucleotides, treatment of N⁴-benzoyl-2′-deoxycytidine (7) with BnBr in the presence of NaH afforded the new N⁴-benzoyl-N³-benzyl-2′-deoxycytidine (5). The formation of compound 5 was rationalized by invoking the selective removal of a proton from the nucleobase with the formation of a resonance-stabilized anion (10a–d) that was readily benzylated at the N³-centered resonance form (10b). Nucleoside 5 provides access for the obtainment of the new N³-benzyl-2′-deoxycytidine derivative, which, after conversion into the corresponding 5′-O-DMT-3′-phosphoramidite, will be exploited for the construction of new short oligonucleotides. The effect of the N³-benzyl substituent on GQ assembly and properties will be also investigated.