Self-Assembly of Functionalized Lipophilic Guanosines into Cation-Free Stacked Guanine-Quartets

Abstract

The hierarchical self-assembly of various lipophilic guanosines exposing either a phenyl or a ferrocenyl group in the C(8) position was investigated. In a solution, all the derivatives were found to self-assemble primarily into isolated guanine (G)-quartets. In spite of the apparent similar bulkiness of the two substituents, most of the derivatives form disordered structures in the solid state, whereas a specific 8-phenyl derivative self-assembles into an unprecedented, cation-free stacked G-quartet architecture.

Introduction

Among DNA bases, guanine (G) exhibits a unique ability to undergo self-assembly, forming various distinct supramolecular structures.^1,2 In particular, lipophilic guanosine derivatives have been reported to generate either supramolecular ribbon-like architectures³⁻⁶ or tetrameric array (G-quartets)-based assemblies.^7,8 The formation of a specific assembly motif is ruled by both the specific chemical structure of the derivative^9,10 and the environmental conditions.^10,11 Among various G assemblies, G-quartet (hereafter G₄)-based supramolecular architectures are by far the most important because of the biological relevance of this structural motif in DNA.^12,13 Many G₄-based lipophilic complexes can be found in the literature, ranging from octamers (two stacked G₄s) and hexadecamers,¹⁴⁻¹⁶ these being the most frequent and studied examples, up to pseudopolymeric stacked assemblies.¹⁷ All these aggregates form spontaneously only in the presence of a metal cation, with the latter playing a crucial role in templating and stabilizing these architectures. For this reason, simple, isolated G₄s are probably the least-described supramolecular structures that originate from guanosine because, in the absence of templating cations, the common self-assembled motif of lipophilic guanosines has a ribbon-like architecture. Isolated G₄s as the main species, either in the absence¹⁸⁻²¹ or in the presence^22,23 of a metal cation, have been indeed seldom reported. Isolated G₄s lacking a templating ion are observed when a bulky substituent is placed at the C(8) position of the nucleobase or, more generally, in cases where the formation of G-ribbons is hindered by the molecular structure. The ability of isolated G₄s, in the absence of a templating cation, to form higher-order organized assemblies has never been reported.

Here, we have focused our attention on both 8-ferrocenyl and 8-phenyl functionalized lipophilic guanosines carrying groups with different bulkiness at the 5′ position (Scheme 1), and we show that subtle modifications in the molecular structure cause a dramatic change in the propensity of the molecules to undergo a hierarchical self-assembly. In particular, lipophilic guanosine derivative 1 (8Ph5Fc) was found to form isolated G₄s, which further self-assemble into stacked cation-free architectures. Conversely, derivatives 2–8, and derivative 8 (8Fc5Ph) especially, where phenyl and ferrocenyl substituents are swapped with respect to 1, exhibit no clear tendency to form supramolecular assemblies arising from piling up of G₄s.

Scheme 1. Chemical Structures of the Investigated Guanosine Derivatives.

Results and Discussion

All investigated G derivatives were synthesized, starting from commercial guanosine, as described in Schemes S1–S4.

The circular dichroism (CD) spectrum of all compounds in CH₂Cl₂ shows only a weak molecular induced circular dichroism (ICD) band, attributed to the chiral perturbation of the guanine chromophore by the chiral sugar residue, and does not change upon the addition of either MeOH or [2.2.2] cryptand: this rules out the presence of templating ions residual from synthetic procedures. In contrast, deliberate addition of KI produces a strong excitonic spectrum, characteristic of G₄ stacking²⁴ (Figure S1).

A typical feature of the ¹H-NMR spectra of G₄ structures is the splitting of the signal for exocyclic amino protons into two signals as hydrogen bonding produces a large downfield shift of the proton involved in G₄ formation.¹⁶ Usually, this signal splitting is not observed at room temperature because the two protons are in fast exchange regime, but it can be easily monitored by lowering the temperature. Figures S2–S9 show the downfield portion of the ¹H-NMR spectrum of the investigated compounds in CD₂Cl₂ at different temperatures in the absence of added metal cations. In all cases, signals that can be ascribed to amino groups start to appear at −5 to −20 °C both in the 9–10 ppm and in the 5–5.5 ppm ranges, and they become progressively sharper upon further lowering the temperature. In the case of 8Ph5Fc (Figure S2), the amino proton signal is baseline-broadened in the CD₂Cl₂ room temperature spectrum. At −5 °C, two signals start to appear at 9.8 and 5.5 ppm for bound-N(2)H and free-N(2)H, respectively, and they shift slightly downfield and upfield, respectively, by further lowering the temperature. Moreover, the N(1)H imino proton signal resonates at 12.6 ppm at room temperature and undergoes deshielding upon lowering the temperature. The same signal resonates at 10.9 ppm in dmso-d₆, suggesting that in CD₂Cl₂, G is H-bonded already at room temperature. All the other investigated compounds show the same behavior, yet they differ in two aspects. First, the two amino signals start to appear only at lower temperatures (compare −20 °C spectra shown in Figures S2–S9). Second, imino and amino signals of the second assembled species (e.g., 12.38 and 9.98 ppm for 8Fc5C10, Figure S6) become visible in most cases (with the exception of the two 5’OH derivatives 8Ph5OH and 8Fc5OH, see below) in the downfield portion of the spectrum. No clear evidence could be obtained on the architecture of these minor species. Nonetheless, this suggests a higher stability for the aggregate formed by 8Ph5Fc, while for derivatives 2–8, other, although minor, self-assembly pathways are viable.

Further evidence of the existence of self-assembled species in solution is given by nuclear Overhauser effect (NOE) spectra (Figures 1 and S10–S16). As the first general observation, in all cases, NOE spectra display cross-peaks having the same phase as the diagonal. This feature is characteristic of a slow tumbling regime, implying that the objects in the solution have a molecular weight above ca. 2000 Da,²⁵ whereas the molecular weight of an isolated molecule of 1–8 falls in the range of 667–929 Da. Another common feature of the main species formed by all of the investigated compounds is their preference for the syn conformation around the glycosidic bond. Figure 1b,c shows selected one-dimensional NOE spectroscopy (1D-NOESY) spectra of 1 recorded at −20 °C as an example. The interaction between “o” and H1’ protons, already strong at room temperature (not shown), and the absence of any correlation between H5’/H5” and phenyl protons points to an exclusive syn conformation for 1, as depicted in the inset of Figure 1a. Spectra reported in Figures S10–S16 point to the same conclusion. In the case of derivatives 8Ph5OH and 8Fc5OH, lacking a bulky substituent at the 5′ position, the syn conformer is possibly stabilized by an intramolecular H-bond between 5’OH and N(3) (see spectrum d in Figure S10).

Figure 1.

(a) Downfield portion of the 600 MHz ¹H-NMR spectrum of 8Ph5Fc in CD₂Cl₂ (4.3 mM) at −20 °C and signal assignment (diastereotopic protons were not assigned); (b) NOESY-1D spectrum of the same sample upon irradiation at 7.83 ppm (“o” protons); (c) NOESY-1D spectrum of the same sample upon irradiation at 5.82 ppm (1′ proton). In each NOE spectrum were used 512 coadded transients, a recycle delay of 0.6 s, a mixing time of 0.6 s, and a 50 Hz shaped pulse. Irradiated frequencies are indicated with an arrow.

It is known²⁶ that a downfield shift of the ribose H2’ proton parallels an increase in the percentage of syn conformer population. In particular, 8-bromoguanosine (δ_H2’ = 5.01 ppm in DMSO-d₆) is ca. 90% syn, while guanosine (δ_H2’ = 4.429 ppm) is only 40% syn. Although a direct comparison with these figures is not possible, an analysis of the observed chemical shifts for derivatives 1–8 (see Table S1) allows some qualitative conclusions. The introduction of ferrocenyl substituent at the 8-position exhibits a marked effect on the chemical shift of both H1’ and H2’ sugar protons, which move downfield by about 1 ppm. On the other hand, the 8-phenyl substituent exhibits a smaller effect and is limited to the H2’ and H3’ protons. Taking this result into account, it is possible to notice that the H2’ proton tends to resonate at lower fields than H1’ for the 8-phenyl derivatives, while 8-ferrocenyl derivatives show the same chemical shift order (δ_H1’ > δ_H2’ > δ_H3’) as the reference compounds (entries h–n in Table S1). This suggests that the phenyl ring has a higher ability to drive the conformational equilibrium toward the syn isomer than the ferrocenyl substituent. In particular, the unique sequence δ_H3’ > δ_H2’ > δ_H1’ shown by 8Ph5Fc in CD₂Cl₂ should be noticed.

NOE spectra also provide unambiguous information on the structure of the main self-assembled species in solution. Another feature common to the main species formed by 1–8 is the dipolar coupling between the protons of the substituent at the C(8) position and the exocyclic amino group. For example, in the case of 8Ph5Fc, the o protons interact with both exocyclic amino and ferrocene protons (Figure 1b). These proximities can only occur at the intermolecular level and rule out G-ribbon assemblies (see Figure S17) but point to a G₄ structure (such as the one presented in Figure 2 in the case of 8Ph5Fc) as the main self-assembled aggregate formed by all the derivatives.

Figure 2.

Proposed model for the isolated G₄ formed by 8Ph5Fc. Arrows indicate selected NOEs. Some atoms are omitted for clarity.

Stabilization of this structure by residual water²⁷ seems unlikely because in no case did we observe in NOE spectra a signal in the 1.5–2.5 ppm region, except for the expected exchange signals with N(1)H and N(2)H.

To gain deeper insights into the propensity of the derivatives to undergo self-assembly, we have extended our study to atomic force microscopy (AFM) imaging. In addition to 8Ph5Fc, derivatives 8Fc5C10 and 8Ph5C10 were studied for comparison. The samples were prepared by drop-casting 150 μL of guanosine solutions in CH₂Cl₂ (concentration 0.6 mg/mL) onto the basal plane of thermally grown SiO₂/Si (230 nm SiO₂ on Si [100]). Prior use, the substrates were cleaned and functionalized with a monolayer of hexamethyldisilazane (HMDS), covalently bonded to the pending SiOH groups, in order to render the surface hydrophobic and to avoid the formation of hydrogen bonds between the molecules and the substrate. Figure 3 displays the topographical AFM images of films of 8Ph5Fc (Figure 3a,c) and 8Fc5C10 (Figure 3b,d) prepared using the same experimental conditions.

Figure 3.

AFM images of supramolecular metal-free G aggregates drop-cast from CH₂Cl₂ 0.6 mg/mL on HMDS-functionalized SiO₂. (a,c) Molecule 8Ph5Fc. (b,d) Molecule 8Fc5C10. Images Z-scales: (a) 10 nm; (b) 140 nm; (c) 7 nm; and (d) 110 nm.

The observed morphologies are markedly different. Molecule 8Fc5C10 forms a disordered network of entangled bundles of fibers, while 8Ph5Fc self-assembles into layers of ordered and well-aligned fibers possessing an average height of 4.1 ± 0.5 nm (measured with respect to the widest empty areas) and an average width of 32 ± 4 nm. Being the lateral size of the fibers comparable to the curvature radius of the used AFM tips (nominal radius 7 nm), the measured width value must be corrected to exclude the broadening artifact given by the mechanical convolution effect. Considering the measured width and an interfiber penetration of the tip of ∼1.8 nm, we can apply well-established models²⁸ to conclude that the real width of such fibers amounts to 19 ± 4 nm. As the radius of the G-quartet structure in columnar aggregates, both in water²⁹ and in organic solvents,¹⁷ amounts to 12.5 Å and a fully elongated decanoyl residue measures ca.12 Å, it is possible to estimate a diameter of 4.8 nm for the G₄s formed by 8Ph5Fc. This value is in good agreement with the average measured height for the fibers. Assuming a 3.3 Å spacing (characteristic of optimal nucleobase stacking³⁰), the cylindrical fibers shown in Figure 3a,c are composed on an average of six stacked G₄s.

To promote the aggregation into more stable and ordered supramolecular structures, the films were then exposed to solvent vapor annealing (SVA) in CH₂Cl₂ for 48 h under ambient conditions. Such a method makes it possible to trigger the molecular reorganization toward the generation of thermodynamically favored architectures. It is noteworthy that this method has previously been successfully used to finely tune the self-assembly of various molecular systems, including n-type perylene nanowires,³¹ p-type pentacene,³² hexa-peri-hexabenzocoronene (HBC)³³ structures, and porphyrins.³⁴Figure 4 portrays the AFM images of the 8Ph5Fc (a,c) and 8Fc5C10 (b,d) fibers formed on the surface after the SVA treatment.

Figure 4.

AFM images of supramolecular metal-free G aggregates drop-cast from CH₂Cl₂ 0.6 mg/mL on HMDS-functionalized SiO₂ after SVA (CH₂Cl₂ for 48 h). (a,c) Compound 8Ph5Fc. (b,d) Compound 8Fc5C10. Images Z-scales: (a) 13 nm; (b) 25 nm; (c) 12 nm; and (d) 20 nm.

Interestingly, the two compounds showed a similar behavior: in both cases, SVA caused an increased entanglement of the fibrillar structures, resulting in an extremely uniform coverage of the underneath substrate, which is no longer exposed. In particular, in the case of 8Ph5Fc, the as-deposited fibers show high rigidity and no tendency to entangle, causing the formation of holes and discontinuities in the resulting films, as shown in Figure 3a,c. After the SVA treatment, the fibers assemble into less-ordered bundles, forming a multilayered uniform film (Figure 4a,c). In the latter case, the fibers feature an average measured width of 41 ± 3 nm, suggesting that the packing at the molecular level is not affected by the SVA treatment, but it is not possible to correct the data with the previously used model because of the multilayer nature of the film after SVA. In the case of compound 8Fc5C10, the SVA process exhibits a strong impact over the morphology of the films that undergo a dramatic decrease in roughness from 23.2 nm (before SVA, Figures 3b,d) to 4.15 nm (after SVA, Figures 4b,d). Moreover, the compound 8Fc5C10 fibers show completely different conformations: before SVA, the fibrillar structure is hardly visible, while after that, the fibrillar units are extremely well defined and form a strongly entangled network.

On the other hand, samples of 8Ph5C10 showed no tendency to form crystalline structures. In this case, AFM characterization has not revealed any well-defined structures, and the films look amorphous (see Figure S18).

Conclusions

In summary, we have designed and synthesized several organic soluble guanosines carrying either a phenyl or a ferrocenyl group at the C(8) position. Their self-assembly has been studied in solution by NMR and in thin films by means of AFM. Derivative 8Ph5Fc shows the exclusive existence of isolated G₄s in the CD₂Cl₂ solution, while compounds 8Fc5C10 and 8Ph5C10 do form isolated G₄s as well, but another aggregated species is present in equilibrium in solution. The hierarchical self-assembly of 8Ph5Fc and 8Fc5C10 leads to the formation of fibers, which can be deposited and characterized on surfaces. Guanosine 8Fc5C10 shows the formation of a disordered film, arising from the coexistence of different self-assembled species (probably a mixture of stacked G₄s and H-bonded dimeric/oligomeric ribbon-like aggregates), whereas 8Ph5Fc cleanly assembles into unprecedented, stacked cation-free G₄ architectures. The ferrocenyl moiety of 8Fc5C10 seems to be not large enough to drive the self-assembly toward the exclusive existence of isolated G₄s in solution, while its shape seems to hamper π-stacking and hence a shift in equilibria toward (stacked) quartets in the solid state. On the other hand, the results obtained for 8Ph5C10 indicate that the self-assembly is not ruled uniquely by the nature of the C(8) substituent: the behavior observed only for 8Ph5Fc suggests that also the nature of the 5′ substituent, although irrelevant for G₄ formation, affects the subsequent self-assembly. These results further expand the range of supramolecular architectures that can be obtained in thin films and provide unambiguous evidence for the key role played by the subtle design of guanosine starting building blocks to control the hierarchical self-assembly motifs that can be attained.

Experimental Procedures

General Methods

All reactions that require anhydrous conditions were carried out under a dry argon atmosphere in oven-dried glassware. Macherey-Nagel polygram silica gel plates (layer thickness 0.20 mm) were used for thin layer chromatography (TLC) analyses. Column chromatography was performed on Geduran silica gel 60 (40–63 μm). Reagents and solvents, including dry solvents, were purchased from Sigma-Aldrich or TCI. Electrospray ionization (ESI) mass spectra were obtained from methanol solutions in either the positive or negative mode, with Micromass ZMD 4000 or ZQ-4000 instruments. High-resolution mass spectrometry (HRMS) spectra were recorded on a Waters Xevo G2-XS QTof system. Nuclear magnetic resonance (NMR) spectra were recorded on Varian Inova (600, 400, or 300 MHz) spectrometers and referenced to the residual solvent resonance (s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, qi = quintet, m = multiplet; coupling constant are in Hz). Structural assignments were made with additional information from gCOSY (gradient-selected correlation spectroscopy), gHSQC (gradient-selected heteronuclear single-quantum correlation), and gHMBC (gradient-selected heteronuclear multiple bond correlation) experiments. Diastereotopic protons/carbons were not assigned. CD spectra were recorded on a Jasco J-710 spectropolarimeter. Synthetic schemes with compound numbering are reported in the Supporting Information (S1–S4)

Microscopy Studies

The AFM study of the self-assembled 1, 4, and 5 was performed using Veeco/Bruker Dimension 3100 that runs with a Nanoscope IV controller. Solutions of investigated molecules were prepared in 0.6 mg/mL CH₂Cl₂ and deposited by drop-casting (150 μL) on clean SiO₂/Si (230 ± 10 nm SiO₂ on [100] Si) functionalized with a monolayer of HMDS. The SVA treatment was performed by placing the compound films in a sealed environment and saturating with CH₂Cl₂ vapor under ambient conditions, for 48 h. The raw AFM data were processed through the application of background flattening.

8-Bromo Guanosine 9

The compound was prepared according to a literature procedure.³⁵ Thus, commercial guanosine 5 (1.00 g, 3.53 mmol) was suspended in 60 mL of an acetonitrile/water 2:1 mixture, and N-bromosuccinimide (943 mg, 5.3 mmol, 1.5 eq) was added in three portions over 20 min. Stirring was continued until TLC (CH₂Cl₂/MeOH 8:2) revealed the disappearance of the starting material (2 h). Solvents were then removed in vacuo, and the pale-yellow solid thus obtained was suspended in acetone (20 mL) and stirred at room temperature for 2 h. The flask was then placed in a refrigerator and left overnight at −20 °C. The precipitate was filtered and washed several times with cold acetone to afford 1.09 g, 3.02 mmol (yield 86%) of 8-bromo guanosine 9 as a white solid.

ESI-MS (m/z): 362.1/363.9 [M + H]⁺; 383.9/386.0 [M + Na]⁺; 360.1/362.0 [M – H]⁻.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₁₀H₁₂BrN₅O₅Na 383.9914; found 383.9919.

¹H-NMR (dmso-d₆, 600 MHz): δ 10.81 (bs, 1H, NH), 6.50 (bs, 2H, NH₂), 5.68 (d, 1H, J = 6.0 Hz, H1’), 5.44 (d, 1H, J = 6.0 Hz, OH2’), 5.08 (d, 1H, J = 5.1 Hz, OH3’), 5.01 (m, 1H, H2’), 4.92 (m, 1H, OH5’), 4.13 (m, 1H, H3’), 3.85 (m, 1H, H4’), 3.63 (m, 1H, H5’), 3.54 (m, 1H, H5’). ¹³C{¹H} NMR (dmso-d₆, 75 MHz): δ 155.9, 153.9, 152.5, 121.5, 118.0, 90.1, 86.3, 70.9, 70.7, 62.5

8-Bromo-5’-O-tert-butyldimethylsilyl Guanosine 10

8-Bromo guanosine 9 (1.274 g, 3.52 mmol) was dried at 50 °C over P₂O₅ in vacuo for 2 h and dissolved in dimethylformaimde (DMF) (17 mL). Imidazole (491 mg, 7.21 mmol, 2.05 eq) was added. To the resulting mixture was then added dropwise a solution of t-butyldimethylsilyl chloride (558 mg, 3.70 mmol, 1.05 eq.) in tetrahydrofuran (THF) (9 mL), and stirring was continued for 2 h. As TLC (CH₂Cl₂/MeOH 8:2) revealed the presence of unreacted starting material, an extra amount of imidazole (120 mg, 1.76 mmol, 0.5 eq.) and t-butyldimethylsilyl chloride (266 mg, 1.76 mmol, 0.5 eq.) was added, and reaction was continued for 2 h. The crude mixture was poured into water (50 mL), and the precipitate was filtered, washed with water and Et₂O, and dried to afford the product (1.04 g, 2.19 mmol, 62%) as a white solid.

RF = 0.6 (CH₂Cl₂/MeOH 8:2).

ESI-MS (m/z): 473.9/475.9 [M – H]⁻; 475.9/477.9 [M + H]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₁₆H₂₇BrN₅O₅Si 476.0959; found 476.0965.

¹H-NMR (CD₃OD, 600 MHz): δ 5.85 (d, 1H, J = 4.2 Hz, H1’), 5.26 (dd, 1H, J = 5.7 Hz, J = 4.2 Hz, H2’), 4.56 (m, 1H, J = 5.7 Hz, H3’), 3.95 (m, 1H, J = 11.4 Hz, J = 5.7 Hz, J = 4.0 Hz, H4’), 3.92 (1H, dd, J = 11.4 Hz, J = 4.0 Hz, H5’), 3.83 (dd, 1H, J = 11.4 Hz, J = 5.7 Hz, H5’), 0.84 (s, 9H, tBuSi), 0.01 and −0.03 (s,s, 6H, SiMe₂). ¹³C{¹H} NMR (CD₃OD, 151 MHz): δ

8-Phenyl-5’-O-tert-butyldimethylsilyl Guanosine 11

PdCl₂(PPh₃)₂ (59.0 mg, 0.084 mmol, 0.2 eq.) was added to a degassed solution of 10 (200 mg, 0.421 mmol), phenylboronic acid (77 mg, 0.630 mmol, 1.5 eq.), and K₃PO₄ (223 mg, 1.05 mmol) in 7 mL of a 6:1 mixture of dioxane/water. The mixture was heated at 95 °C for 22 h in an oil bath. After cooling to room temperature, solvents were removed by distillation, the dark residue was suspended in 50 mL of Et₂O and filtered. The filtrate was washed with brine (40 mL) and 1 N HCl (6 mL) and then dried over MgSO₄. The precipitate was washed with a CH₂Cl₂/MeOH 4:1 mixture; washings were combined with the ethereal residue, and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH 95:5), and the product was obtained in a 61% yield (121 mg, 0.256 mmol) as a white solid.

RF = 0.2 (CH₂Cl₂/MeOH 9:1).

ESI-MS (m/z): 472.4 [M – H]⁻; 474.1 [M + H]⁺; 496.2 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₂₂H₃₂N₅O₅Si 474.2167; found 474.2173.

¹H-NMR (dmso-d₆, 600 MHz): δ 10.73 (bs, 1H, NH), 7.67–7.64 (m, 2H, ArH), 7.54–7.52 (m, 3H, ArH), 6.43 (bs, 2H, NH₂), 5.60 (d, 1H, J = 5.5 Hz, H1’), 5.36 (d, 1H, J = 6.0 Hz, OH), 5.07 (m, 1H), 4.94 (d, 1H, J = 6.0 Hz, OH), 4.13 (m, 1H), 3.84–3.74 (m, 3H, H4’,H5’, H5″), 0.841 (s, 9H, tBuSi), 0.006 and 0.003 (s,s, 6H, SiMe₂). ¹³C{¹H} NMR (dmso-d₆, 75 MHz): δ 157.2 (C), 153.5 (C), 152.7 (C), 147.8 (C), 130.7 (C), 129.9 (CH, Ph), 129.5 (CH, Ph), 129.1 (CH, Ph), 117.4 (C), 89.7 (C1’), 85.3 (C4’), 70.7 (C3’), 70.5 (C2’), 64.1 (C5’), 26.3 (CH₃), 18.5 (C), −4.8 (CH₃), −4.8 (CH₃).

8-Phenyl-5’-O-tert-butyldimethylsilyl-2′,3’-O-didecanoyl Guanosine 3 (8Ph5Si)

8-Phenyl-5’-O-tert-butyldimethylsilyl guanosine 11 (100 mg, 0.211 mmol) was dried in vacuo over P₂O₅ at 50 °C for 1 h and dissolved in 15 mL of an acetonitrile/toluene 2:1 mixture. Decanoic anhydride (163 μL, 0.444 mmol, 2.10 eq.), Et₃N (64 μL, 0.444 mmol, 2.10 eq.), and a small amount of 4-dimethylaminopyridine (DMAP) were then added, and the mixture was heated at 80 °C in an oil bath for 7 h. After cooling to room temperature, MeOH (0.5 mL) was added, and stirring was continued for 20 min. Solvents were removed by distillation, the residue was dissolved in CH₂Cl₂, washed with 5% NaHCO₃ and brine, and dried over MgSO₄. After the removal of solvents, the residue was purified by chromatography on silica gel (CH₂Cl₂/MeOH 99:1), and the product (120 mg, 0.153 mmol) was obtained as a white solid in 72% yield.

RF = 0.36 (CH₂Cl₂/MeOH 97:3).

ESI-MS (m/z): 780.6 [M – H]⁻; 782.6 [M + H]⁺; 804.6 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₄₂H₆₇N₅O₇SiNa 804.4702; found 804.4713.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.38 (bs, 1H, NH), 7.77 (d, 2H, orto-ArH), 7.54–7.59 (m, 3H, ArH), 6.37 (m, 1H, H2’), 6.17 (m, 1H, H3’), 5.77 (d, 1H, J = 3.2 Hz, H1’), 4.14 (m, 1H, H4’), 3.90 (1H, dd, J = 12.0 Hz, 4.2 Hz, H5’), 3.78 (dd, 1H, J = 12.0 Hz, 5.0 Hz, H5’), 2.28 (t, 2H, −CH₂–CO), 2.24 (t, 2H, −CH₂–CO), 1.60 (m, 2H, CH₂–CH₂–CO), 1.52 (m, 2H, CH₂–CH₂–CO), 1.28–1.23 (m, 24H, CH₂), 0.87 and 0.85 (t,t, 6H, CH₃), 0.78 (s, 9H, tBuSi), 0.07 and 0.04 (s,s, 6H, SiMe₂). ¹³C{¹H} NMR (CD₂Cl₂, 151 MHz): δ 172.4 (C=O), 172.3 (C=O), 159.4 (C), 153.6 (C), 152.4 (C4), 148.6 (C8), 130.0 (CH, Ph), 129.5 (C), 129.3 (CH, Ph), 129.1 (CH, Ph), 116.9 (C), 87.5 (C1’), 82.0 (C4’), 72.1 (C2’), 70.3 (C3’), 62.6 (C5’), 34.0 (CH₂), 33.9 (CH₂), 32.01 (CH₂), 31.97 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.4 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 25.6 (CH₃), 24.9 (CH₃), 24.9 (CH₂), 22.8 (CH₂), 22.8 (CH₂), 18.2 (C), 14.0 (CH₃), 14.0 (CH₃), −5.6 (CH₃), −5.7 (CH₃).

8-Phenyl-2′,3’-O-didecanoyl Guanosine 2 (8Ph5OH)

To a solution of 3 (120 mg, 0.153 mmol) in THF (4 mL) were added 72.5 mg (0.230 mmol, 1.5 eq) of tetra-n-butylammonium fluoride (TBAF). The mixture was stirred at room temperature until TLC (CH₂Cl₂/MeOH 92:8) showed complete disappearance of the starting material. The reaction mixture was concentrated, redissolved in CH₂Cl₂, and washed with sat. NaHCO₃ (3 × 10 mL). The organic phase was dried over MgSO₄, and solvents were removed by distillation. The residue was purified on silica gel by first eluting with Et₂O and then with CH₂Cl₂/MeOH 95:5. The product was obtained as a white solid in a 73% yield (75 mg, 0.112 mmol).

RF = 0.28 (CH₂Cl₂/MeOH 92:8).

ESI-MS (m/z): 666.6 [M – H]⁻; 668.4 [M + H]⁺; 690.4 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₃₆H₅₃N₅O₇Na 690.3837; found 690.3846.

¹H-NMR (dmso-d₆, 600 MHz): δ 10.84 (bs, 1H, NH), 7.61–7.58 (m, 2H, o-ArH), 7.55–7.53 (m, 3H, m- and p-ArH), 6.48 (bs, 2H, NH₂), 6.18 (t, 1H, OH), 5.78 (d, 1H, J = 6.0 Hz, 1’H), 5.49 (m, 1H, 2’H), 5.18 (m, 1H, 3’H), 4.08 (m, 1H, 4’H), 3.75 (m, 1H, 5’H), 3.64 (m, 1H, 5’H), 2.35–2.16 (m, 4H, CH₂–CO), 1.48 and 1.40 (qi, qi, 4H, CH₂–CH₂–CO), 1.27–1.12 (m, 24H, CH₂), 0.85 and 0.84 (t,t, 6H, CH₃). ¹³C{¹H} NMR (dmso-d₆, 151 MHz): δ 172.3 (C=O 3′), 171.8 (C=O 2′), 159.6 (C), 153.7 (C), 153.5 (C), 147.4 (C8), 131.9 (CH, p-Ph), 130.5 (C, Ph), 129.9 (CH, o-Ph), 129.2 (CH, m-Ph), 119.6 (C), 88.1 (C1’), 85.1 (C4’), 72.1 (C3’), 71.7 (C2’), 61.6 (C5’), 33.9 (CH₂), 33.6 (CH₂), 31.8 (CH₂), 29.3 (CH₂), 29.3 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.2 (CH₂), 29.2 (CH₂), 29.0 (CH₂), 28.9 (CH₂), 24.8 (CH₂), 24.5 (CH₂), 22.6 (CH₂), 14.0 (CH₃).

8-Phenyl-5’-O-ferrocenoyl-2′,3′-O-didecanoyl Guanosine 1 (8Ph5Fc)

Ferrocene carboxylic acid (77.5 mg, 0.337 mmol, 1.3 eq.) was dried in vacuo at 50 °C for 1 h and dissolved in THF (9 mL). Et₃N (175 μL, 1.217 mmol, 4.7 eq.) was added, and the resulting solution was cooled to 0 °C. Methanesulfonyl chloride (25.5 μL, 0.311 mmol, 1.2 eq.) was added, and the mixture was allowed to warm to room temperature and stirred for 2 h. A solution of vacuum-dried 8-phenyl-2′,3′-O-didecanoyl guanosine 2 (173 mg, 0.259 mmol) in THF (6 mL) was then added, followed by a catalytic amount of DMAP. Stirring was continued for 24 h, MeOH (0.5 mL) was added, and the reaction mixture was concentrated in vacuo. The residue was dissolved in CH₂Cl₂, washed with water, and dried over MgSO₄. The solvent was removed by distillation, and the residue was purified by chromatography on silica gel (gradient from CH₂Cl₂ to CH₂Cl₂/MeOH 99:1), affording the product (85 mg, 0.097 mmol) as a yellow solid in 38% yield.

RF = 0.24 (CH₂Cl₂/MeOH 9:1).

ESI-MS (m/z): 878.6 [M – H]⁻; 880.5 [M + H]⁺; 902.5 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₄₇H₆₁FeN₅O₈Na 902.3762; found 902.3776.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.57 (bs, 1H, NH), 7.81 (d, 2H, J = 7.1 Hz, Ph), 7.56 (t, 2H, J = 7.1 Hz, Ph), 7.49 (t, 1H, J = 7.1 Hz, Ph), 6.48 (dd, 1H, J = 7.8 Hz, 5.4 Hz, H3′), 6.43 (dd, 1H, J = 5.4 Hz, 3.0 Hz, H2’), 5.85 (d, 1H, J = 3.0 Hz, H1’), 4.67 (s, 1H, Fc), 4.62 (dd, 1H, J = 12.0 Hz, 4.3 Hz, H5’), 4.61 (s, 1H, Fc), 4.41 (m, 1H, H4’), 4.34 (dd, 1H, J = 12.0 Hz, 4.3 Hz, H5’), 4.20 (s, 1H, Fc), 4.17 (s, 1H, Fc), 3.97 (s, 5H, Fc), 2.36–2.26 (m, 4H, CH₂–CO), 1.67–1.52 (m, 4H, CH₂–CH₂–CO), 1.33–1.20 (m, 24H, CH₂), 0.88–0.84 (m, 6H, CH₃). ¹³C{¹H} NMR (CD₂Cl₂, 151 MHz): δ 172.5 (C, CO(3’)), ’)), 117.1 (C), 87.6 (CH, C1’), 79.9 (CH, C4’), 72.5 (CH, C2’), 71.8 (CH, Fc), 71.7 (CH, Fc), 70.1 (C, Fc), 70.1 (CH, Fc), 70.0 (CH, C3’), 69.8 (CH, Fc), 61.7 (CH₂, C5’), 34.0 (CH₂), 33.9 (CH₂), 32.0 (CH₂), 32.0 (CH₂), 29.8 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.4 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 25.7 (CH₂), 24.9 (CH₂), 22.8 (CH₂), 14.0 (CH₃)

8-Phenylguanosine 12

To a degassed mixture of 9 (273 mg, 0.754 mmol), phenylboronic acid (110 mg, 0.902 mmol), Na₂CO₃ (160 mg, 1.51 mmol), tris(3-sulfonatophenyl)phosphine (TPPTS) (11 mg, 0.0194 mmol) in H₂O (2.4 mL), and acetonitrile (1.2 mL) were added 44 mg (0.0196 mmol) of Pd(OAc)₂, and the reaction mixture was stirred at 83 °C in an oil bath overnight. TLC (RP18, H₂0/CH₃OH 1:1) confirmed the disappearance of the starting material. The reaction mixture was then cooled to room temperature and diluted with water (20 mL). The pH was adjusted to 7 with 1 N HCl. The resulting suspension was refluxed for a short time, cooled to room temperature, and maintained at 4 °C for 2 h. The precipitate was filtered, washed with water, and air-dried to afford the product as a white solid in 80% yield (216 mg, 0.60 mmol). The compound was sufficiently pure and was used in the following step without further purification.

ESI-MS (m/z): 360.2 [M – H]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₁₆H₁₈N₅O₅, 360.1302; found 360.1308.

¹H-NMR (dmso-d₆, 400 MHz): δ 10.75 (s, 1H, NH), 7.68–7.65 (m, 2H, Ph), 7.54–7.52 (m, 3H, Ph), 6.38 (bs, 2H, NH₂), 5.64 (d, 1H, J = 6.4 Hz, H1’), 5.36 (d, 1H, J = 6.4 Hz, OH_2′), 5.05–5.00 (m, 2H, H2′, OH_5′), 4.96 (d, 1H, J = 5.6 Hz, OH_3′), 4.09–4.06 (m, 1H, H3’), 3.85–3.81 (m, 1H, H4’), 3.70–3.65 (m, 1H, H5’), 3.58–3.52 (m, 1H, H5’).¹³C{¹H} NMR (dmso-d₆, 101 MHz): δ 157.0 (C), 153.5 (C), 152.4 (C), 147.9 (C), 130.5 (C, Ph), 129.8 (CH, Ph), 129.6 (CH, Ph), 129.0 (CH, Ph), 117.6 (C), 89.4 (CH), 86.3 (CH), 71.0 (CH), 70.7 (CH), 62.5 (CH₂).

8-Phenyl-2′,3′,5′-O-tridecanoyl Guanosine 4 (8Ph5C10)

To a stirred suspension of 12 (97 mg, 0.27 mmol) in MeCN (4 mL) were added Et₃N (0.19 mL, 1.35 mmol), decanoic anhydride (0.33 mL, 0.89 mmol), and a catalytic amount of DMAP. The mixture was stirred at room temperature and monitored by TLC (DCM/MeOH 8:2). After 2 h, the mixture was concentrated under reduced pressure, and the resulting oil was partitioned between DCM and water. The organic layer was collected, the solvent was removed by distillation, and the residue was purified by column chromatography on silica (DCM/acetone 9:2.5 to remove decanoic acid and then DCM/MeOH 9:1) to afford triester 4 as a white waxy solid in 59% yield (131 mg, 0.16 mmol).

ESI-MS (m/z): 822.6 [M + H]⁺, 844.6 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₄₆H₇₁N₅O₈Na 844.5195; found 844.5200.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.43 (bs, 1H, NH), 7.74 (m, 2H, o-Ph), 7.58 (m, 2H, m-Ph), 7.53 (m, 1H, p-Ph), 6.29 (dd, 1H, J = 5.6 Hz, 3.8 Hz, H2’), 6.16 (t, 1H, J = 5.9 Hz, H3’), 5.86 (d, 1H, J = 3.7 Hz, H1’), 4.52 (m, 1H, H5’), 4.30 (m, 1H, H4’), 4.26 (m, 1H, H5’), 2.33–2.21 (m, 6H, CH₂–CH₂–CO), 1.64–1.59 (m, 2H, CH₂–CH₂–CO), 1.53–1.49 (m, 4H, 2 CH₂–CH₂–CO), 1.31–1.18 (m, 36H, CH₂), 0.87 (t, 3H, CH₃), 0.85 (t, 3H, CH₃), 0.81 (t, 3H, CH₃).¹³C{¹H} NMR (CD₂Cl₂, 151 MHz): δ 173.4 (CO^5’) 172.4 (CO), 172.2 (CO), 159.2 (C), 153.7 (C), 152.2 (C4), 148.3 (C8), 129.9 (Ph), 129.4 (Ph), 129.3 (Ph), 129.1 (Ph), 117.1 (C5), 87.7 (CH, C1’), 79.4 (CH, C4’), 72.3 (CH, C2’), 70.6 (CH, C3’), 62.8 (CH₂, C5’), 34.0 (CH₂), 34.0 (CH₂), 33.9 (CH₂), 32.0 (CH₂), 32.0 (CH₂), 31.9 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.4 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 24.9 (CH₂), 24.9 (CH₂), 24.9 (CH₂), 22.8 (CH₂), 22.8 (CH₂), 22.7 (CH₂), 14.0 (CH₂), 13.9 (CH₃).

8-Ferrocenylguanosine 13

A mixture of 9 (0.300 g, 0.83 mmol), dimethoxyethane (DME) (12 mL), ferroceneboronic acid (0.286 g, 1.24 mmol, 1.5 eq), and NaOH (3 M, 5.25 mL, 15.75 mmol) was degassed with a stream of Ar for 40 min in an ultrasonic bath. PdCl₂(PPh₃)₂ (0.058 g, 0.1 eq) was added, and the resulting solution was refluxed at 85 °C under argon in an oil bath for 48 h. The solvent was then removed under reduced pressure, the reaction mixture was neutralized with 10% HCl, and the solid was filtered, washed with water, and dried. The product thus obtained was used in the subsequent step without further purification.

An analytical sample was obtained by chromatography on silica (DCM/MeOH 8:2).

RF = 0.17 (DCM/MeOH 9:1).

ESI-MS (m/z): 466.3 [M – H]; 468.1 [M + H]⁺; 490.1 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₂₀H₂₁FeN₅O₅Na 490.0784; found 490.0790.

¹H-NMR (dmso-d₆, 600 MHz): δ 10.67 (bs, 1H, NH), 6.65 (d, 1H, J = 7.2 Hz, H1’), 6.3 (bs, 2H, NH₂), 5.53 (d, 1H, J = 6.0 Hz, OH^2’), 5.36 (d, 1H, J = 6.0 Hz, OH^3’), 5.24 (m, 1H, OH^5’), 5.16 (m, 1H, H2’), 4.68 (s, 2H, Fc), 4.62 (s, 2H, Fc), 4.31 (s, 5H, Fc), 4.19 (m, 1H, H3’), 3.97 (m, 1H, H4’), 3.71 and 3.57 (m, m, 2H, H5’,H5).”¹³C{¹H} NMR (dmso-d₆, 151 MHz): δ 157.8, 153.3, 152.4, 146.9, 117.7, 86.3, 80.4, 75.2, 71.8, 70.8, 70.2, 70.1, 69.9, 69.6, 68.9, 62.8.

8-Ferrocenyl-2′,3′,5’-O-tridecanoyl Guanosine 5 (8Fc5C10)

Crude 8-ferrocenylguanosine 13 (0.430 g, 1.0 eq, 0.92 mmol) was dried over P₂O₅ in vacuo for 2 h at 55 °C and then suspended in 30 mL of an acetonitrile–toluene 1:1 mixture. Decanoic anhydride (443 μL, 3.15 eq, 2.90 mmol) and triethylamine (209 μL, 3.15 eq, 2.90 mmol) were then added, followed by a catalytic amount of 4-dimethylamino pyridine. The mixture was stirred under argon for 14 h at 80 °C in an oil bath. A second aliquot of decanoic anhydride (443 μL, 3.15 eq, 2.90 mmol) and triethylamine (TEA) (209 μL, 3.15 eq, 2.90 mmol) was added, and stirring was continued for 12 h at the same temperature. The solvent was removed under reduced pressure, and the crude was dissolved in dichloromethane and extracted with a sat. NaHCO₃ and brine. The organic layer was dried over MgSO₄. The crude reaction mixture was then applied to a silica gel column packed in DCM and eluted with a mixture of DCM–methanol (from 99:1 to 95:5). The product was obtained as an orange glass in 23% yield (196 mg, 0.21 mmol).

ESI-MS (m/z): 930.5 [M + H]⁺; 952.4 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₅₀H₇₆FeN₅O₈ 930.5038; found 930.5042.

¹H-NMR (dmso-d₆, 600 MHz): δ 10.77 (bs, 1H, NH), 6.75 (d, 1H, J = 6.0 Hz, H1’), 6.58 (t, 1H, J = 6.0 Hz, H2’), 6.40 (bs, 2H, NH₂), 5.74 (dd, 1H, J = 6.0 Hz, 4.2 Hz, H3’), 4.64 (s, 1H, Fc), 4.60 (s, 1H, Fc), 4.50 (bs, 2H, Fc), 4.48 (m, 1H, H5’), 4.37 (m, 1H, H4’), 4.30 (m, 1H, H5’), 4.28 (s, 5H, Fc), 2.45–2.27 (m, 6H, CH₂–CH₂–CO), 1.58 (qi, 2H, J = 6.6 Hz, CH₂–CH₂–CO), 1.47 (m, 4H, 2 CH₂–CH₂–CO), 1.24–1.17 (m, 36H, CH₂), 0.85–0.82 (m, 9H, CH₃). ¹³C{¹H} NMR (dmso-d₆, 151 MHz): δ 175.0 (CO), 173.1 (CO^5’), 172.2 (CO^2’), 156.6 (C), 153.6 (C), 152.6 (C4), 146.1 (C8), 117.3 (C5), 86.6 (CH, C1’), 80.1 (CH, C4’), 74.5 (C, Fc), 71.1 (CH, C3’), 70.6 (CH, C2’), 70.2 (CH, Fc), 70.0 (CH, Fc), 69.8 (CH, Fc), 68.7 (CH, Fc), 68.6 (CH, Fc), 63.0 (CH₂, C5’), 34.1 (CH₂), 33.9 (CH₂), 33.8 (CH₂), 33.6 (CH₂), 31.7 (CH₂), 29.3 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 29.1 (CH₂), 29.0 (CH₂), 29.0 (CH₂), 28.8 (CH₂), 28.8 (CH₂), 25.0 (CH₂), 24.9 (CH₂), 24.8 (CH₂), 24.7 (CH₂), 22.5 (CH₂), 14.4 (CH₃), 14.4 (CH₃).

8-Ferrocenyl-5’-O-tert-butyldimethylsilyl Guanosine 14

A mixture of 13 (0.135 g, 0.29 mmol), imidazole (39 mg, 0.58 mmol), and t-butyldimethylsilyl chloride (48 mg, 0.32 mmol) in DMF (3 mL) was stirred at room temperature for 2 h. After the completion of the reaction (TLC DCM/MeOH 8:2), water (10 mL) was added, and the resulting precipitate was filtered and washed with water. The dry residue was purified by column chromatography (DCM/MeOH 92:8) to afford the product as a white solid in 62% yield (104 mg, 0.18 mmol).

RF = 0.6 (CH₂Cl₂/MeOH 8:2).

ESI-MS (m/z): 580.4 [M – H]⁻; 582.1 [M + H]⁺; 604.2 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₂₆H₃₆FeN₅O₅Si 582.1830; found 582.1835.

¹H-NMR (dmso-d₆, 400 MHz): δ 10.64 (s, 1H, NH), 6.51 (d, 1H, J = 5.6 Hz, H1’), 6.34 (bs, 2H, NH₂), 5.50 (d, 1H, J = 5.6 Hz, OH_2’), 5.27–5.23 (m, 1H, H2’), 5.07 (d, 1H, J = 5.6 Hz, OH_3’), 4.67–4.66 (m, 2H, Fc), 4.47–4.45 (m, 2H, Fc), 4.29 (s, 5H, Fc), 4.27 (m, 1H, H3’), 3.92–3.85 (m, 2H, H5’, H4’), 3.79–3.75 (m, 1H, H5’), 0.82 (s, 9H, tBuSi), −0.014 and −0.010 (s,s, 6H, SiMe₂). ¹³C{¹H} NMR (dmso-d₆, 101 MHz): δ 156.7, 153.1, 152.8, 146.7, 117.4, 89.3, 85.1, 75.2, 71.0, 70.4, 69.9, 69.9, 69.7, 69.2, 68.8, 64.2, 26.2, 18.4, −4.7, −4.8.

8-Ferrocenyl-5’-O-tert-butyldimethylsilyl-2′,3’-O-didecanoyl Guanosine 6 (8Fc5Si)

To a suspension of 14 (71 mg, 0.12 mmol) in MeCN (4 mL) were added Et₃N (45 μL, 0.32 mmol), decanoic anhydride (100 μL, 0.27 mmol), and a catalytic amount of DMAP. The mixture was stirred at room temperature for 4 h. MeOH (0.2 mL) was then added, and solvents were removed under reduced pressure. The residue was partitioned between DCM (20 mL) and water (10 mL). The organic layer was further washed with water (10 mL) and concentrated in vacuo to give an orange glassy solid, which was purified by column chromatography on silica (gradient from pure DCM to DCM/MeOH 97:3). Pure DCM (300 mL ca.) was used to elute decanoic acid, and then, DCM/MeOH 97:3 was used to elute two orange bands: the first one consisting of pure 6 (55 mg, 0.06 mmol, yield 50%), while the second one containing the desilylated derivative 7 formed during work-up.

ESI-MS (m/z): 888.7 [M – H]⁻; 890.5 [M + H]⁺, 912.6 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₄₆H₇₂FeN₅O₇Si 890.4545; found 890.4533.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.33 (bs, 1H, NH), 6.82 (d, 1H, J = 4.7 Hz, H1’), 6.74 (m, 1H, H2’), 6.18 (m,1H, H3’), 4.82 (bs, 2H, Fc), 4.53 (bs, 1H, Fc), 4.48 (bs, 1H, Fc), 4.27 (s, 5H, Fc), 4.28–4.24 (m, 1H, H4’), 3.94 (dd, 1H, J = 11.4 Hz, 4.7 Hz, H5’), 3.83 (dd, 1H, J = 11.4 Hz, 4.7 Hz, H5’), 2.38 (m, 2H, CH₂–CH₂–CO), 2.33 (t, 2H, CH₂–CH₂–CO), 1.70–1.64 (m, 2H, CH₂–CH₂–CO), 1.61–1.56 (m, 2H, CH₂–CH₂–CO), 1.36–1.23 (m, 24H, CH₂), 0.87 (t, 3H, CH₃), 0.84 (t, 3H, CH₃), 0.82 (s, 9H, t-Bu), 0.00 (s, 3H, SiMe), −0.030 (s, 3H, SiMe). ¹³C{¹H} NMR (CD₂Cl₂, 151 MHz): δ 172.5 (CO), 172.2 (CO), 158.8 (C), 153.2 (C), 152.8 (C4), 148.2 (C8), 116.9 (C), 86.4 (CH, C1’), 82.4 (CH, C4’), 73.8 (C, Fc), 71.4 (CH, C2’), 70.7 (CH, C3’), 70.5 (CH, Fc), 70.2 (CH, Fc), 69.8 (CH, Fc), 69.0 (CH, Fc), 68.3 (CH, Fc), 62.6 (CH₂, C5’), 34.2 (CH₂), 34.0 (CH₂), 32.0 (CH₂), 31.9 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 25.6 (CH₃), 25.1 (CH₂), 24.9 (CH₂), 22.8 (CH₂), 22.7 (CH₂), 18.2 (C), 14.0 (CH₃), −5.6 (SiMe).

8-Ferrocenyl-2′,3’-O-didecanoyl Guanosine 7 (8Fc5OH)

To a solution of 6 (55 mg, 0.06 mmol) in 3 mL of THF were added 30 mg (0.09 mmol) of TBAF^.3H₂O, and the mixture was stirred at room temperature for 20 h. The solvent was removed by evaporation, and the residue was dissolved in DCM (15 mL) and washed sequentially with 5% NaHCO₃ (5 mL) and water (5 mL, 4 times). The organic fraction was then dried over Na₂SO₄, and the solvent was removed by distillation. The residue was purified by chromatography on silica gel (gradient from pure DCM to DCM/MeOH 96:4) to afford 7 (33 mg, 0.04 mmol) as yellowish glass in 69% yield.

RF = 0.32 (DCM/MeOH 95:5).

ESI-MS (m/z): 774.5 [M – H]⁻; 776.3 [M + H]⁺, 798.3 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₄₀H₅₇FeN₅O₇Na 798.3500; found 798.3506.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.31 (bs, 1H, NH), 7.22 (d, 1H, J = 13.8 Hz, H1’), 6.98 (bs, 2H, NH₂), 6.50 (bs, 1H, OH), 6.12 (dd, 1H, J = 13.8 Hz, 7.2 Hz, H2’), 5.71 (d, 1H, J = 7.2 Hz, H3’), 4.78 (bs, 1H, Fc), 4.57 (bs, 2H, Fc), 4.52 (bs, 1H, Fc), 4.35 (s, 5H, Fc), 4.37–4.31 (m, 1H, H4’), 4.01–3.96 (m, 1H, H5’), 3.87–3.80 (m, 1H, H5’), 2.47 (t, 2H, CH₂–CO), 2.28 (t, 2H, CH₂–CO), 1.72 (m, 2H, CH₂–CH₂–CO), 1.54 (m, 2H, CH₂–CH₂–CO), 1.40–1.20 (m, 24H, CH₂), 0.88 (t, 3H, CH₃), 0.82 (t, 3H, CH₃). ¹³C{¹H} NMR (CD₂Cl₂, 151 MHz): δ 172.5 (CO), 171.9 (CO), 158.5 (C), 153.5 (C), 151.7 (C), 148.1 (C), 117.5 (C), 86.2 (CH, C1’), 85.7 (CH, C4’), 73.6 (C, Fc), 72.9 (CH, C3’), 71.6 (CH, C2’), 70.9 (CH, Fc), 70.2 (CH, Fc), 69.7 (CH, Fc), 69.5 (CH, Fc), 68.8 (CH, Fc), 63.1 (CH₂, C5’), 34.5 (CH₂), 33.8 (CH₂), 32.0 (CH₂), 31.9 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.1 (CH₂), 25.2 (CH₂), 24.7 (CH₂), 22.8 (CH₂), 14.0 (CH₃).

8-Ferrocenyl-5’-O-benzoyl-2′,3’-O-didecanoyl Guanosine 8 (8Fc5Ph)

Guanosine derivative 7 (54 mg, 0.07 mmol) was dried in vacuo over P₂O₅ at 50 °C for 2 h and then dissolved in DMF (1 mL). To the resulting solution were added Et₃N (15 μL, 0.10 mmol), benzoic anhydride (21 mg, 0.09 mmol), and a catalytic amount of DMAP. The mixture was stirred at room temperature for 8 h, and then, water (10 mL) and DCM (10 mL) were added. The organic phase was washed with water (10 mL), and the combined aqueous fractions were washed with DCM (2 × 5 mL). The organic fractions were combined, and solvents were removed under reduced pressure. The residue was purified by chromatography on silica (DCM/MeOH 96:4) to afford derivative 8 (52 mg, 0.06 mmol) as a yellow–orange solid in 85% yield.

RF = 0.13 (DCM/MeOH 96:4).

ESI-MS (m/z): 878.5 [M – H]⁻; 880.4 [M + H]⁺, 902.4 [M + Na]⁺.

HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₄₇H₆₂FeN₅O₈ 880.3942; found 880.3922.

¹H-NMR (CD₂Cl₂, 600 MHz): δ 12.40 (bs, 1H, NH), 7.92 (bd, 2H, ortho-Ph), 7.40 (bt, 1H, para-Ph), 7.31 (bt, 2H, meta-Ph), 6.90 (d, 1H, J = 3.8 Hz, H1’), 6.63 (dd, 1H, J = 5.7 Hz, 3.8 Hz, H2’), 5.51 (t, 1H, J = 5.7 Hz, H3’), 4.83–4.79 (m, 3H, H5’, 2Fc), 4.58–4.54 (m, 2H, H4’, H5’), 4.51 (bs, 1H, Fc), 4.48 (bs, 1H, Fc), 4.25 (s, 5H, Fc), 2.41–2.37 (m, 4H, 2 CH₂–CO), 1.69 (m, 2H, CH₂–CH₂–CO), 1.62 (m, 2H, CH₂–CH₂–CO), 1.39–1.21 (m, 24H, CH₂), 0.88 (t, 3H, CH₃), 0.85 (t, 3H, CH₃). ¹³C{¹H} NMR (dmso-d₆, 151 MHz): δ 172.2 (CO), 172.2 (CO), 165.8 (CO), 158.5 (C), 153.5 (C), 152.4 (C), 146.1 (C), 133.9 (CH), 129.7 (C), 129.6 (CH), 128.1 (CH), 117.2 (C), 92.2 (CH), 86.7 (CH), 79.7 (C), 74.4 (CH), 71.0 (CH), 70.8 (CH), 69.9 (CH), 69.8 (CH), 68.7 (CH), 68.6 (CH), 63.5 (CH₂), 33.9 (CH₂), 33.6 (CH₂), 31.7 (CH₂), 31.7 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 29.1 (CH₂), 28.9 (CH₂), 28.8 (CH₂), 24.8 (CH₂), 24.7 (CH₂), 22.5 (CH₂), 14.3 (CH₃).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.1c00502.

• Synthetic schemes, VT ¹H-NMR spectra, 1D-NOESY spectra, figures and tables, NMR spectra of compounds 1–14 (PDF)

A. Ciesielski, P. Samorl̀, M. Campitiello and S. Masiero were financially supported by EC through the FP7 project ICT-MOLARNET (318516). A. Ciesielski, M.A. Squillaci, and P. Samorl̀ were supported by the Marie Sklodowska-Curie ETN project SYNCHRONICS (GA 643238). S. Pieraccini, A. Cremonini, and S. Masiero were supported by the University of Bologna (RFO 2018 and RFO 2020). P. Samorl̀ and A. Ciesielski were supported by the Labex projects CSC (ANR-10-LABX-0026 CSC) within the Investissement d’Avenir program ANR-10-IDEX-0002-02 and the International Center for Frontier Research in Chemistry (icFRC).

The authors declare no competing financial interest.