Heterocycle-modified 2′-deoxyguanosine nucleolipid analogs stabilize guanosine gels and self-assemble to form green fluorescent gels

Abstract

Nucleoside-lipid conjugates are very useful supramolecular building blocks to construct self-assembled architectures suited for biomedical and material applications. Such nucleoside derivatives can be further synthetically manipulated to endow additional functionalities that could augment the assembling process and impart interesting properties. Here, we report the design, synthesis and self-assembling process of multifunctional supramolecular nucleolipid synthons containing an environment-sensitive fluorescent guanine. The amphiphilic synthons are composed of an 8-(2-(benzofuran-2-yl)vinyl)-guanine core and alkyl chains attached to 3′-O and 5′-O-positions of 2′-deoxyguanosine. The 2-(benzofuran-2-yl)vinyl (BFV) moiety attached at the C8 position of the nucleobase adopted a syn conformation about the glycosidic bond, which facilitated the self-assembly process through the formation of a G-tetrad as the basic unit. While 3′,5′-diacylated BFV-modified dG analog stabilized the guanosine hydrogel by hampering the crystallization process and imparted fluorescence, BFV-modified dGs containing longer alkyl chains formed a green fluorescent organogel, which transformed into a yellow fluorescent gel in the presence of a complementary non-fluorescent cytidine nucleolipid. The ability of the dG analog containing short alkyl chains to modulate the mechanical property of a gel, and interesting fluorescence properties and self-assembling behavior exhibited by the dG analogs containing long alkyl chains in response to heat and complementary base underscore the potential use of these new supramolecular synthons in material applications.

Graphical Abstract.

Heterocycle-modified 2′-deoxyguanosine nucleolipids stabilize guanosine hydrogels and also form a green fluorescent organogel, which transforms into a yellow fluorescent gel in the presence of a complementary non-fluorescent cytidine nucleolipid.

Introduction

The components of nucleic acids, namely nucleobases, nucleosides, nucleotides, and their derivatives serve as very useful supramolecular synthons to build self-assembled architectures for applications in biomedical and materials research.^[1] These synthons not only retain their recognition (H-bonding) and metal ion binding features but are also easily scalable, which is one of the major challenges associated with nucleic acid-based self-assembled architectures.^[2] Another proven advantage of these building blocks is that they can be conveniently modified to add additional functionalities to facilitate as well as tune the self-assembling process to form various architectures. Assemblies made of such tailor-made hybrids exhibit responsiveness to external stimuli, support the formation of gels, and hence, have been used as biocompatible delivery vehicles for drugs and genes. Notably, among the nucleobases, the ability of guanine and its derivatives to form variety of self-assembled structures has attracted particular attention.^[3]

Much like polymorphic G-quadruplex structures formed by G-rich sequences, guanine and its derivatives utilize combinations of Watson-Crick and Hoogsteen base paring faces to form variety of self-assemblies, which depend on the ionic conditions.^[4] For example, in the absence of any added cations, guanine assembles into G-ribbon I and G-ribbon II architectures.^[3a] Addition of cations like K⁺ and Na⁺ ions facilitate guanine/guanosine to adopt planar G-tetrad structures, which stack one above the other to form hierarchical assemblies.^{[3a, 5]} Similarly, guanine and guanosine derivatives self-assemble in solution^[6] and also support gelation process in the absence and presence of added metal ions.^[3b,7] Such self-assembled systems have been used as drug delivery vehicles^[8] and in the construction of injectable gels,^[8d,9] synthetic ions channels,^[10] sensors^[11] and photoactive materials.^[12] Interestingly, naphthalene diimide and perylene diimide electron acceptors have been conjugated to two guanine electron donors to construct G-quadruplex-based organic frameworks in which donors and acceptors form ordered but segregated π-stacked arrays. This bioinspired system when excited, produced long-lived mobile charge carriers and also acted as a cathode material in a Li-ion battery.^[13] Similarly, self-assembled rods and spheroids were constructed from guanine-containing peptide nucleic acid dimers and monomers, which acted as good organic light-emitting materials and photonic crystals.^[14]

Inspired by these systems, we sought to develop multifunctional nucleoside supramolecular synthons that would enable the construction of hierarchical assemblies with interesting optical properties. Here, we describe the development of novel nucleolipids containing an environment-sensitive fluorescent guanine analog as the head group and fatty acid chains of different lengths attached to 3′-O and 5′-O-positions of the 2′-deoxyribose sugar as the lipophilic tail group. The fluorophore is based on an 8-(2-(benzofuran-2-yl)vinyl)-guanine core, which prefers a syn conformation about the glycosidic bond and favors the formation of G-tetrad as the basic building block. While the fluorescent 2′-deoxyguanosine derivative containing acetyl group did not form gels by itself, it hindered the crystallization process in a guanosine hydrogel, thereby facilitating the formation of a stable cogel with modulated mechanical properties. On the other hand, 2′-deoxyguanosine nucleolipids containing longer fatty acid chains (e.g., myristoyl and palmitoyl) formed stable organogels and retained reasonable fluorescence even in the gel state (λ_em = 497 nm, green color). Intriguingly, addition of a complimentary non-fluorescent cytidine nucleolipid to the above gel resulted in a remarkable red-shift in emission maximum (538 nm, yellow color), which was responsive to changes in temperature.

Results and discussion

Design of fluorescent 2′-deoxyguanosine nucleolipids

We used simple physical organic concepts to assemble multifunction nucleolipid supramolecular synthons. Literature report and our own studies suggest that responsive fluorescent nucleosides could be built by tethering heterocyclic rings onto otherwise non-emissive nucleobases.^[15] For example, benzofuran attached at the C-5 position of uracil ring via a rotatable aryl-aryl bond produced a highly environment-sensitive fluorescent probe.^[16] In the present study, we decided to conjugate benzofuran ring at C-8 position of guanosine via an alkene linker for three reasons (Figure 1). First, modification at C-8 position of guanine favors syn glycosidic conformation, which in turn favors G-tetrad formation.^[17] Second, the conjugated benzofuran, apart from imparting fluorescence, could also aid in self-assembling process via stacking interaction. ^[17a,18] Lastly, isomerizable alkene bond could provide an extra handle to control the self-assembly process as well as the ensuing changes in the photophysical properties by using light as an external stimulus.^[19] Further, coupling of fatty acids at 3′-O- and 5′-O-positions of 2′-deoxyribose sugar could facilitate self-assembly thorough hydrophobic interactions.^[18,20] Based on this synthon design, it is expected that a coordinated interplay of H-bonding, stacking and hydrophobic interactions would facilitate hierarchical self-assembling process, which could be potentially modulated by chemical and physical triggers.

Figure 1.

Design of self-assembling fluorescent deoxyguanosine nucleolipids. Attachment of a heterobicycle to the C8-position of guanine and fatty acid acyl chains to the sugar generates responsive nucleolipid supramolecular synthons. Guanine can invoke multiple H-bonding interactions through Watson-Crick and Hoogsteen faces. Heterobicycle conjugated to the nucleobase via an alkene linker imparts fluorescence and can potentially influence the self-assembling process and photophysical properties.

Synthesis and characterization of fluorescent 2′-deoxyguanosine nucleolipids

Benzofuran-conjugated nucleoside 3 was prepared by performing Suzuki–Miyaura cross-coupling reaction between 8-bromo-2′-deoxyguanosine (1) and (E)-2-(2-(benzofuran-2-yl)vinyl) pinacol boronic ester (2)^[21] in the presence of Pd(OAc)₂ and a water-soluble triphenylphosphine ligand (TPPTS, Scheme 1). 3′- and 5′-hydroxyl groups of the 2′-deoxyribose sugar of nucleoside 3 were reacted with acetic anhydride to yield the acetylated product 4 in good yields. Nucleolipids (5 and 6) with myristoyl and palmitoyl chains were prepared by coupling nucleoside 3 with myristic acid and palmitic acid, respectively, using EDC·HCl (Scheme 2). Large coupling constants for the alkene protons (J = 15.2–15.6 Hz) confirmed the trans configuration of the double bond in the nucleolipids.

Scheme 1.

Synthesis of (E)-diacetyl 8-(2-(benzofuran-2-yl)vinyl)-2′-dG (4). TPPTS = Triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt and DMAP = 4-dimethylaminopyridine.

Scheme 2.

Synthesis of lipophilic 8-(2-(benzofuran-2-yl)vinyl)-2′-dG nucleolipids 5 and 6. EDC·HCl = 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Modifications at C8 position of guanine often result in anti to syn conformational change about the glycosidic bond (N9-C1′).^[22] This change in conformation is because of the steric clash between the substituent at C-8 position and 5′ OH group of sugar residue. Consistent with the reported chemical shift trends for syn conformational, ¹H2′ and ¹³C2′ signals of 3 and 4 displayed a downfield shift and an upfield shift, respectively, as compared to the native nucleoside, which adopts an anti conformation (Figure S1 and Figure S2, Table S1 and S2). ^[23] Furthermore, 2D NOESY analysis of 3 and 4 showed strong cross-peak between anomeric proton (H1′) and vinyl proton (Ha), which further confirmed the syn glycosidic conformation (Figure S3–S6).

Fluorescent 2′-deoxyguanosine nucleolipids are sensitive to solvent polarity changes

Photophysical properties of nucleolipids were investigated in solvents of different polarity such as methanol, dioxane and chloroform. The ground-state electronic spectra of nucleolipids 4–6 exhibited marginal shift in absorption maximum with changes in solvent polarity (Figure S7 and Table 1). The fluorescence properties of the synthons are sensitive to polarity of the medium. In dioxane, the nucleolipids exhibited highest quantum yields (≥94%) and in methanol and chloroform the yields are noticeably lesser. A similar trend in quantum yields was observed in case of previously reported 8-aryl deoxyguanosine derivatives.^[24] The photophysical properties of such emissive nucleosides incorporated into oligonucleotides are influenced by hydrogen bonding, π-π stacking interactions and rigidification of the fluorophore.^[24b,25] We anticipated that such factors could also influence the photophysical properties of heterocycle-modified modified deoxyguanosine nucleolipids during the self-assembly process.

Table 1. Photophysical properties of modified nucleolipids in different solvents.

nucleolipid	solvent	λmax[a] (nm)	λem (nm)	I rel [b]	Φ[c]
4	methanol	378	450	0.82	0.75
	dioxane	380	450	1.00	0.95
	chloroform	380	443	0.24	0.26
5	methanol	378	453	0.88	0.79
	dioxane	380	450	1.00	0.94
	chloroform	380	445	0.61	0.60
6	methanol	378	450	0.83	0.77
	dioxane	380	450	1.00	0.97
	chloroform	380	443	0.67	0.70

^[a]Longest absorption maximum is given.

^[b]Emission intensity relative to the intensity in dioxane is given.

^[c]Quantum yield of nucleolipids in different solvents was measured relative to 9,10-diphenylanthracene as a standard.^[26] Standard deviation for Φ is ≤ 0.04.

Acetylated dG nucleolipid stabilizes guanosine gel

Guanosine and some of its derivatives can form hydrogels in the presence of metal ions, but the major limitation associated with such gels is their poor stability.^[27] For example, crystallization occurs over a period of time, which collapses the gel (Figure S8). Addressing this drawback is very important to advance the utility of guanosine-based hydrogel gels for biomedical and material applications. FESEM image of xerogel of guanosine indicated the presence of long-range ordered-sheets having a width of around 1 μm (Figure 2A). We envisioned that transforming this sheet structure into fibrous network would stabilize guanosine gels. In this context, non-gelling additives, have been used to prevent the crystallization process and stabilize the gel or even provide additional functional properties.^[28] Acetylated 2′-deoxyguanosine 4 has the same recognition feature of the native base and better solubility in water-DMSO mixture as compared to other nucleolipids (5 and 6). Hence, it is likely that addition of 4 to guanosine could impart heterogeneity to the overall self-assembly, thereby disfavoring the crystallization process. Guanosine analog 4 alone does not support gelation in the absence or presence of metal ions (K⁺ and Na⁺) in DMSO or water-DMSO mixture (data not shown). Rewardingly, addition of 4 to a solution of guanosine in the presence 500 mM KCl produced a gel, which was stable for few months (Figure 3). Up to addition of 35 wt% of 4 resulted in the formation of a stable gel and beyond this concentration, the gel collapsed. FESEM image of this cogel revealed a fibrous network, a typical feature that favors stable gel formation (Figure 2B).

Figure 2.

FESEM images of xerogel of (A) 2 wt% guanosine and (B) 2 wt% cogel (35:65, 4:G).

Figure 3.

(A)Visual appearance of 2 wt% cogel made from varying ratios of 4 and guanosine (G) in DMSO-H2O mixture containing 500 mM KCl. (B) Vial showing 2 wt% cogel (35:65, 4:G) is stable and no crystallization was observed after a few months.

Interestingly, a guanosine gel formed in the presence of 4 exhibited a weak fluorescence band, which was markedly red-shifted (λ_em = 510 nm) as compared to the free analog 4 (λ_em = 474 nm). When the gel was transformed into a sol an intense band with a blue-shifted emission maximum (474 nm) was observed (Figure S9). It is well documented that guanosine quenches the fluorescence of several fluorophores via electron transfer process.^[29] However, we don’t have a cogent explanation as to why the emission maximum of 4 is significantly red-shifted in the gel state.

Role of fluorescent additive in the gelation process

During the gelation process it is likely that not all the components are incorporated into a gel network, i.e., some % of the individual components may remain in the solution phase.^[30] Components in solution give sharper NMR signals, whereas the components assembled into the gel network will exhibit broad signals. A mixture of guanosine and 4 was heated and ¹H NMR spectrum was recorded as a function of temperature (85 °C to 25 °C at 10 °C interval). As the temperature was lowered, the cogel started to form leaving behind some amount of the components in the solution phase, which was detected by ¹H NMR (Figure S10 and S11). The amount of individual components left in the sol phase was estimated by comparing the integration of anomeric protons (H1′: 5.68 ppm for guanosine and 6.32 ppm for 4) with that of an internal tert-butyl alcohol standard (singlet at 1.07 ppm for methyl groups, Table S3). The results show that at room temperature ~44% of the available guanosine and ~96% of the available 4 participate in the gel network formation. Although 4 by itself does not support gelation, it is more efficiently incorporated as compared to guanosine in a gel network formed by the mixture. A possible reason behind the incorporation preference of 4 is its ability to adopt a syn-conformation over the anti (Figure S12).^[31] It is reported that syn-conformation of guanosine helps in the gelation process by forming effective cation-templated H-bonded self-assembly of stacked G-quartets.^[17c,32] Whereas anti-conformation of guanosine derivatives hinders the process of gelation. However, after a certain concentration of 4 (beyond 35%), the amount of guanosine left to form a gel is low, and hence, the cogel collapses.

We then investigated the effect of 4 on the mechanical stability of the gel as C8 BFV modification, though helps in tetrad formation, could affect the packing of tetrads. For this, viscoelastic properties of gels formed using different ratios of 4 and guanosine were determined by rheology analysis (Figure S13). Strain sweep experiment shows that the gels are stable till 1% of strain. Upon increasing the amount of 4 (25 to 40 wt %), the storage modulus (G′) was found to decrease progressively. It is possible that the tetrads formed by a heterogeneous combination of guanosine and BFV-modified dG analog 4 do not stack efficiently on each other due to steric hindrance. This could curtail long range ordering resulting in lowering of mechanical strength. This observation is also in line with the SEM images of xerogels made of guanosine alone and a mixture of guanosine and 4 (Figure 2). Taken together, the acetylated version of the fluorescent guanosine is helpful in tuning the gelation process and also imparts interesting fluorescence properties. Such dual component systems could significantly advance the utility of guanosine-based gels in biomedical and materials research by not only countering the inherent crystallization problem associated with guanosine gels but also by providing additional functional properties.

Nucleolipids containing longer fatty acid acyl chains form green fluorescent organogels

Unlike the acetylated guanosine derivative 4, 3′,5′-O-dimyristoyl- and dipalmitoyl-substituted nucleolipids 5 and 6 supported the formation of organogels in DMSO. The nucleolipids dissolved in DMSO by heating, when left to come to room temperature formed gels, which were stable for several months (Figure 4A). The gelation process did not require cations like K⁺ or Na⁺. Gel-sol interconversion was found to be thermo-reversible over several cycles of heating and cooling steps. Further, a hot solution of nucleolipids in DMSO, when subjected to ultrasonication, rapidly formed gels. The gelation ability of nucleolipids was found to depend on the alkyl chain length. Critical gelation concentration (CGC) of nucleolipid 5 containing C14 myristoyl chains (1 w/v %) was found to be higher as compared to nucleolipid 6 containing C16 palmitoyl chains (0.7 w/v %).

Figure 4.

(A) Left pair: photograph of organogels of fluorescent nucleolipids 5 and 6 in DMSO at respective CGC values. Middle and right pairs: The gels show intense green fluorescence upon UV illumination at 254 nm and 365 nm, respectively. (B) Fluorescence spectra of nucleolipid gels 5 and 6 in DMSO (at CGC concentration) at two different temperature 25 °C (gel state, λ_em = 497 nm) and 75 °C (sol state, λ_em = 484 nm). The samples were excited at 387 nm with an excitation and emission slit widths of 1 nm and 1 nm, respectively. (C and D) FESEM images of xerogels of nucleolipids 5 and 6, respectively.

Importantly, in the gel state also nucleolipids exhibited high fluorescence in the green emission region, which is in contrast to the majority of low molecular weight gelators, which lose their fluorescence upon self-assembly due to aggregation induced quenching (Figure 4A).^[33] In nucleolipids 5 and 6, guanine ring is conjugated to the benzofuran ring via an aryl-vinyl bond, which could undergo rotation about the C=C–Ar axis much like in tetraphenylethylene, a well-studied system showing aggregation-induced enhanced emission (AIEE).^[34] If the two ring systems in the nucleolipids are rigidified in the gel state, then it could show enhancement in the fluorescence intensity. To test this, the fluorescence of nucleolipids at respective CGC was recorded in the gel (25 °C) and sol states (75 °C, Figure 4B). In the gel state, the nucleolipids showed an intense fluorescence band centred around 497 nm. Upon increasing the temperature, the gels disassembled and the sols showed 3 to 4-fold quenching in fluorescence intensity with a slight blue-shift in emission maximum (484 nm). To rule out the effect of temperature on fluorescence intensity, we recorded the fluorescence emission of a dilute solution of nucleolipid 5 (2.5 μM) at 25 °C and 75 °C. We did not observe any significant changes in emission intensity with increase in temperature (Figure S14). This result suggests that nucleolipids 5 and 6 likely exhibit AIEE.

In terms of morphology, xerogels of nucleolipids formed entangled ribbon-like structures, which was ascertained by FESEM analysis (Figure 4C and 4D). The mechanical properties of the gels were studied by rheological measurements at constant oscillating frequency with varying shear strain (Figure S15). At low strain values, storage modulus (G′) of 5 and 6 (~2800 and ~1700 Pa) was significantly greater than its loss modulus (G″: ~360 and ~240 Pa). The crossover point of G′ and G″ for 5 and 6, where gel transforms into sol, was observed at around 45% and 20% of the strain, respectively. Increasing the angular frequency at a constant strain of 0.05 % did not significantly affect the G′ and G″ values of the nucleolipid gels. However, both the values were higher for a gel formed by 5 as compared to a gel formed by 6. These results indicate that nucleolipid gel 5 containing C14 myristoyl chains exhibits higher mechanical strength in terms of viscoelastic character compared to 6 containing C16 palmitoyl chains.

Driving force for the supramolecular gelation process

To evaluate the interactions that drive the gelation process, variable temperature ¹H NMR spectra of gels formed using 5 and 6 at their CGC values were recorded. As the temperature of gels was increased from 25 °C to 75 °C, the N1 imino and N2 amino hydrogens exhibited progressive upfield shift in their signal during the course of gel to sol transition (Δδ = 0.19 and Δδ = 0.20 ppm, respectively, Figure 5 and Figure S16). Aromatic C-H of benzofuran moiety also showed a small upfield shift and sharper signal during gel to sol transition (Δδ ~ 0.04). Importantly, in the gel state of 5 we observed a peak at 11.95 ppm corresponding to the N1 imino H characteristic of a G-tetrad assembly (Figure 5).^{[11g, 35]} This peak disappeared when the temperature was increased from 25 °C to 75 °C. The progressive shift in proton signals as a function of increasing temperature is due to loosening or breaking of the respective H-bonds as the gel is transformed into solution.^[18,36] These results indicate that N1-H and N2-H of guanine base and to some extent C-H of benzofuran ring drives the self-assembling process by possibly forming G-tetrad as the basic unit. This notion is further substantiated by CD analysis of nucleolipids in gel and sol states. CD spectrum of organogels of 5 and 6 recorded at 25 °C revealed two opposite signed bands at 269 nm and 256 nm, which suggests the presence of G-tetrad motif in the gel network (Figure S17).^[7c] A negative peak at 446 nm corresponds to the (2-(benzofuran-2-yl)vinyl)-chromophore. Upon transforming the gel to sol, the CD signals almost disappeared indicating the disassembling of the gel network. Formation of such cation-free stacked guanine quartets without adding any template ions have been previously reported.^[37] Usually, G-quartets are formed in the presence of templating cations (e.g., Na⁺, K⁺, Ag⁺). However, bulky substituents at the C-8 position of guanine orient ribose sugar in the syn conformation with respect to the nucleobase. Syn conformation forces guanine moiety to adopt a quartet structure even in the absence of any added cations.

Figure 5.

Partial ¹H NMR spectra of nucleolipid organogels 5 in d₆-DMSO as a function of increasing temperature. N1-H (denoted in blue) and N2-H (denoted in red), which could participate in strong hydrogen bonding interactions, exhibited a significant upfield shift in their proton signals during gel to sol transition. The aromatic C-H proton of benzofuran ring (denoted in magenta) also showed small upfield during gel-sol transition.

Further insights on the molecular arrangement of nucleolipids in the gel network was gained by PXRD analysis. PXRD spectrum of xerogels of nucleolipids 5 and 6 displayed a prominent diffraction peak corresponding to an interplanar distance of 4.04 nm and 3.75 nm, respectively (Figure S18). Notably, a xerogel of nucleolipid 5 containing myristoyl chains showed diffraction peaks corresponding to layer spacings in the ratio of 1:1/2:1/3:1/4:1/6, which is indicative of an ordered lamellar arrangement.^[18,38] Layer spacings in the range of 4.1 and 3.8 Å were also observed, which could arise due to tetrads held by π-π stacking interaction.^[39] Collectively, the results obtained from a battery of experiments suggest a hierarchical self-assembling process, wherein nucleolipids come together to form a G-tetrad by a coordinated H-bonded network, which stacks and propagates into a lamellar arrangement resulting in fibers and then entangled ribbons (Figure 6).

Figure 6.

Pictorial representation of the possible mechanism for the hierarchical self-assembly of the nucleolipids (e.g., 5) leading to the formation of organogel. The nucleolipid assembled into G-quartet unit even in the absence of cation which furthers assembles via π-π stacking giving G-quadruplex structure. These interactions eventually lead to the formation of entangled sheets resulting in the immobilization of the solvent.

In general, gelators containing longer alkyl chains pack well and immobilize solvents more efficiently compared to gelators containing shorter alkyl chains.^[40] While both the nucleolipids 5 and 6 form entangled ribbon-like structures (Figure 4), nucleolipid 6 containing palmitoyl chains has a lower CGC value compared to nucleolipid 5 containing myristoyl chains. This observation is also supported by PXRD spectrum of xerogels. Nucleolipids 5 and 6 displayed an inter-layer spacing of 4.04 nm and 3.75 nm, respectively (Figure S18). However, 5 exhibits higher strength in terms of viscoelastic character compared to 6 as optimal hydrophilic-hydrophobic balance and type of packing is required to form a strong gel. Xerogels of nucleolipid 5 containing myristoyl chains showed diffraction peaks corresponding to an ordered lamellar arrangement. But this kind of ordered packing structure was not evident in the case of nucleolipid 6.

Addition of a complementary nucleolipid transforms green fluorescent gel to yellow fluorescent gel

We next studied the effect of complementary nucleobase interaction on the gelation process and ensuing changes in the photophysical properties. Addition of cytidine (5 equiv.) to nucleolipid 5 did not affect the gel formation and its fluorescence (Figure S19). On the other hand, addition of 2′,3′-O-dimyristoyl-substituted cytidine nucleolipid 7 to guanosine nucleolipid 5 resulted in an interesting observation. 1 and 2.5 equivalents of cytidine nucleolipid 7 did not show detectable changes in gelation process and fluorescence as monitored by inverted vial method and UV irradiation (data not shown). Addition of 5 equivalents of 7 did not disrupt the organogel formed by 5, but exhibited remarkable red-shift in emission maximum (497 nm to 543 nm), thereby uniquely converting the green fluorescence gel to yellow fluorescent gel, albeit with reduced fluorescence intensity (Figure 7). When the yellow fluorescent cogel was disassembled by heating at 75 °C, it showed blue shift in emission band (480 nm) corresponding to the free nucleolipid 5. It is to be noted that cytidine nucleolipid 7 by itself does not form organogels. This change in emission wavelength and emission intensity endows a fluorochromic property to the nucleolipids as they efficiently respond to a non-fluorescent chemical additive and changes in temperature, and such systems could be utilized in developing chemical and temperature sensors.^[41]

Figure 7.

Effect of addition of cytidine nucleolipid 7 on the gelation ability and fluorescence of guanosine nucleolipid 5. (A) Picture of vials showing changes in emission colour after the addition of 7. (B) Fluorescence spectra of cogel made of nucleolipid 5 and cytidine dimyristate 7 (1:5 millimolar ratio) in DMSO (at CGC of nucleolipid 5) at two different temperatures: 25 °C (gel) and 75 °C (sol). The samples were excited at 387 nm with an excitation and emission slit widths of 1 nm and 2 nm, respectively.

In order to understand the possible reasons for the observed changes in emission properties upon addition of cytidine nucleolipid, we performed ¹H NMR, PXRD and FE-SEM experiments. The proton signal at 11.95 ppm corresponding to N1 imino of G-tetrad of guanosine gel 5 disappeared upon addition of cytidine nucleolipid 7. Further, a small downfield-shift in the N1 imino and N2 amino hydrogens (Δδ = 0.15 and Δδ = 0.07 ppm, respectively) was observed. This could be possibly due to the ability of cytidine nucleolipid to form hydrogen bonding interactions with the guanosine nucleolipid, which then disturbs the original self-assembly pattern of the guanosine nucleolipid 5. PXRD analysis also indicated changes in self-organization, wherein the lamellar arrangement exhibited by xerogel 5 was altered by the addition of 7 (Figure 8). Individually, a xerogel of nucleolipid 5 formed long-range tapes and a drop-casted sample of 7 formed flower-like structures (Figure S20). A SEM image of the xerogel formed using the mixture showed twisted tapes of guanosine nucleolipid 5 and more pronounced flower-like structures of cytidine nucleolipid 7, which interestingly grew on the tapes. Though we do not have a cogent experimental evidence for the observed bathochromic shift in the emission wavelength of the gel after addition of cytidine nucleolipid, we speculate that changes in the arrangement of fluorescent nucleolipid 5 in the assembled state in the presence of a complementary nucleolipid could be responsible for the fluorescence outcome. The influence of fluorophore arrangement in the supramolecular matrix leading to changes in optical properties have been reported previously.^[14a]

Figure 8.

PXRD of xerogels of (A) 5, (B) 5 + 7 mixture, and (C) 7 alone.

Conclusion

We have developed environment-sensitive fluorescent 2′-deoxyguanosine supramolecular synthons, which show interesting self-assembly behavior and emission properties. Mixing acetylated dG nucleolipid (nongelator) with guanosine (gelator) hinders the crystallization process and supports the formation of a thermodynamically stable guanosine gel. This method could be used in tuning the morphology and mechanical properties of guanosine gels, which would make them more useful in biomedical applications. We also demonstrated that upon attaching fatty acid chains to the sugar residue, the BFV-modified nucleolipid synthons self-assemble to form supramolecular gels exhibiting intense green fluorescence. Interestingly, addition of a complimentary cytidine dimyristate nucleolipid to the fluorescent guanosine nucleolipid resulted in the formation of a gel that displayed a remarkable change in emission maximum and emission intensity, i.e., the green fluorescent gel transformed into yellow fluorescent gel. An added advantage of this nucleolipid design is that the heterobicycle is conjugated to the nucleobase via an alkene linker, which could exhibit stimuli-induced isomerization process. Such a transformation could influence the self-assembly process and photophysical properties, which we plan to study in future. Taken together, this bioinspired system could be useful in developing organic light-emitting materials for sensor applications.

Experimental section

Gelation test

Cogel made up of OAcBFVdG (4) and guanosine: A weighed amount of 4 and guanosine in a glass vial was dissolved in DMSO:H2O system by heating. The samples were cooled to room temperature and the gel formation was confirmed by inverting the vial. Stable gels were formed in 30 min. Repeated heating and cooling steps were performed to confirm the thermo-reversibility of the formed gels. All experiments were performed at least in duplicate.

Organogel made of nucleolipid 5 and 6: A weighed amount of deoxyguanosine nucleolipids in a glass vial was dissolved in DMSO by heating. The samples were cooled to room temperature and the gel formation was confirmed by inverting the vial. Nucleolipids 5 and 6 formed stable gels in 30 min. Repeated heating and cooling steps were performed to confirm the thermo-reversibility of the organogels. All experiments were performed at least in triplicate. For all the experiments detailed below, fresh gels were made prior to analysis.

FESEM analysis

The morphology of assemblies formed by individual nucleolipids and different mixtures of nucleolipids and nucleosides was characterized by FESEM. Gel samples in respective solvents were drop-casted on a silicon wafer and dried in a vacuum desiccator for ~15 h. Samples were gold-sputtered before analysis. The FESEM images were analyzed by using ImageJ 1.46r software.

Rheology studies

Rheology measurements were carried out in an Anton Paar MCR 302 instrument by using 15 mm diameter parallel plate. Measurements were carried out at 25 °C. For all the experiments, around 200 μL of the hot solution was placed on the plate.

Cogel made up of OAcBFVdG (4) and guanosine (G): A strain sweep experiment at a constant frequency (10 rad/sec) was performed in the 0.01–500% range to determine the linear viscoelastic region of the gel sample. A hot solution of the gel was loaded on to the plate. The hot solution was allowed to form a stable gel for 30 min prior to the measurement. The gap was fixed at 500 μm for the parallel plate apparatus.

Organogel made up of nucleolipid 5 and 6: A strain sweep experiment at a constant frequency (10 Hz) was performed in the 0.01–100% range to determine the linear viscoelastic region of the gel sample. Frequency sweep experiment at constant strain (0.05 %) was performed in the 0.1–100 rad/sec range. A hot solution of the gel was loaded on to the plate and allowed to form gel for 30 min prior to measurement. The gap was fixed at 1 mm for the parallel plate apparatus.

Variable temperature ¹H NMR

¹H NMR was recorded on a Jeol 400 MHz NMR instrument as a function of temperature.

Cogel made of OAcBFVdG (4) and guanosine: A 2 wt% mixture of 4 and guanosine (35:65) containing 500 mM KCl in 0.5 mL of d6-DMSO:D₂O (1:1) was warmed to obtain a clear solution and to this hot solution an internal standard tBuOH (2 μL) was added. Then the solution was transferred into a hot NMR tube, which was immediately transferred into a Jeol 400 MHz NMR instrument at 85 °C and was allowed to equilibrate for 15 min. NMR spectra were recorded at 85 °C, after which the sample was cooled to 75 °C and again allowed to equilibrate for 15 min. This process was repeated until the final measurement (at 25 °C) was completed.

Organogel made up of nucleolipid 5 and 6: Gels of 5 (1 w/v %) and 6 (0.7 w/v %) in d₆-DMSO at respective CGC were formed in individual NMR tubes by heating and cooling steps. The temperature of the sample was elevated from 25 °C to 75 °C with an increment of 10 °C and equilibration time of 15 min.

PXRD analysis

Gels of 5 and 6 in DMSO at respective CGC were formed on a glass slide by drop-casting method. The glass slide was placed in a vacuum desiccator and dried under vacuum for nearly 15 h to obtain xerogels. PXRD spectrum was recorded using Bruker D8 Advance diffractometer with CuKα source (1.5406 Å). Diffraction data were collected at 2θ angle from 1° to 30° using a 0.01° step size and 0.5 s per step. Low angle diffraction data was collected by keeping the motorized divergence slit in automatic mode so as to maintain the X-ray beam footprint on the sample to 12 x 12 mm. Further, the position sensitive detector (Lynxeye) channels were reduced to minimize the background X-ray scattering entering the detector.

Circular Dichroism analysis

Spectra were collected from 600 to 200 nm on a Jasco J-815 CD spectrometer using 1 nm bandwidth at 25 °C and 75 °C. Sample kept for 10 min at each temperature for equilibration. Experiments were performed in duplicate wherein each spectrum was an average of three scans. The spectrum of DMSO without nucleolipid was subtracted from all the sample spectra.

Variable temperature fluorescence experiment

Spectra were recorded in a micro fluorescence cuvette (Hellma, path length 1.0 cm) using Fluoromax-4 fluorescence spectrometer (Horiba Jobin Yvon).

Cogel made up of OAcBFVdG (4) and guanosine: A hot solution of a 2 wt% mixture of 4 and guanosine (35:65) containing 500 mM KCl in 0.5 mL of DMSO:H₂O (1:1) was taken in a micro fluorescence cuvette and was allowed to stand at RT until a stable cogel was formed, cuvette was inverted to test the gel formation. Fluorescence spectra of gel samples were recorded at two different temperature 25 °C and 85 °C. Sample was excited at 387 nm with an excitation slit width of 1 nm and emission slit width of 2 nm with an equilibration time of 15 min.

Organogel 5 and 6: A hot solution of the nucleolipid at respective CGC was taken in a micro fluorescence cuvette and was allowed to stand at RT until a stable gel was formed, cuvette was inverted to test the gel formation. Fluorescence spectra of gel samples were recorded at two different temperature 25 °C and 75 °C. Sample was excited at 387 nm with an excitation slit width of 1 nm and emission slit width of 1 nm with an equilibration time of 10 min.