Construction of Supramolecular Organogel with Circularly Polarized Luminescence by Self-Assembled Guanosine Octamer.

Gel formation using guanosine self-assembly is an important process in supramolecular chemistry. Here, we report the stepwise construction of circularly polarized luminescent supramolecular organogels from self-assembled guanosine quadruplexes. A lipophilic guanosine derivative (aldG) is designed and synthesized for the formation of a well-defined G8-octamer. The diamine linkers are used to connect G8-octamer units by imine formation to facilitate the construction of the supramolecular gel networks. 1H NMR experiments show that the pre-assembled aldG8-octamer remains intact and is crucial for transparent and stiff organogel formation. With extended conjugation, the aldG organogels exhibit strong green fluorescence emission and circularly polarized properties without the assistance of any external fluorescent dyes, suggesting an alternative approach to construct molecular probes for biological and material applications.

INTRODUCTION

Guanosine (G) is a type of important nucleoside originating from RNA. It can form different self-assembled species through intermolecular H-bonding, including G-dimer, G-ribbon, G-quartet, and G-quadruplex.[1,2] Self-assemblies of guanosine to form well-defined G-quadruplexes have been extensively studied and applied in functional material development and biomedical research.[3-14] One particularly interesting application in G-quadruplexes is the formation of gels for tissue engineering, cell culture, and drug delivery.[15-17] The fibrous gel network can be constructed through the dynamic supramolecular self-assembly of guanosine. As one of the representative examples, Davis and coworkers[18] introduced borate ester linkages into guanosine self-assembly with the formation of G4-hydrogels in 2014 (Scheme 1A, left). Using this strategy, several functional G4-hydrogels were developed.[19-22] Other G-hydrogel systems with various rheological[23-25] and mechanical[26-28] properties were reported, which further demonstrated their applications in analytical,[29,30] biological,[31-33] optical,[34,35] and chemical research.[36,37] Compared with G-hydrogels, G-organogels are less studied possibly due to the limited functional sites to fulfill both gelation and solubility requirements.[38-44] In general, lipophilic modification on ribose is often required to provide additional hydrophobic interaction to stabilize the fibrous network (Scheme 1A, right). Also, random G-ribbon is the predominant species in these organogels without the formation of G4-quartet.

Scheme 1.

Structural Design

(A) Previous guanosine gel systems.

(B) Design and schematic representation of stepwise construction G8-octamer organogel.

In the previously reported G-gel systems, ribose modification is the dominant approach for the construction of a fibrous network.[15-17,45,46] The guanine part only serves as a H-bonding carrier. The only variable synthetic handle on guanine, C-8 position, remained less developed for gelation (Scheme 1A). Gel formation using C-8 modified guanosine derivatives has been reported; the substituents at the C-8 position affected the conformation of guanosine (syn- and anti- ratio) and further influenced the gel property.[47,48] Recently, our group reported the self-assembly properties of 8-aryl modified lipophilic guanosine derivatives.[49,50] The 8-aryl substituent did not interrupt the H-bond interactions within a G4-quartet unit but did lead to the formation of stable and well-defined G8-octamers with the assistance of alkali and alkali earth metal cations. These studies provided the opportunity for functional group installation at the C-8 position for gel network construction. The 8-aryl substituent further extended the conjugated system of the electron-rich guanine. Incorporation of an electron-withdrawing group (EWG) on the C-8 aryl guanosine may open a new avenue toward the fluorescence (FL)-active guanosine as a potential molecular probe. The luminescent guanosine may also offer the possibility for circularly polarized emission. Circularly polarized luminescence (CPL), which is widely used in biomaterials, CPL sensing and probing, CPL switches, and CPL display, comes from the deexcitation of a chiral excited state molecule.[51-53] With chirality dictated by the ribose, guanosine with extended conjugation on guanine presumably could serve as a chiral FL chromophore to induce CPL.

Based on our previous studies, we designed 8-(4-formyl phenyl)guanosine (aldG) as shown in Scheme 1B. Our rationale for supramolecular G-gel formation is based on the following: (1) with C-8 substitution, aldG could form stable G8-octamers with the metal cations; (2) diamines could be applied to react with aldehyde forming imine under the dynamic equilibrium, and the resulting imine could serve as the linker between G8-octamers for the fibrous gel network formation; and (3) the imine could also facilitate the photoluminescent properties of guanosine due to its electron-withdrawing nature, leading to the rare intrinsically fluorescent guanosine and possible CPL. With this new linking strategy, the ribose could be “free” for lipophilic modification to enhance the solubility in the organic phase and to trap organic solvents to achieve the more challenging guanosine-based organogel. Here, we report the construction of organogels with green FL emission through the condensation of self-assembled aldG8 and various diamines.

RESULTS AND DISCUSSION

Self-Assembling Study of aldG with KPF6

Using our previously developed method, aldG was synthesized and applied in G-quadruplex formation with alkali and alkali earth metal cations (see Supplemental Experimental Procedures for synthetic procedures and Figure S9 for NMR spectra). Since the H-bond in a G-quadruplex could be interrupted by other H-bond competitors, incorporation of the carbonyl group (a new H-bond receptor) on C-8 aryl could lead to the formation of other H-bond complexes/oligomers besides the desired G4-quartet. As observed in previous studies, dissolving guanosine “monomer” in a nonpolar organic solvent, such as CDCl3, usually gives broad and messy 1H NMR signals due to the formation of various H-bonded isomers.[49,50,54] Dissolving the aldG ligand in CDCl3 gave a rather clean 1H NMR spectrum, with characteristic H-bond formation of N1-H at 11.8 ppm (Figure 1A). None of the self-assembled guanosine complexes we tested matches this spectrum in Figure 1A, implying that the guanosine monomer exists as metal-free guanosine assemblies in non-polar solvents.[3-10] Although the formation of the metal-free H-bond isomers could influence the construction of the desired G8-octamers, treating aldG ligand with KPF6 gave 1 set of signals in the NMR spectra (1H, 31P, and 19F). The chemical shift of N1-H at 12.8 ppm suggested the formation of a characteristic H-bond in a G-quartet[49,50] (see Figure S1 for other self-assembling NMR spectra). Finally, the structure of [aldG8K]+(PF6)− was confirmed by single-crystal X-ray crystallography (Figure 1B; Table S3). As shown in the crystal structure (Figure 1B, side view), the phenyl aldehydes for each layer are parallel on the side of the G8-octamer. The conformation is ideal and provides the desired synthetic handles to react with the diamine for gel network construction in both vertical and horizontal directions. With the structure of [aldG8K]+(PF6)− confirmed, the gelation studies were performed under different reaction conditions.

Figure 1.

Characterization of aldG8-Octamer

(A) 1H NMR spectra in CDCl3:CD3CN = 10:1.

(B) Single-crystal X-ray diffraction (some tert-butyldimethylsilyl ethers (OTBS) groups were excluded for clearance).

Gelation Studies Using [aldG8K]+(PF6)−

The ethylenediamine (1a) was first used as the co-gelator to react with the pre-assembled [aldG8K]+(PF6)− complex in different solvents for the evaluation of gel formation. Notably, G8-complexes were not completely soluble in 1,2-dichloroethane (DCE), even at elevated temperatures. Treating amine 1a and the [aldG8K]+(PF6)− complex with a 4:1 ratio in DCE (25 mM) at 80°C, a clear light solution was formed within 5 min. Cooling down the mixture to room temperature allowed the formation of transparent organo Gel-1 (Figure 2A). Besides DCE, tetrahydrofuran (THF) and dimethylformamide (DMF) were also found to be suitable solvents for this gelation process (see Figure S2 for the 1H NMR spectra of [aldG8K]+(PF6)− with heating and cooling). Other solvents, including DMSO, CH3OH, CH3CN, and acetone, failed to form gels in this system (see Tables S1 and S2). With the initial success in gel formation using [aldG8K]+(PF6)−, we wondered whether the pre-formation of G8-octamer is necessary for this process. The monomeric aldG ligand was then applied under the gelation conditions by reacting with 1a. As shown in Figure 2A (right), no gel was formed with aldG ligand in either DCE or DMF, even with prolonged heating at 80°C. The further addition of KPF6 solution to the above system could not give any gel formation. This observation strongly suggested that the pre-assembled G8-octamer is essential for this gel-formation process. G8-octamer is an indispensable unit in the gel network in DCE or DMF. Although aldG ligand could form organogel in THF upon treatment with 1a, the resulting gel is neither clear nor stiff. The mechanical strength of this gel is much weaker than the aldG8-octamer gel, suggesting the essential role of pre-assembled H-bonds within aldG8-octamer in the formation of this new G-organogel.

Figure 2.

Gelation Studies

(A) Evaluation of gelation using aldG8-octamer and aldG ligand.

(B) Monitoring the gelation process by time-dependent 1H NMR spectra in CDCl3:CD3CN = 10:1.

With these observations, monitoring the gelation process by time-dependent 1H NMR spectra was carried out (Figure 2B). The 1H NMR indicated that the aldehyde proton (H13) disappeared within 30 min, demonstrating a new peak at ~8.5 ppm corresponding to the imine formation (H13′). The ratio of integration between H13 (aldehyde) and H13′ (imine) to non-exchangeable H1′ is ~1, suggesting the near-stoichiometric conversion of aldehyde to imine with no other guanosine by-products formed. It is noteworthy that in the previously reported guanosine-based gel systems, guanosine monomer, gelator, and template cation were mixed without the preformation of G-quadruplexes.[15-17] The actual assembling forms of guanosine in those systems are difficult to confirm. In the present work, a stepwise gelation approach used preformed G8-octamer as one of the gelators, followed by imine condensation. The H-bond proton H1 signal remained at 12.6 ppm, suggesting the retention of G-quartet throughout the entire process. This structural information could provide insights into future design in new functional material development based on the self-assembled G-quartets. In comparison, aldG ligand was subjected and monitored under the same reaction condition. The 1H NMR showed that the aldehyde converted to imine; however, the H1 proton signal disappeared upon the addition of 1a, suggesting that the H-bonding in the original metal-free H-bonded guanosine ligands were disrupted. This could account for the previous observation that aldG-monomer ligand failed to form stable organogels of fair quality. With the gelation process identified by 1H NMR experiments, it is clear that [aldG8K]+(PF6)− remained as a G8-octamer during the gelation and was the predominant and necessary guanosine species in this new organogel system. These results again highlighted the importance of pre-assembled H-bonded subunits toward the formation of high-quality supramolecular structural networks (see Figures S3 and S4 for gelation in THF-d and DMF-d).

To fully investigate the gelation process of this new system, other diamines were used to react with [aldG8K]+(PF6)−. The results are summarized in Figure 3A. Aliphatic diamines with increased chain length were tested (1b–1d). However, all of these extended diamine linkers gave homogeneous solutions instead of desired gels. The reaction between p-xylylenediamine (1e) and [aldG8K]+(PF6)− led to the successful formation of a white organogel (Gel-2) in DCE. p-Phenylenediamine (1h) was also tested, although with a much less reactive aromatic amine group. As expected, transparent yellow organogels (Gel-3) were achieved, despite that a longer reaction time is needed due to the reduced amine reactivity. Chiral diamine 1f and 1g were also tested with an initial intention for potential chirality matching. No gel was obtained after both reactions, resulting in clear solutions. This result could be attributed to the improved solubility from the additional phenyl groups.

Figure 3.

Gelation Screenings

(A) Screening of diamines.

(B) Gel-1, Gel-2, and Gel-3 in DCE, THF, and DMF at lowest gelation concentration.

The lowest gelation concentrations of various diamines in different solvents were determined. The results are summarized in Figure 3B. Gel-1 showed a similar lowest gelation concentration in all three solvents. In general, 1e could form gel at a lower concentration compared to 1a. The Gel-3 from DCE showed the lowest gelation concentration among all of the tested samples. This observation could be reasoned on the slow formation of aromatic-aromatic imine. The slow reaction rate increased the homogeneity of the gel network to retain more solvents. This result suggested a potential new strategy to further extend G-quartet networks to achieve significantly larger networks through similar condensation with well-designated coupling partners. Finally, the scanning electron microscopy (SEM) of Gel-1 from DCE was conducted, confirming the formation of a fibrous network (see Figure S5).

Luminescent Properties of aldG8-Octamer Gels

After the studies of the gel formation, FL properties of all of these gels from different gelators and solvents are evaluated (see Figure S6). In the DCE system, Gel-1 and Gel-2 gave the same emission with λem = 471 nm (λex at 403 and 415 nm, respectively). This result suggested the similar FL active groups in both gel systems (G8 with aliphatic amine linkers). Gel-3 showed a blue-shifted emission spectrum, with λem = 427 nm, which is consistent with the formation of aromatic imines. Comparisons of FL emissions between gel and G8-complexes in different solvents are shown in Figures 4A-4C. In DCE and THF, Gel-1 showed a slightly blue-shifted emission compared with aldG8-octamer (Figures 4A and 4B). Enhanced FL emission of Gel-1 (compared with G8) was obtained in DMF, with λem = 503 nm (Figure 4C). Compared with DCE, the emission of both complex and gel showed a dramatic red shift, giving green emission, which suggested promising potential applications in a biocompatible environment. These λem trends fit the typical twisted intramolecular charge transfer (TICT) mechanism, in which more polar solvents cause red shift.[55] Circular dichroism (CD) spectra were measured (Figure 4E), revealing the additional peaks in Gel-1 compared with [aldG8K]+(PF6)−, as suggested in the additional absorption band shown in the UV-visible (UV-vis) spectrum (see Figure S7 for CD spectra and Figure S8 for UV-vis spectra). This result suggested that a new chiral chromophore was formed in the organogel, likely associated with the newly formed imine containing 8-modified guanine. The CPL spectra of Gel-1 in DMF showed that the gel gave CPL with dissymmetric factor g ≈ +1.5 × 10−3 at ~500 nm (Figure 4F). This result suggested the successful chirality induction of excited aldG by ribose. With green-light circularly polarized emission, this aldG octamer organogel system could provide new avenues for broader application in both bioorganic chemistry and chiral sensing and probing.

Figure 4.

Optical Spectra of aldG8-Gels

(A) FL spectra of aldG8-octamer and Gel-1 in DCE.

(B) FL spectra of aldG8-octamer and Gel-1 in THF.

(C) FL spectra of aldG8-octamer and Gel-1 in DMF.

(D) Photograph of Gel-1 under excitation at 365 nm.

(E) CD spectra of Gel-1 in DMF.

(F) CPL spectra of Gel-1 in DMF.

In summary, we demonstrated the stepwise construction of FL-active supramolecular organogels from self-assembled guanosine quadruplexes. The modification of the guanosine C-8 aryl group with aldehyde introduced new synthetic handles for further network extension without interrupting the self-assembled structures. Simple preparation, good stability, and green FL emission without the assistance of any external fluorescent dyes suggest the strong potential of this new supramolecular organogel for applications in biomedical and materials-related research.

EXPERIMENTAL PROCEDURES

Resource Availability

Lead Contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Xiaodong Shi (xmshi@usf.edu).

Materials Availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and Code Availability

The authors declare that the data supporting the findings of this study are available within the article and the Supplemental Information. All other data are available from the Lead Contact upon reasonable request. The accession number for the crystallographic dataset reported in this paper is Cambridge Crystallographic Data Centre (CCDC): 2000805.

Preparation of [aldG8K]+(PF6)−

aldG-monomer (10 mg, 0.018 mmol), KPF6 (1 mg, 0.005 mmol, 0.28 equiv) was added to a mixture of CH2Cl2 (0.5 mL), CH3CN (0.05 mL), and deionized water (0.5 mL). The mixture was stirred overnight. The water phase was removed, and the organic layer was washed with deionized water (0.5 mL × 3). The solvent was removed under vacuum to give [aldG8K]+(PF6)− as a yellow solid.

Preparation of aldG8-Octamer Gels

[aldG8K]+(PF6)− (20 mg) was added into a selected organic solvent, followed by ultrasonication for 30 s. Diamine 4 equiv was added to the solution, followed by ultrasonication for another 30 s. Then, the vial was heated at 80°C for 5 min (ethylenediamine, Gel-1, and p-xylylenediamine, Gel-2) or 10 min (p-phenylenediamine, Gel-3). Finally, the mixture was cooled to room temperature and stood for a certain period of time until gel formation.

Preparation of UV-Vis, CD, FL, and CPL samples

aldG-monomer (solid), [aldG8K]+(PF6)− (solid), and Gel-1 (gelation concentration: 30 mM) were loaded between 2 KBr films for UV-vis, CD, and CPL measurement. Gel-1 in various solvents (30 mM) were prepared in a 1-cm cuvette and directly used for FL measurement. [aldG8K]+(PF6)− solutions in various solvents (30 mM) were prepared in a 1-cm cuvette and directly used for FL measurement.

Preparation of Time-Dependent 1H NMR Experiment of Gels and [aldG8K]+(PF6)−

[aldG8K]+(PF6)− (10 mg) and solvent (0.5 mL) were added to an NMR tube, followed by ultrasonication for 30 s. Ethylenediamine 1 (0.6 μL) was then added to the NMR tube and manually shaken several times before being subjected to the NMR spectrometer.

Preparation of SEM Samples

The SEM samples were prepared by drying Gel-1 (in DCE) under vacuum on conducting resin before gold sputtering.

Determination of Melting Temperature

Gel-1 in DCE, THF, and DMF were prepared following the general procedure of gelation. The samples were placed in a heating plate. The temperature was increased at every 5°C interval and allowed to equilibrate for 3 min before inversion. At each temperature, the vial was physically inverted to assess melting. The sample was considered “melted” when the gel flowed upon inversion and could no longer be self-supporting. DMF Gel-1 showed that the melting temperature was between 60°C and 65°C. Heating DCE and THF gels to 130°C gave only boiling solvent inside the gel, but the gels did not lose the self-supporting property.