Gelation-Driven Component Selection in the Generation of Constitutional Dynamic Hydrogels Based on Guanine-Quartet Formation

Gelation-driven component selection in the generation of constitutional dynamic hydrogels based on guanine-quartet formation

Nampally Sreenivasachary and Jean-Marie Lehn*

Laboratoire de Chimie Supramolećulaire, Institut de Science et d’Ingeńierie Supramolećulaires (ISIS), Universite´Louis Pasteur, 8 Alleé Gaspard Monge, BP 70028, 67083 Strasbourg Cedex, France

Contributed by Jean-Marie Lehn, March 1, 2005 The guanosine hydrazide 1 yields a stable supramolecular hydrogel Such processes form the basis of the recently developed dynamic based on the formation of a guanine quartet (G-quartet) in pres- combinatorial chemistry (5, 6), in which molecular recognition ence of metal cations. The effect of various parameters (concen- events have been implemented toward the generation of optimal tration, nature of metal ion, and temperature) on the properties of binding agents toward artificial or biological (7) molecular recep- this gel has been studied. Proton NMR spectroscopy is shown to tors through a target-driven shift in the distribution of the library allow a molecular characterization of the gelation process. Hydra- constituents toward the best binder(s). Changes in library constit- zide 1 and its assemblies can be reversibly decorated by acylhy- uents may also be caused by redistribution of components induced drazone formation with various aldehydes, resulting in formation by metal ion binding (8, 9) or environmental factors such as of highly viscous dynamic hydrogels. When a mixture of aldehydes temperature and pH (N. Giuseppone and J.-M.L., unpublished is used, the dynamic system selects the aldehyde that leads to the data). most stable gel. Mixing hydrazides 1, 9 and aldehydes 6, 8 in 1:1:1:1 The amplification of a given constituent of a CDL under the ratio generated a constitutional dynamic library containing the pressure of a self-organization process, such as the formation of an four acylhydrazone derivatives A, B, C, and D. The library consti- organized phase (for example, a gel), would be of special interest. tution displayed preferential formation of the acylhydrazone B It would represent a process of self-organization by selection (3, 4) that yields the strongest gel. Thus, gelation redirects the acylhy- by which the formation of a structured phase drives the selection of drazone distribution in the dynamic library as guanosine hydrazide the components that make up the dynamic constituent producing 1 scavenges preferentially aldehyde 8, under the pressure of the most highly organized and most stable assembly. gelation because of the collective interactions in the assemblies of Gels attract much current interest for their potential as intriguing G-quartets B, despite the strong preference of the competing materials (10, 11) and as substrates for biomedical applications (12, hydrazide 9 for 8. Gel formation and component selection are 13). In particular, hydrogels formed from low-molecular-weight thermoreversible. The process amounts to gelation-driven self- compounds that respond to pH are suitable candidates for oral drug organization with component selection and ampliﬁcation in con- delivery as well as biosensor technology, especially when biochem- stitutional dynamic hydrogels based on G-quartet formation and ical components are involved. Hydrogels have also been shown to reversible covalent connections. The observed self-organization respond to ligand-receptor molecular recognition (14, 15) and and component selection occur by means of a multilevel self- redox stimuli (16). assembly involving three dynamic processes, two of supramolecu- Here, we describe our studies of a system in which the formation lar and one of reversible covalent nature. They extend constitu- of a supramolecular hydrogel drives the selection of the compo- tional dynamic chemistry to phase-organization and phase- nents that form the constituent leading to the most stable gel. It transition events. embodies a process of self-organization by selection under the pressure of gelation. It presents triple constitutional dynamics, two dynamic combinatorial chemistry ͉ component selection ͉ at the supramolecular level and a third one of covalent dynamic supramolecular chemistry nature, which involves selection by covalent self-assembly of the component that generates the hydrogel of highest cohesive upramolecular entities present the ability to reversibly modify strength. Stheir constitution through exchange and rearrangement of their The system brings together several features of particular interest, molecular components because of the lability of the noncovalent namely (i) self-organization and dynamics at both the supramo- interactions that hold them together (1, 2). Similar features may be lecular and molecular levels; (ii) generation of dynamic hydrogels; imported into molecular species if reversible covalent bonds are (iii) dynamic selection of the optimal components; (iv) implemen- introduced into their structure, allowing cleavage and formation of tation of biochemical components; and (v) adaptive behavior in interatomic connections with fragment exchange under specific response to external factors. conditions. Thus, entities, capable of reversible modification of their consti- Materials and Methods tution, define a constitutional dynamic chemistry on both the Instrumental Techniques. NMR spectra were recorded on a 400- supramolecular and the molecular levels (3). Because the consti- MHz spectrometer (Bruker, Wissembourg, France). The chemical tutional changes may be expected to respond to external factors, shifts are reported in ppm downfield from tetramethylsilane; cou- constitutional dynamic chemistry is the basis for the design and pling constants are given in Hz. Electrospray ionization (ESI)-MS development of adaptive chemical systems. It generates constitu- was carried out on a Bruker MicroTOF MS coupled with liquid tional dynamic libraries (CDLs) whose constituents are in dynamic chromatography. Samples were prepared at a concentration of 200 equilibrium, such that they can exchange their components and ␮M in Milli-Q water or in 0.5 M ammonium acetate buffer. Before express all of the entities that are potentially accessible through recombination by means of reversible covalent bonds and noncovalent interactions. The CDL may then adapt to (internal or) Abbreviations: G-quartet, guanine quartet; CDL, constitutional dynamic library; pD, p2H. external physical factors or chemical effectors by selection (3, 4) of *To whom correspondence should be addressed. E-mail: [email protected]. the appropriate components for the optimal constituent. © 2005 by The National Academy of Sciences of the USA

5938–5943 ͉ PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501663102 Downloaded by guest on September 30, 2021 Fig. 3. Temperature of gelation Tgel determined visually as a function of hydrazide 1 concentration in buffer (0.5 M) of the acetates of various cations ϩ ϩ ϩ ϩ as follows. (}), Na ;(■), K ;(Œ), NH4 ;({), Me4N .(a) Tgel values (in °C for 10 Fig. 1. Guanine derivatives self-assemble to G-quartets in the presence of ϩ ϩ ϩ ϩ and 50 mM, ion): (33, 65, Na ), (59, 87, K ), (45, 73, NH4 ), and (54, 79, Me4N ). metal ions. ϩ (b) Tgel of an aqueous solution of 1 (15 mM) as a function of K concentration (in mM, °C): (15, 41), (30, 51), (45, 61), (60, 61), and (90, 61). injection, a small aliquot of the sample was diluted 20-fold and used for ESI-MS. The following mild conditions were used to detect the signal with respect to the internal reference gave the amount of 1 supramolecular assembly of guanine quartets (G-quartets): dry still free in the gelled solution. The gel was destroyed by addition heater temperature was set at 120°C, ion polarity was positive, of a drop of concentrated DCl giving a clear solution, and then the nebulizer pressure was 0.4 bar, capillary voltage was 4,000 V, end Ϫ ͞ proton NMR spectrum was recorded; the integration of H-8 of 1 plate offset voltage was 400 V, and dry gas flow was 3.0 liters min. (100% free in the solution) with respect to the internal dioxane Viscosity measurements were performed on a digital rheometer reference gave the amount of 1. The difference between the integral (model DV-III; Brookfield, Middleboro, MA) fitted with a CPE-40 of total free 1 (100% in solution) and the integral of free 1 in the spindle model of 4 cm in diameter and a 1° angle. gelled solution yielded the percentage of gelation. The integration of the internal dioxame signal was also checked against an external CHEMISTRY Preparation of Gels and Measurement of Gel Melting Temperature. dioxane reference contained in a capillary. The guanosine hydrazide 1 was dissolved in 0.5 M acetate buffer ␮ (pH 6, 500 l) to make up a concentration of 15 mM. The container Dynamic Combinatorial Library Generation. Stock solutions (150 was heated until guanosine hydrazide 1 dissolved completely, and mM) of the aldehydes (6–8) and hydrazides (9 and 10) were 2 it was then cooled to room temperature. Gelation was observed, prepared by dissolving a given compound in H2O or deuterated and the gel melting temperature (Tgel) was determined visually by buffer solution [0.5 M sodium acetate or potassium acetate, p2H the vial-inversion method. The sample vials were immersed in an (pD) 6.0]. Guanosine hydrazide 1 (2.3 mg) was dissolved in a 500-␮l inverted position in an oil bath, and the temperature was increased buffer in an NMR tube to make up a 15 mM solution. Then, we slowly. Tgel was taken as the point at which the gel started to flow. added 50 ␮l of hydrazide (9 or 10) solutions and 50 ␮l of aldehyde (6 and 8) solutions from the stock solutions. The NMR tube was Determination of the Gelled Fraction by Proton NMR Spectroscopy. gently heated to 50–60°C for 5–6 h to reach equilibrium. It was then 2 1 We added guanosine hydrazide 1 (2.3 mg), H2O (450 ␮l), 45 ␮lof cooled to room temperature, and the H-NMR spectrum was KCl stock solution (1 M), and 5 ␮l of dioxane stock solution (300 recorded once the solution was fully gelled. The CHAN proton mM) to an NMR tube, yielding 15 mM 1͞90 mM KCl͞3mM signals of the anti and syn isomers of each acylhydrazone (Ϸ75% dioxane solution. The NMR tube was gently heated until guanosine anti and 25% syn Ϯ 10%, depending on the compound) were hydrazide dissolved, and it was then cooled to room temperature. integrated, giving the fraction of the library constituents present as The 1H-NMR spectra were recorded when the sample was fully free in solution. The fraction of guanosine acylhydrazone in the gel gelled. The percentage of free hydrogelator was determined by was obtained by difference. Although the acylhydrazone (1ϩ6) integrating the proton signal of H-8 on the guanine group with does not give a gel, a small amount (Յ3%) of it could be trapped respect to the internal dioxane reference. The proton signal of H-8 in the gel formed by the acylhydrazone (1ϩ8) in the mixtures of is sharp for free 1 in solution, whereas that for 1 engaged in the gel (1ϩ9 or 10)with(6ϩ8). The spectra of the individual acylhydra- is broadened beyond detection. Integration of the observable H-8 zones (15 mM) showed a weak signal (Յ5%) of unreacted aldehyde

2 1 Fig. 4. Investigation of the gelation of 1 (15 mM, H2O) by H-NMR spectroscopy. (a) Fraction of free 1 (Œ), 1 engaged in the gel (‚) as function of Kϩ Fig. 2. Hydrogel formed from 1.(a) Picture showing that the sample does not concentration (lines drawn through the experimental data points). (b) Frac- flow when the vial is inverted at 15 mM, 23°C in 0.5 M sodium acetate buffer tion of free 1 in the presence of 90 mM KCl as a function of temperature. at pH 6.0. (b) Transmission electron microscopy images of fibers forming the Experimental data points and the calculated best fit to a sigmoidal curve are gel. given. (Inset) Derivative curve giving the transition temperature Tt.

Sreenivasachary and Lehn PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5939 Downloaded by guest on September 30, 2021 Fig. 5. Reversible decoration of guanosine hydrazide 1 and of its G-quartet assembly Q1 by condensation with various aldehydes (2–8).

proton. On heating, the CHAN signals broadened both for the Compounds containing guanine groups are well known to individual compounds and for the mixtures, and the signals due to undergo quadruple association into G-quartets through the anti and syn forms merged progressively. Hoogsten-type hydrogen-bonding forming supramolecular mac- rocycles that stack into G4 assemblies in the presence of cations ϩ ϩ ϩ Synthesis. All commercial reagents were purchased from Aldrich such as Na ,K , and NH4 with formation of hydrogels (Fig. 1) and used without any further purification. (21, 22). Organo-soluble derivatives of guanine have also been shown to form organized materials such as liquid crystals (23, Guanosine-5؅-Hydrazide 1. We added hydrazine hydrate (80 mg, 1.6 24). These results led us first to explore the physicochemical mmol) to a suspension of guanosine-5Ј-methylester (17) (100 mg, properties of 1. 0.32 mmol) in methanol (150 ml), and the mixture was refluxed for 12 h. The reaction mixture was concentrated to one-third of its Gelation Properties of Guanosine Hydrazide 1. Guanosine hydrazide volume, filtered, and dried under a vacuum to give 76 mg of 1, although not soluble by itself in pure water, was found to form hydrazide 1 (yield, 76%; white solid; mp, 241–243°C): 1H NMR (400 stable free-standing gels at 15 mM in the presence of Naϩ,Kϩ, ϩ ϩ MHz, DMSO-d6): 10.8 (br s, 1H), 10.6 (br s,1H),7.95(s, 1H), 6.57 and NH4 , as well as of the much larger Me4N cation at neutral (br s, 2H), 5.78 (d, J ϭ 7.6 Hz, 1H), 5.73 (m, 1H), 5.54 (br s, 2H), pH (phosphate buffer). The hydrogels were strong enough not 4.52 (m,1H),4.45(m, 1H), 4.33 (d, J ϭ 1.2 Hz, 1H), 4.1 (s, 1H). to flow on inversion of the container (Fig. 2a) and were found 13 C-NMR (100.6 MHz, DMSO-d6): ␦ 169.2, 157.1, 153.9, 150.3, to be stable at room temperature for several days. They are 137.7, 117.8, 88.0, 84.6, 73.5, 72.6. ESI-MS m͞z 312.1 [MϩH]ϩ; pH-sensitive, forming around neutral and slightly acidic pH high-resolution MS (fast atom bombardment MS) found 312.1083, (acetate buffer) but not at pH 8.0 or 9.0 (borate buffer). (C10H14N7O5 calculated 312.0978). CHN analysis calculated for Electrospray MS showed a peak for (G4ϩNa) at m͞z 1,267.4, C10H13N7O5 0.5 H2O: C, 37.57; H, 4.39; N, 30.67; found: C, 37.01; confirming the identity of the G-quartet. The transmission H, 4.35; N, 30.67.

Results and Discussion In an ongoing exploration of the dynamic combinational chemistry of derivatives of biological substances [amino acids, peptoids (18), carbohydrates (19, 20), nucleic acid components (D. T. Hickman, N. Sreenivasachary, and J.-M.L., unpublished data)], we synthesized the guanosine-5Ј-hydrazide derivative 1 by treatment of the corresponding methylester (17) with hydrazine.

Fig. 6. Viscosity of the gels formed from the acylhydrazone derivatives obtained by reacting guanosine hydrazide 1 with the aldehydes 7 (Œ) and 8 (‚).

5940 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501663102 Sreenivasachary and Lehn Downloaded by guest on September 30, 2021 Fig. 7. Generation of a dynamic library of acylhydrazones C, D and of the acylhydrazone G-quartets A and B from hydrazides 1, 9 and aldehydes 6 and 8.

electron microscopy observation of the gel prepared from 1 However, the present gels are much more stable than reported revealed fibers of several micrometers in length (Fig. 2b). for guanosine (21). T The temperatures of gelation, gel, were measured at pH 6 in CHEMISTRY sodium acetate buffer (0.5 M) as a function of gelator concen- Proton NMR Determination of Guanosine Hydrazide 1 Gelation. The tration (Fig. 3a). The results indicated that hydrazide 1 was able fraction of 1 engaged in the hydrogel was determined by to gelate the buffer solution even at a concentration as low as 10 1H-NMR spectroscopy. Indeed, whereas the signal of H-8 on the guanine group is sharp for free 1, that for 1 engaged in the gel mM [i.e., Ϸ0.3% (wt), giving a Tgel value of 33°C]. At 50 mM, Tgel was 65°C, and increasing the concentration up to 100 mM gave is broadened beyond detection. Integration of the observable product precipitation. The formed hydrogel was thermally re- H-8 signal with respect to an internal reference signal (3 mM versible but unstable to shear. dioxane) gave the fraction of 1 free and in the gel (by difference) as a function of Kϩ concentration (Fig. 4a) (see Materials and Determination of Tgel as a function of the concentration of 1 ϩ Methods). It was found that practically total gelation (Ն98%) for different cations showed that K was the most efficient occurred above Ϸ45 mM KCl. Thus, NMR spectroscopy offers gelator͞G4 assembler. The other cations showed the sequence of ϩ Ͼ ϩ Ͼ ϩ a general method for studying the efficiency of the gelation gelation efficiency Me4N NH4 Na (Fig. 3a), in line with processes. Similarly, the variation of the fraction of free 1 as a the results reported for guanosine gels (21). function of temperature was followed by integration of the H-8 The gelation temperature Tgel of 1, determined visually by ϩ proton signal (Fig. 4b). It displays a sigmoidal shape similar to container inversion, as a function of K concentration, remained the melting curve of double-stranded DNA, yielding a transition unchanged at 61°C above 45 mM and up to 90 mM salt, indicating temperature Tt of 43°C. that it was independent of ion concentration when gelation was The difference between the visually determined Tgel (61°C) complete (Fig. 3b). and Tt (43°C) may be ascribed to the fact that they concern two According to previous studies (21), the gelation properties different events. Different physical methods refer to different of compound 1 may be attributed to the formation of G- microscopic processes (25). Tt refers to the variation at the quartets and subsequent stacking into columns induced by molecular level of the amounts of free and bound (in the gel) binding of metal cations between the G4 species (Fig. 1). states of 1, possibly involving motions within fibrils without

Table 1. Equilibrium distribution of acylhydrazones A–F in the dynamic mixtures generated from the hydrazides 1, 9, and 10 and the aldehydes 6 and 8 Hydrazides Aldehydes Acylhydrazones at the equilibrium, %

Entry 191068A B C D E F

1 11 0 118394211—— 2 10 1 11937——4012 3 0 1 0 1 1 — — 15 85 — — 4 0 2 0 1 1 — — 50 50 — — 511001—87—13—— 6 101 01—96——— 4 7 110 1048—52——— 8 1 0 1 1 0 51 — — — 49 — 9 0 1 1 1 1 — — 25 25 25 25

All compound fractions have been determined by proton NMR spectroscopy. The concentration of all compounds was 15 mM in sodium acetate buffer at pD 6 and 25°C (see Materials and Methods).

Sreenivasachary and Lehn PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5941 Downloaded by guest on September 30, 2021 as documented (18, 20, 26–28). As a result, reversible decoration of 1, its G4 derivative Q1 (Fig. 5), and the gel-forming assemblies by diverse groups becomes possible, with the perspective of modulating the properties of the organized phase and inducing the selection of a specific group. Thus, we conducted an exten- sive investigation involving hydrazide 1 and a series of aldehydes 2–8 (Fig. 5; in all cases, 15-mM solutions of each component in 2 sodium acetate buffer in H2O at pD 6.0 and 25°C). The addition of stoichiometric amounts of the aldehydes 2–4 to the gel formed by 1 disrupted the gel and resulted in product precipitation, whereas aldehydes 5 and 6 gave solutions at the same concentration. The reaction of 1 with 1-formyl furan-3- sulfonic acid 7 and with pyridoxal-5-phosphate 8 yielded highly viscous gels. The condensation reactions with 5–8 were followed by 1H-NMR spectroscopy observing the disappearance of the aldehyde proton signal (see above). The acylhydrazones generated from 1 with 5 or 6 each showed two imine proton signals corresponding to the syn and anti imine isomers. Fig. 8. Distribution of the acylhydrazones A–D in the CDL generated from a Rheological measurements on the gels formed by the acylhy- mixture of the hydrazides 1, 9 and the aldehydes 6 and 8 (all 15 mM, sodium drazone derivatives Q2 obtained from aldehydes 7 and 8 indi- 2 acetate buffer, pD 6 in H2O) as a function of temperature after reaching cated that they had a much higher viscosity (2,400 and 1,900 equilibrium, as calculated from integration of the CHAN 1H-NMR signals. At mPa, respectively, at 0.38 turns sϪ1 shear rate) than the gel 25°C: A, 8%; B, 39%; C, 42%; D, 11%; at 55°C: A, 15%; B, 35%; C, 35%; D, 15%; formed by the hydrazide 1 itself and that they presented both at 80°C: A, 22%; B, 28%; C, 28%; D, 22%. thermal and shear stress reversibility (Fig. 6). Similar shear stress reversibility is observed for supramolecular polymers (ref. 29 and E. Kolomiets and J.-M.L., unpublished data). depolymerization. Tgel describes the gel-to-sol transition at the macroscopic level, when the material flows under gravity shear because of loss of cohesion of the assembly. Also, the NMR data Gelation-Driven Component Selection. The promising results ob- indicate full melting of the gel at Ϸ55–60°C (onset of the plateau tained for the formation of highly viscous hydrogels by the Q 1 of the curve in Fig. 4b). Such data may be of much interest for acylhydrazone quartet derivatives 2 of guanosine hydrazide prompted us to explore whether the gelation process would drive the understanding of the relationship between microscopic and the selection, within a dynamic library of constituents, of the macroscopic collective events in gels and organized phases in component generating the constituent that yields the strongest general. They also point to the fact that motion within or gel. It would represent a process of self-organization by selection, exchange in and out of an organized phase may occur before driven by the cohesive strength due to the collective interactions phase transition, a feature of significance for delivery processes. in the resulting assembly. On a related note, size-selective synthesis of oligomers may be induced by folding (30). Formation of Dynamic Gels Derived from Guanosine Hydrazide 1. The To investigate the evolution of the system toward the ‘‘best-fit’’ G-quartets formed by hydrazide 1 are potentially interesting dynamic hydrogel, the aldehydes 6, 8 and the hydrazides 1, 9 were scaffolds for dynamic decoration. This feature offered the selected (Fig. 7). The dynamic library was generated at 15 mM possibility of exploring the effect of side chains attached through concentration for each compound, at pD 6 in sodium acetate reversible condensation with various aldehydes (in particular, of buffer (0.5 M). It consists of four acylhydrazones, each present- biological type) on the properties of the hydrogel. More specif- ing two configurational isomers, undergoing interchange con- ically for the present purposes, 1 displays a hydrazide function- tinuously by acylhydrazone bond formation and cleavage in ality that may engage into the formation of reversible acylhy- aqueous medium (18, 20, 26–28). drazones by condensation with various aldehyde (or ketone) A mixture of the hydrazides 1, 9 and the aldehydes 6, 8 was compounds, thus generating dynamic libraries of acylhydrazones prepared (see Materials and Methods) in a molar ratio of 1:1:1:1

Fig. 9. Dynamic library of acylhydrazones C–F generated from hydrazides 9, 10 and aldehydes 6 and 8.

5942 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501663102 Sreenivasachary and Lehn Downloaded by guest on September 30, 2021 (Fig. 7 and Table 1, entry 1). After gently heating the mixture at giving 87% acylhydrazone B and 13% of D at equilibrium (Table 60°C and then cooling it to room temperature, the 1H-NMR 1, entry 5). Together, these results stress the ability of gelation spectra were recorded when equilibrium was attained (Ͻ6 h). to redirect the acylhydrazone distribution because hydrazide 1 is The distribution of acylhydrazone components at 25°C was able to scavenge 8 from 9 in D despite the strong preference of analyzed by 1H-NMR spectroscopy, by integrating the CHAN 9 for 8 (entry 3). Similarly, compounds 1, 10 and 8 in a 1:1:1 ratio imine protons of the free (nongelated) acylhydrazones, which, yielded acylhydrazones B and F at 96% and 4% respectively, although broadened, could be clearly identified for each con- indicating again strong selection when there is gelation (Table 1, stituent of the mixture (Table 1). A markedly uneven distribution entry 6). Reacting 1, 9 with 6 (1:1:1 ratio) or 1, 10 with 6 (1:1:1 was obtained (Fig. 8). The guanosine hydrazide 1 gave 8% and ratio) gave almost equal distribution of imines because no 39% of the acylhydrazones A and B, resulting from its reaction gelation occurs to drive a selection (Table 1, entries 7 and 8). with aldehyde 6 and pyridoxal monophosphate 8, respectively. Similarly, when 9, 10 were mixed with 6, 8 in a 1:1:1:1 ratio, there Similarly, the serine hydrazide 9 reacted with aldehydes 6 and 8 was no component selection and the fractions of acylhydrazones to give Ϸ42% of C as well as 11% of D. C, D, E, and F were found to be equal (Fig. 9 and Table 1, entry When the 1H-NMR spectra were measured at 55°C, the 9). These experiments highlight that the guanosine hydrazide 1 distribution of acylhydrazones was found be become less uneven. cation-templated self-assembly drives component selection en- On further temperature increase up to 80°C, the gel was melted forced by the ability of the supramolecular assembly B to form completely and the distribution of acylhydrazones was close to a stable hydrogel. equal (Fig. 8). Cooling the reaction mixture slowly over a period Conclusions of 60 min back to 25°C restored the initial distribution, indicating that a selection process occurred, by which the mixture evolved The pH-sensitive guanosine hydrazide 1 was found to be a to favor the constituent B forming a thermodynamically stable powerful hydrogelator, capable of undergoing dynamic decora- dynamic hydrogel over constituents A, C, and D, which do not tion through formation of reversible acylhydrazone bonds with give such an organized phase. As indicated by the 1H-NMR data, various aldehydes. It is a versatile building block for combina- two acylhydrazones B (in the gel) and C (free in solution) clearly tions presenting various gelation abilities depending on the dominate in the CDL at equilibrium. The latter is expressed as appended group. It opens up the possibility to generate biolog- ‘‘image’’ of B, as a consequence of D being depressed by the ically relevant dynamic hydrogels by means of the constitutional dynamic approach. New gelators, bearing in particular biologi-

trapping of pyridoxal monophosphate 8 in B in the gel. Note that CHEMISTRY the dynamic selection is reversible and depends on the temper- cally relevant recognition groups, can be explored systematically ature; at high temperature, when the gel has melted, the for the generation of aqueous gels that may find useful applications in the area of medicinal chemistry and material science. selection disappears, whereas it operates at 25°C when the The gelation process results from a multilevel self-assembly medium is gelled. Thus, a self-organization by selection occurs, based on the cation-templated self-assembly of quartets of driven by gelation and inducing the preferential formation of the guanosine acylhydrazone derivatives. In a broader perspective, most stable hydrogel. Its generality is shown on replacement of within the frameworks of self-organization by selection and serine hydrazide 9 by alanine hydrazide 10, which results in constitutional dynamic chemistry (3), the present results dem- almost the same distribution of acylhydrazones (Table 1, onstrate the ability of a physical parameter, the formation of a entry 2). cohesive organized phase (here, a hydrogel), to direct a consti- Several control experiments were performed (Table 1). tutional dynamic system toward the selection of the components Equimolar amounts of hydrazide 9 and aldehydes 6 and 8 (1:1:1 that lead to the expression of the ‘‘fittest’’ constituent for the ratio) gave 15% of acylhydrazone C and 85% of acylhydrazone directing process in a function-driven fashion. D (Table 1, entry 3), indicating that hydrazide 9 forms preferentially D with aldehyde 8. As expected, hydrazide 9 and We thank Dr. Marc Schmutz (Institut Charles Sadron, Strasbourg, aldehydes 6 and 8 in a 2:1:1 ratio generate equal amounts of C France) for the transmission electron microscopy pictures. N.S. was and D at equilibrium (Table 1, entry 4). Aldehyde 8 and supported by postdoctoral fellowships from the French Ministry of hydrazides 1, 9 in a 1:1:1 molar ratio resulted in gel formation, Research and the Colle`ge de France.

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