Langmuir 2007, 23, 5283-5285
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Supramolecular Polymers Based on the Quadruplex Formation of Ditopic Guanosine Macromonomers in Nonaqueous Media Ga´bor Pinte´r,† Gyula Batta,† Pa´l Horva´th,† Istva´n Lo¨ki,† Tibor Kurta´n,‡ Sa´ndor Antus,‡ Sa´ndor Ke´ki,§ Miklo´s Zsuga,*,§ Ga´bor Nagy,| Ja´nos Aradi,⊥ Tama´s Gunda,† and Pa´l Herczegh*,† Department of Pharmaceutical Chemistry, UniVersity of Debrecen and Research Group for Chemistry of Antibiotics of the Hungarian Academy of Sciences, Departments of Organic Chemistry, Applied Chemistry, and Zoology, UniVersity of Debrecen, and Department of Biochemistry and Molecular Biology, UniVersity Medical School, H-4010 Debrecen, Hungary ReceiVed January 4, 2007. In Final Form: March 14, 2007 The formation of supramolecular polymeric aggregates with a molecular mass of 100 kDa in a nonaqueous solution from a telechelic dimer of isopropylidene guanosine in the presence of K+ ions is reported. The possible structure of macromonomers resulting from the development of G4 quartets was deduced from DOSY NMR, circular dichroism spectra, and dynamic light scattering measurements.
Introduction G quartets (G4’s) are self-assembling, planar, guanosine tetramers formed from four guanine bases by Hoogsteen-type hydrogen bonding (Figure 1a), and these associates are often stabilized by a central alkali metal (Na+ or K+) ion.1,2 Sequences rich in guanosine have been found, for example, in the telomeric regions of chromosomes, which have essential roles in cell multiplication. Telomerase enzymes that are able to resynthesize such sequences are present in high quantities in tumor cells; therefore, they are one of the important subjects in cancer chemotherapy.3,4 The G-containing DNA lines constitute supramolecular units, the G-quadruplexes, which are stabilized by the alkali metal. In the middle of the 1990s, Gottarelli and Davis showed that in nonaqueous media the lipophilic guanosine derivatives are assembled into quartets or quadruplexes.5-9 During such an assembly, the G4 cation-dipoles (formed by hydrogen bonding) may transform into an octamer by π-stacking (Figure 1b), which can be associated into a hexadecamer or columnar aggregates with the aid of the potassium ion. * Corresponding authors. (M.Z.) E-mail:
[email protected]. Fax: 36-52-348173. (P.H.) E-mail:
[email protected]. Fax: 36-52512914. † Department of Pharmaceutical Chemistry, University of Debrecen and Research Group for Chemistry of Antibiotics of the Hungarian Academy of Sciences. ‡ Department of Organic Chemistry, University of Debrecen. § Department of Applied Chemistry, University of Debrecen. | Department of Zoology, University of Debrecen. ⊥ University Medical School. (1) Davis, J. T. Angew. Chem. 2004, 116, 684-716. Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668-698. (2) Guschlbauer, W.; Chantot, J. F.; Thiele, D. J. Biomol. Struct. Dyn. 1990, 11, 491-511. (3) Neidle, S.; Harrison, R. J.; Reszka, A. P.; Read, M. A. Pharm. Ther. 2000, 85, 133-139. (4) Neidle, S.; Parkinson, G. N. Curr. Opin. Struct. Biol. 2003, 13, 275-283. (5) Gottarelli, G.; Masiero, S.; Spada, G. P. J. Chem. Soc., Chem. Commun. 1995, 2555-2557. (6) Davis, J. T.; Tirumala, S.; Jenssen, J.; Radler, E.; Fabris, P. J. Org. Chem. 1995, 60, 4167-4176. (7) Forman, S. L.; Fettinger, J. C.; Pieraccini, S.; Gottarelli, G.; Davis, J. T. J. Am. Chem. Soc. 2000, 122, 4060-4067. (8) Pieraccini, S.; Gottarelli, G.; Mariani, P.; Masiero, S.; Saturni, L.; Spada, G. P. Chirality 2001, 13, 7-12. (9) Giorgi, T.; Grepioni, F.; Manet, I.; Mariani, P.; Masiero, S.; Mezzina, E.; Pieraccini, S.; Saturni, L.; Spada, G. P.; Gottarelli, G. Chem.sEur. J. 2002, 8, 2143-2150.
Figure 1. (a) Structure of a G quartet. (b) [G8] K+ octamer.
Research on the properties of noncovalent, supramolecular polymers has become very intense during the past few years because their ionophoric properties have made their practical application appear to be promising. Recently, Lehn and Ghoussoub10 synthesized a bis-guanine derivative that, in aqueous solution, formed K+-stabilized polymeric hydrogels.
Results and Discussion The goal of the present work was to investigate the formation of supramolecular polymers11 by utilizing quadruplex formation of the lipophilic guanosine derivatives in nonaqueous media. Thus, covalent coupling of the hemisuccinate12 of 1 to R,ω-diamino-poly(ethylene glycol)13 (DPEG) furnished telechelic macromonomer 2 (Scheme 1), whose structure was supported by NMR assignment and matrix-assisted laser desorption ionization timeof-flight (MALDI-TOF) mass spectrometry. The characteristics of the MALDI-TOF MS results are summarized in Table 1. As it turns out from the data of Table 1, both DPEG and 2 have a very narrow molecular weight distribution. (Other details of the MALDI-TOF MS can be found in Supporting Information.) Circular dichroism (CD) spectroscopy has been frequently used to detect the supramolecular assembly of lipophilic guanosine derivatives.14-16 (10) Lehn, J. M.; Ghoussoub, A. Chem. Commun. 2005, 5763-5765. (11) Brunsveld, L.; Folmer, B. J. B.; Meijer, E. W.; Sijbesma, P. Chem. ReV. 2001, 101, 4071-4097. (12) Defrancq, E.; Leterme, A.; Pelloux, A.; Lhomme, M. F.; Lhomme, J. Tetrahedron 1991, 5725-5736. (13) Mongondry, P.; Bonnans-Plaisance, C.; Jean, M.; Tassin, J. F. Macromol. Rapid Commun. 2003, 24, 1091-1100.
10.1021/la070019g CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007
5284 Langmuir, Vol. 23, No. 10, 2007
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Scheme 1. Synthesis of Supramolecular Macromonomer 2
Figure 2. CD spectra of 2 in acetonitrile (-) and chloroform (‚‚‚) without the addition of K+ and after extraction with an aqueous solution of potassium picrate (--).
Table 1. Number-Average Molecular Weight (Mn), Polydispersity (Mw/Mn), and nl, nh, and np Values for r,ω-Diamino-poly(ethylene glycol) (DPEG) and 2 Determined by MALDI-TOF MS. DPEG 2
nl, nha
npb
Mn
Mw/Mnc
24, 41 24, 42
31 31
1470 2260
1.007 1.009
a nl, nh: the lowest and the highest numbers of ethylene oxide (EO) units of oligomers appeared in the MALDI-TOF mass spectrum, respectively. b np: the number of EO units of the most abundant oligomer. c Mw: weight-average molecular weight.
Even in the presence of other chromophores,15-16 an intense negative CD couplet centered at about 260 nm is diagnostic of an aggregate composed of at least two G quartets chirally rotated with respect to each other (G8).8 The CD couplet is attributed to the interaction of the long-axis-polarized transition of the G chromophore, and its negative sign indicates a left-handed helical arrangement.17 We could observe this negative couplet in the CD spectra of 2 taken in CHCl3 or CH3CN solution with low intensity without the addition of K+, probably as a result of a small amount of Na+ or K+ contamination. After the chloroform solution of 2 was equilibrated with potassium picrate, the resulting potassiumcontaining solution showed an intense negative exciton couplet that indicated the presence of left-handed helical stacked G quartets and thus, informally, the appearance of supramolecular polymer aggregates (Figure 2). In contrast to other lipophilic G aggregates,8 in our case the picrate anion is not held very tightly because its characteristic CD transitions at 365 and 413 nm are completely missing. In water or DMSO, no exciton couplet was observed, indicating that there is no aggregate formation in solvents that can form competitive hydrogen bonds. The apparent molecular weights (in fact these are upper limits) referenced to 318 showed that the solution contains three components: one in the 6-9 kDa, another in the 20-30 kDa, (14) Kaucher, M. S.; Lam, Y. F.; Pieraccini, S.; Gottarelli, G.; Davis, J. T. Chem.sEur. J. 2005, 11, 164-173. (15) Masiero, S.; Gottarelli, G.; Pieraccini, S. Chem. Commun. 2000, 19951996. (16) Kotch, F. W.; Sidorov, V.; Lam, Y. F.; Kayser, K. J.; Li, H.; Kaucher, M. S.; Davis, J. T. J. Am. Chem. Soc. 2003, 125, 15140-15150. (17) Gottarelli, G.; Masiero, S.; Spada, G. P. Enantiomer 1998, 3, 429-438. (18) 3: Phenylthio-2,3,4,6-tetra-O-benzyl-R-D-glucopyranoside was used as a reference compound.
Figure 3. Distribution of supramolecular aggregates of 2 determined with DOSY.
Figure 4. (a) TEM image of a supramolecular polymer of 2. (b) Possible structure of the G4-(PEG)4-G4 macromonomer of the supramolecular polymer (a bead in the string).
and the third in the 100-400 kDa region (Figure 3). A composition is suggested on the basis of the mass ratios. In this sample, about 12-16 TMS (tetramethylsilane) molecules are encapsulated in one G4-(PEG)4-G4 bead. This hydrophobic ligand-binding potential was proved by DOSY and 1H NMR integration of the bound TMS signal, which is shifted by +0.07 ppm with respect to free TMS. In another experiment, the CH2Cl2 solution of 2 was equilibrated with potassium picrate in water for 12 h. During the monitoring period, the apparent diffusion coefficients were changing, and only triple exponential fits worked well for the monitored signals in DOSY. In this case, the molar mass of the components was extended to the 10 MDa region when referenced to the internal picrate signal at 8.90 ppm. These DOSY spectra clearly showed that picrate was not present in the aggregates, corroborating the former CD results.
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Langmuir, Vol. 23, No. 10, 2007 5285
The size of the aggregates was also substantiated by the dynamic light scattering experiments. In CHCl3, they showed the presence of aggregates with a bimodal distribution centered at about 0.1 and 1 µm in diameter. Owing to the very narrow molecular weight distribution (i.e., very low dispersion in n) of 2, the bimodal distribution observed by DLS probably is not due to the polydispersity of 2. The TEM (transmission electron microscopy) image made with a sample containing potassium picrate after solvent evaporation shows stringlike aggregates, and the estimated diameter of the repeating spheroid formations is about 10 nm (Figure 4a). We suppose that this smallest structural unit (i.e., a bead in the string) can be represented by two G4 quartets linked with four PEGs as shown in Figure 4b, which are strung to chains by the G8 complex-forming effect of the potassium ion. The estimated geometrical size of the assembly consisting of two G4 quartets and four PEG chains by quantum mechanical calculation is in good agreement with those obtained by TEM.
Conclusions We have prepared a lipophilic, telechelic polyethylene glycol dimer of guanosine that, in the presence of the potassium ion, assembles into a dynamic supramolecular polymer. Supported by CD, DOSY, light scattering photometry, and TEM, we could depict the macromonomer structure as G4-(PEG)4-G4. Studies on the possibility of using our polymer as an ion channel or encapsulating agent are in progress. Acknowledgment. This article is dedicated to Professor Gyo¨rgy Hajo´s on the occasion of his 60th birthday. We thank the Hungarian National Science Foundation and the Hungarian Academy of Sciences for the generous support (grants OTKA T42512, K62213, T42567, N-61336, T049436, F043536, and RET 006/2004). Supporting Information Available: Experimental and selected spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org. LA070019G
