Article pubs.acs.org/Macromolecules
Highly Efficient Transfer of Amino Groups to Imidazolium Entities for Polymer Coupling and Cross-Linking Kai-Steffen Krannig, Davide Esposito, and Markus Antonietti* Department of Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, 14424 Potsdam, Germany ABSTRACT: It is shown that pendant amino groups on polymers can be conveniently transferred into chemically and thermally very stable imidazolium linkages by using a simple modification of the Debus− Radziszewski synthesis, a cascade reaction involving two amines, one methylglyoxal and one formaldehyde monomer. It is shown that this reaction is so effective that it can be used either for the coupling of two terminal amino groups onto PEO chains or the high density x-linking of poly(L-lysine)s into poly(L-lysine)−imidazolium gels. In the latter case, it is shown that sterical hindrances restricts the conversion of amines to about 60 mol %, creating highly interlinked poly(L-lysine− imidazolate) microgels.
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INTRODUCTION Convenient coupling or cross-linking of polymers are omnipresent tools, both in biology as well as in synthetic chemistry. Inner S−S linkages based on cysteine for instance, stabilize the tertiary structure of proteins or lead to larger protein adducts, while clicking of appropriately modified polymers onto other polymers, surfaces or proteins generates protein conjugates, functionalized polymer surfaces or also just block and graft copolymers.1 Synthetic use of those coupling reactions has brought enormous progress, but it is safe to state that new reactions are still cordially welcomed, especially when they are highly effective, based on simple and available functionalities and result in very stable and biocompatible, yet sustainable bonding schemes. In a recent publication, we have used a practically “forgotten” reaction, the Debus−Radziszewski synthesis,2 to couple two primary amino groups from amino acids via methylglyoxal and formaldehyde into an imidazolium moiety, there for the purpose to generate ionic liquids.3 The reaction was conducted in water, at very mild conditions, following the rules of green chemistry, and the yields wereeven for sterically demanding amino acidspartly extremely high (Scheme 1). Considering that no less than four molecules are involved in this coupling reaction, the high yields could be only explained considering the stability of the aromatic system generated at the end of a cascade reaction where an amine is reversibly activated by a
methylglyoxal, which then reacts in an accelerated fashion with a second amine and finally formaldehyde to form the imidazolium cycle (Scheme 1). The resulting linkage is thermally and chemically extremely stable, as confirmed by DSC analysis of the corresponding ionic liquids. It is the special advantage of such cascade reactions involving reversible steps that they can also be performed in a slight excess of coupling agents, here formaldehyde and methylglyoxal. Interestingly, similar imidazolium cross-links involving lysine and arginine residues are responsible for protein aggregation during some degenerative processes.4 Furthermore, similar mechanisms have been evoked in the formation of in vivo extracellular matrix cross-linking involving collagen.5 It is the purpose of the present paper to extend these findings to polymer chemistry and to employ this reaction to the water based coupling or cross-linking of polymers. As model reactions, we choose here the end-to-end linkage of two amino functionalized poly(ethylene glycol) chains as well as the massive cross-linking of lysine toward poly(L-lysine)-imidazolium micro- and macrogels. The latter reaction is to be understood as a model reaction for all amino functionalized proteins, as steric and pH demands are expected to be rather general.
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MATERIALS AND METHODS
All chemicals were purchased from Sigma-Aldrich unless otherwise noted. H−Lys(Z)−OH was obtained from Novabiochem, triphosgene (98%) and THF (extra dry over molecular sieve, 99.5%) from Alfa Aesar. Trifluoroacetic acid (TFA, 99+%) and methylglyoxal (40% in water) came from Acros Organics and heptane from Roth. 1Hexylamine was supplied from Aldrich (99.5%) and distilled prior to
Scheme 1. Reaction Scheme of the Debus−Radziszewski Imidazole Synthesis Using Methylglyoxal and Formaldehyde
Received: February 4, 2014 Revised: March 14, 2014 Published: March 26, 2014 © 2014 American Chemical Society
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use. N,N-Dimethylformamide (DMF, puriss.) was used as supplied and under inert atmosphere. NMR measurements were carried out at room temperature using a Bruker DPX-400 spectrometer operating at 400.1 MHz for 1H NMR. Deuterated TFA and D2O were used as solvents (Sigma-Aldrich); signals were referenced to the residual signal of TFA δ = 11.52 ppm and D2O δ = 4.79 ppm, respectively. Melting points were determined using a MEL-TEMP apparatus from Lab Devices INC, USA with a Fluke 51 thermometer. Size exclusion chromatography (SEC) with simultaneous UV/RI detection was performed with N-methyl-2pyrrolidone (NMP + 5 wt % LiBr) as the eluent at +70 °C using a set of two 300 × 8 mm2 PSS-GRAM columns with average particle sizes of 7 μm and porosities of 100 and 1000 Å. Calibration was done with PMMA standards (PSS, Mainz, Germany). SEC in water was performed with 0.1 M aqueous acetate buffer (pH 4.5) containing 20% methanol using a combination of two PSSNovema columns 300 × 8 mm2 with particle sizes of 10 μm and porosities of 30 and 3000 Å. Aqueous Calibration was done with PEO standards (PSS, Mainz, Germany) (Z)-Lysine-N-carboxyanhydride (NCA). The NCA was synthesized using a modified procedure previously described for γ-benzyl-Lglutamate NCA: 8.0 g H−Lys(Z)−OH (28.5 mmol, 1.0 equiv) were suspended in freshly distilled THF (250 mL) and heated to 50 °C. At this temperature 3.38 g triphosgene (11.4 mmol, 0.4 equiv) were added. A clear solution usually formed within 45 min, otherwise additional triphosgene (0.05 equiv/30 min) was added. After 4 h, argon was bubbled through the reaction mixture for 10 min to remove excess phosgene. The solution was concentrated to one-third of the volume and precipitated into a 10-fold excess of heptane. The yellowish precipitate was collected, redissolved in THF and precipitated from heptanes two more times. The NCA was obtained as colorless powder, 7.8 g (25.5 mmol, 88%). 1 H NMR (400.1 MHz, CDCl3): δ (ppm) = 7.37 (m, 5H, Ar−H), 6.85 (s, 1H, NH), 5.12 (m, 2H, H2C−Ar), 4.94 (m, 1H), 4.28 (m, 1H, H2C−CH-NH), 3.21 (m, 2H, O−C(O)−CH2), 1.97 (m, 1H diast., H2C−CH−NH), 1.81 (m, 1H diast., H2C−CH−NH), 1.66−1.23 (m, 4H, −H2C−H2C−CH-NH). Mp: 97−99 °C (rep. 101 °C).6 Polymerization. A 7.0 g (22.9 mmol, 1 equiv) sample of (Z)lysine−NCA was dissolved in 70 mL of DMF (10 wt %), cooled to 5 °C. 0.46 mL of a 0.1 M solution of freshly distilled 1-hexylamine in DMF (457 μmol, 0.02 equiv), were added. After 1 h, the vessel was evacuated (0.5 mbar) and the reaction-mixture stirred at room temperature for 5 d. The polymerization was terminated by precipitation into 10× H2O, and thus the precipitate was filtered and thoroughly washed using water and methanol. The polymer was further dissolved in THF and precipitated from heptane, collected and dried for 2 d in vacuo to obtain 5.6 g (21.4 mmol, 93%) of the product as colorless solid. SEC (NMP, 70 °C, PMMA standard): Mn 15.682, Đ = 1.13 Deprotection. A 2.0 g poly(Z)-lysine (7.6 mmol, 1.0 equiv) sample was dissolved in 25 mL of TFA and 5.4 mL of HBr in acetic acid (33%) (30 mmol, 4.0 equiv) were added under vigorous stirring. After 1 h the reaction mixture was poured into 10× Et2O and collected by centrifugation. The centrifugate was redispersed in saturated NaHCO3 solution and extensively dialyzed (RC 1000) against deionized water for 3 days and finally freeze-dried to obtain the deprotected polymer as a colorless solid. Imidazolium x-Linking in Solution. A 100 mg poly(L-lysine) sample was dispersed in the respective amount of 10 vol % acetic acid in deionized water. Thus, 60 μL of methylglyoxal (390 μmol, 0.5 equiv) and 29 μL of formaldehyde (390 μmol, 0.5 equiv) were added. After 16 h, the reaction mixture was diluted and extensively dialyzed against deionized water for 3 days. After freeze-drying the cross-linked polymer was obtained as a yellowish powder. Imidazolium x-Linked Films. A 100 mg sample of poly(L-lysine) was dissolved in 1 mL of 10 vol % acetic acid in deionized water and 60 μL of methylglyoxal (390 μmol, 0.5 equiv) and 29 μL of formaldehyde (390 μmol, 0.5 equiv) were added. The mixture was directly transferred into the polymerization form and left on the shaker (175 rpm) for 12 h. The resulting gels were brown.
Article
RESULTS AND DISCUSSION In a first set of model experiments, we coupled two poly(ethylene oxide) (PEO) chains with terminal aminogroups to test the general validity of the approach. Therefore, commercially available amine terminated PEO (Mn = 11.150 g/ mol, cNH2 = 97 μmol/g) was dispersed in water to give a 10 wt % solution (cNH2 = 9.7 × 10−6 mol/L) and methylglyoxal and formaldehyde were added. The reaction was conducted at room temperature for 11 days and each day aliquots were taken for SEC analysis (Figure 1). The SEC elugrams show two
Figure 1. SEC traces (eluent = N-methyl-2-pyrrolidone + 0.5 g/L LiBr, temperature 70 °C, calibration = PEO) of the PEO coupling reaction at different times over 11 days. The elugrams were normalized to “100%” for the uncoupled PEO.
distinctive signals, the signal at lower retention times having a molecular weight twice as high as the starting polymer, indicating successful coupling. Although the conjunction of the two polymer chains is a sterically complicated, bimolecular reaction at comparably low concentrations, we were able to couple no less than 45% in this very simple, nonoptimized reaction layout. Considering the very low concentration of end groups in the here analyzed specific solution, this value is comparably high and is a straight proof for the efficiency of this coupling reaction even under ambient reaction conditions. We also tried to elaborate on the kinetics of this process, but found neither clear first nor second order reaction rates. This is rather typical for cascade reactions with unclear rate determining step. Besides Methylglyoxal, also simple glyoxal was tested in a number of cases, however the reaction is chemically more uniform with the mixed aldehyde−ketone species. This work on low molecular species will be presented in a forthcoming contribution. In a second set of experiments, poly(L-lysine) (DP = 54) was cross-linked and carefully analyzed to get spectroscopic evidence on the nature of the final product. This reaction was first performed in dilute solutions (2.5 wt %), resulting in soluble products. GPC of the polymer before and after imidazolization (Figure 2) essentially revealed the preservation of the monodisperse distribution, thus excluding intermolecular cross-linking. The slightly higher retention time indicates a more compact packing of the polymer, easily explainable by intramolecular cross-linking. The fact, that practically only intramolecular and no intermolecular cross-linking is observed, speaks for a fast and thereby local reaction step. It is remembered that cross-linking of linear chains in diluted solutions always results in molecular species, known as 2351
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of the imidazolium. Integration of all signals is consistent with a degree of cross-linking of 60 mol %, which is much higher than what we expected. We assume that the resulting steric hindrances prevent the remaining amino groups from further reactions. Nevertheless, this partial conversion gives nice evidence for the chemical nature of the cross-linking reaction, while other side reactions are practically not observed. When the reaction was performed at higher concentrations, exceeding 10 wt %, rather strong, but highly swellable poly(Llysine) gels were formed, supporting our assumption that the cross-links at lower concentration are only intramolecular, which however can be shifted to partial intermolecular imidazolization above an overlap concentration. The gels show, rather rare for protein based gels, good reversible swelling properties over a volume factor of 20, and are even after cycling free of cracks or other swelling/drying defects (Figure 4). The mechanical properties of these gels depend manly on concentration and can be adjusted between shear moduli of 105 to 103 Pa, depending on polymer concentration. A detailed rheolological anylsis however is out of the scope of this primary work and has to follow.
Figure 2. SEC traces (eluent = 0.1 M aqueous acetate buffer (pH 4.5) containing 20% methanol, temperature 25 °C, calibration = PEO) of poly(L-lysine) before and after imidazolization of a 2.5 wt % solution. The cross-linked product remains its monomodal distribution and is slightly shifted toward lower retention times, an indication that intramolecular cross-linking occurred exclusively.
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CONCLUSION In this article, we reported on use of the rediscovered Debus− Radziszewski reaction for intra- and intermolecular polymer coupling by formation of a chemically and thermally stable imidazolium linkage under eventually green conditions. Amineterminated PEO was used to demonstrate the general validity of the coupling reaction allowing for up to 45% end-to-end coupling efficiency. Moreover, the reaction was conducted using poly(L-lysine) as the substrate. In diluted solution (≤5 wt %) the formation of microgels was observed. Cross-linking densities of up to 60 mol % were observed which happened to be exclusively intramolecular, that means more than every second monomer of the polypeptide could be cross-linked. At higher concentrations (≥10 wt %) strong, macroscopic gels
microgels, which in the case of polystyrene, for instance, could be kept at the primary molecular weight.7 1H NMR analysis was used to confirm the formation of the aromatic imidazolium ring for these soluble species, to quantify the degree of cross-linking and get further insights into the ring-forming mechanism (Figure 3). New signals in the spectrum can be found at 8.7, 7.2, and 2.2 ppm confirming the formation of the imidazolium ring. It is noteworthy that the signal of the protons adjacent to the amine in the side-chain are shifted downfield and overlap with the signal of the asymmetric carbon in the backbone, giving further evidence for the incorporation of the amine group into the aromatic system. Quantification of the crosslinking was achieved by comparison of the aliphatic signals between 1.8 and 0.8 ppm (internal reference) with the signals
Figure 3. 1H NMR (400.1 MHz, TFA-d) of the cross-linked poly(L-lysine). The spectrum was referenced to the aliphatic signals between 0.8−1.8 ppm. It is noteworthy that k′ was shifted downfield and overlaps with the signal of the asymmetric carbon in the polymer backbone. The degree of cross-linking can be derived from the integration of the imidazolium specific peaks (l and m) or the adjacent methyl group (n). 2352
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Figure 4. Photograph of a poly(L-lysine) network cross-linked with imidazolium entities via the Debus−Radziszewski reaction. The gel is in the dried state, proving the crack-free shrinking by a factor of 20 of this poly(amino acid) network.
were obtained which showed remarkable swelling potential. In perspective, we focus on the use of this easy, efficient, and green reaction for the posttranslational functionalization of peptides as well as the use of the microgels for drug delivery systems.
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AUTHOR INFORMATION
Corresponding Author
*(M.A.) E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) (a) Wang, A.; Lu, S.; Mark, D. Science 1984, 224 (4656), 1431− 1433. (b) Bonduelle, C.; Huang, J.; Ibarboure, E.; Heise, A.; Lecommandoux, S. Chem. Commun. 2012, 48 (67), 8353−8355. (c) Li, M.; De, P.; Gondi, S. R.; Sumerlin, B. S. Macromol. Rapid Commun. 2008, 29 (12−13), 1172−1176. (d) Cottenye, N.; Syga, M.I.; Nosov, S.; Muller, A. H. E.; Ploux, L.; Vebert-Nardin, C. Chem. Commun. 2012, 48 (20), 2615−2617. (e) Helms, B.; Mynar, J. L.; Hawker, C. J.; Fréchet, J. M. J. J. Am. Chem. Soc. 2004, 126 (46), 15020−15021. (f) Tsarevsky, N. V.; Bencherif, S. A.; Matyjaszewski, K. Macromolecules 2007, 40 (13), 4439−4445. (2) (a) Debus, H. Justus Liebigs Ann. Chem. 1858, 107 (2), 199−208. (b) Arduengo, A. J. Acc. Chem. Res. 1999, 32 (11), 913−921. (3) Esposito, D.; Kirchhecker, S.; Antonietti, M. Chem. - Eur. J. 2013, 19 (45), 15097−15100. (4) Lederer, M. O.; Klaiber, R. G. Bioorg. Med. Chem. 1999, 7 (11), 2499−2507. (5) Siani, A.; Tirelli, N. Antioxid. Redox Signaling 2013, DOI: 10.1089/ars.2013.5724. (6) Fasman, G. D.; Idelson, M.; Blout, E. R. J. Am. Chem. Soc. 1961, 83 (3), 709−712. (7) (a) Antonietti, M. Angew. Chem., Int. Ed. 1988, 27 (12), 1743− 1747. (b) Antonietti, M.; Bremser, W.; Schmidt, M. Macromolecules 1990, 23 (16), 3796−3805. (c) Antonietti, M.; Sillescu, H.; Schmidt, M.; Schuch, H. Macromolecules 1988, 21 (3), 736−742.
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