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Enhanced Accessibility of Peptide Substrate toward Membrane-Bound Metalloexopeptidase by Supramolecular Structure of Polyrotaxane Tooru Ooya, Masaru Eguchi, and Nobuhiko Yui* School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan Received September 26, 2000; Revised Manuscript Received December 21, 2000
A L-phenylalanlylglycylglycine- (H-L-PheGlyGly-) terminated polyrotaxane in which many R-cyclodextrins (R-CDs) are threaded onto poly(ethylene oxide) (PEO) was synthesized to evaluate the effect of R-CD threading on the degradation of the terminal H-L-PheGlyGly by a membrane-bound metalloexopeptidase (aminopeptidase M). The threading of R-CDs and introducing H-L-PheGlyGly to the terminals were confirmed by gel permeation chromatography and 1H NMR spectroscopies. In vitro degradation and kinetic studies revealed that the supramolecular structure of the polyrotaxane enhanced the accessibility toward aminopeptidase M despite the higher molecular weight of the polyrotaxane (Mn: ∼16 000). This finding provides a new design of biodegradable polymers for biomedical applications with controlled degradation profile. Controlling enzymatic degradation of polymeric materials is a key factor to achieve a site-specific drug release1 and the disappearance of tissue scaffolds in response to specific enzymes expressed by regenerated tissue.2 Conjugation of oligopeptides that have an appropriate sequence against target enzymes with a hydrophilic polymeric chain has been a major approach to the regulation of enzymatic degradability. However, it is suggested that such the conjugation reduces accessibility of enzymes to the oligopeptide chains due to the association of them in physiological conditions.3 From this perspective, we have proposed biodegradable polyrotaxanes in which a lot of chemically modified R-cyclodextrins (R-CDs) are threaded onto a poly(ethylene oxide) (PEO) chain capped with an amino acid.4 Because dethreading of R-CD derivatives can be triggered by enzymatic terminal hydrolysis, it occurred to us that this could be the basis for new type of drug delivery systems.5 Recently, such the new molecular architecture of polyrotaxanes4,5 and dendrimers6 has been getting a fascinating field of biomedical materials. Particularly, polymeric neoglycoconjugates including a comblike glycodendrigraft, a glycohyperbranched polymer and a glycodendrimer may become new drugs based on the enhanced affinity to cell receptors. Our next aim is to achieve dual-function for these compounds as new biomaterials: controlling the multivalency that is becoming interested in the rational design of new drugs such as virus inhibitors7 and enzymatic hydrolysis. The biodegradable polyrotaxanes have a potential to introduce a variety of ligands, which can simultaneously bind to multivalent receptors on a cell surface, into many hydroxyl groups of R-CDs. Multivalent binding of the polyrotaxanes and the following dissociation of the polyrotaxanes via enzymatic * To whom correspondence should be addressed. Telephone: +81-76151-1640. Fax: +81-761-51-1645. E-mail:
[email protected].
hydrolysis may lead to both programmed multivalent binding and elimination of the degradation products (R-CD derivative, the oligopeptide or amino acid, and PEO) from human body. In our previous study, we found that the supramolecular structure of the polyrotaxane did not induce steric hindrance of the accessibility of enzymes to the terminal pepetide.4e Our present concern is controlling the terminal degradation of polyrotaxanes by membrane-bound zinc metalloexopeptidases such as aminopeptidase M (E.C. 3.4.11.2) that is expressed in intestinal and kidney brush border membranes and the other mucosal surfaces.8 However, peptide substrates with higher molecular weight are unlikely to be hydrolyzed by aminopeptidase M because the accessibility of the substrate toward the enzyme is strongly decreased when the substrate becomes longer than a hexapeptide.9 Here, we report on enhanced degradation behavior of a tripeptide-terminated polyrotaxane consisting of R-CDs and PEO by the action of aminopeptidase M. We synthesized and characterized L-phenylalanlylglycylglycine- (H-L-PheGlyGly-) terminated polyrotaxane by gel permeation chromatography (GPC) and 1H NMR spectroscopies. The degradation of the terminal H-L-PheGlyGly by aminopeptidase M was evaluated comparing a model compound (H-L-PheGlyGly-terminated PEO). The synthesis of a H-L-PheGlyGly-terminated polyrotaxane was carried out by the following three steps (Scheme 1): (i) capping reaction between succinimide ester of tertbutyloxycarbonyl (Boc-) L-PheGlyGly 1 (14 mmol) and an inclusion complex consisting of R-[1-(1-aminopropoxy)-1methylethoxy]-ω-[2-(2-aminopropoxy)-2-methylethoxy]polyoxyethylene (bisaminopropoxy-PEO) (product name: Jeffermine ED-2001, Mn ) 1992, Texaco Chemical Co., TX) and R-CDs, 2 (the number of R-CDs: ca. 17) (0.14 mmol)
10.1021/bm005618f CCC: $20.00 © 2001 American Chemical Society Published on Web 01/23/2001
Enhanced Accessibility by Polyrotaxane
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Scheme 1. Synthesis of H-L-PheGlyGly-Terminated Polyrotaxane
in dimethyl sulfoxide (DMSO) for 3 days at room temperature in heterogeneous conditions to obtain 3,10 (ii) activation of hydroxyl groups of R-CDs in 3 using N,N′-carbonyldiimidazole (CDI), followed by the condensation with 2-aminoethanol,11 and (iii) deprotection of the Boc group using 30% trifluoroacetic acid (TFA) in dichloromethane (DCM). The activation of hydroxyl groups in 3 using CDI was confirmed by colorimetric determination of imidazole (absorbance at 207 nm) after the alkaline hydrolysis of N-acylimidazole groups. The degree of activation was controlled by changing the feed ratio of CDI and the hydroxyl groups, to a maximum of ca. 14 per one R-CD molecule. The free imidazole increased by adding 2-aminoethanol, indicating the condensation reaction proceeded. Final purification was carried out by GPC on Sephadex G-50 column using DMSO as an eluent. Figure 1 shows GPC charts of the product on the final purification (a) and H-L-PheGlyGlyterminated PEO (b). It was found that elution volume of the obtained product was smaller than that of H-L-PheGlyGly-
Figure 1. GPC traces of (a) the product on the final purification process and (b) L-PheGlyGly-terminated PEO. Column: Sephadex G-50 (3 × 100 cm). Eluent: DMSO. Flow rate: 0.3 mL/min. Detection: refractive index.
terminated PEO. Finally, the main product in the peak in Figure 1a was recovered and analyzed by a 1H NMR measurement (solvent: DMSO-d6). The 1H NMR spectrum showed the existence of R-CDs (e.g., C1H: δ ) 4.75 ppm), PEO (CH2: δ ) 3.56 ppm), and H-L-PheGlyGly (aromatics, δ ) 7.25 ppm; peptide bonds, δ ) 8.12 and 8.72 ppm). The complete deprotection of Boc group was confirmed by
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Table 1. 1H NMR Peak Integration on Cycled NOE Difference Experimenta protons of R-CD in H-L-PheGlyGly-terminated polyrotaxane absolute values of the NOE differenceb
C1H
C2H
C3H
C4H
C5H
C6H
0.05
0.29
0.80
0.20
1.00
0.54
a Irradiation point is CH peak (3.56 ppm) of PEO. b These values 2 indicate differences in two peak integrals between before and after irradiation.
Figure 2. Terminal cleavage of H-L-PheGlyGly-terminated polyrotaxane (circle), H-L-PheGlyGly-terminated PEO (triangle), and H-LPheGlyGly-OH (square) by aminopeptidase M (final concentration: 0.04 nmol/mL).
disappearance of a peak at δ ) 1.27 ppm (CH3 of Boc). These results suggest that the purified sample is the polyrotaxane consisting of hydroxyethylcarbamoyl-R-CDs and H-L-PheGlyGly-terminated PEO. The threading of hydroxyethylcarbamoyl-R-CDs was confirmed by the nuclear Overhauser effect (NOE) difference in 0.01 M NaOD (Table 1). When a pulse was irradiated to CH2 peak of PEO (3.56 ppm), the absolute value of the NOE difference attributed to the protons at C3 and C5 positions of R-CD was significantly larger than the other proton peaks of R-CDs. Since the protons of C3 and C5 positions were located in the cavity, the larger value of the NOE difference suggests the threading of R-CDs onto the PEO chain. The number of R-CDs in the polyrotaxane and the average number of hydroxyethylcarbamoyl groups per R-CD were calculated to be ca. 8 by the 1H NMR spectrum. An in vitro degradation experiment was carried out by adding 50 µL of aminopeptidase M stock solution (1.0 mg/ mL) to 2 mL of 50 mM phosphate buffer (pH 7.4) containing the polyrotaxane (48 mg, content of terminal H-L-PheGlyGly; 6.0 µmol) at 37 °C. Terminal peptide bond cleavage was determined by monitoring degradation products (H-LPhe-OH) in high performance liquid chromatography (HPLC) with a reversed-phase column.12 The cleavage of terminal peptide bond between H-L-Phe and Gly in the polyrotaxane was enhanced in comparison with H-LPheGlyGly-terminated PEO, although the cleavage rate of H-L-PheGlyGly-OH was faster than that of the polyrotaxane (Figure 2). The hydrolytic behavior of the mixture between R-CD (or hydroxypropylated R-CD) and H-LPheGlyGly-terminated PEO was similar to that of H-LPheGlyGly-terminated PEO in Figure 2 (data not shown),
indicating that free R-CDs or the modified R-CDs did not act in the vicinity of aminopeptidase M. Since the chain length of peptides as the substrates of aminopeptidase M is limited from di- to heptapeptide9 (Mn:
