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Bioconjugate Chem. 2002, 13, 582−587
Inhibitory Effect of Supramolecular Polyrotaxane-Dipeptide Conjugates on Digested Peptide Uptake via Intestinal Human Peptide Transporter Nobuhiko Yui,*,† Tooru Ooya,† Tomokatsu Kawashima,† Yoshimasa Saito,‡ Ikumi Tamai,‡ Yoshimichi Sai,‡ and Akira Tsuji*,‡ School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan and Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa 920-0934, Japan. Received October 17, 2001; Revised Manuscript Received March 13, 2002
The effect of polyrotaxane-dipeptide (Val-Lys) conjugates on the uptake of a model dipeptide (GlySar) was examined via human peptide transporter (hPEPT1) on HeLa cells. Here, Val-Lys groups are introduced to R-CDs, which are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups (polyrotaxane). The Gly-Sar uptake via hPEPT1 was significantly inhibited in the polyrotaxane conjugates, and this inhibitory effect was not explained by the sum of interaction between hPEPT1 and R-CD-Val-Lys conjugates. Further, the inhibition was significantly greater than those observed in dextran-Val-Lys conjugates. Therefore, our data clearly suggests that supramolecular structure in the polyrotaxane conjugates contributes considerably to the inhibitory effect via multivalent binding of Val-Lys groups with hPEPT1.
INTRODUCTION
Proteins are enzymatically digested to di- or tripeptides and/or amino acids, and these protein-digestive products are absorbed via specific transporters from the small intestine. Multiple transporters are involved in intestinal membrane transports for amino acids, while di- and tripeptides are transported only via peptide transporter, PEPT1. Besides, in terms of the intake of proteins and the absorption of some drugs, peptide transporter is expected to be more important than amino acid transporters. In particular, conjugation of poorly absorbable drugs with PEPT1-recognizable molecules such as di- or tripeptides is a powerful approach to exploit the substrate affinity of PEPT1. Kamo et al. synthesized dipeptide analogues conjugated at the -amino group of Lys in Val-Lys or Lys-Sar with fluorescent dyes as model hydrophobic drugs (1). They also reported that the hydrophobicity of dipeptide conjugates was an important parameter recognized by PEPT1 (2). The objective of their study was to enhance drug absorption through PEPT1, and thus conjugates of lower molecular weight were a minimum requirement. Dietary protein restriction is now recommended for patients of chronic renal disease (CRD),1 but compliance is not typically high due to the effect on quality of life (QOL). In this sense, it is logical to temporarily inhibit the absorption of digested proteins via PEPT1 to improve QOL of patients. The inhibitor should be recognized by PEPT1 and have nonabsorbable properties so as to preclude kidney damage. In these points of view, multivalent ligands * To whom correspondence should be addressed. N. Yui: Phone: +81-761-51-1640. Fax: +81-761-51-1645. E-mail: yui@ jaist.ac.jp. A. Tsuji: Phone: +81-76-234-4479. Fax: +81-76-2344477. E-mail:
[email protected]. † Japan Advanced Institute of Science and Technology. ‡ Kanazawa University.
that display multiple sites of recognition by PEPT1 on intestinal surfaces are advantageous. Such multivalent ligands maycooperatively interact with PEPT1 and inhibit their absorption. Cooperative affinity of ligands using synthetic or natural polymers such as tabacco mosaic virus (3), sugarballs (4), and comb-branched or dendrigraft neoglycopolymers (5) has been proposed as a new drug design strategy. The important factors that control the binding features include valency, architecture of the scaffolds, and orientation of the ligands. Such multivalent ligands can act as inhibitors of receptor-ligand interactions and activators of biological systems (6). For example, Baker, Jr. et al. studied the effect of ligand architecture, including dendrimers, comb-branched copolymers, dendrigraft copolymers, and linear-dendron architectural copolymers, on inhibitory activity and specificity (5). Arimoto et al. reported that a multivalent polymer of vancomycin, synthesized by ring-opening metathesis polymerization (ROMP), showed enhanced antibacterial activity against vancomycin-resistant bacteria (7), suggesting that multivalent ligands might be promising tools in the fight against multiresistant bacteria. Accordingly, the design of the scaffold structure is important not only for clarifying the multivalent mechanism in nature but also for designing new drugs, such as multivalent viral and toxin inhibitors. 1 Abbreviations: CRD, chronic renal disease; QOL, quality of life; ROMP, ring-opening metathesis polymerization; R-CDs, R-cyclodextrins; Gly-Sar, glycylsarcosine; PEO-BA, R-(3-aminopropyl)-ω-(3-aminopropyl)polyoxyethylene, CDI, N,N′-carbonyldiimidazole; DIPEA, N,N′-diisopropylethylamine, HOBt, 1hydroxybenzotriazole; WSC‚HCl, water-soluble carbodiimide [1-ethyl-3-(dimethylamino)carbodiimide]; DMAP, (dimethylamino)pyridine; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HBSS, Hanks balanced salt solution; MES, 2-(N-morpholino)ethanesulfonic acid; HEPES, 2-[4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid.
10.1021/bc015567z CCC: $22.00 © 2002 American Chemical Society Published on Web 04/17/2002
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Scheme 1. Synthesis of Polyrotaxane-Val-Lys Conjugates
We have systematically studied the design of supramolecular-structured polymers as new biomaterials and drug carriers. One of the representatives is a biodegradable polyrotaxane, 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 or an oligopeptide (8). The biodegradable polyrotaxanes may have a variety of ligands, which can simultaneously bind to multivalent receptors on a cell surface, because many hydroxyl groups are available for introducing ligands on the R-CDs located along the polyrotaxane structure. In our previous study, polyrotaxane-biotin conjugates were synthesized to demonstrate the potential of multivalent ligands (9, 10). On the basis of this architectural design, we have proposed the development of multivalent ligands using the polyrotaxanes as a specific inhibitor of PEPT1. In this study, polyrotaxane-dipeptide (valyllysine, Val-Lys) conjugates are synthesized and characterized, and the inhibitory effect of the conjugates on [3H]glycylsarcosine ([3H]Gly-Sar, a model peptide transported by PEPT1) transport in HeLa cells stably expressing human PEPT1 (hPEPT1) is evaluated in comparison with R-CDVal-Lys and dextran-Val-Lys conjugates. Our final goal is to inhibit absorption of digested peptides in the intestinal tract via hPEPT1 transporter through multivalent binding of PEPT1-recognizable polymers. MATERIALS AND METHODS
Materials. R-CD was purchased from Bio-Research Corporation of Yokohama (Yokohama, Japan). R-(3Aminopropyl)-ω-(3-aminopropyl)polyoxyethylene (PEOBA: Mn ) 4000) was kindly supplied by Sanyo Chemical Co. (Kyoto, Japan). Benzyloxycarbonyl-L-phenylalanine (Z-L-Phe), 2-aminoethanol, N,N′-carbonyldiimidazole (CDI), and N,N′-diisopropylethylamine (DIPEA) were purchased from Wako Pure Chemical Co. Ltd., (Osaka, Japan). N-Hydroxysuccinimide, 1-hydroxybenzotriazole (HOBt), and Boc-Val were purchased from Peptide Institute,
Inc. (Osaka, Japan). Lys-(Cbz)-OtBu‚HCl was purchased from BACHEM (Bubendorf, Switzerland). Water-soluble carbodiimide (1-ethyl-3-(dimethylamino)carbodiimide, WSC‚HCl) and (dimethylamino)pyridine (DMAP) were purchased from DOJINDO Labs (Kumamoto, Japan). Dextran from Leuconostoc mesenteroids (Mn ) 1500020000) and Gly-Sar were purchased from Sigma Chemical Co. (St. Louis, MO). N,N-Dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were purchased from the Wako Pure Chemical Co. Ltd. and distilled by the usual method. All other chemicals used were of reagent grade. [3H]Glycylsarcosine ([3H]Gly-Sar) was purchased from Moravek Biochemicals Inc. (Brea, CA). Other chemaicals used were of the highest purity available. Synthesis of Polyrotaxane-Val-Lys Conjugates (Scheme 1). Boc-Val-Lys-OtBu‚HCl was synthesized and characterized according to the previous report by Kamo et al. (1). A polyrotaxane, in which many R-CDs are threaded onto a PEO-BA (Mn ) 4000) capped with Z-LPhe, was prepared according to our method (9, 11). Briefly, an inclusion complex of R-CDs and PEO-BA was prepared by simply mixing a saturated aqueous solution of R-CDs with a PEO-BA aqueous solution (1/44 equiv of R-CD). Then the succinimide ester of Z-L-Phe, prepared by a condensation reaction of Z-L-Phe and N-hydroxysuccinimide, was allowed to heterogeneously react with the terminal amino groups in the inclusion complex in DMSO. The chemical structure was characterized by 750 MHz 1H NMR using a FT-NMR spectrometer (Varian FT-NMR Gemini750, Palo Alto, CA). The number of R-CDs was determined to be ca. 23 from the 1H NMR spectra based on the comparison of integration of the signals at 4.75 (C1H of R-CD) with those at 3.49 (CH2CH2O of PEO). The obtained polyrotaxane (0.17 mmol) and CDI (e.g., 15.6 mmol) were dissolved in dry DMSO (10 mL) and stirred at room temperature for 3 h. The reaction mixture was poured into excess ether, and the resulting precipitate was filtered and dried in vacuo to obtain CDIactivated polyrotaxanes. The number of N-acylimidazole groups per polyrotaxane molecule was determined by
584 Bioconjugate Chem., Vol. 13, No. 3, 2002
Yui et al.
Table 1. Synthesis of CDI-Activated Polyrotaxanes
sample codea
OH groups in polyrotaxane (mmol)
CDI (mmol)
CDI-activation (%)b
27aIM-R/E4-PHE-Z 46aIM-R/E4-PHE-Z 54aIM -R/E4-PHE-Z 57aIM -R/E4-PHE-Z
3.1 3.1 3.1 3.1
3.1 6.1 9.2 15.4
27 46 54 57
b
a aIM ) N-acylimidazole group, R/E4-PHE-Z ) polyrotaxane. Calculated from the equation (A).
measuring the absorbance at 207 nm after hydrolysis of N-acyl carbonate groups using 2 M NaOH aqueous solution. Degree of the activation was calculated by the following equation (A):
CDI-activation (%) ) [Im]/[OH]prx × 100
(A)
where [Im] and [OH]prx are, respectively, concentrations of the N-acylimidazole groups and total hydroxyl groups in the polyrotaxanes. The synthetic conditions and results were summarized in Table 1. The CDI-activated polyrotaxane (N-acylimidazole groups per polyrotaxane molecule: 0.97 mmol), Boc-Val-LysOtBu‚HCl (e.g., 3.3 mmol), and DIPEA (1.1 equiv of Boc-Val-Lys-OtBu‚HCl) were dissolved in DMSO (2 mL) and stirred at room temperature for 24 h. The feed ratio of the CDI-activated polyrotaxane and Boc-Val-LysOtBu‚HCl was varied (Table 2). After that, 2-aminoethanol (33 mmol) was added to the solution and further stirred for 24 h. The solution was dialyzed against water and lyophilized to obtain a polyrotaxane-Boc-Val-LysOtBu conjugate as white powder. Finally, the protecting groups of Boc and t-Bu were removed in the mixture of dichloromethane and trifluoroacetic acid (7:3), precipitated in excess ether, and dried in vacuo to obtain a polyrotaxane-Val-Lys conjugate as white powder. Yield: ∼10%. 1H NMR D2O + NaOD, ppm: δ 7.357.18 (aromatics of Z-L-Phe), 7.15-6.80 (-OCONH- of carbamoyl linkages), 5.02 (C1H of R-CD), 4.25-3.30 (C3H, C5H, C6H2, C4H, and C2H of R-CD), 4.19 (R-CH of Lys), 3.62 (CH2CH2O of PEO), 3.75 (R-CH of Val), 3.17 (CH2 of hydroxyethyl carbamoyl groups), 2.15 (β-CH of Val), 1.38 (β-, γ-, and δ-CH2 of Lys), 0.98 (CH3 of Val). Synthesis of r-CD-Val-Lys Conjugates. R-CD (0.31 mmol) and CDI (5.56 mmol) were dissolved in dry DMF (5 mL), and the solution was stirred at room temperature for 3 h. Boc-Val-Lys-OtBu‚HCl (0.62 mmol) and DIPEA (1.1 equiv of Boc-Val-Lys-OtBu‚HCl) were added to the solution and stirred at room temperature for 24 h. Then, 2-aminoethanol (5.6 mmol) was added to the solution and further stirred for 24 h. The solution was dialyzed against water and lyophilized to obtain an R-CD-Boc-Val-LysOtBu conjugate as white powder. Finally, the protecting groups of Boc and t-Bu were removed in a manner similar to the polyrotaxane-Val-Lys conjugates. Yield: ∼80%. 1H NMR D2O, ppm: δ 7.10-6.81 (-OCONH- of carbamoyl linkages), 4.93 (C1H of R-CD), 4.00-3.33 (C3H, C5H, C6H2, C4H, and C2H of R-CD), 4.35 (R-CH of Lys), 3.67 (R-CH of Val), 3.13 (CH2 of hydroxyethylcarbamoyl groups), 2.01 (β-CH of Val), 1.32 (β-, γ-, and δ-CH2 of Lys), 0.91 (CH3 of Val). Synthesis of Dextran-Val-Lys Conjugates. Hydroxyl groups of dextran were activated using p-nitrophenyl chloroformate according to our previous method (12). The activated dextran (0.01 mmol, the number of p-nitrophenyl carbonate group per 100 glucose units: 20) was dissolved in dry DMSO (10 mL), and then Boc-ValLys-OtBu‚HCl (e.g., 0.4 mmol) was added to the solution.
The reaction mixture was stirred at room temperature for 48 h. The feed ratio of the activated dextran and BocVal-Lys-OtBu‚HCl was varied according to the reaction conditions (Table 2). Then, the solution was dialyzed against water and lyophilized to obtain a dextran-BocVal-Lys-OtBu conjugate as white powder. Finally, the protecting groups of Boc and t-Bu were removed in a manner similar to the polyrotaxane-Val-Lys conjugates. Yield: ∼50%. 1H NMR DMSO-d6, ppm: δ 4.91-4.76 (O2H, O3H of dextran), 4.64 (C1H of dextran), 4.59-4.40 (O4H of dextran), 4.19 (R-CH of Lys), 3.92-3.19 (C3H, C5H, C6H2, C2H, and C4H of dextran), 3.75 (R-CH of Val), 2.13 (β-CH of Val), 1.33 (β-, γ-, and δ-CH2 of Lys), 0.98 (CH3 of Val). Characterization. Purity of the conjugates was confirmed by gel permeation chromatography (GPC) using a column of Sephadex G-75, 0.1 M phosphate buffer, pH 7.4 as an eluent. The peaks on the GPC were detected by refractive index. The structure of the conjugates was characterized by 750 MHz 1H NMR using the FT-NMR spectrometer. The number of R-CDs in the polyrotaxane was determined by the 1H NMR spectra. The number of Val-Lys per one conjugate molecule was determined by the following equation (B):
NVK ) [Val]/[polyrotaxane], [polyrotaxane] ) [Phe]/2 (B) where NVK is the number of Val-Lys per one conjugate molecule, [Val] the concentration of Val in the conjugate, [polyrotaxane] the concentration of the polyrotaxane, and [Phe] the concentration of the terminal Z-L-Phe in the polyrotaxane, which were determined by amino acid analysis using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Cell Culture and Uptake Experiments Using HeLa-hPEPT1. HeLa cells that were stably transfected with human peptide transporter PEPT1-cDNA (HeLahPEPT1) or vector DNA (pcDNA3, Invitrogen, San Diego, CA) alone (HeLa-pcDNA3) were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FCS, 2mM glutamine and 1.0 mg/mL geneticine (G418, Sigma Chemicals, St. Louis, MO), as described previously (13). For the uptake study, each cell line was seeded at a density of 105 cells/cm2 on multidishes (NUNC, Multidish 4 wells, Denmark) and were grown for 4 days. Uptake of [3H]Gly-Sar (0.48 µM) by the cultured cells was examined at 37 °C according to our previous study (13). The uptake of [3H]Gly-Sar by the cells was usually measured using Hanks balanced salt solution (HBSS) consisting of 0.952 mM CaCl2, 5.36 mM KCl, 0.441 mM KH2PO4, 0.812 mM MgSO4, 136.7 mM NaCl, 0.385 mM Na2HPO4, 25 mM D-glucose, and 10 mM MES adjusted to pH 6.0 by HEPES. The cells cultured on the dishes were washed by HBSS and preincubated with HBSS for 5 min. Then, the uptake was initiated by changing the medium with HBSS containing radiolabeled Gly-Sar and each test compound. The uptake was terminated by removing the uptake solution and three times washing with ice-cold HBSS. The cells were dissolved in 0.25 mL aliquots of 5 N NaOH for 2 h, neutralized by 5 N HCl (0.25 mL), and mixed with the scintillation fluid Cleasol-I (Nacalai tesque, Kyoto, Japan), and then the associated radioactivities were measured with a liquid scintillation counter (LSC-5100, Aloka, Tokyo, Japan). Protein was measured by the protein-dye binding method (14).
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Bioconjugate Chem., Vol. 13, No. 3, 2002 585
Table 2. Synthesis of Polyrotaxane-Val-Lys Conjugates sample codea
feed ratio ([VK]/[activated compd])
no. of Val-Lys/molb
no. of R-CD/molc
no. of HE/molc
total Mnc
111HE-R/E4-PHE-Z 1VK71HE-R/E4-PHE-Z 2VK57HE-R/E4-PHE-Z 11VK36HE-R/E4-PHE-Z 46VK98HE-R/E4-PHE-Z 3VK-Dextran 6VK-Dextran 10VK-Dextran 36VK-Dextran 16VK8HE-Dextran 1VK3HE-RCD
0 88 97 153 683 3 13 23 40d 40 2
0 1 2 11 46 3 6 10 36 16 1
23 22 16 9 21 -
111 71 57 36 98 0 0 0 0 8 3
33200 30560 24100 18240 43200 17900 18800 20000 26600 21800 1400
a VK ) Boc-Val-Lys-OtBu‚HCl, HE ) hydroxyethylcarbamoyl groups, R/E4-PHE-Z ) polyrotaxane. b Calculated from amino acid analysis. Calculated from 750 MHz 1H NMR spectra for the polyrotaxane-Val-Lys and R-CD-Val-Lys conjugates, and from GPC with a calibration using pullulan standard. d The reaction was carried out at 40 °C.
c
RESULTS AND DISCUSSION
Synthesis. To introduce Val-Lys groups using the hydroxyl groups of R-CDs in the polyrotaxane, the -amino group of lysine in Val-Lys was used because the R-carboxyl group and R-amino group of Val-Lys are essential for better recognition of PEPT1 (1). The hydroxyl groups of R-CDs in the polyrotaxane were activated by CDI. As shown in Table 1, the percentage of the activation increased with the amount of CDI. The IR spectrum of the sample displayed a strong peak around 1700 cm-1 after activation. These results indicate that N-acylimidazole groups were actually introduced to the polyrotaxane. Maximum number of the activation was found to be nine per R-CD molecule in the polyrotaxane. From the 1H NMR spectrum of the product after coupling Val-Lys with the CDI-activated polyrotaxane, the peaks attributed to Z-L-Phe, R-CDs, PEO, hydroxyethylcarbamoyl group, and Val-Lys were confirmed. There were no peaks attributed to N-acylimidazole groups in the spectrum, suggesting that the coupling reaction and the following washing procedure were successful. Similarly, all the peaks attributed to the Val-Lys conjugates were confirmed with regard to the R-CD-Val-Lys conjugate and the dextran-Val-Lys conjugate. Table 2 summarizes the synthesis of the polyrotaxane-Val-Lys, the R-CD-Val-Lys, and the dextran-Val-Lys conjugates. The number of Val-Lys per polyrotaxane molecule increased with the amount of Val-Lys in the feed. The maximum number of Val-Lys was ca. 46 per polyrotaxane molecule, indicating that two Val-Lys groups were introduced to every R-CD molecule. Figure 1 shows GPC chromatograms of the polyrotaxane-Val-Lys conjugate and the polyrotaxane (a), the R-CD-Val-Lys conjugate and R-CD (b), and the dextran-Val-Lys conjugate and dextran (c), respectively. It was found that the elution volume of the polyrotaxane-Val-Lys conjugate was similar to that of the hydroxyethylcarbamoyl-polyrotaxane, and there was no peak attributed to R-CD. This result indicates that free R-CD and its derivatives are not contaminated in the polyrotaxane-Val-Lys conjugate. The elution time of the R-CD-Val-Lys conjugate was shorter than that of R-CD, presumably due to association of the conjugates. Such an association behavior was not observed in the polyrotaxane-Val-Lys conjugate. Perhaps, due to the rodlike structure of the polyrotaxane, the association may be reduced. The association property of either the polyrotaxane or a drug-polyrotaxane conjugate was studied by light scattering measurements in our previous studies (15, 16). The association number of the polyrotaxane molecules decreases with increasing the number of R-CDs
Figure 1. GPC traces of the conjugates. (a) Polyrotaxane-ValLys conjugate (46VK98HE-R/E4-PHE-Z, solid line) and hydroxyethyl carbamoyl polyrotaxane (dashed line), (b) R-CD-Val-Lys conjugate (1VK8HE-R-CD, solid line) and R-CD (dashed line), and (c) dextran-Val-Lys conjugate (16VK8HE-Dextran, solid line) and dextran (dashed line). Column: Sephadex G-70, Eluent: 0.1 M phosphate buffer, pH 7.4.
in the polyrotaxane. Our previous study suggests that the rodlike structure of polyrotaxanes in physiological environments contribute to their reduced association. As for the dextran-Val-Lys conjugate, the elution time was found to be a little bit shorter than the peak of dextran (Figure 1c). Mn of the dextran calculated from GPC with a calibration using pullulan standard was ca. 17100, and Mn of all the dextran-Val-Lys conjugates (Table 2) was larger than that of the dextran. These results indicate that the dextran-Val-Lys conjugate exhibits an association under buffered conditions. Inhibition of PEPT1-Mediated Transport of [3H] Gly-Sar by the Conjugates. In vitro efficacy of the polyrotaxane-Val-Lys conjugates as an inhibitor of peptide transporter was evaluated by the uptake of Gly-Sar by HeLa cells stably expressing hPEPT1. Figure 2 shows the inhibitory effect of the polyrotaxaneVal-Lys conjugate and the R-CD-Val-Lys conjugate (46VK98HE-R/E4-PHE-Z and 1VK3HE-RCD in Table 2, respectively) on the uptake of [3H]Gly-Sar. In Figure 2,
586 Bioconjugate Chem., Vol. 13, No. 3, 2002
Figure 2. Inhibition of [3H]Gly-Sar uptake via hPEPT1 by polyrotaxane-Val-Lys conjugate (46VK98HE-R/E4-PHE-Z), R-CD-Val-Lys conjugate (1VK3HE-RCD), hydroxyethylcarbamoyl-polyrotaxane (111HE-R/E4PHE-Z) and R-CD. Mean ( SEM (n ) 4). * Significantly different (p < 0.05) from 111HE-R/ E4PHE-Z.
hydroxyethylcarbamoyl-polyrotaxane (111HE-R/E4-PHEZ) and R-CD were used as references, and the concentration was calculated based on Val-Lys residue. The uptake of [3H]Gly-Sar was strongly reduced in the presence of the polyrotaxane-Val-Lys conjugate, while such a reduction was not observed for the polyrotaxane itself (111HE-R/E4-PHE-Z). The R-CD-Val-Lys conjugate showed a slight inhibitory effect with increasing Val-Lys concentration (data not shown), while R-CD itself did not. These results indicate that Val-Lys moiety immobilized on the polyrotaxane or R-CD is actually recognized by hPEPT1. Interestingly, the inhibitory effect of the polyrotaxane-Val-Lys conjugates was much greater than predicted from the Val-Lys dependency of the R-CD conjugates. The R-CD-Val-Lys conjugate can interact with one hPEPT1. On the other hand, Val-Lys moieties introduced to the polyrotaxane backbone has potential to interact with multiple hPEPT1s expressed on HeLa cells. Thus, it is considered that Val-Lys introduced to the polyrotaxane cooperatively interacts with hPEPT1. Since the molecular size of the polyrotaxane-Val-Lys conjugates is much larger than that of the R-CD-ValLys conjugate, the kinetics of Val-Lys-PEPT1 binding may be an important issue in the cooperative interactions. To examine this issue, the conjugates were incubated with HeLa cells at 37 °C for 30 min, prior to the measurement of [3H]Gly-Sar uptake. The inhibitory potency of the polyrotaxane-Val-Lys conjugate increased by the preincubation, as opposed to not using preincubation (Figure 3). On the contrary, the preincubation of the R-CD-Val-Lys conjugate reduced the inhibitory effect. Kiessling et al. reported the enhanced interaction of neoglycopolymers with concanavalin A by preincubation (17). In our case, the polyrotaxane conjugate is considered to rebind with PEPT1 during the incubation, which has been kinetically proved using a model ligand and binding protein (10). Therefore, our results suggest the multivalent binding and/or cooperative interaction of the polyrotaxane-Val-Lys conjugates with hPEPT1. A key feature of our designed polyrotaxane conjugates is the contribution of the supramolecular structure to the inhibitory effect. Figure 4 summarizes the inhibitory effect of the polyrotaxane-Val-Lys conjugates in comparison with the dextran-Val-Lys conjugates. The
Yui et al.
Figure 3. Effect of preincubation on the inhibition of [3H]GlySar uptake via hPEPT1. Mean ( SEM (n ) 4). * Significantly different (p < 0.05) from control. ** Significantly different (p < 0.05) from nonpreincubation. Black bar: simultaneous addition. Slash bar: preincubation of 30 min.
Figure 4. Inhibition of [3H]Gly-Sar uptake via hPEPT1 as a function of the number of Val-Lys in one conjugate molecule. The concentration of each conjugate is 1 mM as Val-Lys basis. The polyrotaxane-Val-Lys conjugates (circle) and dextran-ValLys conjugate (triangle). Mean ( SEM (n ) 4). * Significantly different (p < 0.05) from control.
concentration of Val-Lys in each conjugate was adjusted to be 1 mM. [3H]Gly-Sar uptake in both conjugates decreased with increasing the number of Val-Lys in one conjugate molecule. The inhibition of [3H]Gly-Sar uptake with the polyrotaxane-Val-Lys conjugates was significantly greater than those of the dextran-Val-Lys conjugates. In the case of conjugates between the oligopeptide and the water-soluble polymer, such as dextranVal-Lys conjugates, the hydrophobic oligopeptide (ValLys) groups are likely to associate each other, owing to the flexibility of the polymer backbones, which is related with the result in Figure 1. Such association may prevent access of PEPT1 toward the Val-Lys moieties. In contrast, the Val-Lys moieties introduced in the polyrotaxane are expected to be well arrayed along the supramolecular rodlike backbone, as to easily face PEPT1 on the membrane, considering the result of Figure 1 and our previous studies. Therefore, it is suggested that the supramolecular structure of polyrotaxanes has a suitable architecture for designing polymeric inhibitors capable of multivalent binding between Val-Lys and hPEPT1. CONCLUSIONS
The polyrotaxane-Val-Lys conjugates were found to exhibit great inhibitory effect on the uptake of a model oligopeptide, Gly-Sar, via hPEPT1. Special interest should be paid to the facts that this inhibitory effect was not explained from the monovalent interaction of hPEPT1
Polyrotaxane−Dipeptide Conjugates
with Val-Lys moiety and that the polyrotaxane was superior to dextran as a conjugate pair. These results strongly suggest both the multivalent interaction of the polyrotaxane conjugate with hPEPT1 and the contribution of the supramolecular structure to this effect. The polyrotaxane-Val-Lys conjugates have been confirmed not to be absorbed from the intestine in the alternative in vivo study (18), suggesting that reabsorption in renal epithelial cells via PEPT2 kidney can be avoided. The polyrotaxane used in this study was not specially designed aiming at the practical use but prepared for the first fundamental research, and the in vivo efficacy of the polyrotaxane-Val-Lys conjugates is not so high (data not shown). As mentioned before, in the polyrotaxane-ValLys conjugates, the R-CD-Val-Lys conjugate and a linear PEO chain are noncovalently bound via mechanical locking. Sliding and rotational motion of concentrated Val-Lys groups along the polyrotaxane might contribute to the inhibitory effect reported in this study. The solubility of the polyrotaxane is also an important issue because the introduction of Val-Lys groups into the polyrotaxane reduced the solubility. Our recent study succeeded to prepare carboxyethyl ester-introduced polyrotaxanes as a calcium-chelating agent for peptide drug delivery, and these polyrotaxanes displayed excellent solubility in PBS (19). In this point of view, to clarify the relation of the inhibitory effect with the specific structure of the polyrotaxane, a variation of the polyrotaxane-ValLys conjugates is now being prepared in terms of the molecular weight of PEO, the number of R-CDs, the number of Val-Lys groups, and the degree of carboxyethyl ester groups. Therefore, our design approach on the conjugation of PEPT1-recognizable oligopeptides with supramolecular-structured polyrotaxanes is expected to be useful for retarding the progression of chronic renal disease that requires dietary protein restriction . ACKNOWLEDGMENT
The authors are grateful to Mr. Koichi Higashimine (Japan Advanced Institute of Science and Technology) for 750 MHz 1H NMR measurements. A part of this study was financially supported by Grants-in-Aid from Micromachine Center, Japan, from NOVARTIS Foundation for the Promotion of Science, Japan, and for Scientific Research on Priority Areas “Molecular Synchronization for Design of New Materials System” from the Ministry of Education, Science, Sports and Culture, Japan. LITERATURE CITED (1) Abe, H., Satoh, M., Miyauchi, S., Shuto, S., Matsuda, A., and Kamo, N. (1999) Conjugation of dipeptide to fluorescent dyes enhances its affinity for PepT1 in human intestinal Caco-2 cells. Bioconjugate Chem. 10, 24-31. (2) Takeoka, R., Abe, H, Miyauchi, S., Shuto, S., Matsuda, A., Kobayashi, M., Miyazaki, K., and Kamo, N. (2001) Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1). Bioconjugate Chem. 12, 485-492.
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