Conjugation of Dipeptide to Fluorescent Dyes Enhances Its Affinity for

ACS2GO © 2019. ← → → ←. loading. To add this web app to the home screen open the browser option menu and tap on Add to homescreen...
0 downloads 0 Views 164KB Size
Download PDF
24

Bioconjugate Chem. 1999, 10, 24−31

Conjugation of Dipeptide to Fluorescent Dyes Enhances Its Affinity for a Dipeptide Transporter (PEPT1) in Human Intestinal Caco-2 Cells Hiroshi Abe,† Momoko Satoh,† Seiji Miyauchi,† Satoshi Shuto,‡ Akira Matsuda,‡ and Naoki Kamo*,† Laboratory of Biophysical Chemistry and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812 Japan. Received May 22, 1998; Revised Manuscript Received November 5, 1998

Dipeptide transporters in small intestine have a very wide substrate specificity, so that the transporter sometimes serves as a carrier for peptide-like compounds. We have synthesized dipeptide analogues conjugated at an -amino group of Lys in Val-Lys or Lys-Sar with fluorescent compounds such as fluorescein isothiocyanate and coumarin-3-carboxylic acid. Uptakes of these peptide analogues were examined by measuring intracellular accumulations into monolayers of the human intestinal epithelial cell line Caco-2 expressing the dipeptide transporter PEPT1. Kinetic analysis and effects of addition either of uncoupler (protonophore) or by Gly-Sar, one of the good substrates of PEPT1, revealed that fluorescent dipeptides were taken up by passive diffusion. In contrast, these analogues remarkably inhibited the Gly-Sar uptake by Caco-2 cells. Among the fluorescent analogues synthesized in this paper, Val-Lys(Flu) was the most potent competitive inhibitor against the Gly-Sar uptake with an inhibition constant of 5 µM. This value is the smallest among those ever reported: Val-Lys(Flu) has the highest affinity for PEPT1 among chemicals ever reported. The importance of the hydrophobic part of the substrate was pointed out.

INTRODUCTION

Dipeptide transporters in small intestine play an important role in the efficient absorption of small peptides following the digestion of dietary proteins (1). Due to the structural diversity of these oligopeptides, the peptide transporter is believed to have very broad substrate specificity. In some cases, this transporter serves as a carrier for pharmacological-active peptidelike compounds, which are exemplified by oral β-lactam antibiotics (2-4), the anti-neoplastic agent bestatin (5), and angiotensin-converting enzyme (ACE) inhibitors (6, 7). From a study on structure-activity relationship, Kramer et al. (4) proposed the essential structure of a substrate that is recognized and transported by the intestinal dipeptide/oligopeptide transporter. The requisite structure has the peptide core with a free negatively charged C-terminal group; a large extent of bulkiness of the side chain of the C-terminal amino acid is allowed, as exemplified by one of the substrates having high affinity, ACE inhibitor captopril (6, 7). An N-terminal amino group or a weakly basic group at the N-terminals also seems essential (8); a wide variety in chemical structure or bulkiness of the side chain is allowed as true of the C-terminal. The dipeptide transporter with large capacity and broad specificity could be used as a shuttle system to increase intestinal absorption of poorly absorbable drugs by conjugation of these drugs to a dipeptide or tripeptide core. On the basis of this idea, we synthesized conjugated * Corresponding author. Fax +81-11-706-4989. E-mail, [email protected]. † Laboratory of Biophysical Chemistry. ‡ Laboratory of Medicinal Chemistry.

dipeptide analogues: the -amino group of Lys in ValLys or Lys-Sar was conjugated with fluorescent dyes such as fluorescein isothiocyanate (FITC, abbreviated as Flu) or coumarin-3-carboxylic acid (Coum).1 We chose these two peptides as a dipeptide core, since they might not be digested by peptidase in the intestine (9). The reason for our choice of Lys is the existence of an amino group at the -position to which we could easily add the fluorescent dye, a model of a poorly absorbing drug. The fluorescence of the bioconjugate allows easy assay of the accumulation of these peptide analogues into cells. Unfortunately, contrary to our expectations, these fluorescent peptide analogues were not transported by PEPT1 in Caco-2 cells. Quite interestingly, however, the affinity of these analogues to PEPT1 was very high; ValLys(Flu) competitively inhibited the Gly-Sar uptake by Caco-2 cells with an inhibition constant of 5 µM. This figure is the smallest of all reported values for inhibitors and substrates of PEPT1: this fluorescent peptide analogue has the highest affinity for PEPT1 among chemicals ever reported. MATERIALS AND METHODS

Materials. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), Coum, and glycylsarcosine were purchased from Sigma Chemical Co. (St. Louis, MO). [14C]glycylsarcosine ([14C]Gly-Sar) (1.78 GBq/mmol) was purchased from Daiichi Pure Chemicals Co., Ltd. (Ibaraki, Japan). 1 Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; Coum, coumarin-3-carboxylic acid; FITC, fluorescein isothiocyanate; Flu, fluorescein; Gly-Sar, glycylsarcosine; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; MES, 2-(Nmorpholino)ethanesulfonic acid; Tris, Tris(hydroxymethyl)aminomethane; WSCD, water-soluble carbodiimide [1-ethyl-3-(3dimethylaminopropyl)carbodiimide].

10.1021/bc980049i CCC: $18.00 © 1999 American Chemical Society Published on Web 12/31/1998

Interaction of Fluorescent Dipeptide with PEPT1

The culture medium (RPMI 1640) and fetal bovine serum (FBS) were purchased from GIBCO BRL Life Technologies (Grand Island, NY). THF was purchased as a dehydrated solvent from Kanto Chemical Co. Inc. (Tokyo, Japan). DMF was distilled from CaH2 at reduced pressure. Toluene and Et2O were distilled from sodium metal/ benzophenone ketyl. Unless otherwise stated, starting materials were obtained from commercial suppliers and were used without further purification. All amino acids used were L-isomer. Boc-Val and Boc-Lys(Cbz) were purchased from Peptide Institute, Inc. (Osaka, Japan). Lys(Cbz)-Ot-Bu, Lys(Cbz)-OMe and Sar-Ot-Bu were purchased from Kokusan Chemical Co. Inc. (Tokyo). FITC was purchased from Molecular Probes Inc. (Eugene, OR). Other chemicals used were of highest purity available. Synthesis. General Procedures. 1H NMR spectra were obtained on a JEOL EX270 or Bruker ARX500 and are given in parts per million (δ) relative to either tetramethylsilane or the solvent signal as internal reference (CHCl3, 7.27 ppm; DMSO, 2.49 ppm). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Analytical thin-layer chromatography was carried out on silica gel 60 (Merck, 70-230 mesh or 230-400 mesh). All reactions were performed under a nitrogen atmosphere, unless stated otherwise. Synthesis of Dipeptide Analogues. Boc-Val-Lys(Cbz)Ot-Bu (5). A mixture of Boc-Val (3.26 g, 15 mmol), Lys(Cbz)-Ot-Bu‚HCl (5.05 g, 15 mmol), HOBt (6.19 g, 30 mmol), and DMAP (1.83 g, 15 mmol) in DMF (120 mL) were stirred at 0 °C for 15 min; then WSCD‚HCl (2.93 g, 15.3 mmol) was added. After 30 min, the reaction mixture was warmed to room temperature and stirred for a further 2 h. The mixture was diluted with EtOAc (300 mL) and washed successively with 0.6 M citric acid (150 mL), H2O (150 mL), aqueous saturated NaHCO3 (150 mL), H2O (150 mL), and brine (100 mL), then dried (Na2SO4) and concentrated. The resulting residue was purified by column chromatography (SiO2, 75:1 CHCl3MeOH) to give 5 (7.02 g, 87% as a white amorphous powder). 1H NMR (CDCl3, 270 MHz): 0.84 (3 H, d, J ) 6.6 Hz, Val-CH3), 0.93 (3 H, d, J ) 6.6 Hz, Val-CH3), 1.20-1.87 (24 H, m, Lys β, γ, δ -CH2, 2 × t-Bu), 2.062.11 (1 H, m, Val β-CH), 3.11-3.25 (2 H, m, Lys -CH2), 4.00 (1 H, dd, J ) 6.2 Hz, Val R-CH), 4.44 (1 H, ddd, J ) 3.8, 7.6, 8.7 Hz, Lys R-CH), 5.09 (2 H, s, PhCH2), 5.21 (1 H, m, Val R-NH), 5.34 (1 H, br s, Lys -NH), 6.64 (1 H, d, J ) 7.6 Hz, Lys R-NH), 7.35-7.38 (5 H, m, Ph). Boc-Val-Lys-Ot-Bu‚HCl (6). A solution of 5 (6.3 g, 11.8 mmol) in AcOH (250 mL) containing 10% Pd/C (500 mg) was stirred under atmospheric pressure of hydrogen for 4 h. The catalyst was filtered off by Celite filtration. The filtrate was diluted with toluene and concentrated until no AcOH remained. The crude oil was dissolved in MeOH-H2O (10:1, 100 mL), and the solution was put on a Diaion WA-30 resin column (HCl form), which was developed with MeOH-H2O (10:1). The eluent was concentrated, and the resulting residue was azeotroped with toluene (×2) to give 6 (5.0 g, 100% as a white solid). 1 H NMR (CDCl3, 270 MHz): 0.93 (3 H, d, J ) 6.6 Hz, Val-CH3), 1.00 (3 H, d, J ) 6.6 Hz, Val-CH3), 1.14-1.89 (24 H, m, Lys β, γ, δ-CH2, 2× t-Bu), 2.08-2.15 (1 H, m, Val β-CH), 3.07 (2 H, br, Lys -CH2), 4.24 (1 H, dd, J ) 5.3, 8.6 Hz, Val R-CH), 4.37-4.39 (1 H, m, Lys R-CH), 5.58 (1 H, d, J ) 9.9 Hz, Val R-NH), 7.74 (1 H, d, J ) 7.3 Hz, Lys R-NH), 8.27 (3 H, br, Lys -NH3). Boc-Val-Lys(Flu)-Ot-Bu (9a). FITC (269 mg, 0.690 mmol) was added to a solution of 6 (308 mg, 0.726 mmol) in DMF (20 mL), followed by Et3N (101 µL, 0.724 mmol)

Bioconjugate Chem., Vol. 10, No. 1, 1999 25

at 0 °C, and the mixture was stirred at the same temperature for 15 min and then at room temperature for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 0.6 M citric acid (40 mL), H2O (30 mL), and brine (20 mL), then dried (Na2SO4) and concentrated. The resulting residue was purified by column chromatography (SiO2 15:1:0.08 CHCl3MeOH-AcOH) to give 9a (525 mg, 96% as a yellow powder). 1H NMR (DMSO-d6, 500 MHz): 0.83 (3 H, d, J ) 6.9 Hz, Val-CH3), 0.87 (3 H, d, J ) 6.9 Hz, Val-CH3), 1.23-1.77 (24 H, m, Lys β, γ, δ -CH2, 2× t-Bu), 1.94 (1 H, m, Val β-CH), 3.51 (2 H, br, Lys -CH2), 3.85 (1 H, dd, J ) 7.9 Hz, Val R-CH), 4.14 (1 H, dd, J ) 7.9, 13.4 Hz, Lys R-CH), 6.57 (2 H, dd, J ) 2.0, 8.7 Hz, xanthene H-2′, H-7′), 6.60 (1 H, d, J ) 8.8 Hz, Val R-NH), 6.61 (2 H, d, J ) 8.7 Hz, xanthene H-1′, H-8′), 6.68 (2 H, d, J ) 2.0 Hz, xanthene H-4′, H-5′), 7.18 (1 H, d, J ) 8.3 Hz, H-3), 8.05 (2 H, m, Lys R-NH, Lys -NH3), 8.24 (1 H, br, H-6), 9.85 (1 H, br, NH), 10.11 (2 H, s, 2× OH). FAB-HRMS calcd for [C41H50N4O10S + H]+: 791.3325. Found: 791.3307. Val-Lys(Flu)‚HCl (10a). A mixture of 9a (467 mg, 0.591 mmol) in THF (5 mL) and 4 M HCl/dioxane solution (30 mL) was stirred at room temperature for 30 min and evaporated under reduced pressure. The resulting residue was triturated with Et2O and dried in vacuo to give 10a (397 mg, 100% as an orange powder). 1H NMR (CD3OD, 270 MHz): 1.09 (3 H, d, J ) 7.3 Hz, Val-CH3), 1.12 (3 H, d, J ) 7.3 Hz, Val-CH3), 1.47-2.03 (6 H, m, Lys β, γ, δ -CH2), 2.26 (1 H, m, Val β-CH), 3.66 (2 H, m, Lys -CH2), 3.82 (1 H, d, J ) 5.5 Hz, Val R-CH), 4.47 (1 H, m, Lys R-CH), 7.20 (2 H, dd, J ) 2.2, 9.1 Hz, xanthene H-2′, H-7′), 7.34 (2 H, d, J ) 2.2 Hz, xanthene H-4′, H-5′), 7.40 (1 H, d, J ) 8.3 Hz, Ar H-3), 7.61 (2 H, d, J ) 9.1 Hz, xanthene H-1′, H-8′), 8.12 (1 H, dd, J ) 2.2, 8.3 Hz, Ar H-4), 8.57 (1 H, d, J ) 2.2 Hz, Ar H-6). FAB-HRMS calcd for [C32H34N4O8S + H]+: 574.3128. Found: 574.3143. Boc-Val-Lys(Coum)-Ot-Bu (9b). WSCD‚HCl (150 mg, 0.783 mmol) was added to a mixture of 6 (316 mg, 0.745 mmol), coumarin-3-carboxylic acid (149 mg, 0.783 mmol), and HOBt (300 mg, 1.49 mmol) in DMF (15 mL) at 0 °C. After 10 min, Et3N (103 µL, 0.745 mmol) was added, and the mixture was stirred at the same temperature for 30 min. After the reaction mixture was stirred at room temperature for 2 h, it was diluted with EtOAc (40 mL) and washed successively with 0.6 M citric acid (30 mL), H2O (20 mL), and brine (20 mL), then dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, 40:1 CHCl3-MeOH) to give 9b (394 mg, 92% as a white amorphous). 1H NMR (CDCl3 270 MHz): 0.93 (3 H, d, J ) 6.7 Hz, Val-CH3), 0.98 (3 H, d, J ) 6.7 Hz, Val-CH3), 1.33-1.95 (24 H, m, Lys β, γ, δ -CH2, 2 × t-Bu), 2.16 (1 H, m, Val β-CH), 3.46 (2 H, br, Lys -CH2), 4.05 (1 H, m, Val R-CH), 4.40 (1 H, m, Lys R-CH), 5.18 (1 H, d, J ) 8.6 Hz, Val R-NH), 6.68 (1 H, d, J ) 7.3 Hz, Lys R-NH), 7.35-7.41 (2 H, m, H-8, H-6), 7.66 (1 H, m, H-7), 7.77 (1 H, m, H-5), 8.87 (1 H, m, Lys -NH2), 8.96 (1 H, s, H-4). FAB-HRMS calcd for [C30H43N3O8 + H]+: 574.3128. Found: 574.3143. Val-Lys(Coum)‚HCl (10b). The desired product 10b (243 mg, 100% as a white powder) was obtained from 9b (308 mg, 0.662 mmol) in a similar manner as described for the synthesis of 10a. 1H NMR (CD3OD, 270 MHz): 1.07 (3 H, d, J ) 7.1 Hz, Val-CH3), 1.11 (3 H, d, J ) 7.1 Hz, Val-CH3), 1.50-2.01 (6 H, m, Lys β, γ, δ -CH2), 2.23 (1 H, m, Val β-CH), 3.44 (2 H, m, Lys -CH2), 3.74 (1 H, d, J ) 5.5 Hz, Val R-CH), 4.46 (1 H, dd, Lys R-CH), 7.417.47 (2 H, m, H-6, H-8), 7.75 (1 H, ddd, J ) 1.6, 7.1, 8.7 Hz, H-7), 7.85 (1 H, dd, J ) 1.6, 7.9 Hz, H-5), 8.84 (1 H,

26 Bioconjugate Chem., Vol. 10, No. 1, 1999

s, H-4), 9.05 (1 H, m, Lys R-NH). FAB-HRMS calcd for [C21H27N3O6 + H]+: 418.1978. Found: 418.1976. Boc-Lys(Cbz)-Sar-Ot-Bu (7). Compound 7 was prepared from Boc-Lys(Cbz) (2.2 g, 5.78 mmol) and Sar-Ot-Bu‚HCl (1.0 g, 5.5 mmol) using the procedure described for the synthesis of 5. The crude product was purified by column chromatography (SiO2, 10:1 CHCl3-MeOH) to give 7 (2.79 g, 100% as a white solid). 1H NMR (CDCl3, 270 MHz) 1.13-1.89 (24 H, m, Lys β, γ, δ -CH2, 2× t-Bu), 2.903.30 (5 H, m, Lys -CH2, N-CH3), 3.54-4.38 (2 H, m, Sar-CH2), 4.39-4.75 (1 H, m, Lys R-CH), 5.00-5.20 (3 H, m, PhCH2, Lys -NH), 5.24-5.49 (1 H, m, Lys R-NH), 7.26-8.06 (5 H, m, Ph). Boc-Lys-Sar-Ot-Bu‚HCl (8). The desired product 8 (2.10 g, 100% as a yellow oil) was obtained from 7 (2.6 g, 5.13 mmol) in a similar manner as described for the synthesis of 6. 1H NMR (DMSO-d6, 270 MHz): 1.16-1.76 (24 H, m, Lys β, γ, δ -CH2, 2 × t-Bu), 2.73 (2 H, m, Lys -CH2), 2.81, 3.06 (3 H, m, N-CH3), 3.62-4.18 (2 H, m, Sar-CH2), 4.23-4.47 (1 H, m, Lys R-CH), 6.77-7.06 (1 H, m, Lys R-NH), 7.11-7.27 (5 H, m, Ph), 8.02 (3 H, br, NH3). Boc-Lys(Flu)-Sar-Ot-Bu (11a). Compound 11a was prepared from 8 (1.1 g, 2.68 mmol) and FITC (954 mg, 2.45 mmol) using the procedure described for the synthesis of 9a. The crude product was purified by column chromatography (SiO2, 15:1:0.016 CHCl3-MeOH-AcOH) to give 11a (1.32 g, 71% as a yellow powder). 1H NMR (DMSO-d6, 500 MHz) 1.20-1.70 (24 H, m, Lys β, γ, δ -CH2, 2 × t-Bu), 2.83, 3.09, 3.18 (3 H, m, N-CH3), 3.49 (2 H, m, Lys -CH2), 3.76-4.20 (2 H, m, Sar R-CH2), 4.42 (1 H, m, Lys R-CH), 6.54 (2 H, dd, J ) 1.7, 8.9 Hz, xanthene H-2′, H-7′), 6.61 (2 H, d, J ) 1.7 Hz, xanthene H-4′, H-5′), 6.67 (2 H, d, J ) 8.9 Hz, xanthene H-1′, H-8′), 6.95 (1 H, m, Lys R-NH), 7.15 (1 H, d, J ) 8.3 Hz, H-3), 7.75 (1 H, d, J ) 7.9 Hz, H-4), 8.29 (1 H, s, H-6), 8.46 (1 H, m, Lys -NH), 10.27 (1 H, br, Ar-NH); FAB-HRMS calcd for [C39H46N4O10S + H]+ 763.3012, found 763.3011. Lys(Flu)-Sar‚HCl (12a). The desired product 12a (97 mg, 100% as an orange powder) was obtained from 11a (122 mg, 160 µmol) in a similar manner as described for the synthesis of 10a. 1H NMR (DMSO-d6, 270 MHz): 1.35-1.85 (6 H, m, Lys β, γ, δ -CH2), 2.89, 3.10 (3 H, m, N-CH3), 3.50 (2 H, m, Lys -CH2), 3.94-4.23 (2 H, m, Sar-CH2), 4.43 (1 H, m, Lys R-CH), 6.48-6.67 (4 H, m, xanthene H-1′, H-8′, H-2′, H-7′), 6.74 (2 H, d, J ) 1.7 Hz, xanthene H-4′, H-5′), 7.19 (1 H, d, J ) 8.3 Hz, H-3), 7.86 (1 H, dd, J ) 1.7, 8.3 Hz, H-4), 8.24 (3 H, br s, NH3), 8.42 (1 H, s, H-6), 8.68 (1 H, br s, Lys -NH), 10.8 (1 H, br s, Ar-NH). FAB-HRMS calcd for [C30H30N4O8S + H]+: 607.1862. Found: 607.1870. Boc-Lys(Coum)-Sar-Ot-Bu (11b). Compound 11b was prepared from 8 (702 mg, 1.71 mmol) and coumarin-3carboxylic acid (341 mg, 1.79 mmol) using the procedure described for the synthesis of 9b. The crude product was purified by column chromatography (SiO2, 40:1 CHCl3MeOH) to give 11b (790 mg, 85% as a white amorphous powder). 1H NMR (CDCl3, 270 MHz): 1.42-1.90 (24 H, overlapping m, Lys β, γ, δ -CH2, 2 × t-Bu), 2.97, 3.13 (3 H, m, N-CH3), 3.46 (2 H, m, Lys -CH2), 3.73-4.29 (2 H, m, Sar-CH2), 4.41-4.69 (1 H, m, Lys R-CH), 5.31 (1 H, m, Lys R-NH), 7.33-7.41 (2 H, overlapping m, H-8, H-6), 7.63-7.70 (2 H, m, H-7, H-5), 8.81 (1 H, m, Lys -NH), 8.90 (1 H, s, H-4). FAB-HRMS calcd for [C28H39N3O8 + H]+: 546.2815. Found: 546.2804. Lys(Coum)-Sar‚HCl (12b). The desired product 12b (260 mg, 100% as a white powder) was obtained from 11b (333.7 mg, 6.12 mmol) in a similar manner as described for the synthesis of 10a. 1H NMR (DMSO-d6, 270 MHz): 1.53-1.79 (6 H, m, Lys β, γ, δ -CH2), 2.88, 3.09 (3 H, m,

Abe et al.

N-CH3), 3.39-3.30 (2 H, m, Lys -CH2), 3.94, 4.18 (2 H, m, Sar-CH2), 4.41 (1 H, m, Lys R-CH), 7.45 (1 H, ddd, J ) 1.0, 7.6, 7.9 Hz, H-6), 7.52 (1 H, dd, J ) 1.0, 8.6 Hz, H-8), 7.76 (1 H, ddd, J ) 1.3, 7.6, 8.6 Hz, H-7), 8.00 (1 H, dd, J ) 1.3, 7.6, 8.6 Hz, H-5), 8.71 (1 H, m, Lys -NH), 8.86 (1 H, s, H-4). FAB-HRMS calcd for [C19H23N3O6 + H]+: 390.1665. Found: 390.1671. Boc-Val-Lys(Cbz)-OMe (15). A mixture of Boc-Val (6.67 g, 30.7 mmol), Lys(Cbz)-OMe‚HCl (8.47 g, 25.6 mmol), HOBt (10.5 g, 50.9 mmol), and 4-methylmorpholine (2.81 mL, 25.6 mmol) in DMF (170 mL) was stirred at 0 °C for 15 min. Then, WSCD‚HCl (6.38 g, 33.3 mmol) was added to the solution. After 30 min, the reaction mixture was warmed to room temperature and stirred for 10 h. It was diluted with EtOAc (350 mL) and washed successively with 0.6 M citric acid (200 mL), H2O (150 mL), aqueous saturated NaHCO3 (200 mL), H2O (150 mL), and brine (100 mL), dried (Na2SO4), and concentrated. The resulting residue was purified by column chromatography (SiO2, 80:1 CHCl3-MeOH) to give 15 (10.56 g, 83.7%) as white solid. 1H NMR (DMSO-d6, 500 MHz) 0.75 (3 H, d, J ) 6.7 Hz, Val-CH3), 0.79 (3 H, d, J ) 6.7 Hz, Val-CH3), 1.15-1.70 (15 H, Lys β, γ, δ -CH2, t-Bu), 1.85 (1 H, m, Val β-CH), 2.90 (2 H, m, Lys -CH2), 3.53 (3H, s, CO2CH3), 3.75 (1 H, dd, J ) 9.0 Hz, Val R-CH), 4.14 (1 H, dt, J ) 5.9, 7.2 Hz, Lys R-CH), 5.53 (2 H, s, PhCH2), 6.52 (1 H, d, J ) 9.0 Hz, Val R-NH), 7.14 (1 H, t, J ) 5.0 Hz, Lys -NH), 7.22-7.35 (5 H, m, Ph), 8.07 (1 H, d, J′ ) 7.2 Hz, Lys R-NH). Boc-Val-Lys-OMe (16). A solution of 15 (4.96 g, 10.06 mmol) in MeOH (80 mL) containing 10% Pd/C (500 mg) was stirred under atmospheric pressure of hydrogen for 2 h. The catalyst was filtered off by Celite filtration. The filtrate was concentrated to give 16 (3.61 g, 100% as a white amorphous). 1H NMR (DMSO-d6, 500 MHz): 0.75 (3 H, d, J ) 6.7 Hz, Val-CH3), 0.79 (3 H, d, J ) 6.7 Hz, Val-CH3), 1.15-1.64 (15 H, m, Lys β, γ, δ -CH2, t-Bu), 1.85 (1 H, m, Val β-CH), 3.00 (2 H, m, Lys -CH2), 3.54 (3 H, s, CO2CH3), 3.75 (1 H, dd, J ) 8.0 Hz, Val R-CH), 4.16 (1 H, dt, J ) 8.3 Hz, Lys R-CH), 6.56 (1 H, m, Val R-NH), 8.11 (1 H, m, Lys R-NH). Boc-Val-Lys(Flu)-OMe (17a). Compound 17a was prepared from 16 (2.80 g, 7.80 mmol) and FITC (3.04 g, 7.81 mmol) using the procedure described for the synthesis of 9a. The crude product was purified by column chromatography (SiO2, 13:1:0.14 CHCl3-MeOH-AcOH) to give 17a (4.13 g, 70.7% as a yellow amorphous): 1H NMR (CD3OD, 500 MHz) 0.93 (3 H, d, J ) 6.7 Hz, Val-CH3), 0.97 (3 H, d, J ) 6.7 Hz, Val-CH3), 1.38-1.94 (15 H, m, Lys β, γ, δ -CH2, t-Bu), 2.02 (1 H, m, Val β-CH), 3.61 (2 H, br s, Lys -CH2), 3.69 (3 H, s, CO2CH3), 3.91 (1 H, d, J ) 6.9 Hz, Val R-CH), 4.45 (1 H, m, Lys R-CH), 6.54 (2 H, dd, J ) 2.3, 8.7 Hz, xanthene H-2′, H-7′), 6.68 (2 H, m, xanthene H-1′, H-8′), 7.08-7.21 (3 H, m, Ar H-3, xanthene H-4′, H-5′), 7.75 (1 H, m, Ar H-4), 8.14 (1 H, br s, Ar H-6). [C38H44N4O10S + H]+: 749.2856. Found: 749.2812. Val-Lys(Flu)-OMe‚HCl (18a). The desired product 18a (445 mg, 94% yellow powder) was obtained from 17a (500 mg, 0.668 mmol) in a similar manner as described for the synthesis of 10a. 1H NMR (CD3OD, 270 MHz): 1.09 (3 H, d, J ) 6.9 Hz, Val-CH3), 1.11 (3 H, d, J ) 6.9 Hz, Val-CH3), 1.35-2.10 (6 H, m, Lys β, γ, δ-CH2), 3.39 (1 H, m, Val β-CH), 3.65 (3 H, s, CO2CH3), 3.70 (2 H, m, Lys -CH2), 3.83 (1 H, d, J ) 5.7 Hz, Val R-CH), 4.50 (1 H, m, Lys R-CH), 7.19 (2 H, dd, J ) 2.3, 9.2 Hz, xanthene H-2′, H-7′), 7.33 (2 H, d, J ) 2.3 Hz, xanthene H-4′, H-5′), 7.39 (1 H, d, J ) 8.3 Hz, Ar H-3), 7.59 (2 H, d, J ) 9.2 Hz, xanthene H-1′, H-8′), 8.12 (1 H, dd, J ) 2.1, 8.3 Hz,

Interaction of Fluorescent Dipeptide with PEPT1

Ar H-4), 8.58 (1 H, d, J ) 2.1 Hz, Ar H-6). FAB-HRMS calcd for [C33H36N4O8S + H]+: 649.2332. Found: 649.2343. Boc-Val-Lys(Flu) (19a). NaOH (5 M, 0.44 mL) was added dropwise to a solution of 17a (1.5 g, 2.0 mmol) in MeOH/dioxane (8/2 mL) at 0 °C, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc (200 mL) and washed successively with 0.6 M citric acid (150 mL), H2O (100 mL), and brine (80 mL), dried (Na2SO4), and concentrated. The resulting residue was purified by flash silica gel column chromatography (4.5:1:0.1 CHCl3-MeOH-AcOH) to give 19a (1.3 g, 88% as a yellow powder). 1H NMR (CD3OD, 270 MHz): 0.92 (3 H, d, J ) 6.6 Hz, Val-CH3), 0.97 (3 H, d, J ) 6.6 Hz, Val-CH3), 1.28-2.06 (16 H, m, Lys β, γ, δ-CH2, t-Bu, Val β-CH) 3.63 (2 H, m, Lys -CH2), 3.94 (1 H, d, J ) 6.8 Hz, Val R-CH), 4.43 (1 H, m, Lys R-CH), 6.54 (2 H, dd, J ) 2.4, 8.7 Hz, xanthene H-2′, H-7′), 6.66 (1 H, m, Ar H-3), 6.68 (2 H, d, J ) 1.9 Hz, xanthene H-4′, H-5′), 7.14 (2 H, d, J ) 8.2 Hz, xanthene H-1′, H-8′), 7.76 (1 H, m, Ar H-4), 8.14 (1 H, br, Ar H-6). FAB-HRMS calcd for [C37H42N4O8S + H]+: 735.2699. Found: 735.2723. Cell Culture. Caco-2 cells (ATCC HTB-37) at passage 28 were purchased from the American Type Culture Collection. They were passaged in 75 cm2 culture flasks (FALCON, Becton Dickinson and Co., Lincoln Park, NJ) in culture medium consisting of RPMI 1640 supplemented with FBS (15%), HEPES (5 mM), and NaHCO3 (2 g/L) without antibiotics. The cells were maintained at 37 °C in an atmosphere of 5% CO2. Cells between the 30th and 40th passage were used in this study. At approximately 80% confluence, cells were seeded using 0.02% EDTA and 0.05% trypsin at a density of 53 000 cells/cm2 on 60 mm plastic culture dishes. The cells were fed fresh medium every 3 days and were used between the 14th and 16th day for the transport studies. Uptake Experiments in Monolayer Caco-2 Cells. The uptake of [14C]Gly-Sar by Caco-2 cells was measured. The composition of the incubation medium was 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose, 5 mM MES (pH 6.0), or HEPES (pH 7.4). After removal of the culture medium, each dish was washed once with 5 mL of the incubation medium (pH 7.4) prewarmed at 37 °C and incubated with 2 mL of the same medium for 10 min at 37 °C. The cells were then incubated with 2 mL of incubation medium (pH 6.0) containing [14C]Gly-Sar in the absence or the presence of inhibitors for a designated period at 37 °C. The medium was then aspirated off, and the dishes were rapidly rinsed twice with ice-cold incubation medium (pH 7.4). The cells were solubilized in 1 mL of 1 M NaOH followed by neutralization by 1 mL of 1 M HCl. The cellassociated radioactivity was determined in an ACSII scintillation counting cocktail (Amersham, Buckinghamshire England) by a liquid scintillation counter (LSC 2500, Packard). The uptake of fluorescent dipeptide analogues was measured, unless otherwise indicated, in a similar manner as described above. The cells were solubilized in 1.5 mL of 1 M NaOH using a rubber policeman and neutralized by 1.5 mL of 1 M HCl. The extracted solution was centrifuged in a tabletop microfuge (Hitachi, Tokyo). The supernatant was diluted with 25 mM Tris-buffer (pH 9.6). The fluorescent intensity due to cell accumulation of fluorescein derivatives was determined with a Hitachi MPF-1 fluorometer (Hitachi, Tokyo) with emission at 520 ( 5 nm and excitation at 489 ( 5 nm. The fluorescent intensity by coumarin-3-carboxy derivatives was determined with emission at 410 ( 5 nm and excitation at 320 ( 5 nm. When the uptake of drugs was measured in

Bioconjugate Chem., Vol. 10, No. 1, 1999 27

the presence of CCCP, the cells were preincubated for 30 min at 37 °C with 1.5 mL of incubation medium (pH 6.0) containing 40 µM CCCP, and then incubated for the designated period at 37 °C with 1.5 mL of the incubation medium (pH 6.0) containing various drugs with 40 µM CCCP. The cells were then treated as described above. The uptake of cepharosporins (cefadroxil and cephradine) was measured, unless otherwise indicated, in a similar manner as described above. The accumulation of cepharosporins was linear within 15 min, then the uptake rates were estimated from the slope. The cells were scraped with a rubber policeman into 1 mL of extraction solution (30 mM phosphate buffer pH 7.0/methanol, 50: 50) and maintained for 1 h at room temperature. The extraction solution was centrifuged at 15 000 rpm in a tabletop microfuge (Hitachi, Tokyo Japan) for 15 min, and the supernatant was passed through a Millipore filter (SJGVL, 0.22 µm). The concentrations of cepharosporins in the filtrate were determined by a highperformance liquid chromatography L-6000 (Hitachi, Tokyo Japan) equipped with an UV detector L4000 (Hitachi Co.). The HPLC conditions were column, ERC ODS 6.0 mm inside diameter × 100 mm (ERC Inc., Saitama Japan); mobile phase, 50 mM phosphate buffer (pH 7.0)/methanol, 90:10 for cefadroxil and 70:30 for cephradine; flow rate, 0.8 mL/min; wavelength, 262 nm; injection volume, 20 µL; room temperature. In all uptake measurements, unless otherwise indicated, values were corrected for the protein content. The protein content of the cell monolayers solubilized in 1.0 mL of 1 M NaOH was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as a standard. Kinetic Analysis of Gly-Sar Uptake. The uptake data of Gly-Sar were fitted to a Michaelis-Menten type equation with a nonsaturable component by an iterative nonlinear least-squares method using a MULTI program (12),

v0 )

Vmax[S] Km + [S]

+ Kd[S]

(1)

where v0 represents the initial uptake rate of Gly-Sar; Vmax, the maximum uptake velocity; [s], the concentration of Gly-Sar; Km, Michaelis constant; Kd, the coefficient of passive diffusion. RESULTS AND DISCUSSION

Synthesis of the Fluorescent Dipeptide Analogues. Kramer et al. (4) reported that the minimum requirement of the chemical structure of substrates for recognition by the dipeptide transporter is the existence of the peptide bond, a free carboxyl group, and an R-amino group in N-terminal. Considering these structural requirements and the ease of preparing bioconjugates as described in the Introduction, Val-Lys and LysSar were selected as the core of the dipeptide analogues, although recently it is shown that the existence of the peptide bond is not essential (13, 14). It is also expected that these dipeptides are highly resistant to hydrolysis by peptidase owing to the bulky group of Val and to methylated amino groups of Sar. The strategy of the synthesis of fluorescent dipeptide analogues was conjugation of a fluorescent dye to -amino group of the dipeptides (see Scheme 1). Here, a represents FITC (Flu), and b, Coum. These fluorescent dyes were selected from the point that FITC is relatively large in the molecular size and Coum, small.

28 Bioconjugate Chem., Vol. 10, No. 1, 1999

Abe et al.

Scheme 1a

a Reagents: i, WSCD‚HCl, HOBt, DMAP, DMF; (ii), 10% Pd/C, AcOH, then ion exchanger resin; iii, FITC, Et N, DMF; iv, Coumarin3 3-carboxylic acid, WSCD‚HCl, HOBt, Et3N, DMF; v, 4 M HCl/dioxane.

Using proptected amino acids which are commercially available, desired products 10a, 10b, 12a, 12b, 18a, and 19a were synthesized by the usual reactions of peptide

condensation and deprotection. Overall yield was 10a, 84%; 10b, 80%; 12a, 71%; 12b, 85%; 18a, 56%; 19a, 52%. It is noted that for removal of Cbz-protecting group from

Interaction of Fluorescent Dipeptide with PEPT1

5, 7, and 15, we chose acetic acid as a reaction solvent since the resulting product was unstable in a neutral solution and led to an undesired reaction of isomerization. After removal of Cbz-protecting group, the product was applied to a weakly acidic ion-exchange resin column to obtain 6, 8, and 16 as a hydrochloride salt. Protected ValLys(Flu), 18a, and 19a were synthesized to investigate the effect of amino or carbonyl group in Val-Lys(Flu) on the recognition by the dipeptide transporter. Since the high purity of compounds 10a, 10b, 12a, 12b, 18a, and 19a was confirmed by NMR analysis, these compounds were used in the transport experiment without further purification. Fluorescein (Flu) is strongly fluorescent either in the conjugated or nonconjugated state. Although Coum itself has a little fluorescence, conjugation to dipeptide enhanced the quantum yield. The fluorescent dipeptide analogues of Val-Lys(Flu) and (Flu)Lys-Sar were digested in the presence of cytosolic enzymes in Caco-2 cells, and analysis with aid of their fluorescence showed that the hydrolysis of Val-Lys or Lys-Sar occurred. The half-time was about 25 min for both. These analogues showed their inhibition on GlySar uptake when they were added (see below): not the digested products but the analogues themselves exerted the inhibition. Accumulation of Fluorescent Dipeptide Analogues by Caco-2 Cells. The uptake of (Flu)Lys-Sar and Val-Lys(Flu) was linear with respect to time within 20 min (data not shown); the rate of uptake was estimated by the accumulation in 15 min. (Coum)Lys-Sar and ValLys(Coum), on the other hand, were rapidly degraded to coumarin-3-carboxylic acid inside cells. We therefore were unable to determine the initial uptake of Coum analogues. The kinetics of the Flu analogues was measured. The uptake of (Flu)Lys-Sar and Val-Lys(Flu) increased linearly and showed no saturation up to 2 mM with the increase in concentration: for (flu)Lys-Sar, uptake (pmol/ mg/15 min) ) (0.624 ( 0.016) × (concn in µM), R ) 0.996, and for Val-Lys(Flu), uptake (pmol/mg/15 min) ) (0.521 ( 0.02) × (concn in µM), R ) 0.993. In Caco-2 cells, dipeptide is taken up by a symporter (co-transporter) with H+ that is energized by an inwardly electrochemical potential difference of proton. If these Flu analogues are accumulated by the symporter, the uptake should cease or at least decrease with the addition of an uncoupler that dissipates the proton electrochemical potential. On the contrary, however, CCCP, the uncoupler, did not decrease the accumulation (see Figure 1). Gly-Sar is a substrate of a dipeptide transporter, PEPT1, that is expressed in Caco-2 cells (10, 11). Gly-Sar did not inhibit the uptake of these analogues (see Figure 1). These three observations imply that (Flu)Lys-Sar and Val-Lys(Flu) are accumulated into cells via a passive diffusion. Figure 1 shows that addition of CCCP and Gly-Sar increased the accumulation of (Flu)Lys-Sar and Val-Lys(Flu). A plausible reason for this increase will be discussed later. Kinetic Analysis of Active Gly-Sar Accumulation by Caco-2 Cells. Although Gly-Sar is known to be a substrate of PEPT1, we ourselves characterized Gly-Sar uptake by Caco-2 cells. The initial uptake rates were estimated by the slope of the total accumulation vs time; this slope was constant up to 15 min. Analysis with eq 1 gave the following values: Vmax ) 2.4 nmol/h/mg of protein, Km ) 0.86 mM, and Kd ) 1.2 nmol/h/mg of protein. These values were consistent with those reported by others (15). Figure 1 shows that CCCP decreased the uptake of Gly-Sar, implying that this uptake requires a proton electrochemical potential. It is worth noting that

Bioconjugate Chem., Vol. 10, No. 1, 1999 29

Figure 1. Effect of Gly-Sar and CCCP on the uptake of ValLys(Flu) and (Flu)Lys-Sar by Caco-2 cells. (Leftmost columns) Uptake of 100 µM Gly-Sar was measured in the absence (unfilled column) or presence (filled column) of 40 µM CCCP. (Middle and rightmost columns) Uptake of 200 µM Val-Lys(Flu) or (Flu)Lys-Sar was measured in the absence (unfilled column) or the presence(filled column) of 40 µM CCCP or of 10 mM GlySar (striped column). The uptake was determined in 15 min incubation, then normalized by that for the value of corresponding control experiments. Each column represents the mean ( S. E. of three experiments. (***) P < 0.005; (**) P < 0.01; (*) P < 0.05, significant differences from the control using Student’s t-test.

a single Michaelis-Menten term is enough to describe the uptake kinetics aside from the passive diffusion term. This would suggest the existence of only one transporter that transports Gly-Sar. This is consistent with the reported fact that Caco-2 cells functionally express PEPT1 as the only dipeptide transporter revealed by the Northern blot analysis (16). Remarkable Inhibition of Gly-Sar Uptake by the Fluorescent Dipeptide Analogues. As described above, the fluorescent dipeptide analogues were not taken up by any transporters although these have a peptide bond. We next examined whether these analogues are recognized by the dipeptide transporter PEPT1 whose substrate is Gly-Sar. Inhibition of Gly-Sar uptake by these fluorescent dipeptide analogues was measured, and results (Figure 2) showed that these analogues strongly inhibited the uptake. It is noted that 1 mM Val-Lys(Flu) completely inhibited the Gly-Sar uptake. Cefalexin and cefadroxil are known to be transported by PEPT1 (11, 16, 17), but their inhibition was much weaker than the analogues. To evaluate the kinetic type of inhibition by fluorescent dipeptide analogues, we performed Dixon plot analysis of Gly-Sar uptake by Val-Lys(Flu). Results are detailed in Figure 3 where the three lines cross at one point, indicating the competitive inhibition of Gly-Sar by ValLys(Flu) (18, 19). The inhibition constant (Ki) was calculated to be 5 µM, which is the smallest value reported so far. The values of Ki or Km reported for various peptide transport systems are in the range 1-10 mM (17, 20, 21). In other words, this compound has the highest affinity for PEPT1 among both inhibitors and substrates that have ever been reported. We do not yet know the molecular mechanism of the inhibition by fluorescent analogues; do the analogues inhibit the binding of Gly-Sar or its intraprotein translocation? Two facts that (1) fluorescent analogues are transported by a passive diffusion mechanism and (2)

30 Bioconjugate Chem., Vol. 10, No. 1, 1999

Figure 2. Effect of various fluorescent dipeptide-analogues, dipeptides or antibiotics on the uptake of Gly-Sar, a substrate of PEPT1. Uptake rate of 100 µM Gly-Sar was measured in the absence or the presence of 1 mM various fluorescent dipeptideanalogues, dipeptides or antibiotics. Initial uptake rates were determined using a linear regression of the linear portion in Gly-Sar uptake (measured at 5, 10, and 15 min). The uptake rate was normalized by that for the absence of fluorescent dipeptide-analogues. Each column represents the mean ( SE of three experiments. (***) P < 0.005; (**) P < 0.01; (*) P < 0.05, significant differences from the control using Student’s t-test.

Figure 3. Dixon-Webb plot analysis for the inhibition of GlySar uptake by Val-Lys(Flu) . The experiments were performed as described in the legend to Figure 1 in the presence of various concentrations of Val-Lys(Flu). Circles, squares and triangles represent 100, 200, and 300 µM Gly-Sar uptake rate, respectively. These rates were corrected for the passive diffusion (see eq 1). The inhibition of Gly-Sar upatake by Val-Lys(Flu) was analyzed by Dixon-Webb plot, showing the competitive inhibition. The Ki value was determined to be 5 µM from the intersection of the three lines. Each point represents the mean ( SE of three experiments.

inhibition is competitive suggest that these analogues strongly inhibit the binding of Gly-Sar to the transporter: the fluorescent analogues can bind the binding site of the transporter but they are not transported owing to their bulkiness. In this respect, Otto et al. (22) reported that a fluorescent dipeptide β-Ala-Lys-N-AMCA (AMCA is 7-amino-4-methylcoumarin-3-acetic acid) is transported by a dipeptide transporter of adenohypophysial folliculostellate cells. The reason that this fluorescent dipeptide is transported and our fluorescent dipeptide is not transported is not known. What Chemical Features Enhance the Recognition by PEPT1? Inhibition of Gly-Sar uptake by several

Abe et al.

Figure 4. Effect of modification of the amino or carboxyl group in Val-Lys(Flu) on the Gly-Sar uptake. The experiments were performed as described in the legend to Figure 1. The uptake rate of 20 µM Gly-Sar was measured in the presence of 100 µM of each compound and was corrected for the passive diffusion. The uptake rate was normalized by that obtained in the absence of compounds. Each column represents the mean ( SE of three experiments. (***) P < 0.01; (**) P < 0.05; (*) P < 0.1, significant differences from the control using Student’s t-test.

derivatives of Val-Lys(Flu) or related compounds was measured. The fluorescein (uranine) itself had no effect on the uptake, indicating that the peptide core of ValLys(Flu) is recognized by PEPT1. Figure 2 shows that an inhibition of Gly-Sar by Val-Lys is about 35% while the conjugation of Val-Lys to Flu inhibited it appreciably. Why does this conjugation increase the affinity although Flu is not recognized at all? The answer to this remains unclear. We can only point out the importance of the hydrophobicity of the dipeptide analogues. This tendency seems to appear in Figure 2; among Gly-X peptides, the order of inhibition by X is Phe > Leu > Pro > Glu, an order corresponding to hydrophobicity, although the differences are small. An experiment on the importance of hydrophobicity is now in progress in this laboratory. We prepared two derivatives from Val-Lys: N-terminalmodulated Boc-Val-Lys(Flu) and C-terminal-modulated Val-Lys(Flu)-Ot-Bu. As seen in Figure 4, the order of the inhibition is Val-Lys(Flu) > Boc-Val-Lys(Flu) > ValLys(Flu)-Ot-Bu. This means that the carboxyl group might be more essential in the recognition by the peptide transporter than the amino group. This is consistent with the study on the intestinal absorption of cefixime and aminocephalosporin that do not have an R-amino group (23). Possible Use of This Strong Inhibitor. We described here our successful preparation of a strong inhibitor of PEPT1. The inhibition constant is the smallest of those yet reported. The primary use of this strong inhibitor is to examine the possibility that some drugs can be transported via PEPT1. Our preliminary experiments were the initial uptake rate of 1 mM cefadroxil was 0.218 ( 0.004 nmol/min/mg of protein (mean ( SE, n ) 3) and the addition of 200 and 500 µM Val-Lys(Flu) reduced 0.079 ( 0.001 (36.4% of the control) and 0.047 ( 0.001 (22.5%) nmol/min/mg of protein, respectively. Similarly, the rate of 1 mM cephradine was 0.200 ( 0.003 nmol/min/mg of protein (mean ( SE, n ) 3) and the addition of 200 and 500 µM Val-Lys(Flu) reduced 0.091 ( 0.002 (45%) and 0.067 ( 0.001 (30%) nmol/min/mg of protein, respectively. These suggest that the present fluorescent analogue is the useful tool for this purpose. Second, the high affinity of the dipeptide analogues synthesized is applicable to the resin of the affinity chromatography; results in Figure 4 suggest that the

Interaction of Fluorescent Dipeptide with PEPT1

N-terminal should be linked to the resin. The high affinity of these analogues is also a powerful tool for purification of the dipeptide transporter. Plausible Reasons Why CCCP Increases the Uptake of These Analogues. Figure 1 shows that CCCP increases the uptake of Val-Lys(Flu) and (Flu)Lys-Sar. If the uptake process requires energy, CCCP should show the reverse effect. There is no reason for the enhancement except the following: Val-Lys(Flu) is expelled by an energy-requiring efflux transporter in Caco-2 cells; the existence of an efflux transporter has been suggested (24, 25). Results of Figure 1 suggest the high affinity of ValLys(Flu) to the efflux transporter, because this analogue is transported by passive diffusion so that its intracellular concentration might not be large. The reason Gly-Sar increases the uptake of Val-Lys(Flu) and (Flu)Lys-Sar is not yet clear. We are now analyzing this efflux transporter. CONCLUDING REMARKS

We conjugated the dipeptide to fluorescent dyes. The resulting conjugates showed the highest affinity to the dipeptide transporter among those reported to date, although the conjugates are not transported by the transporter. Use of the high affinity of the analogues might help to characterize the transporter. As one reason for the high affinity, we pointed out the hydrophobicity; we should determine on a molecular basis whether this hypothesis is correct. We unfortunately failed to synthesize here the conjugated drugs that are transported by the transporter, and further study is needed to synthesize such chemicals which can improve the poor absorption in intestine. ACKNOWLEDGMENT

We thank Dr. M. Sugawara in Department of Pharmacy, Hokkaido University Hospital for his guidance of HPLC experiments and Miss R. Tateoka for her assistance. This investigation was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan. LITERATURE CITED (1) Adibi, S. A., and Mercer, D. W. (1973) Protein digestion in human intestine as reflected in luminal, mucosal, and plasma amino acid concentrations after meals. J. Clin. Invest. 52, 1586-1594. (2) Okano, T., Inui, K., Maegawa, H., Takano, M., and Hori, R. (1986) H+ uphill transport of aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border membranes. J. Biol. Chem. 261, 14130-14134. (3) Tsuji, A., Terasaki, T., Tamai, I., and Hirooka, H. (1987) H+ gradient-dependent and carrier-mediated transport of cefixime, a new cephalosporin antibiotic, across brush-border membrane vesicles from rat small intestine. J. Pharmacol. Exp. Ther. 241, 594-601. (4) Kramer, W., Dechent, C., Girbig, F., Gutjahr, U., and Neubauer, H. (1990) Intestinal uptake of dipeptides and β-lactam antibiotics. I. The intestianl uptake system for dipeptides and β-lactam antibiotics is not part of a brush border membrane peptidase. Biochim. Biophys. Acta 1030, 41-49. (5) Inui, K., Tomita Y., Katsura, T., Okano, T., Takano, M., and Hori, R. (1992) H+ coupled active transport of bestatin via the dipeptide transport system in rabbit intestinal brushborder membranes. J. Pharmacol. Exp. Ther. 260, 482-486. (6) Hu, M., and Amidon, G. L. (1988) Passive and carriermediated intestinal absorption components of captopril. J. Pharm. Sci. 77, 1007-1011.

Bioconjugate Chem., Vol. 10, No. 1, 1999 31 (7) Swaan, P. W., Stehouwer, M. C., and Tukker, J. J. (1995) Molecular mechanism for the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat and lisinopril. Biochim. Biophys. Acta 1236, 31-38. (8) Taylor M. D., and Amidon G. L., Eds. (1995) in Peptide-based Drug Design: Controlling Transport and Metabolism, pp 1-567, American Chemical Society, Washington, DC. (9) Woodley, J. F. (1994) Enzymatic barriers for GI peptide and protein delivery. Crit. Rev. Ther. Drug Carrier Syst. 11, 6195. (10) Dantzig, A. H., and Bergin, L. (1990) Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochim. Biophys. Acta 1027, 211-217. (11) Inui, K., Yamamoto, M., and Saito, H. (1992) Transepithelial transport of oral cephalosporins by monolayers of intestinal epithelial cell line Caco-2: Specific transport systems in apical and basolateral membranes. J. Pharmacol. Exp. Ther. 261, 195-201. (12) Yamaoka, K., Tanigawara, Y., Nakagawa, T., and Uno T. (1981) A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-Dyn. 4, 879-885. (13) Ganapathy, M. E., Huang, W., Wang, H., Ganapathy V., and Leibach, F. H. (1998) Valacyclovir: A substrate for the interstinal and renal peptide transporters PEPT1 and PEPT2. Biochem. Biophys. Res. Commun. 246, 470-475. (14) Enjoh, M., Hashimoto, K, Arai, S., and Shimizu, M. (1996) Inhibitory effect of arphamenin A on intestinal dipeptide transort. Biosci. Biotechnol. Biochem. 60, 1893-1895. (15) Brandsch, M., Miyamoto, Y., Ganapathy, V., and Leibach, F. H. (1994) Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line. Biochem. J. 299, 253-260. (16) Ganapathy, M. E., Brandsch, M., Prasad, D., Ganapathy V., and Leibach, F. H. (1995) Differential recognition of β-lactam antibiotics by intestinal and renal peptide transporters, PEPT1 and PEPT2. J. Biol. Chem. 270, 2567225677. (17) Dantzig, A. H., Tabas, L. B., and Bergin, L. (1992) Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and comparison to cephalexin uptake. Biochim. Biophys. Acta 1112, 167-173. (18) Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. J. 55, 170-171. (19) Bornish-Bowden, A. (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and noncompetitive inhibitors. Biochem. J. 137, 143-144. (20) Ganapathy, V., Burckhardt, G., and Leibach, F. H. (1984) Characteristics of glycylsarcosine transport in rabbit intestinal brush-border membrane vesicles. J. Biol. Chem. 259, 8954-8959. (21) Dantzig, A. H., Duckworth, D. C., and Tabas, L. B. (1994) Transport mechanism responsible for the absorption of loracarbef, cefixime, and cefuroxime exetil into human intestinal Caco-2 cells. Biochim. Biophys. Acta 1191, 7-13. (22) Otto, C., Dieck, S., and Bauer, K. (1996) Dipeptide uptake by adenohypophysial folliculostellate cells. Am. J. Physiol. 271, C210-C217. (23) Bai, P. F., Subramanian, P., Mosberg, H. I., and Amidon, G. L. (1991) Structural requirements for the intestinal mucosal-cell peptide transporter: the need for N-terminal R-amino group. Pharm. Res. 8, 593-599. (24) Elferink, R. P. L., Bakker, C. T. M., and Jansen, P. L. M. (1993) Glutathione-conjugate transport by human colon adenocarcinoma cells (Caco-2 cells). Biochem. J. 290, 756-764. (25) Collington, G. K., Hunter, J., Allen, C. N., Simmons, N. L., and Hirst, B. H. (1992) Polarized efflux of 2,7-bis(2carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers. Biochem. Pharm. 44, 417-424.

BC980049I