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Bioconjugate Chem. 2004, 15, 536−540
Design, Synthesis, and Evaluation of 5′-S-Aminoethyl-N6azidobenzyl-5′-thioadenosine Biotin Conjugate: A Bifunctional Photoaffinity Probe for the es Nucleoside Transporter James K. Addo and John K. Buolamwini* Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, Tennessee 38163. Received September 11, 2003; Revised Manuscript Received March 12, 2004
A bifunctional biotinylated photoaffinity label for the nitrobenzylmercaptopurine riboside (NBMPR)sensitive (es) nucleoside transporter (ENT1) has been synthesized and evaluated. This new probe, 5′-S-aminoethyladenosine-N6-azidobenzyl-5′-thioadenosine biotin conjugate (SAEATA-14-biotin), exhibited high-affinity binding to the es transporter in K562 cells as determined by flow cytometry, with a Ki of 2.69 nM in competition against 5-(SAENTA)-x8-fluorescein. It also exhibited covalent linking to the es transporter in BeWo cell membranes upon UV irradiation. This new bifunctional probe is a potential tool for determining the amino acid residues involved in ligand binding at the NBMPR-binding site of the ENT1 nucleoside transporter, as well as for the purification of the transporter.
INTRODUCTION
Nucleoside transporters are integral membrane glycoproteins required for the cellular uptake of physiological nucleosides such as adenosine and their synthetic analogues. Two families of nucleoside transporters have been identified: (i) equilibrative (facilitated diffusion) transporters, ENTs, and (ii) concentrative (sodium ioncoupled) transporters, CNTs (1-5). The equilibrative transporters are found in most mammalian cells and tissues studied, whereas the concentrative transporters are limited to specialized epithelial tissues and some cultured cell lines. The ENT1 transporter also known as the es transporter is by far the predominant nucleoside transporter of most mammalian cells examined and can be distinguished from the other equilibrative transporter, ENT2, also known as ei transporter, by its high sensitivity to inhibition by S6-(4-nitrobenzyl)mercaptopurine riboside (NBMPR, 1). Inhibition of the es transporter by NBMPR arises from high-affinity binding (Kd 0.1-1.0 nM) (6). The ei transporter is at least 1000-fold resistant to inhibition by NBMPR (4, 7).
regulation of tissue adenosine levels and thereby modulation of its signal transduction functions in cardioprotection and neuroprotection. Thus, nucleoside transport inhibitors (NTIs) have potential therapeutic uses in several therapeutic areas including ischemic heart disease and stroke, cancer and viral chemotherapy, and the preservation of donor organs for transplantation (8). The known inhibitors are mostly unsuitable for clinical applications, and therefore novel inhibitors are needed. Several nucleoside transporters have been cloned (9), but structural biology studies of these transporters have been hampered by the lack of the appropriate chemical probes. Devising appropriate molecular probes for elucidation of the amino acid residues involved in inhibitor-binding will go a long way to promote these studies. In the absence of a 3D structure of the es transporter or its complex with an inhibitor, photoaffinity labeling offers a useful approach to identifying the amino acid residues involved in ligand binding. In this regard, NBMPR and its analogues, which bind with high affinity and high specificity to the ENT1 transporter, are excellent templates for designing affinity labels of this transporter. In this paper, we describe the design, synthesis, and evaluation of such an ENT1 transporter probe, a biotinylated azido analogue of NBMPR, termed SAEATA-x14-biotin (compound 2).
Interest in nucleoside transporters has been on the increase because of their involvement in the efficacy of anticancer and antiviral nucleoside drugs, as well as their * Address correspondence to this author at Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Sciences Center, 847 Monroe Ave., Suite 327, Memphis, TN 38163. Phone (901) 448-7533. Fax (901) 448-6828. E-mail:
[email protected].
10.1021/bc034165j CCC: $27.50 © 2004 American Chemical Society Published on Web 05/04/2004
Bifunctional Photoaffinity Probe EXPERIMENTAL SECTION
Synthesis. Thin-layer chromatography (TLC) was conducted on silica gel F254 plates (Analtech). Compounds were visualized by UV light or 5% H2SO4 in EtOH spraying reagent. 1H and 13C spectra were recorded on Bruker ARX (500 MHz) instrument, using CDCl3, CD3OD, (CD3)2SO, or CD3COCD3 as solvents and tetramethylsilane (TMS) as internal standard. Flash column chromatography was performed on Fisher silica gel (170400 mesh). Melting points were determined using a Fisher-Johns melting point apparatus and are reported uncorrected. Mass spectra were obtained on a BrukerHP Esquire-LC mass spectrometer, and IR spectra with a Perkin-Elmer System 2000 FT-IR spectrometer. All solvents and reagents were bought from Sigma-Aldrich Chemical Co. and used without further purification, unless where specified. HPLC was performed on a GILSON Instrument with UV detection. Biotinylated SAENTA (SAENTA-x14-biotin, 3) was prepared by reacting Biotin-xx SE (Molecular Probes, Eugene, OR) with SAENTA phthalhydrazide salt (10) in dimethylformamide (Buolamwini and Paterson, unpublished results). [3H]NBMPR (23 Ci/mmol, 1 Ci ) 37 GBq) was purchased from Moravek Biochemicals (Brea, CA). 5-(SAENTA)-x8fluorescein was prepared as previously reported (11). 5′-S-(2-(6-(6-Biotinamidohexanoyl)aminohexanoyl)aminoethyl)-N6-(4-aminobenzyl)-5′-thioadenosine (4). A mixture of SAENTA-x14-biotin (3, 100 mg, 0.109 mmol), activated carbon (20.0 mg, G-60, 100 mesh), ferric chloride hexahydrate (10.00 mg, 0.036 mmol), and methanol (5 mL) was refluxed for 10 min with stirring. To this boiling mixture was added hydrazine hydrate (40 mg, 1.25 mmol, 99.1%) dropwise, and the mixture was stirred under reflux for an additional 12 h, cooled, and filtered, and the residue was washed with methanol (3 × 5 mL). The filtrate was evaporated to give a solid residue. The residue was recrystallized from EtOAc to give compound 4 as colorless crystals: 88 mg, 91% yield, mp 184-185 °C. 1H NMR (500 MHz, CDCl3) δ 1.166-2.022 (m), 2.484-2.571 (m), 2.782-3.316 (m), 3.98-4.01 (m), 4.084.14 (m), 4.26-4.31 (m), 4.511 (bs), 4.712 (bs), 5.33 (d, J ) 6.0, 1H), 5.49 (d, J ) 5.56 5.56, 1H), 5.88 (d, J ) 5.56 Hz, 1H), 6.350 (s), 6.41 (s), 6.49 (d, J ) 8.29 Hz, 1H), 7.01 (d, J ) 8.29 Hz, 1H), 7.70-7.74 (m), 7.88-7.89 (m), 8.14-8.19 (m), 8.34 (bs), 9.39 (bs); IR (neat) νmax (3255, 2933, 2340, 1623, 1541, 1245, 1036, 920 cm-1. MS (ESI) m/z 915 (M + H)+. 5′-S-(2-(6-(6-Biotinamidohexanoyl)aminohexanoyl)aminoethyl)-N6-(4-azidobenzyl)-5′-thioadenosine (2, SAEATA-x14-biotin). A solution of sodium nitrite (5.64 mg, 0.082 mmol) in cold water (0.20 mL) at 0 °C was made and added with stirring to a cold (0 °C to -5 °C) solution of compound 4 (65 mg, 0.074 mmol) in glacial acetic acid (0.5 mL). The mixture was then stirred at 0 °C for another 20 min. To the resulting solution of diazonium salt, at 0 °C, was added a solution of NaN3 (5.33 mg, 0.082 mmol) in cold water (0.20 mL). The mixture was then allowed to stir at 0 °C for an additional 1.5 h in the dark. The reaction mixture was then evaporated to give a solid residue. H2O (1 mL) was then added, and the reaction mixture was extracted with EtOAc (3 × 5 mL). The combined organic extracts were dried (anhydrous Na2SO4) and filtered. The solvent was evaporated in vacuo to give a crude solid residue. This residue was washed three times with H2O (3 × 5 mL) and the solid obtained dried in vacuo and recrystallized in MeOH/EtOAc (1:1) to give the target compound 2 (57 mg, 85.20% yield): mp 115-116 °C. 1H NMR (500 MHz, CDCl3) δ 1.22- 2.11 (m), 1.18-2.02 (m), 2.54-3.17 (m),
Bioconjugate Chem., Vol. 15, No. 3, 2004 537
3.92-4.11 (m), 4.15 (bs), 4.22-4.28 (m), 4.66-4.70 (m), 4.79-4.80 (m), 5.88 (d, J ) 5.857, 1H), 7.036 (d, J ) 8.785, 1H), 7.36 (d, J ) 8.785 Hz, 1H) 8.19 (bs), 8.37 (s); IR (neat) νmax 3303, 2925, 2854, 2363, 2110, 1763, 1631, 1541, 1507, 1466, 1294, 1211, 1120, 1053 cm-1. MS (ESI) m/z 910 (M + H)+. The purity was checked by reversed phase HPLC using a C18 column and two different solvent systems: (1) methanol/acetonitrile (1:1 v/v) and (2) solvent methanol/water (4:1 v/v) (Fisher HPLC grade). Cell Culture. K562 chronic myelogenous leukemia and BeWo choriocarcinoma cells were purchased from American Type Culture Collection (Rockville, MD). Stock cultures were grown in antibiotic-free growth media consisting of RPMI 1640 supplemented with 10% fetal bovine serum for K562 cells, or RPMI 1640 supplemented with 5% fetal bovine serum plus 5% Nuserum type IV for BeWo cells. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2 in air. Flow Cytometry. K562 suspension cells maintained in exponential growth (0.5 × 105 to 5 × 105 cells/mL) were used in flow cytometric evaluation of probe binding to the ENT1 (es) nucleoside transporter as previously described (11). Briefly, cells were washed once and suspended at 4 × 105 cells/mL in phosphate-buffered saline at pH 7.4 and incubated with 5-(SAENTA)-x8fluorescein (25 nM) in the presence or absence of varying concentrations of test compounds at room temperature for 45 min. Flow cytometric measurements for cellassociated fluorescence were performed at the end of the incubation period with a FACSCalibur (Becton Dickinson, San Jose, CA) equipped with a 15 mW argon laser (Molecular Resources Flow Cytometry Facility, University of Tennessee Health Sciences Center). In each assay, 5000 cells were analyzed. The units of fluorescence were arbitrary mean channel numbers. Percentage (%) of control (es transporter-specific fluorescence in the presence of SAENTA-fluorescein without test compounds) was calculated for each sample by eq 1.
% control )
(SFs) (SFf)
× 100
(1)
where SFs is the es transporter-specific fluorescence of test samples, and SFf is the es transporter-specific fluorescence of the SAENTA-fluorescein ligand. The inhibition constant (Ki) values were calculated from the IC50 value obtained by nonlinear regression fitting of the concentration-effect curves using eq 2.
Ki ) IC50 /(1 + [L]/KL)
(2)
where [L] and KL are the concentration and the Kd value of the SAENTA-fluorescein ligand, respectively. The IC50 value was obtained by regression fitting to the data in Figure 1 using the Prism program (GraphPad, San Diego, CA). BeWo Cell Membrane Preparation. BeWo cell membranes were prepared according to the protocol described in Boumah et al. (12). Cells were harvested by trypsinization and disrupted by sonication in the presence of a protease inhibitor (2 mM AEBSF, a water soluble analogue of the protease inhibitor PMSF). The lysates were fractionated in a 20% Percoll gradient and washed twice by centrifugation at 40000g, for 15 min at 4 °C in 50 mM Tris/HCl buffer (pH 7.4). Protein concentration was determined by the Lowry method (13). Photolabeling. Membranes with 10-50 µg of protein were incubated with 100 nM of compound 2 in the
538 Bioconjugate Chem., Vol. 15, No. 3, 2004
Addo and Buolamwini Scheme 1. Synthetic Route to Compound 2a
Figure 1. Flow cytometric analysis of the binding of nbmpr and biotinylated analogues to the es transporter of k562 leukemia cells. Cells were incubated with graded concentrations of test compounds in the presence of 25 nM 5-(SAENTA)-x8flourescein at 22 °C for 45 min. Each data point represents the mean ( SEM for three different experiments.
presence or absence of 10 µM NBMPR in Tris/HCl buffer in the presence of AEBSF for 30 min at 22 °C, cooled to 4 °C, and then irradiated with shortwavelength UV light for 3-5 min at a distance of 3 cm. The reaction was stopped by addition of an equal volume of Tris buffer containing 12% trichloroacetic acid (TCA). The membranes were pelleted and washed twice with Tris/HCl containing TCA. For indirect determination of photocross-linking by compound 2, the membranes were washed and used in [3H]NBMPR binding as described below. [3H]NBMPR Binding to Membranes. Membrane samples containing 10 µg of protein each were incubated with [3H]NBMPR (1.0 nM; 15 Ci/mmol) in binding medium (50 mM Tris/HCl, pH 7.4, 20 mM dithiothreitol) in the presence or absence of 10 µM nonradioactive NBMPR for 30 min at 22 °C. Bound and free [3H]NBMPR were then separated from each other by rapid filtration with Whatman GF/B filters. The radioactivity that was retained on the filters, i.e., bound [3H]NBMPR was measured by liquid scintillation counting. Streptavidin Detection of Photo-Cross-Linked Compound 2. To detect photo-cross-linking of protein by SAEATA-x14-biotin (2), BeWo cell membranes (50 µg) photolabed with the probe were subjected to SDS-PAGE electrophoresis, after which the protein was electroblotted onto polyvinylidine fluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked in BLOTTO (5% nonfat dried milk) in TBS-T: (20mM TrisHCl, pH 7.6, 137mM NaCl, and 0.1% Tween-20) at 4 °C overnight. This was followed by incubation with horseradish peroxidase-conjugated streptavidin diluted in BLOTTO (0.5µg/mL) for 1 h. After three washes in TBST, the blot was treated with Enhanced ChemiLuminescence (ECL, Amersham) detection reagents as described by the manufacturer. Biotinylated proteins were visualized by exposure to X-ray film and development. RESULTS AND DISCUSSION
Our bifunctional probe design took into consideration the following: (1) Experimental evidence shows that nucleoside analog es transporter inhibitors such as NBMPR do not require a free 5′-OH group on the ribose ring, and that with an appropriate spacer, the 5′-position of inhibitors is very tolerant of bulky substituents (10, 11, 14). For example, bulky fluorescein derivatives can be tethered to the 5′-position and still retain high affinity
a Reagents and conditions: (a) NH NH .‚H O/FeCl ‚6H O/ 2 2 2 3 2 activated carbon/MeOH/reflux; (b) (i) NaNO2/aq AcOH, 0 °C, (ii) NaN3, 0 °C.
binding with Kd values as low as 2 nM (11). Therefore, we decided to tether a biotin moiety via an appropriate spacer chain to the 5′-position of the nucleoside portion of the probe for avidin or streptavidin affinity purification and detection purposes. (2) The NO2 group in NBMPR was replaced by an azido photoreactive group to use for photo-cross-linking to the transporter. The azidobenzyl moiety, like the nitrobenzyl moiety of NBMPR, affords high-affinity binding at the es transporter (15). Furthermore, p-azidobenzyladenosine (ABA) has been used successfully to photolabel the es transporter of human erythrocytes (16). Although NBMPR itself is a photolabeling agent, the radical species for covalent linking to the protein is produced by cleavage of the sulfur-benzyl bond (17), which means that before covalent linking, the high-affinity conferring nitrobenzyl group is lost. This is disadvantageous for specific labeling, because the loss of the nitrobenzyl moiety could cause a dissociation of the bound inhibitor (18) from the binding site prior to crosslinking with the protein, which increases the likelihood of labeling residues not involved in binding. Replacing the sulfur by NH prevents the loss of the benzyl portion upon irradiation and increases the likelihood of bindingsite specific photo-cross-linking. The synthesis of the probe followed Scheme 1 and began with the derivatizable high-affinity es transporter ligand 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA). In SAENTA, a 2-aminoethylthio (H2NCH2CH2S-) has replaced the 5′-OH group to provide a handle for tethering a reporter or other group (10). Compound 3, SAENTA-x14-biotin, was obtained by a procedure involving the reaction of the phthalhydrazide salt of SAENTA with (6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid succinimydyl ester (biotin-xx, SE)
Bifunctional Photoaffinity Probe
Bioconjugate Chem., Vol. 15, No. 3, 2004 539
Table 1. Ki Values for Inhibition of SAENTA-fluor Binding to the es Transporter (ENT1) in K562 Cells compound
Ki (nM)a
SAEATA-x14-biotin (2) SAENTA-biotin (3) 4 NBMPR
2.69 ( 0.36 1.03 ( 0.40 14.99 ( 0.53 1.2 ( 0.22
a Data points are the mean ( standard deviation from three separate experiments.
in the presence of Et3N to give this biotinylated derivative (Buolamwini and Paterson, unpublished, results). Scheme 1 was then followed for the rest of the synthesis. Reduction of the nitro group of compound 3 was effected by treatment with FeCl3/H2N-NH2‚xH2O in the presence of activated carbon in methanol to obtain the amino compound, 4. Diazotization of compound 4 using aqueous NaNO2 in acetic acid and subsequent displacement of the diazonium group with the azido group by treatment with NaN3 yielded the target bifunctional photaffinity label, compound 2. Biological Evaluation. Binding at the es Transporter. The ability of this new bifunctional es transporter label to bind to the transporter was determined by a flow cytometric ligand-binding assay using a SAENTAfluorescein conjugate, 5-(SAENTA)-x8-fluorecein (11) as the fluorescent es transporter competitive ligand. The results of the flow cytometric evaluation are presented in Figure 1, which shows potent concentration-dependent displacement of the competitive ligand by all the compounds but the amino compound (4), which is consistent with the known structure-activity relationship (15). The Ki values used to compare how tightly the ligands bound to the ENT1 transporter are presented in Table 1. These results indicate that the novel bifunctional photoaffinity label, compound 2, has a high affinity for the ENT1 transporter (Ki 2.69 nM), which makes it suitable for photolabeling and probing the transporter. Photolabeling. For the purpose of determining if compound 2 will cross-link at the NBMPR-binding site of the ENT1 transporter, cell membranes prepared from BeWo cells, known to express large numbers of ENT1 (12), were incubated with compound 2 in the absence or presence of the potent ENT1 inhibitor NBMPR, and the samples were irradiated with shortwave UV light. The membranes were then washed to get rid of noncovalently bound ligands and then used for [3H]NBMPR specific binding analysis. The [3H]NBMPR binding results for the irradiated and washed membranes are shown in Figure 2. They indicate that in the absence of the ENT1 competitive ligand NBMPR, compound 2 covalently bound to (cross-linked) the membranes and could not be completely washed off. In the samples that contained the NBMPR, cross-linking and covalent blocking of the NBMPR-binding site on ENT1 by compound 2 was inhibited. This demonstrates that the new photoaffinity probe can photo-cross-link ENT1 at the NBMPR binding site and will be a useful tool in identify amino acid residues at the NBMPR-binding site of the ENT1 (es) nucleoside transporter. Streptavidin Detection of Cross-Linked Compound 2. To demonstrate that the pendant biotin group of compound 2 can be detected and used as a handle in streptavidin affinity purification as well as probe detection, BeWo cell membranes were photolabeled with compound 2, subjected to SDS-PAGE, blotted unto PVDF membranes, and probed with streptavidin horseradish peroxidase conjugate. The results shown in Figure 3 show that not only can compound 2 be used to
Figure 2. [3H]NBMPR binding to BeWo membranes that were washed after incubation with test compounds followed by irradiation with UV light. Membranes were from samples incubated with 100 nM SAEATA-x14-biotin, samples incubated with 100 nM SAEATA-x14-biotin in the presence of 10 µM NBMPR. The data represent the mean and minimum and maximum values from two separate experiments.
Figure 3. Streptavidin detection of compound 2 photocrosslinked to BeWo cell membranes. BeWo cell membrane preparation (50 µg protein) irradiated with UV light was loaded onto 12% SDS-polyacrylamide gel and electrophoresis carried out. Protein was electroblotted unto polyvinylidine fluoride membranes (Immobilon-P; Millipore, Bedford, MA), blocked with 5% nonfat dried milk in TBS-T: (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated streptavidin (0.5 µg/mL) in BLOTTO for 1 h. The membrane was washed in TBS-T, and treated with Enhanced ChemiLuminescence (ECL, Amersham) detection reagents as described by the manufacturer. Biotinylated proteins were visualized by exposure to X-ray film and development.
photolabel amino acids at the NBMPR-binding site on the ENT1 transporter (Figure 2), but it can also be detected by binding it to streptavidin. Figure 3 confirms that the probe cross-links protein and suggests that compound 2 can be used as a bifunctional probe to crosslink the ENT1 transporter and be used for affinity isolation of ENT1 polypeptides. Efforts are underway to use this novel probe for photolabeling and identification of the amino acid residues of the ENT1 transporter involved in ligand binding, by mass spectrometric means similar to a recent characterization of the dexniguldipine binding site of P-glycoprotein (19). ACKNOWLEDGMENT
Financial support of this work came from the National Heart, Lung and Blood Institute, NIH, Grant No. HL 67479. Yaqin Zhang, Bing Chen, and Ja’Wanda Grant are credited for technical support. LITERATURE CITED (1) Plagemann, P. G. W., Wohlhueter, R. M., and Woffendin, C. (1988) Nucleoside and nucleobase transport in animal cells. Biochim. Biophys. Acta 947, 405-443. (2) Gati, W. P., and Paterson, A. R. P. (1989) Nucleoside transport. In The Red Cell Membrane: Structure, Function,
540 Bioconjugate Chem., Vol. 15, No. 3, 2004 and Clinical Implications (Agre, P., Parker, J. C., Eds.) pp 635-661, Marcel Dekker, New York. (3) Belt, J. A., Marina, N. M., Phelps, D. A., and Crawford, C. R. (1993) Nucleoside transport in normal and neoplastic cells. Adv. Enzyme Regul. 33, 235-252. (4) Cass, C. E. (1995) Nucleoside transport. In Drug Transport in Antimicrobial and Anticancer Chemotherapy (Georgopapadakou, N. H., Ed.) pp 403-451, Marcel Dekker, New York. (5) Young, J. D., Cheeseman, C. I., Mackey, J. R., Cass, C. E., and Baldwin, S. A. (2000) Molecular mechanisms of nucleoside and nucleoside drug transport. In Current Topics in Membranes (Barrett, K. E., Donowitz, M., Eds.) pp 329-378, Vol. 50, Academic Press, San Diego, CA. (6) Paterson, A. R. P., Jacobs, E. S., Harley, E. R., Fu, N.-W., Robins, M. J., and Cass, C. E. (1983) Inhibition of nucleoside transport. In Regulatory Functions of Adenosine (Berne, R. M., Rall, T. W., Rubio, R., Eds.) pp 203-220, Martinus, Nijhoff, The Hague, (7) Thorn, J. A., and Jarvis, S. M. (1996) Adenosine transporters. Gen. Pharmacol. 27, 613-620. (8) Buolamwini, J. K. (1997) Nucleoside transport inhibitors: structure-activity relationships, and potential therapeutic applications. Curr. Med. Chem. 4, 35-66. (9) Cass, C. E., Young, J. D., and Baldwin, S. A. (1998) Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem. Cell. Biol. 76, 761-770. (10) Agbanyo, F. R., Vijayalakshmi, D., Craik, J. D., Gati, W. P., McAdam, D. P., Asakura, J.-I., Robins, M. J., Paterson, A. R. P., and Cass, C. E. (1990) 5′-S-(2-Aminoethyl)-N6-(4nitrobenzyl)-5′-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport: partial purification of the nitrobenzylthioinosinebinding protein of pig erythrocytes by affinity chromatography. Biochem. J. 270, 605-614. (11) Buolamwini, J. K., Wiley, J. S., Robins, M. J., Craik, J. D., Cass, C. E., Gati, W. P., Paterson, A. R. P. (1994) Conjugates of fluorescein and SAENTA (5′-S-(2-aminoethyl)N6-(4-nitrobenzyl)-5′-thioadenosine): flow cytometry probes
Addo and Buolamwini for the es transporter elements of the plasma membrane. Nucleosides Nucleotides 13, 737-751. (12) Boumah, C. E., Hogue, D. L, and Cass, C. E. (1992) Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) Cells. Biochem. J. 288, 987-996. (13) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. (14) Ziemnicka-Merchant, B., Aran, J. M., Plagemann, P. G. W., Krafft, G. A. (1992) Effects of chemical modification of nitrobenzylthioinosine on its binding to high-affinity membrane binding sites and inhibition of nucleoside transport. Biochem. Pharmacol. 44, 1577-1583. (15) Robins, M. J., Asakura, J.-I., Kaneku, M., Shibuya, S., Jacobs, E. S., Agbanyo, F. R., Cass, C. E., and Paterson, A. R. P. (1994) Synthesis of substituted-benzyl and sugarmodified analogues of 6-N-(4-nitrobenzyl)adenosine and their interactions with “ES” nucleoside transport systems. Nucleosides and Nucleotides 13, 1627-1646. (16) Young, J. D., Jarvis, S. M., Robins, M. J., and Paterson, A. R. P. (1983) Photoaffinity labeling of the human erythrocyte nucleoside transporter by N6-(P-azidobenzyl)adenosine and nitrobenzylthioinosine. Evidence that the transporter is a Band 4.5 polypeptide. J. Biol. Chem. 258, 2202-2208. (17) Fleming, S. A., and Jensen, A. W.(1993) Photocleavage of benzyl-sulfide bonds. J. Org. Chem. 58, 7135-7137. (18) Paul, B., Chen, M. F., and Paterson, A. R. P. (1975) Inhibitors of nucleoside transport: structure-activity relationships. J. Med. Chem. 18, 968-973. (19) Borchers, C., Boer, R., Klemm, K., Figala, V., Denzinger, T., Ulrich, W.-R., Haas, S., Ise, W., Gekeler, V., and Przybylski, M. (2002) Characterization of the dexniguldipine binding site in the multidrug resistance-related transport protein P-glycoprotein by photoaffinity labeling and mass spectrometry. Mol. Pharmacol. 61, 1366-1376.
BC034165J
