Synthesis and Characterization of a New Bioactivated Paramagnetic

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Bioconjugate Chem. 2007, 18, 1716–1727

ARTICLES Synthesis and Characterization of a New Bioactivated Paramagnetic Gadolinium(III) Complex [Gd(DOTA-FPG)(H2O)] for Tracing Gene Expression Yu-Ton Chang,† Chiu-Min Cheng,‡ Yu-Zheng Su,† Wei-Thung Lee,† Jui-Sheng Hsu,§ Gin-Chung Liu,| Tian-Lu Cheng,*,‡ and Yun-Ming Wang*,† Faculty of Medicinal and Applied Chemistry, Faculty of Biomedical Science and Environmental Biology, Department of Radiology, Kaohsiung Municipal Hsiao-Kang Hospital, Department of Medical Imaging, Kaohsiung Medical University Hospital, and Department of Radiology, Kaohsiung Medical University, and 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Received January 18, 2007; Revised Manuscript Received June 7, 2007

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a β-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The 17O NMR experiments 298 298 were conducted to estimate the water exchange rate k298 ex and rotational correlation timeτR . The kex value of [Gd(DOTA-FPG)(H2O)] is similar to that of [Gd(DO3A-bz-NO2)(H2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H2O)] is dramatically longer than that of [Gd(DOTA)(H2O)]- Relaxometric studies show that the percentage change in the T1 value of [Gd(DOTA-FPG)(H2O)] decreases dramatically in the presence of β-galactosidase and human serum albumin. The T1 change percentage of [Gd(DOTA-FPG)(H2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-β-D-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H2O)] in the presence of human serum albumin and β-galactosidase (2670 ( 210) is significantly higher than that of [Gd(DOTA-FPG)(H2O)] in the sodium phosphate buffer solution (1490 ( 160). In addition, the MR images show a higher-intensity enhancement in CT26/β-gal tumor with β-galactosidase gene expression but not for the CT26 tumor without β-galactosidase gene expression. We conclude that [Gd(DOTAFPG)(H2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.

INTRODUCTION Molecular imaging is providing a sensitive and specific method for the detection and localization of the biochemical appearance in ViVo. Magnetic resonance imaging (MRI) offers several advantages over other clinical diagnostic techniques for molecular imaging, including high spatial resolution, noninvasiveness, high anatomical contrast, and lack of harmful radiation. However, sensitivity of MRI to depicting a small molecule is constrained by the ubiquitous protons in the body, resulting in a high background and low signal to noise ratio (SNR). Hence, * Corresponding authors. Dr. Yun-Ming Wang, Kaohsiung Medical University, Faculty of Medicinal and Applied Chemistry, 100 ShihChuan 1st Road, Kaohsiung 807, Taiwan, Tel 886-7-3121101ext. 2218, Fax 886-7-3125339, E-mail address [email protected]; Dr. TianLu Cheng, Kaohsiung Medical University, Faculty of Biomedical and Environmental Biology, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, Tel 886-7-3121101ext. 2697, Fax 886-7-3227508, E-mail [email protected]. † Faculty of Medicinal and Applied Chemistry. ‡ Faculty of Biomedical Science and Environmental Biology. § Department of Radiology, Kaohsiung Municipal Hsiao-Kang Hospital. | Department of Medical Imaging, Kaohsiung Medical University Hospital, and Department of Radiology, Kaohsiung Medical University.

the alternative amplification strategies using smart contrast agents are required to yield a higher sensitivity (1). Nonspecific contrast agents such as [Gd(DTPA)(H2O)]2(H5DTPA ) 3,6,9-tri(carboxymethyl)-3,6,9-triazaundecanedioic acid) (Magnevist, gadopentetate), [Gd(DTPA-BMA)(H2O)] (H3DTPA-BMA ) 1,7-bis[(N-methylcarbamoyl)methyl]1,4,7-tris(carboxymethyl)-1,4,7-triazaheptane) (Omniscan, gadodiamide), and [Gd(DOTA)(H2O)]- (H4DOTA ) 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Dotarem, gadoterate) are widely available for clinical use. They provide a nonspecific distribution pattern that allows measurement of tissue profusion, vascular permeability, or vascular volume. These parameters can be fitted by fast pharmacologic modeling and imaging techniques or steady-state imaging techniques (2, 3). However, the true molecular targets cannot be ideally displayed with these approaches. Different signal characteristics upon interacting with the specific target by smart contrast agents could provide new ways for detecting the stages of biological molecules. Theoretically, smart contrast agents exhibit a strong signal alteration upon target interaction whether there is a bioactivated effect or not. This makes them ideal candidates for molecular imaging because they have the highest SNR for molecular target identification.

10.1021/bc070019s CCC: $37.00  2007 American Chemical Society Published on Web 10/13/2007

Bioactivated Paramagnetic Gadolinium(III) Complex

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Scheme 1. Synthetic Scheme of DOTA-FPGa

a Reagents and conditions: (i) NaOH, TBAB, H2O, CH2Cl2, 73%; (ii) DAST, CH2Cl2, 94%; (iii) H2, Pd/C, ethyl acetate, 85% (35); bromoacetyl bromide, dimethylamine, CH2Cl2, 32%; (v) DO3A-tris-tbutyl ester (7), triethylamine, CH2Cl2 (36); NaOCH3, CH3OH; (vii) TFA, 45% for three steps.

The smart MR contrast agents carry out structural changes upon target interaction, which significantly change their signal properties (e.g., increasing the number of inner-sphere water molecules, q) (4). Enzyme-activated contrast agents for MRI have been developed and characterized (1, 5). These gadolinium(III) chelates developed an MR signal amplification strategy based on oxidoreductase-mediated polymerization of paramagnetic monomer into oligomers of higher relaxivity. Other enzyme motifs such as β-galactosidase, often used as a reporter gene in molecular biology, have been explored for a smart contrast agent for MRI (4, 6, 7). In this system, the route of water exchange to the gadolinium chelate is blocked by an enzymatically cleavable galactopyranoside substrate. β-Galactosidase activity results in an increase in T1 relaxivity, and thus, the signal intensity increased in the T1-weighted image. The feasibility of detecting β-galactosidase expression in Xenopus laeVis embryos has been evaluated (6, 8). The synthesis and characterization of Gd(III) complexes with poly(amino carboxylate) had been investigated in our previous studies (9–12). In this study, we design, synthesize (Scheme 1), and characterize the enzymatic contrast agent [Gd(DOTAFPG)(H2O)] (H3DOTA-FPG ) 1-(2-difluoromethyl-4-(1-(4,7,10triscarboxymethyl-(1,4,7,10-tetraazacyclodecyl))acetamido)phenyl)-β-D-galactopyranose), which can be activated by β-galactosidase. In an attempt to achieve improvements in enzyme-mediated relaxivity enhancement, we decided to link a bioactivated residue onto gadolinium(III) chelate in order to achieve conjugation and, therefore, a higher molecular weight of the final product obtained. This chelate consists of a gadolinium(III) complex and an enzymatic moiety, 2-difluoromethylphenyl-β-galactopyranoside, which can be cleaved when there is the existence of β-galactosidase. The nucleophile

of human serum albumin (HSA) and β-galactosidase will attack [Gd(DOTA-FP)(H2O)] (H3DOTA-FP ) 1-(6-fluoromethylene4-(1-(4,7,10-triscarboxymethyl(1,4,7,10-tetraazacyclodecyl)) acetamido)cyclohexa-2,4-dienone) complex to form biomacromolecules (13) [Gd(DOTA-FP)(H2O)]-HSA and [Gd(DOTAFP)(H2O)]-β-galactosidase. The higher relaxivity (r1) of the [Gd(DOTA-FP)(H2O)]-HSA is achieved as a result of the larger rotational correlation time value of the macromolecule [Gd(DOTA-FP)(H2O)]-HSA as shown in Scheme 2. 17O NMR relaxation rates and angular frequencies of the free Gd(III) complex in the presence and absence of HSA and β-galactosidase at variable temperatures will be reported. Furthermore, the effect of β-galactosidase on the longitudinal relaxation time (T1) of Gd(III) complex will be investigated. Finally, the MR images of Gd(III) chelate in the presence and absence of β-galactosidase and HSA, animal model with and without β-galactosidase expression will be performed.

EXPERIMENTAL PROCEDURES Materials. 1-(2-Difluoromethyl-4-aminophenyl)-2,3,4,6-tetraacetyl-β-D-galactopyranose (13) and 4,7,10-(tris-tbutylcarboxymethyl)-(1,4,7,10-tetraazacyclodecane) (DO3A-tris-tbutyl ester) (14, 15) were prepared by the previously published method. D(+)-Galactose was purchased from Acros. 2-Hydroxy5-nitrobenzaldehyde, tetra-N-butylammonium bromide (TBAB), and diethylaminosulfur trifluoride (DAST) were purchased from Lancaster. All other reagents used for the synthesis of the ligand were purchased from commercial sources, unless otherwise noted. 17O-enriched water (20.3%) was purchased from Isotec Inc. Human serum albumin (HSA, product no. A-1653, fraction V powder 96–99%) and β-galactosidase (product no. G5635–5KU, grade VIII from Escherichia coli) were purchased from Sigma

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Chang et al.

Scheme 2. Proposed Mechanism of HSA to Bind to [Gd(DOTA-FPG)(H2O)]

and used without any further purification. The molecular weights were assumed to be 66.5 kDa (HSA) and 465 kDa (βgalactosidase). β-Gal Staining Kit was purchased from Invitrogen. The 100 mM sodium phosphate buffer solution was used to maintain the pH of all solutions (pH ) 7.4) containing HSA. 1 H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Varian Gemini-400 spectrometer with 5 mm sample tubes. The 13C NMR spectra were referenced internally relative to 3-(trimethylsilyl)-1-propanesulfonic acid for D2O. The concentration of Gd(III) complex was determined by ICP-MS with a Perkin-Elmer OPTIMA 2000. Synthesis.1-(2-Formyl-4-nitrophenyl)-2,3,4,6-tetraacetyl-β-D-galactopyranose (3). A solution of 2-hydroxy-5-nitrobenzaldehyde (8.29 g, 49.61 mmol) in CH2Cl2 (100 mL) was stirred at room temperature with an aqueous solution of 1 N NaOH (50 mL) and tetra-N-butylammonium bromide (TBAB) (10.99 g, 33.1 mmol). A solution of 2,3,4,6-aceto-R-D-bromogalactopyranose (13.60 g, 33.07 mmol) was added to this stirred mixture at room temperature. The synthesis of 2,3,4,6-aceto-R-D-bromogalactopyranose was prepared by the previously published method. The mixture solution was stirred for 3 days. The mixture solution was evaporated and yielded a bright yellow oil. Then, the oil was dissolved in CH2Cl2 and washed with 2 N NaOH solution (4 × 250 mL) and water several times. After collecting the organic phase, the goal compound was obtained by evaporating the solvent. Purification by chromatography (silica, CH2Cl2) gave a yellow viscous solid. The yellow solid was crystallized in diethyl ether, and this led to the production of a white solid. Collected solid and evaporated to dryness, yield 11.98 g (72.82%). 1H NMR (400 MHz, CDCl3): δ 10.31 (s, 1H, -CHO),

8.66 (dd, 1H, ArH), 8.38 (dd, 1H, ArH), 7.27 (d, 1H, ArH), 5.60 (dd, 1H, sugar, -CH-), 5.50 (d, 1H, sugar, -CH-), 5.30 (d, 1H, sugar, -CH-), 5.18 (dd, 1H, sugar, -CH-), 4.20 (m, 3H, sugar, -CH- and -CH2OAc), 2.06 (m, 12H, -OAc). 13C NMR (100 MHz, CDCl3): δ 20.47, 20.56, 20.61, 61.26, 66.56, 68.06, 70.23, 71.76, 98.77, 115.76, 124.34, 125.75, 130.10, 143.28, 162.04, 169.29, 169.94, 170.03, 170.23, 187.02. ESI-MS: calcd m/z 497.4, found 498.8 [M + H]+. Anal. Calcd for C21H23NO13: C 50.71, H 4.66, N 2.82. Found: C 50.33, H 4.78, N 2.90. 1-(2-Difluoromethyl-4-nitrophenyl)-2,3,4,6-tetraacetyl-βD-galactopyranose (4). Diethylaminosulfur trifluoride (DAST) (3.81 mL) was added to a solution of 1-(2-formyl-4-nitrophenyl)2,3,4,6-tetraacetyl-β-D-galactopyranose (11.98 g, 24.08 mmol) in dry CH2Cl2 (300 mL). The reaction was stirred at ambient temperature under N2 for 6.5 h. The reaction mixture was quenched by the addition of ice and extracted twice with CH2Cl2 (100 mL). The combined organic layers were washed successively with water and brine and dried over MgSO4. The above solution was filtered and concentrated by rotary evaporation, which yielded a yellow crude compound. Purification by chromatography (silica, CH2Cl2) collected the first point and gave a yellow solid (11.82 g, 94.49%). 1H NMR (400 MHz, CDCl3): δ 8.47 (dd, 1H, ArH), 8.32 (dd, 1H, ArH), 7.23 (d, 1H, ArH), 6.70–6.98 (t, 1H, -CF2H), 5.56 (dd, 1H, sugar, -CH-), 5.50 (d, 1H, sugar, -CH-), 5.15(m, 2H, sugar, -CH-), 4.20 (m, 3H, sugar, -CH- and -CH2OAc), 2.06 (m, 12H, -OAc). 13C NMR (100 MHz, CDCl3): δ 20.49, 20.56, 20.61, 30.88, 61.31, 66.58, 67.74, 70.18, 71.72, 99.25, 107.54, 109.91, 112.28, 114.91, 122.73, 122.77, 122.81, 122.86, 127.68, 143.07, 158.65, 158.72, 169.50, 169.95, 170.03, 170.23. ESI-MS: calcd m/z 519.4, found

Bioactivated Paramagnetic Gadolinium(III) Complex

520.0 [M + H]+. Anal. Calcd for C21H23F2NO12: C 48.56, H 4.46, N 2.70. Found: C 48.36, H 4.55, N 2.71. 1-(2-Difluoromethyl-4-aminophenyl)-2,3,4,6-tetraacetyl-β-Dgalactopyranose (5). Pd/C (10% Pd, 0.22 g) was added to a stirring solution of 1-(2-difluoromethyl-4-nitro-phenyl)-2,3,4,6tetraacetyl-β-D-galactopyranose (11.23 g, 21.62 mmol) in ethyl acetate under 1.5 atm H2. This step was repeated until the pressure stopped decreasing. Purification by chromatography (silica, CH2Cl2) can produce a yellow solid (8.98 g, 84.86%) and concentrated the pure compound in a vacuum. 1H NMR (400 MHz, CDCl3): δ 6.66–6.97 (m, 4H, ArH and -CF2H), 5.45 (m, 2H, sugar, -CH2-), 5.09 (dd, 1H, sugar, -CH-), 4.87 (d, 1H, sugar, -CH-), 4.24 (dd, 1H, sugar, -CH-), 4.16 (dd, 1H, sugar, -CH-), 4.02 (t, 1H, sugar, -CH-), 2.06 (m, 12H, -OAc). 13C NMR (100 MHz, CDCl3): δ 20.54, 20.61, 20.63, 61.31, 66.82, 68.34, 70.69, 71.01, 101.04, 108.75, 111.10, 112.51, 112.54, 112.58, 112.62, 113.45, 117.81, 118.35, 141.99, 169.60, 170.09, 170.21, 170.34. ESI-MS: calcd m/z 489.4, found 490.8 [M + H]+. Anal. Calcd for C21H25F2NO10: C 51.54, H 5.15, N 2.86. Found: C 51.34, H 5.28, N 2.75. 1-(2-Difluoromethyl-4-(1-bromoacetamido)phenyl)-2,3,4,6tetraacetyl-β-D-galactopyranose (6). 1-(2-Difluoromethyl-4aminophenyl)-2,3,4,6-tetraacetyl-β-D-galactopyranose (8.94 g, 18.27 mmol) was dissolved in CH2Cl2 (60 mL) and treated with 40% dimethylamine (2.31 mL, 18.30 mmol). Bromoacetyl bromide (1.59 mL, 18.30 mmol) was dissolved in CH2Cl2 (40 mL) and dropped into the above solution in an ice bath overnight. Then, the reaction was treated with dilute HCl, dilute NaHCO3 solution, and water. The organic solution was evaporated and washed with diethyl ether to give a pure white solid (3.61 g, 32.38%). 1H NMR (400 MHz, CDCl3): δ 8.19 (s, 1H, ArH), 7.79 (d, 1H, ArH), 7.58 (s, 1H, -NH-), 7.13 (d, 1H, ArH), 6.98–6.70 (t, 1H, -CF2H), 5.50 (m, 2H, sugar, -CH2-), 5.12 (dd, 1H, sugar, -CH-), 5.01 (d, 1H, sugar, -CH-), 4.24 (dd, 1H, sugar, -CH-), 4.16 (dd, 1H, sugar, -CH-), 4.02 (t, 1H, sugar, -CH-), 4.03 (s, 2H, -CH2Br), 2.06 (m, 12H, -OAc). 13C NMR (100 MHz, CDCl3): δ 20.55, 20.64, 20.67, 29.24, 61.37, 66.73, 68.04, 70.48, 71.27, 100.13, 108.31, 110.67, 113.02, 116.17, 118.26, 118.30, 118.34, 124.00, 132.55, 132.54, 163.51, 169.61, 170.06, 170.17, 170.34. ESI- MS: calcd m/z 610.4, found 611.7 [M + H]+. Anal. Calcd for C23H26BrF2NO11: C 45.26, H 4.29, N 2.29. Found: C 45.02, H 4.44, N 2.36. 1-(2-Difluoromethyl-4-(1-(4,7,10-triscarboxymethyl-(1,4,7,10tetraazacyclodecyl))-acetamido)phenyl)-β- D -galactopyranose (DOTA-FPG, 8). DO3A-tris-tbutyl ester (1.05 g, 2.04 mmol), KI (0.0354 g, 0.21 mmol), and triethylamine (0.545 mL, 3.93 mmol) were dissolved in CH3CN (50 mL); 1-(2-difluoromethyl-4-(1-bromoacetamido)phenyl)-2,3,4,6-tetraacetyl-β-Dgalactopyranose (1.18 g, 1.93 mmol) was added to the solution, and the mixture was stirred for 2 h. The synthesis of DO3Atris-tbutyl ester was prepared by the previously published method. After 2 h, the mixture was warmed to 50 °C and stirred for 24 h. The solvent was removed by rotary evaporation. The acetyl group was removed by reaction with NaOCH3 and dry CH3OH (50 mL) for 2 h. Deprotection of tert-butyl ester group was achieved by stirring at room temperature in trifluoroacetic acid (TFA) (20 mL) for 12 h. After 12 h, the solution was quenched with water and adjusted to pH 11 with ammonium hydroxide. Next, this compound was purified by AG 1 × 8 anion exchange resin column (200–400 mesh, HCO2- form, eluted first with H2O (2 L) and then with a gradient of formic acid) and then collected and concentrated 0.02–0.03 N formic acid solution to give major product. The trace of formic acid was removed by coevaporation with water (200 mL) five times, yielding 0.65 g (45.02%) of DOTA-FPG as a yellow viscous oil. 1H NMR (400 MHz, D2O): δ 7.61 (s, 1H, ArH), 7.48 (d, 1H, ArH), 7.18 (d, 1H, ArH), 7.03 (t, 1H, -CF2H), 4.98 (d, 1H,

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sugar, -O-CH-O), 3.92 (d, 1H, sugar, -CH-), 3.61 (m, 9H, sugar and DOTA, -CH- and -CH2-), 3.35 (s, 8H, DOTA, -N-CH2CO-), 3.02 (m, 12H, DOTA, -CH2-). 13C NMR (100 MHz, D2O): δ 7.40, 22.43, 18.01, 48.16, 50.90, 51.32, 53.30, 55.25, 55.56, 56.40, 60.67, 61.31, 68.39, 70.37, 72.53, 75.46, 101.11, 109.43, 111.76, 114.10, 116.33, 117.48, 19.04, 120.53, 123.49, 123.71, 125.38, 125.81, 129.78, 130.94, 132.14, 151.37, 156.84, 169.85, 170.17, 174.33, 197.37. HRMS (FAB+): m/z 708.0 [M + H]+. Anal. Calcd for C29H43F2N5O13: C 49.22, H 6.12, N 9.90. Found: C 49.42, H 6.08, N 9.95. Preparation of Lanthanide Complexes. The Gd(III) and Eu(III) complexes were prepared by dissolving the DOTA-FPG (0.58 mmol) in H2O (10 mL) and adjusting the pH of the solution to 6.5 with dilute NaOH. LnCl3 (0.57 mmol, dissolved in 5 mL H2O and brought to pH ) 6.5 with NaOH) was added dropwise, maintaining pH at 5.5–6.0 with dilute NaOH periodically. The mixture solution was stirred at room temperature. After 2–3 days, the pH showed no change and the formation of Ln(III) chelate was considered complete. The pH was brought to 8.0, and the solution was centrifuged to remove excess lanthanide ions as Ln(OH)3 and verified by the xylenol orange test. The trace Ln(OH)3 was removed by filtration through a 200 nm nylon filter, and the solution was then evaporated under reduced pressure. The purity of [Gd(DOTA-FPG)(H2O)] and [Eu(DOTA-FPG)(H2O)] was determined by HPLC and identified by mass. The HPLC chromatograms of these complexes were deposited as Supporting Information (Figures 1S–2S). Reversed-Phase High-Performance Liquid Chromatography (HPLC) Method. The HPLC experiments were per¨ KTAbasic 10 instrument equipped formed on an Amersham A with an Amersham UV-900 detector and Amersham Frac-920 fraction collector. Supelco RP-C18 column (5 µm, 4.6 × 250 mm; and 5 µm, 10 × 250 mm) were used. 17 O NMR Measurements. For the variable-temperature 17O NMR measurements of [Gd(DOTA-FPG)(H2O)], [Gd(DOTAFP)(H2O)], and [Gd(DOTA-FP)(H2O)]-HSA, the components and pH values of the utilized solutions were as follows: 20 mM [Gd(DOTA-FPG)(H2O)] in 100 mM sodium phosphate buffer solution, 5.5% 17O isotope, pH ) 7.4; 20 mM [Gd(DOTAFPG)(H2O)] in 100 mM sodium phosphate buffer solution, 2 µM β-galactosidase, 5.5% 17O isotope, pH ) 7.4; 20 mM [Gd(DOTA-FPG)(H2O)] in 100 mM sodium phosphate buffer solution, 2 µM β-galactosidase, 4.5% (w/v) human serum albumin (HSA), 5.5% 17O isotope, pH ) 7.4. The measurement of the 17O longitudinal, transverse relaxation rates and chemical shift were carried out with a Varian Gemini-400 (9.4 T, 54.2 MHz) spectrometer equipped with a 10 mm probe using an external D2O lock. The Varian 600 temperature control unit was used to stabilize the temperature in the range 278–323 K. The samples were sealed in glass spheres adapted into 10 mm NMR tubes to eliminate susceptibility corrections to the chemical shift. Data Analysis. The simultaneous least-squares fitting of 17O NMR data was determined by fitting the experimental data using the program SCIENTIST for WINDOWS by Micromath, version 2.0. MR Experiments. Relaxation Time Measurement. The longitudinal relaxation times (T1) in different buffer solutions of Gd(III) complex were measured to determine relaxivity (r1). The measurements were made using a NMR relaxometer operating at 20 MHz and 37.0 ( 0.1 °C (NMR-120 Minispec, Bruker). Before each measurement, the relaxometer was tuned and calibrated. The values of r1 were determined from five data points generated by an inversion–recovery pulse sequence. To study the effect of β-galactosidase enzyme on the r1 value of [Gd(DOTA-FPG)(H2O)] solutions, β-galactosidase isolated from Escherichia coli was used. The Escherichia coli enzyme was reconstituted with 0.1 M sodium phosphate buffer, pH ) 7.4 at

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25.0 ( 0.1 °C, and proved its activity with p-nitrophenyl-β-Dgalactopyranoside (PNPG). For the longitudinal relaxation time (T1) measurements of [Gd(DOTA-FPG)(H2O)] in the presence and absence of β-galactosidase and HSA, the components of the utilized solutions were as follows: (a) 0.5 mM [Gd(DOTAFPG)(H2O)] in 100 mM sodium phosphate buffer solution; (b) 0.5 mM [Gd(DOTA-FPG)(H2O)] and 2 µM β-galactosidase in 100 mM sodium phosphate buffer solution; (c) 0.5 mM [Gd(DOTA-FPG)(H2O)] and 0.5 mM HSA in 100 mM sodium phosphate buffer solution; (d) 0.5 mM [Gd(DOTA-FPG)(H2O)], 0.5 mM HSA and 2 µM β-galactosidase in 100 mM sodium phosphate buffer solution. The percentage change of T1 value in these solutions was plotted against the incubation time. These measurements were made in triplicate to reduce systematic error in the relaxation time (T1) measurements. Cells and Animal Model. CT26 colon carcinoma cells were obtained from American type Culture Collection (Manassas, VA). Recombinant β-galactosidase retrovirus was packaged by cotransfection of pVSVG with pLNCX-LacZ into GP2-293 cells (Clontech, BD Biosciences, USA). After 48 h, the culture medium was filtered, mixed with 8 µg/mL polybrene, and added to CT26 colon carcinoma cells. The cells were selected in G418 and reclone to generate CT26/β-gal cells. Cells were grown in Dulbecco’s Minimal Essential Medium (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated bovine calf serum, 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5% CO2. 6–8 week old female Balb/c mice were purchased from National Laboratory Animal Center, Taipei, Taiwan. Animal experiments were performed in accordance with institutional guidelines. The CT26 and CT26/β-gal tumors were conducted with three Balb/c mice via subcutaneous injection of 100 µl (106 cells) phosphate buffer solution suspension into the right or left shoulder regions of mouse. The CT26 and CT26/β-gal tumors took one to three weeks to reach a predetermined size. The experiment was performed three weeks after implantation, at which time the tumors measured at least 0.5 cm in diameter. Functional β-galactosidase in Vitro. Functional expression of β-galactosidase in cells was measured by staining CT26 or CT26/β-gal cells with β-galactosidase activity by the β-Gal Staining Kit according to the manufacturer’s instructions. Cells were grown in 6 well plates and fixed with 3 mL fixation solution at room temperature for 10 min. The plate was washed twice with phosphate buffer solution, and 2 mL staining solution (1 mg/mL X-gal) was added at room temperature until the cell stained blue. Cells were examined on an upright microscope (Olympus BX41, Japan). Cytotoxicity of Gd(III) Chelate. CT-26 cells were seeded overnight in 96-well plates. Graded concentrations of [Gd(DOTA-FPG)(H2O)] were added into CT-26 cells in the presence and absence β-galactosidase (2 µg/well) in triplicate for 24 h at 37 °C. The cells were subsequently incubated for 48 h in fresh medium. Cell viability was determined by the 3Hthymidine incorporation assay. Cells were harvested with a filtermate apparatus and incorporated radioactivity was determined on a Top-Count scintillation counter. Results are expressed as percent inhibition of 3H-thymidine incorporation as compared to untreated cells by the following formula: % inhibition ) (cpmsample – cpmbackground)/(cpmcontrol – cpmbackground) × 100 Histological Analysis of Functional β-galactosidase in Vivo. CT26 and CT26/β-gal tumors were excised and embedded in Tissue-Tek OCT in liquid nitrogen and sectioned into 10 µm slices. The tumor sections were stained for β-galactosidase activity by the β-Gal Staining Kit as described above and counterstained with nuclear fast red. All sections were examined under phase contrast microscope.

Chang et al.

In Vitro MR Studies of [Gd(DOTA-FPG)(H2O)]. For the MR images of [Gd(DOTA-FPG)(H2O)] and its enzymatic cleavage, the components and pH values of the utilized solutions were as follows: (a) 0.8 mM [Gd(DOTA-FPG)(H2O)], 2 µM β-galactosidase, and 0.8 mM HSA in 100 mM sodium phosphate buffer solution pH ) 7.4; (b) 0.8 mM [Gd(DOTA-FPG)(H2O)] and 0.8 mM HSA in 100 mM sodium phosphate buffer solution pH ) 7.4; (c) 0.8 mM [Gd(DOTA-FPG)(H2O)] in 100 mM sodium phosphate buffer solution pH ) 7.4; (d) 0.8 mM [Gd(DOTA-FPG)(H2O)] and 2 µM β-galactosidase in 100 mM sodium phosphate buffer solution pH ) 7.4; (e) sodium phosphate buffer solution. The solutions were transferred to 1.5 mL test tubes for MR imaging. MR imaging was performed with a clinical 1.5T superconductive MR scanner (Gyroscan ACS-NT; Philips Medical Systems, Best, The Netherlands) and a knee coil. MR pulse sequence included T1-weighted (TR/TE 100/10 ms) two-dimensional spin–echo sequence. Mean signal intensities for the test tubes were measured in the central section of the imaging volume with use of operator-defined regions of interest with a minimum of 10 pixels per region. To compare differences in SI data among various test tubes, analysis of variance for repeated measurements was used. Mean data were compared with the Scheffe test. A P value of less than 0.05 was considered to indicate a statistically significant difference. In Vivo MR Studies of [Gd(DOTA-FPG)(H2O)] for Animal Model. The Balb/c mice (n ) 3) established CT26 and CT26/β-gal tumors in the left and right shoulder regions, respectively, were anesthetized with 40–50 mg/kg intraperitoneal sodium pentobarbital. The mouse was then placed in an animal coil. MR imaging was performed using a clinical 3.0 T superconductive MR scanner (Signa; GE Medical Systems, Milwaukee, WI). T1-weighted (TR/TE 100/13 ms) transaxial images were obtained before and after intravenous injection of 0.3 mmol/kg [Gd(DOTA-FPG)(H2O)]. The following parameters were used: two excitation, slice thickness ) 3 mm, matrix ) 256 × 224, field of view ) 8 cm, one signal acquisition, and imaging time ) 1.6 min. Postcontrast scans were obtained every 5 min for half an hour.

RESULTS Synthesis of the Ligand. The starting material 2,3,4,6-acetoR-D-bromogalactopyranose was synthesized by methods in the literature (4). The ligand DOTA-FPG was prepared as follows. First, 2-hydroxy-5-nitrobenzaldehyde was successfully alkylated on the hydroxyl group with 2,3,4,6-aceto-R-D-bromogalactopyranose. Second, the aldehyde group was fluorinated, and the nitro group was hydrogenated to the amino group. Then, the bromoacetyl group was used as a linker to bind DO3A-trist butyl ester and 2-difluoromethylphenyl-β-galactopyranose. Finally, the ligand DOTA-FPG underwent hydrolysis with NaOCH3 and TFA. The ligand DOTA-FPG was analyzed by HPLC and showed one peak, which was identified by ESI-MS to confirm its purity. The HPLC chromatogram of this ligand is deposited as Supporting Information (Figure 3S). Luminescence Method for Establishing Solution Hydration States. Luminescence lifetime data have been obtained for the complex [Eu(DOTA-FPG)(H2O)] to determine the number of inner-sphere water molecules in the aqueous solution (16–18). The luminescence lifetime (τ) has been determined in both H2O and D2O τH O ) 0.590 ms and τD O ) 1.90 ms). The 2 2 [Eu(DOTA-FPG)(H2O)] complexes containing 1.23 and 1.10 inner-sphere water (Table 1) were calculated by eqs 1 and 2 as follows q ) 1.05[τH-12O - τD-12O]

(1)

q ) 1.2 [( τH-12O - τD-12O) - 0.25]

(2)

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Bioconjugate Chem., Vol. 18, No. 6, 2007 1721

Table 1. Relaxivity r1 of [Gd(DOTA-FPG)(H2O)], [Gd(DOTA-FPG)(H2O)]/β-gal, [Gd(DOTA-FPG)(H2O)]/HSA, [Gd(DOTA-FPG)(H2O)]/β-gal + HSA, [Gd(HP-DO3A)(H2O)], [Gd(DOTA)(H2O)]-, and [Gd(DTPA)(H2O)]2- in 100 mM sodium phosphate buffer solution at 37.0 ( 0.1 °C and 20 MHz complex

relaxivity r1/mM-1 s-1

pH

[Gd(DOTA-FPG)(H2O)] [Gd(DOTA-FPG)(H2O)]/β-gal a [Gd(DOTA-FPG)(H2O)]/HSA b [Gd(DOTA-FPG)(H2O)]/β-gal + HSA [Gd(HP-DO3A)(H2O)]d [Gd(DOTA)(H2O)]-e [Gd(DTPA)(H2O)]2-f

c

7.4 7.4 7.4 7.4 7.5 7.3 7.6

( ( ( ( ( ( (

0.1 0.1 0.1 0.1 0.1 0.1 0.1

3.96 3.77 7.58 15.5 3.65 3.56 3.89

( ( ( (

0.04 0.09 0.06 0.2

( 0.03

a

[Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase (2 µM). [Gd(DOTA-FPG)(H2O)] in the presence of human serum albumin (HSA) (0.5 mM). c [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase (2 µM) and human serum albumin (HSA) (0.5 mM). d Data obtained from ref (23). e ref (22). f ref (24). b

where q is the number of water molecules bound to metal ions, τH2O is the luminescence half-life in H2O solution and τD O is 2 the luminescence half-life in D2O solution. The q value is obtained from the luminescence study. Relaxometric Studies of the Gd(III) Complex. The longitudinal water proton relaxivity (r1p) results from the contribution arising from water molecules in the inner and outer coordination spheres as in eq 3 r1p ) Ris1p + Ros 1p

(3)

Ros 1p

The outer sphere term, , describes the contribution arising from the water molecules diffusing near the paramagnetic chelate (19). For compounds of similar size, it can be assumed that the outer-sphere mechanism makes a similar contribution at high magnetic flux densities (>0.1 T). Therefore, the small differences in relaxivity can be ascribed to differences in Ris1p . The contribution arising from the exchange of the water molecules directly coordinated to the paramagnetic metal ion is obtained by eq 4 as follows (20, 21) Ris1p ) [C]q/[55.6(T1M + τM)]

(4)

where [C] represents the molar concentration of the gadolinium(III) complex, τ1M is the longitudinal relaxation time of the inner-sphere water protons and τM is the residence lifetime of the bound water. The longitudinal relaxivity (r1) value of [Gd(DOTA-FPG)(H2O)] in the presence and absence of β-galactosidase and HSA compared with [Gd(DOTA)(H2O)]- (22), [Gd(HP-DO3A)(H2O)] (23) (H3HP-DO3A ) 1,4,7,10-tetraazacyclododecane-1-(2-hydroxylpropyl)-4,7,10-triacetic acid), and [Gd(DTPA)(H2O)]2- (24) (the structural formula shown in Chart 1) are shown in Table 1. The relaxivity (r1) of [Gd(DOTAFPG)(H2O)] is slightly higher than those of [Gd(DOTA) (H2O)]-, [Gd(HP-DO3A)(H2O)], and [Gd(DTPA)(H2O)]2-. It is quite clear that introducing 2-difluoromethylphenyl-β-galactopyranoside into DOTA increases the relaxivity value. The relaxivity of [Gd(DOTA-FPG)(H2O)]/β-gal is slightly lower than that of [Gd(DOTA-FPG)(H2O)] because of the β-galactopyranoside residue removed from [Gd(DOTA-FPG)(H2O)] by β-galactosidase. The relaxivity of [Gd(DOTA-FP)(H2O)]/β-gal + HSA (15.5 ( 0.2 mM-1 s-1) is about 4-fold higher than that of [Gd(DOTA-FPG)(H2O)]. This is due to cleavage of [Gd(DOTAFPG)(H2O)] by β-galactosidase and subsequent conjugation to proteins to form the macromolecules [Gd(DOTA-FP)(H2O)]β-gal and [Gd(DOTA-FP)(H2O)]-HSA. The presence of proteins, small molecules, or ions in Gd(III) chelate solution can affect the relaxivity of the Gd(III) complex. Relaxivity (r1) measurements obtained in solutions of varying species not only describe how the Gd(III) complex responds to

that composition but also provide coordinate information occurring at the Gd(III) center. The native lysosomal environment of β-galactosidase is acidic, with maximum activity observed between pH 4 and 5. The relaxivity values of [Gd(DOTA-FPG)(H2O)] in two different buffer solutions were thus measured at pH ) 5.0. The [Gd(DOTA-FPG)(H2O)] chelate under 10 mM pyridine and 100 mM sodium chloride buffer solution at pH ) 5.0 has a relaxivity (r1) of 3.88 ( 0.02 mM-1 s-1. However, for this chelate in 100 mM acetate buffer solution at pH ) 5.0, the r1 value is 3.63 ( 0.01. The slight difference between these buffer solutions may be attributed to the coordinating ability of the buffer constituents. The coordinating ability of pyridine is weaker than that of acetate buffer (7). The r1 values at pH ) 5.0 in different buffer solutions are only slightly lower than that of sodium phosphate buffer solution at pH ) 7.4, which indicates that [Gd(DOTA-FPG)(H2O)] is still stable in a native lysosomal environment. In this study, the [Gd(DOTA-FPG)(H2O)] under the sodium phosphate buffer solution in the absence or presence of β-galactosidase and HSA, the longitudinal relaxation time (T1) change percentage was investigated under different incubation times as shown in Figure 1. The relaxation time (T1) of [Gd(DOTA-FPG)(H2O)] under the sodium phosphate buffer solution maintains constant T1 values over 45 min. This result implicates that the [Gd(DOTA-FPG)(H2O)] chelate is cleaved by β-galactosidase to form macromolecules [Gd(DOTAFP)(H2O)]-β-gal and [Gd(DOTA-FP)(H2O)]-HSA in the presence of β-galactosidase and HSA. The bound relaxivity (r1b) (25.0 mM-1 s-1) for the noncovalent [Gd(DOTA-FPG)(H2O)]/ HSA adduct was also obtained by the proton relaxation enhancement method (25, 26) and ultrafiltration studies. (27–29) The studies of [Gd(DOTA-FPG)(H2O)] noncovalently bound to HSA were deposited as Supporting Information (Figures S2–S4). Proof of the Proposed Mechanism by LC/MS. In order to mimic HSA or enzyme proteins to bind to the Gd(III) chelate, L-lysine was used to prove this proposed mechanism. The HPLC chromatograms of (a) 1.0 mM [Gd(DOTA-FPG)(H2O)] alone, (b) 1.0 mM [Gd(DOTA-FPG)(H2O)] in the presence of 2 µM β-galactosidase, and (c) 1.0 mM [Gd(DOTAFPG)(H2O)] in the presence of 2 µM β-galactosidase and excess L-lysine in pH 7.4 Tris buffer were shown in Figure 2a–c, respectively. From Figure 2a, [Gd(DOTA-FPG)(H2O)] was detected at 19.9 min and confirmed by LC-MS (ESI+): calcd m/z 861.9, found m/z 862.6 [M + H]+. A peak of [Gd(DOTA-FP)(H2O)] at 24.6 min as shown in Figure 2b was obtained when [Gd(DOTA-FPG)(H2O)] was incubated with β-galactosidase and was proven by LC-MS (ESI+): calcd m/z 679.75, found m/z 680.22 [M + H]+. From Figure 2c, a peak of [Gd(DOTA-FP)(H2O)]-L-lysine was detected at 30.2 min and confirmed by LC-MS (ESI+): calcd m/z 825.94, found m/z 827.28 [M + H]+. These results indicate that [Gd(DOTA-FPG)(H2O)] can bind generally to protein in the presence of β-galactosidase and HSA. Water-Exchange Rate and Rotational Correlation Time Studies of Gd(III) Complexes. The experimental 17O NMR data for [Gd(DOTA-FPG)(H2O)] complex in the presence and absence of β-galactosidase and HSA is deposited as Supporting Information (Tables 1S–3S); that is, the 17O NMR chemical shifts (∆ω, longitudinal (1/T1r) and transverse (1/T2r) relaxation rates were analyzed simultaneously (eqs 1S–9S used in Supporting Information S1). The results are plotted in Figure 3 with the corresponding curve representing the results of the best fitting of the data according to the equations. As illustrated by Table 2, there are a large number of parameters influencing the data obtained by the different techniques. The 17O NMR technique has the advantage that the outer-sphere contributions

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Chart 1

to the relaxation rates are negligibly small, which is a consequence of the oxygen nucleus being closer to the paramagnetic center when bound to the inner-sphere. Cytotoxicity of Gd(III) Chelate. The cytotoxicity of [Gd(DOTA-FPG)(H2O)] was also investigated by incubating various concentrations of [Gd(DOTA-FPG)(H2O)] with CT26 cells. After 72 h, cell viability was examined by the 3Hthymidine incorporation into cellular DNA. Figure 4 shown that the IC50 value of [Gd(DOTA-FPG)(H2O)] to CT26 cells was larger than 100 µM, demonstrating that [Gd(DOTAFPG)(H2O)] displayed low-cytotoxicity to CT26 cells after β-galactosidase activation. MR Imaging Studies of Enzymatic Activation of Gd(III) Chelate. The percentage change in r1 generated by enzymatic conversion of [Gd(DOTA-FPG)(H2O)] can be visualized by

MR images. The MR images of [Gd(DOTA-FPG)(H2O)] solution placed in 1.5 mL (11 mm × 39 mm) microtubes in the presence and absence of β-galactosidase and HSA were shown in Figure 5 (p < 0.05). The signal intensity values of the MR images for these Gd(III) complex solutions were also given in Figure 5, indicating that a high signal intensity of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA was obtained. Characterization of Functional β-Galactosidase in Vitro and in Vivo. To verify the enzymatic activity of the β-galactosidase in cells, CT26 and CT26/β-gal cells were stained with X-gal and the cells examined under a microscope as shown in Figure 6. The results indicated that CT26/β-gal cells, but not the parental CT26 cells, converted X-gal to a blue color, demonstrating that β-galactosidase on the CT26/

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Bioconjugate Chem., Vol. 18, No. 6, 2007 1723

intensity change of the tumor were shown in Figure 8, indicating that [Gd(DOTA-FPG)(H2O)] can act as a MRI contrast agent for noninvasive imaging of gene expression in vivo.

DISCUSSION

Figure 1. Change in T1 (%) of enzyme-catalyzed hydrolysis of [Gd(DOTA-FPG)(H2O)] measured at 20 MHz 37.0 ( 0.1 °C. (0) 0.5 mM [Gd(DOTA-FPG)(H2O)] in 100 mM sodium phosphate buffer solution; (2) 0.5 mM [Gd(DOTA-FPG)(H2O)] and 2 µM β-galactosidase in 100 mM sodium phosphate buffer solution; (0) 0.5 mM [Gd(DOTA-FPG)(H2O)] and 0.5 mM HSA in 100 mM sodium phosphate buffer solution; (0) 0.5 mM [Gd(DOTA-FPG)(H2O)], 2 µM β-galactosidase, and 0.5 mM HSA in 100 mM sodium phosphate buffer solution.

Figure 2. The HPLC chromatograms of [Gd(DOTA-FPG)] incubated with 2 µM β-galactosidase and bound to L-lysine. (a) 1.0 mM [Gd(DOTAFPG)(H2O)] alone, (b) 1.0 mM [Gd(DOTA-FPG)(H2O)] in the presence of 2 µM β-galactosidase, and (c) 1.0 mM [Gd(DOTA-FPG)(H2O)] in the presence of 2 µM β-galactosidase and excess L-lysine in pH 7.4 Tris buffer and 298 K. HPLC conditions: gradient of 100% to 0% water in CH3OH as organic phase; flow rate 0.5 mL/min; wavelength set at 254 nm; reversephase C18 column used.

β-gal cells was functionally active. To investigate whether the β-galactosidase activity was still functionally active in vivo, the tumor sections were also stained with X-gal and the sections examined under a microscope as shown in Figure 7. Histological staining for β-galactosidase activity revealed a strong blue color in CT26/β-gal tumors but not in parental CT26 tumors. This result shows that β-galactosidase on the CT26/β-gal tumors was functionally active in vivo. MR Images Studies in Vivo. To investigate whether sites of β-galactosidase expression could be noninvasively imaged, Balb/c mice bearing established CT26 and CT26/β-gal tumors in their left and right shoulder regions, respectively, were intravenously injected with 0.3 mmol/kg [Gd(DOTAFPG)(H2O)]. The MR images of the animal and time-signal

As shown in Figure 1, the [Gd(DOTA-FPG)(H2O)] complex is cleaved to form a smaller molecular weight [Gd(DOTA-FP) (H2O)] under the sodium phosphate buffer solution at pH ) 7.4 in the presence of β-galactosidase, and then conjugated to the β-galactosidase to form the macromolecule [Gd(DOTAFP)(H2O)]-β-galactosidase. However, there is only a small amount of cleaved [Gd(DOTA-FPG)(H2O)] conjugated to β-galactosidase due to the concentration of β-galactosidase (2 µM) being significantly lower than that of [Gd(DOTAFPG)(H2O)] (0.5 mM). Thus, the percentage change in longitudinal relaxation time (T1) increases about 15%. Furthermore, for the [Gd(DOTA-FPG)(H2O)] in human serum albumin under sodium phosphate buffer solution at pH ) 7.4 in the presence of β-galactosidase, the percentage change in longitudinal relaxation time (T1) significantly decreased by about 60%. The reason is that [Gd(DOTA-FPG)(H2O)] is cleaved to form a small molecule [Gd(DOTA-FP)(H2O)], and then conjugated to β-galactosidase and human serum albumin to form the macromolecules [Gd(DOTA-FP)(H2O)]-HSA and [Gd(DOTA-FP)(H2O)]β-galactosidase. Therefore, the relaxation time (T1) is dramatically lower than those of the above two conditions. In comparison with the bioactivated contrast agent 4,7,10-tri(acetic acid)-1(2-β-galactopyranosylethoxy)-1,4,7,10-tetraazacyclododecane gadolinium (Egad) (4) and gadolinium(III)-1-(4-(2-(1-(4,7,10triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))ethylcarbamoyloxymethyl)-2-nitrophenyl)-β-D-glucopyronuronate (7), the T1 change percentages were 20% and 15%, respectively. Therefore, the T1 change percentage of [Gd(DOTA-FPG)(H2O)] is significantly higher than those of the above two bioactivated contrast agents. In addition, for the [Gd(DOTA-FPG)(H2O)] and human serum albumin in sodium phosphate buffer solution at pH ) 7.4 in the absence of β-galactosidase as a control experiment, longitudinal relaxation time is slightly decreased by about 25%, which is a result of the liphophilic [Gd(DOTAFPG)(H2O)] noncovalently bound to human serum albumin. The lower binding constant (KA) (9.0 ( 0.1 × 102 M–1 and bound fraction (33.8%) of [Gd(DOTA-FPG)(H2O)] to HSA were obtained by E and M titrations (25, 26) and used to explain the result of the percentage change in relaxation time (T1) of [Gd(DOTA-FPG)(H2O)] chelate in HSA without β-galactosidase decreasing by only 25%. As shown in Table 2, the scalar coupling constant values of [Gd(DOTA-FPG)(H2O)] only and [Gd(DOTA-FPG)(H2O)] in the presence and absence of β-galactosidase and HSA, A/p , are very similar to those obtained for other Gd(III) complexes (-3.4 × 106 and -3.8 × 106 rad s-1 for [Gd(DOTA)(H2O)](30) and [Gd(DO3A-bz-NO2)(H2O)] (H3DO3A-bz-NO2 ) 1-(4nitrobenzyl)methylcarbamoyl-4,7,10-tri(acetic acid)tetraazacyclododecane) (31), respectively) with one inner-sphere water molecule. Over the whole temperature range, the transverse 17O relaxation rates (1/T2r) increase with increasing temperature, indicating that this system is in the slow exchange regime. However, there is degradation of the protein at temperatures higher than 50 °C for the [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA in 17O NMR experiments. Therefore, the temperatures of these 17O NMR experiments are in the range 278–323 K. 1/T2r is determined by the relaxation rate of the coordinated water molecule (1/T2m), which is influenced by the water residence lifetime (τM ) 1/kex), the longitudinal electronic relaxation rate (1/T1e), and the scalar 6 -1 coupling constant (A/p ). The k298 ex (2.54 × 10 s ) value of

1724 Bioconjugate Chem., Vol. 18, No. 6, 2007

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Figure 3. Temperature dependence of (A) transverse and (B) longitudinal 17O relaxation rates; (C) 17O chemical shifts at B ) 9.4 T for 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate, 5.5% 17O isotope (left); 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate, 2 µM β-galactosidase, 5.5% 17O isotope (middle); 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate, 2 µM β-galactosidase, 4.5% (w/v) HSA, 5.5% 17O isotope (right). The lines represent simultaneous least-squares fits to all data points displayed. Table 2. Parameters Obtained from Simultaneous Fitting of parameter 298 kex (106 s-1) ∆H‡ (kJ mol-1) ∆S‡ (J mol-1k-1) A/p (106 rad s-1) τR298 (ps) Cos ER (kJ mol-1) method

17

O NMR Relaxation Time and Chemical Shift Dataa

[Gd(DOTA-FPG) [Gd(DOTA-FP) [Gd(DOTA-FP) [Gd(DOTA) [Gd(TRITA-bz-NO2) [Gd(DO3A-bz-NO2) [Gd(DTPA-N-MA) (H2O)]- f (H2O)]g (H2O)]b (H2O)]c (H2O)]-HSAd (H2O)]- e (H2O)]- h 2.54 ( 0.1 53.4 ( 0.7 57.0 ( 1.4 -3.8 ( 0.1 228 ( 13 0 24.2 ( 3.8 17 O

1.32 ( 0.8 41.8 ( 1.5 12.6 ( 5.6 -3.8 ( 0.1 188 ( 6 0.23 15.1 ( 5.8 17 O

1.64 ( 0.4 31.4 ( 4.0 -20.6 ( 6.9 -3.8 ( 0.1 1068 ( 39 0.23 26.5 ( 1.2 17 O

4.8 ( 0.4 48.8 ( 1.6 46.6 ( 6.0 -3.4 ( 0.3 90 ( 15 0.25 ( 0.08 17 ( 3 17 O

120 ( 20 35.5 ( 1.9 21 ( 5 -3.7 ( 0.1 225 ( 7 23.3 ( 1 NMRD, 17O

1.6 ( 0.1 40.9 ( 2.5 11.1 ( 8 -3.8 ( 0.2 210 ( 10 0.06 ( 0.04 17.7 ( 0.1 17 O

1.3 ( 0.1 48.6 ( 1.1 35.7 ( 4 -4.2 ( 0.2 143 ( 4 0.16 ( 0.03 19.8 ( 0.6 17 O, EPR

a The bold values were fixed in the fitting procedure. b 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate buffer solution, 5.5% 17O isotope. c 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate buffer solution, 2 µM β-galactosidase, 5.5% 17O isotope. d 20 mM [Gd(DOTA-FPG)(H2O)] under 100 mM sodium phosphate buffer solution, 2 µM β-galactosidase, 4.5% (w/v) HSA, 5.5% 17O isotope. e Data obtained from ref (30). f ref (33). g ref (31). h ref. (32).

Figure 4. Cytotoxicity of [Gd(DOTA-FPG)(H2O)] to CT26 cells. CT26 (O) or CT-26 in the presence of β-galactosidase (•) was incubated with graded concentrations of [Gd(DOTA-FPG)(H2O)]. Cellular DNA synthesis was measured 72 h later. Results represent the mean of results from three separate wells. Error bars show the standard error of the mean.

[Gd(DOTA-FPG)(H2O)] is similar to those of [Gd(DOTAFPG)(H2O)] in the presence of β-galactosidase ([Gd(DOTAFP)(H2O)]) (1.32 × 106 s-1) and [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA ([Gd(DOTA-FP)(H2O)]HSA) (1.64 × 106 s-1) These k298 ex values are also similar to

those of the Gd(III) complexes with DO3A- and DTPAmonoamide derivatives ([Gd(DO3A-bz-NO2)(H2O)] (1.6 × 106 s-1) (31) and [Gd(DTPA-N′-MA)(H2O)]- (H4DTPA-N′-MA ) N-(methylcarbamoylmethyl)diethylenetriamine-N,N′,N-tetraacetic acid) (1.9 × 106 s-1) (32). As has been observed above, the water exchange rate decreases from [Gd(TRITA-bz-NO2)(H2O)]- (H4TRITA-bzNO2 ) 12-(p-nitrobenzyl)-1,4,7,10-tetraazacyclo-tridecane1,4,7,10-tetraacetic acid) (120 × 106 s-1) (33) to the [Gd(DOTAFPG)(H2O)] (2.54 × 106 s-1) with one inner-sphere water molecule. This rate of decrease is accompanied by an increase in ∆Hq. Since the result for the Gd(III) complex with DOTAFPG obeyed this trend, we conclude that water exchange on the [Gd(DOTA-FPG)(H2O)] complex takes place most probably via a limiting dissociative D mechanism (34). At 298 K, the τ298 R value of [Gd(DOTA-FPG)(H2O)] (228 ps) shown in Table 2 is significantly higher than that of [Gd(DOTA)(H2O)]- (90 ps) (30) but is similar to [Gd(DO3Abz-NO2)(H2O)] (210 ps) (31). It indicates that the introduction of the 2-difluoromethylphenyl-β-galactopyranoside in DOTA increases the τ298 R value and causes the relatively high relaxivity of the [Gd(DOTA-FPG)(H2O)] complex, compared to the previous results for [Gd(DOTA)(H2O)]- and [Gd(HP-DO3A) (H2O)]. A similar relationship between the τ298 R value and the relaxivity (r1) was observed in Gd(III) complexes with TTDA

Bioactivated Paramagnetic Gadolinium(III) Complex

Figure 5. Representative T1-weighted (TR/TE 100/10 ms) MR images of solutions and the signal intensity in test tubes at 1.5 T MR scanner: (a) sodium phosphate buffer solution with [Gd(DOTA-FPG)(H2O)], β-galactosidase and HSA; (b) sodium phosphate buffer solution with [Gd(DOTA-FPG)(H2O)] and HSA; (c) sodium phosphate buffer solution with [Gd(DOTA-FPG)(H2O)]; (d) sodium phosphate buffer solution with [Gd(DOTA-FPG)(H2O)] and β-galactosidase; (e) sodium phosphate buffer solution.

Figure 6. Cell display of functional β-galactosidase. CT26 cells (left) and CT26/β-gal cells (right) were stained with β-galactosidase activity by the β-Gal Staining Kit (1 mg/mL X-gal) before examination under an upright microscope.

Figure 7. Histological analysis of functional β-galactosidase in vivo. Sections of CT26 (left) and CT26/β-gal (right) tumor were stained with β-galactosidase activity by the β-Gal Staining Kit and viewed under phase contrast microscope.

(H5TTDA ) 3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid) (11) and TTDA-BOM (H5TTDA-BOM ) 6-carboxymethyl-benzyloxymethyl-3,10-di(carboxymethyl)-3,6,10triazadodecanedioic acid) (12). The attainment of high relaxivities can be expected when the molecular reorientational correlation time of the complexes is lengthened to the nanosecond range

Bioconjugate Chem., Vol. 18, No. 6, 2007 1725

Figure 8. Representative T1-weighted (TR/TE 100/13 ms) MR images of animal model at 3.0 T MR scanner: (A) precontrast images; (B) at 10 min after intravenous injection of 0.3 mmol/kg [Gd(DOTAFPG)(H2O)] intensity enhancement of CT26/β-gal tumor; (C) timesignal intensity change of the CT26 and CT26/β-gal tumors after injection of 0.3 mmol/kg [Gd(DOTA-FPG)(H2O)].

(25). At 298 K, the τ298 R value of [Gd(DOTA-FPG)(H2O)] is higher than that of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase, but is significantly lower than that of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA. These results can be employed to explain the percentage change in T1 values of Gd(III) chelate increasing by 15% in the presence of β-galactosidase and decreasing by 60% in the presence of β-galactosidase and HSA. The signal intensity of [Gd(DOTA-FPG)(H2O)] only in 100 mM sodium phosphate buffer solution (1490 ( 160) is higher than that of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase enzyme (1200 ( 120) (Figure 5) attributed to the molecular reorientational correlation time (τ298 ) decreasing R after the galactopyranoside residue was removed as shown in Table 2. The nucleophile of β-galactosidase attacks the Gd(III) chelate after the galactopyranoside residue is removed; however, the concentration of β-galactosidase is significantly lower than that of the Gd(III) complex. In addition, the signal intensity of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA (2670 ( 210) is significantly higher than that of [Gd(DOTA-FPG)(H2O)] in the sodium phosphate buffer solution. The nucleophile of the higher molecular weight HSA (66.5 kDa) mainly attacks the Gd(III) chelate and the high relaxivity of the Gd(III) chelate conjugate is obtained. The T1-mediated enhancement of [Gd(DOTA-FPG)(H2O)] was altered by the action of β-galactosidase yielding the expected high signal intensity for MR imaging. These results are in agreement with the previous percentage change in relaxation time (T1) decreasing by 60%. The [Gd(DOTA-FPG)(H2O)] complex under the sodium phosphate buffer solution in the presence of HSA was conducted as a control experiment. The signal intensity of its MR imaging is slightly higher than that of [Gd(DOTAFPG)(H2O)] in sodium phosphate buffer solution but is significantly lower than that of [Gd(DOTA-FPG)(H2O)] in the presence of HSA and β-galactosidase. Therefore, the expected high signal intensity of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA obtained is attributed to HSA and β-galactosidase conjugated to Gd(III) chelate.

1726 Bioconjugate Chem., Vol. 18, No. 6, 2007

The signal intensity of the CT26/β-gal tumor (3210 ( 190) is significantly higher than CT26 tumor (1830 ( 80) (p < 0.05) at 10 min (Figure 8). This intense enhancement clearly indicates that [Gd(DOTA-FPG)(H2O)] can be activated by CT26/β-gal tumors. These results are consistent with the previous percentage change in T1 significantly decreasing in the presence of β-galactosidase and HSA. The main reason is attributed to its higher rotational correlation time (τ298 ) of [Gd(DOTAR FP)(H2O)]-HSA after the galactopyranoside residue is removed and conjugated to protein. Therefore, [Gd(DOTA-FPG)(H2O)] may be a potential MRI contrast agent for imaging gene expression. In conclusion, a novel bioactivated magnetic resonance imaging contrast agent containing galactopyranoside residue, [Gd(DOTA-FPG)(H2O)], was designed and synthesized. The percentage change in longitudinal relaxation time (T1) decreased to 60% of enzymatic cleavage of [Gd(DOTA-FPG)(H2O)] in the presence of β-galactosidase and HSA. This indicates that the HSA and β-galactosidase conjugate to the Gd(III) chelate after the galactopyranoside residue is removed. A significant signal intensity enhanced by MR images was also observed for [Gd(DOTA-FPG)(H2O)] solution in the presence of β-galactosidase and HSA. Moreover, the signal intensities of tumors with β-galactosidase gene expression are significantly higher than those of tumors without β-galactosidase gene expression in vivo. Therefore, [Gd(DOTA-FPG)(H2O)] possesses enzymatic cleavage and a longer rotational correlation time, as well as higher percentage change in relaxation time (T1) and higher signal intensity of the MR image in the presence of β-galactosidase and protein. This might result in a new type of contrast agent to trace gene expression by MRI.

ACKNOWLEDGMENT We are grateful to the National Science Council of the Republic of China for financial support under Contract Nos. NSC 95-2627-M-037-001 and NSC 95-2623-7-037-001-NU. This research was also supported in part by grants from National Health Research Institutes under Contract No. NHRI-EX-959424EI. Supporting Information Available: Additional experimental data and spectra as outlined in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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