Complexes for Sensing Glucose by MRI - American Chemical Society

and The Rogers Magnetic Resonance Center, Department of Radiology, University of Texas Southwestern. Medical Center, 5323 Harry Hines Boulevard, Dalla...
0 downloads 0 Views 299KB Size
Download PDF
Bioconjugate Chem. 2004, 15, 1431−1440

1431

Cyclen-Based Phenylboronate Ligands and Their Eu3+ Complexes for Sensing Glucose by MRI Robert Trokowski,† Shanrong Zhang,§ and A. Dean Sherry†,§,* Department of Chemistry, The University of Texas at Dallas, P.O. Box 830688, Richardson, Texas 75083-0688, and The Rogers Magnetic Resonance Center, Department of Radiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9085. Received April 25, 2004; Revised Manuscript Received August 4, 2004

Novel cyclen-based phenylboronate ligands and their corresponding Eu3+ complexes have been examined as glucose sensors using chemical exchange saturation transfer (CEST) MR imaging for detection. Two isomeric bis-phenylboronate complexes, Eu(4) and Eu(10), and a mono-phenylboronate complex, Eu(12), had been prepared and characterized by UV and circular dichroism spectroscopy, mass spectrometry, and CEST imaging. Both the free ligands and their Eu3+ complexes bind to simple sugars, but their selectivity and binding affinities vary with sugar structure. Interestingly, the free ligands, 4 and 10, are selective for fructose over glucose, but this selectivity order switches in the respective Eu3+ complexes. Of the complexes examined, Eu(4) shows the highest selectivity and binding affinity for glucose (2275 ( 266 M-1 at pH 10.2 and 339 ( 29 M-1 at pH 7). Glucose acts as a “capping” moiety in the Eu(4)‚glucose binary complex and modulates water exchange between a single Eu3+bound water molecule and bulk water, an effect that can be detected by CEST imaging. Thus, Eu(4) represents a new class of metabolite-specific imaging agents that may allow mapping of metabolites by MRI of the bulk water signal.

INTRODUCTION

Scheme 1

Sugars play important roles in energy production and storage in living organisms so methods to access their concentration and distribution in vivo are becoming increasingly important. A variety of techniques have been reported for quantification of glucose, the most abundant sugar in mammalian cells, but few provide information about its distribution in tissues (1). Among the detection methods, the most common are based on the enzymatic detection by glucose oxidase (2) or chemical sensors based on complexation of cis-hydroxy groups of sugars by phenylboronic acids. Such binding interactions have been detected by using a variety of spectroscopic techniques, including UV, fluorescence, or circular dichroism (CD) or electrochemical methods (3-7). Unfortunately, most of these methods cannot be applied to detect sugars in vivo (8). Magnetic resonance imaging (MRI) or spectroscopy (MRS) techniques have been used to obtain maps of common metabolites such as lactate, creatine, and N-acetylaspartate by 1H detection and high energy metabolites by 31P detection (9). Free glucose has been measured in brain by 1H NMR (10, 11) and by 13C NMR (12) although 1H detection is complicated by the numerous overlapping resonances in the 2-4 ppm range from other endogenous molecules. Recently, a 13C-edited 1H T-SEDOR technique had been used to image glucose in brain of living mice (13) but this requires double resonance techniques not commonly available on clinical MR scanners and the infusion of a high cost tracer (13Cenriched glucose). Thus, a simple method for mapping this common metabolite in vivo, especially from tissues deep within the body, remains an important goal. * To whom correspondence should be addressed: Phone: (972) 883 2907. E-mail: [email protected]. † The University of Texas at Dallas. § University of Texas Southwestern Medical Center.

It is known that simple phenylboronic acids form reversible, cyclic ester bonds with cis-diols in a variety of sugars (14) and that the binding constants for these 1:1 complexes is highly dependent upon the orientation of the cis-diol groups in the sugar, typically decreasing in the order of D-fructose . D-galactose > D-mannose > D-glucose (15, 16). Sensors having two phenylboronic acid functionalities in juxtaposition form intramolecular 1:1 complexes with both “ends” of a sugar molecule (Scheme 1a) and often show a different stability order related to the specific spatial position of two boronic acid groups. In such complexes, a sugar can be selectively captured by appropriate positioning two phenylboronic acids in the same sensor molecule. The goal of this work was to create a sensor for glucose that might be used to image this important metabolite in tissues by magnetic resonance imaging (MRI). On the basis of prior experience with paramagnetic chemical exchange saturation transfer (PARACEST) agents (17), we anticipated that lanthanide complexes derived from tetraaza-tetraamide ligands with two appended phenylboronic acid groups might capture a glucose by forming a “bridge” above a single Ln3+-bound water molecule and, in doing so, the encapsulated glucose might slow the rate at which the Ln3+-bound water molecule exchanges with bulk water. In prototype experiments reported elsewhere (18), we demonstrated that upon binding of glucose to Eu(4) (see structure of ligand

10.1021/bc0498976 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/12/2004

1432 Bioconjugate Chem., Vol. 15, No. 6, 2004

Trokowski et al.

4 in Scheme 2), water exchange is indeed about 2-fold slower and this was sufficient to image glucose in vitro by MRI. Here, we describe details of the synthesis and characterization of two isomeric bis-phenylboronate sensors, Eu(4) and Eu(10), and a mono-phenylboronate sensor, Eu(12). The binding constants between various sugars and these sensors were determined by UV and CD spectroscopy, and their potential as PARACEST imaging agents were compared. EXPERIMENTAL PROCEDURES

Instrumental Methods. Column chromatography was performed using Mallinckrodt Silicar silica gel-150 (60-200 mesh). Hydrogenations were performed on a Parr hydrogenation apparatus. ECI and MALDI TOF mass spectral analyses were obtained from HT Laboratories in San Diego. FAB mass spectral analyses were obtained from University of Kansas Mass Spectrometry Lab. CD spectra were recorded on AVIV Circular Dichroism Spectrometer Model 202. NMR spectra were recorded on JEOL Eclipse 270 MHz. UV-vis spectra were recorded on Hewlett-Packard 8453 UV-vis spectrophotometer. MRI images were recorded on a 4.7T Varian imager. A 2.5 cm surface coil was used to excite and to receive the magnetic resonance signal. A spin-echo pulse sequence was modified by adding a frequency-selective a presaturation pulse before the imaging pulses. Typically, a presaturation period of 1 s was used. The saturation power was set to 36 db (corresponding to B1 field of 1020 Hz). The saturation frequency was set to 50 ppm (on the Eu3+-bound water resonance), 30 ppm (between the Eu3+bound water and the bulk water), and -50 ppm (opposite to the Eu3+-bound water), respectively. The CEST images were obtained by pixel by pixel subtraction the image at 50 ppm from that at 30 ppm, respectively. All other imaging parameters were presented in the corresponding figure captions. Data Analysis. Data fitting was performed by using the commercial software, PSI-PLOT version 7.5, Poly Software International, Inc.. Binding constants were obtained by nonlinear fitting the experimental data to a 1:1 binding model (28).

Aexp )

A0 + AlimKa[sugar] 1 + Ka[sugar]

(1)

where Ka is the binding constant, A0, Alim, and Aexp are the initial, equilibrium, and experimental values, either the relative UV, CD signal intensities, or MRI CEST ratios. Reagents. 1,4,7,10-Tetraazacyclododecane was purchased from Strem Chemicals, Inc. (Newburyport, MA); phenylboronic acid and 10% Pd/C were purchased from Aldrich Chemical Co. (Milwaukee, WI). All other reagents used were of analytical grade. Syntheses. 3-Nitrophenylboronic acid and 3-aminophenylboronic acid was prepared as described by Seaman and Johnson (26) and Groziak et al. (49), respectively. 2-Bromo-N-methylacetamide was prepared by the method of Valtancoli et al. (50) and 1,7-bis(benzyloxycarbonyl)1,4,7,10-tetraazacyclododecane was prepared by methods described by Kovacs and Sherry (21, 22). 3-Bromoacetylaminophenylboronic Acid. To a mixture of 3-aminophenylboronic acid (1.77 g; 12.19 mmol) and sodium carbonate (2.74 g; 24.38 mmol) in THF (85 mL) cooled in an ice bath was added bromoacetyl bromide (3.4 mL; 36.57 mmol). The mixture was stirred at 0 °C for 30 min and at 25 °C for 1 h. The solution was filtered and the filtrate was evaporated under vacuum.

Water (10 mL) was added to the residue, and the solid was filtered, washed with water (10 mL), and dried to yield 2.63 g (79%) of white solid. 1H NMR (CD3OD); δ (ppm): 3.95 (s; 1H); 7,29 (t; 1H; J ) 7.9); 7,47 (b.s; 1H); 7.62 (d; 1H; J ) 7.9); 7,80 (s; 1H); 13C NMR (CD3OD); δ (ppm): 28.46; 121.95; 125.39; 127.85; 129.89; 137.29; 166.36; ECI mass spectrum: 719 (M - H+; anhydrous trimer). 1,7-Bis(benzyloxycarbonyl)-4,10-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (2). To a solution of 1,7-bis(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (2,0 g; 4,54 mmol) and 2-bromo-Nmethylacetamide (1,8 g; 11,48 mmol) in acetonitrile (70 mL) was added sodium carbonate (0.96; 9.05 mmol). The mixture was refluxed for 18 h. The remaining solids were removed by filtration, and filtrate was rotary evaporated in a vacuum. The residue was loaded on silica gel column (250 g), and the product was eluted with chloroform: methanol (5:0,2). The fractions containing the product were combined, and the solvent was removed in a vacuum to give 2.24 g (84%) of colorless oil. 1H NMR (CDCl3); δ (ppm): 2.58-2.85 (m; 14H); 3.12 (br.s; 4H); 3.39 (br.s; 8H); 5.07 (s; 4H); 7.29-7.31 (m; 10H); 13C NMR (CDCl3); δ (ppm): 25.58; 48.9; 55.4; 67.56; 128.32; 128.46; 128.73; 136.48; 157.05; 171.5. 1,7-Bis(methyl(carbamoylmethyl))-1,4,7,10tetraazacyclododecane (3). A mixture of 1,7-bis(benzyloxycarbonyl)-4,10-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (2.24 g; 3.85 mmol) and 10% Pd/C (0.51 g) in absolute ethanol (60 mL) was shaken under 45 psi of H2 for 3 days. The catalyst was removed by filtration, and the filtrate was rotary evaporated to dryness in a vacuum to give 1.18 g (98%) of white solid. 1 H NMR (CD3OD); δ (ppm): 2.79 (s; 6H); 2.84-2.89 (m; 8H); 2.97-3.03 (m; 8H); 3.35 (s; 4H); 13C NMR (CD3OD); δ (ppm): 25.19; 44.10; 51.10; 57.35; 172.74. 1,7-Bis((m-dihydroxyborylphenyl)carbamoylmethyl)-4,10-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (4). To a solution of 1,4-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (1.18 g; 3.77 mmol) and 3-bromoacetylaminophenylboronic acid (2.04 g; 7.99 mmol) in methanol:acetonitrile (25:25 mL) was added sodium carbonate (0.80 g; 7.54 mmol). The mixture was refluxed overnight. The solvent was removed by rotary evaporation in a vacuum, and the remaining residue was dissolved in 0.5 M HCl (60 mL) and subsequently extracted with ethyl acetate (3 × 50 mL). The aqueous layer was neutralized with 2 M NaOH to pH 7, and the solid that precipitated at this point was filtered and dried to yield 2.01 g (80%) of a white solid. 1 H NMR (CD3OD); δ (ppm): 2.25-2.45 (m; 14H); 2.66 (br.s; 8H); 3.05 (br.s; 4H); 3.27 (br.s; 4H); 7.22 (t; 1H; J ) 8.1 Hz); 7.42 (br.s; 1H); 7.69 (d; 1H; J ) 8.1 Hz); 7.79 (br.s; 1H); 13C NMR (CD3OD); δ (ppm): 25.10; 50.68; 56.55; 57.59; 125.35; 127.77; 129.49; 137.79; 170.98; 173.05; Fab mass spectrum: 781.4 (M + H+; glycerol complex). 11,12-dioxo-1,4,7,10-tetraazabicyclo[8.2.2]tetradecane (5); 4,7-bisbenzyl-1,4,7,10-tetraaza bicycle[8.2.2]-tetradecane-11,12-dione (6) and 1,4dibenzyl-1,4,7,10-tetraazacyclododecane (7) were prepared according to the method of Handel at al. (23). 1,4-Bisbenzyl-7,10-bis(methyl(carbamoylmethyl))1,4,7,10-tetraazacyclododecane (8). To a solution of 1,4-dibenzyl-1,4,7,10-tetraazacyclododecane (3.5 g; 9.94 mmol) and 2-bromo-N-methylacetamide (3.5 g; 23.02 mmol) in acetonitrile (300 mL) was added sodium carbonate (2.1 g; 19.81 mmol). The mixture was refluxed overnight. The remaining solid was filtered and the

Phenylboronate Eu3+ Complexes for Glucose Sensing

solvent removed by rotary evaporation in a vacuum. The residue was dissolved into chloroform, loaded onto silica gel column (270 g), and eluted with chloroform:methanol (5:0.4). The fractions containing the product were combined, and the solvent was removed in a vacuum to give 4.06 g (83%) of a colorless oil. 1H NMR (CDCl3); δ (ppm): 2.54 (br.s; 8H); 2.71-2.79 (m; 14H); 3.1 (s; 4H); 3.55 (s; 4H); 7.12-7.33 (m; 10H); 13C NMR (CDCl3); δ (ppm): 26.09; 51.73; 53.41; 53.55; 54.53; 58.98; 59.90; 127.32; 128.35; 129.27; 137.7; 172.11. 1,4-Bis(methyl(carbamoylmethyl))-1,4,7,10tetraazacyclododecane (9). A mixture of 1,4-bis-benzyl-7,10-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (2.55 g; 5.16 mmol) and 10% Pd/C (0.7 g) in absolute ethanol (60 mL) was shaken under 45 psi of H2 for 3 days. The catalyst was removed by filtration and the solvent removed by rotary evaporation in a vacuum to give 1.5 g (92.5%) of a yellow oil. 1H NMR (CDCl3); δ (ppm): 2.51-2.57 (m; 8H); 2.63-2.72 (m; 10H); 2.81 (s; 4H); 3.06 (s; 4H); 13C NMR (CDCl3); δ (ppm): 18.25; 25.84; 44.91; 46.32; 52.33; 53.92; 58.06; 58.51; 171.88. 1,4-Bis((m-dihydroxyborylphenyl)carbamoylmethyl)-7,10-bis(methylcarbamoylmethyl)-1,4, 7,10tetraazacyclododecane (10). To a solution of 1,4-bis(methyl(carbamoylmethyl))-1,4,7,10-tetraazacyclododecane (0.95 g; 3.02 mmol) and (m-dihydroxyborylphenyl) bromo acetylamide (1.7 g; 6.6 mmol) in methanol:acetonitrile (25:25 mL) was added sodium carbonate (0.64 g; 6.04 mmol). The mixture was refluxed overnight. The solvent was removed by rotary evaporation in a vacuum, and the residue was dissolved in 0.5 M HCl (50 mL) and extracted with ethyl acetate (3 × 50 mL). The aqueous layer was neutralized with 2 M NaOH to pH 7, and the precipitate that formed was filtered and dried to yield 1.27 g (64%) of a white solid. 1H NMR (CD3OD); δ (ppm): 2.20-2.65 (m; 14H); 2.70-2.85 (m; 8H); 3.1 (br.s; 4H); 3.25 (br.s; 4H); 7.08 (d; 1H; J ) 8 Hz); 7.27 (br.s; 1H); 7.56 (t; 2H; J ) 8 Hz); 13C NMR (CD3OD); δ (ppm): 24.96; 50.47; 56.57; 57.22; 124.93; 127.56; 129.14; 137.64; 170.58; 172.75. Fab mass spectrum: 781.3 (M + H+; glycerol complex). 1-(m-Dihydroxyborylphenyl)carbamoylmethyl)1,4,7,10-tetraazacyclododecane (11). 3-Bromoacetylaminophenylboronic acid (1.0 g; 3.87 mmol) in methanol (150 mL) was added dropwise to a solution of 1,4,7,10tetraazacyclododecane (4.8 g; 27.86 mmol) in acetonitrile (350 mL) over 2 days at room temperature. The reaction mixture was stirred for an additional day at room temperature. The solvent was removed by rotary evaporation in a vacuum. Acetonitrile as added to the remaining residue and the precipitate that formed was filtered, washed twice with acetonitrile, and dried to yield 1.0 g (60%) of a hydrobromide salt. 1H NMR (CD3OD); δ (ppm): 2.76-2.99 (m; 16H); 3.43 (s; 2H); 7.15 (t; 1H; J ) 7.4 Hz); 7.31 (d; 1H; J ) 7.4 Hz); 7.41 (s; 1H); 7.61 (d; 1H; J ) 7.4 Hz). 13C NMR (CD3OD); δ (ppm): 44.15; 44.36; 45.29; 51.85; 57.88; 118.19; 124.82; 126.75; 129.38; 136.48; 170.87. 1-((m-Dihydroxyborylphenyl)carbamoylmethyl)4,7,10-tris(methylcarbamoylmethyl)-1,4,7,10-tetraazacyclododecane (12). To a solution of 1-(m-dihydroxyborylphenyl)carbamoyl methyl)-1,4,7,10-tetraazacyclododecane (1.0 g; 2.32 mmol) and 2-bromo-N-methylacetamide (2.12 g; 13.95 mmol) in methanol:acetonitrile (25:25 mL) was added sodium carbonate (1.48 g; 13.95 mmol). The mixture was refluxed 2 days. The remaining solids were removed by filtration, and solvent was removed by rotary evaporation in a vacuum. The resulting residue was extracted first with chloroform:methanol

Bioconjugate Chem., Vol. 15, No. 6, 2004 1433 Scheme 2

(50:0.5) and then by acetonitrile and finally removed by filtration, washed with cold water, washed twice with acetonitile, and dried to yield 0.9 g (69%) of a white solid. 1H NMR (CD OD); δ (ppm): 2.31-2.49 (m; 4H); 2.683 2.81 (m; 15H); 3.05-3.14 (m; 6H); 3.35 (s; 8H); 7.11 (t; 1H; 7.7 Hz); 7.22-7.36 (m; 2H); 7.69 (d; 1H; 7.7 Hz). 13C NMR (CD3OD); δ (ppm): 25.04; 50.54; 56.66; 56.89; 57.53; 117.78; 124.52; 126.42; 129.33; 136.57; 170.29; 172.66; 172.74. Fab mass spectrum: 619.3 (M + H+; glycerol complex). Eu(4), Eu(10), and Eu(12) Preparation. Typically, ligand (10% excess) was added to an aqueous solution of EuCl3 (0.2 M), and the resulting solution was stirred at room-temperature overnight to ensure full complexation. If necessary, a small amount of concentrated NaOH was added to maintain the pH near 7 until free Eu3+ could no longer be detected using xylenol orange as colorimetric indicator (in 1 M NaAc/HAc buffer solution at pH 5.3). Excess free ligand was filtrated, and the residue was freeze-dried. RESULTS

Synthesis and Characterization of Phenylboronic Acid-Appended Macrocycles. Lanthanide cyclen complexes with amide pendant arms exhibit high kinetic inertness even in very acidic condition (19) and consequently have been found to have very important application as MRI contrast agents via a novel route based on chemical exchange saturation transfer (CEST) mechanism (20). We therefore focused on preparing new ligands based on tetramide derivatives of cyclen with phenylboronic acids groups as pendant arms. For bissubstitutions, two possibilities exist: one where the phenylboronic acids are trans to one another across the cyclen ring (ligand 4) and another where they are cis to one another (ligand 10). 1,7-Substituted cyclen was achieved by following a procedure reported by Kovacs and Sherry (21, 22) through protection of trans-amines in cyclen by using benzyl chloroformate (Scheme 2). Treatment of 1 with 2-bromo-N-methylacetamide and sodium carbonate in MeCN followed by hydrogenolysis afforded 3 with methyl amide arms selectively substituted at positions 1 and 7. In the final step, reaction of 3 with 3-bromoacetylaminophenylboronic acid in MeCN:MeOH/ sodium carbonate afforded the target ligand 4. As these phenylboronate products have low solubility in water at pH 7 in water, they were easily purified by precipitation. Solid 4 was isolated in 80% yield.

1434 Bioconjugate Chem., Vol. 15, No. 6, 2004 Scheme 3

Selective protection of adjacent nitrogens of the cyclen ring was achieved following the procedure reported by Handel and co-workers (23) through formation of the oxamide by condensation with diethyl oxalate. This was followed by alkylation with benzyl bromide and deprotection by selective hydrolysis of the bicyclic diamide to afford 7 (Scheme 3). Reaction of 7 with 2-bromo-Nmethylacetamide in MeCN/sodium carbonate yielded 8 and hydrogenolysis afforded the 1,4 methyl diamide derivative 9 in 92% yield. The reaction of 9 with 3-bromoacetylaminophenylboronic acid in MeCN:MeOH/ sodium carbonate followed by precipitation from water at pH 7 yielded the cis-ligand 10 in 64% yield. Finally, selective monoalkylation of cyclen can be run using two main strategies, either direct alkylation of excess of cyclen with the appropriate alkyl halide (24) or selective N-functionalization followed by alkylationdeprotection steps (25). We found that the best experimental conditions to perform the monoalkylation of cyclen required the use of 7 equiv of cyclen in MeCN in room temperature. In this way, we could isolate compound 11 in satisfactory yield (60%) without using chromatography (Scheme 4). Once the monoalkylated product 11 was isolated, conversion to the corresponding N-monophenylboronate compound 12 was accomplished Scheme 4

Trokowski et al.

by reaction of 11 with 2-bromo-N-methylacetamide in MeCN:MeOH/sodium carbonate. 12 was isolated as a white solid from water at pH 7 in 69% yield. All compounds were characterized by 1H and 13C NMR and mass spectroscopy. Elemental analyses of these boroncontaining products were unsuccessful due to formation of incombustible residues (26) and the possibility of existences of mixture of acids and anhydrides that are common moieties of boronic acids. Europium complexes were formed by reaction with EuCl3 at pH 7 and room temperature. The complexes were conveniently purified by filtering off the excess ligand from water at pH 7. pH Titrations. The UV absorption spectra of phenylboronic acids undergo characteristic changes along with the sample pH that reflect conversion of neutral B(OH)2 to anionic B(OH)3- species. Typical absorption spectra of Eu(4) at various pH values are shown in Figure 1. Even though Eu(4) has two boronic acids, the single isobestic point near 253 nm indicates that the two groups titrate independently and have identical pKa values. For this reason, plots of A271 versus pH for Eu(4), Eu(10), and Eu(12) were fit to a single protonation step to give the pKa values shown in Table 1. Upon addition of excess sugar (D-fructose in this case), there is a shift in apparent pKa of these systems to lower values that reflects a decreased boron-oxygen bond angle in the -B(OR)2(OH)- species and an increase in acidity of the boron center. D-Fructose was chosen as the reference sugar in this experiment because it has been reported that this sugar provides the largest pKa shift upon binding to boronic acids (14, 27). The UV spectra of Eu(4), Eu(10), and Eu(12) collected as a function of pH in the presence of excess D-fructose shows that all three phenylboronate derivatives form complexes with sugar and experience a similar pKa shift upon binding to D-fructose. Sugar Titrations. Changes also occur in the UV spectrum of phenylboronic acid upon addition of sugars (at constant pH) and these may be monitored to determine a conditional binding constant characteristic of that pH value (28). Initial binding experiments for Eu(4), Eu(10), and Eu(12) were performed at pH 10.2 (well above the pKa of the boronic acids in these systems) with the three most abundant monosaccharides in human plasma, D-glucose (∼5 mM), D-fructose (∼0.05 mM), and Dgalactose (∼0.05 mM) (29). Although D-fructose is known to display the highest affinity among these monosaccharides for mono-phenylboronic acid derivatives, some bisphenylboronic acid derivatives bind selectively with D-glucose, providing that the phenylboronic acid functionalities are appropriately positioned (30-33). Typical changes in absorbance of Eu(4) upon addition of Dfructose and D-glucose are shown in Figure 2. In most experiments with monosaccharides, a general increase in absorbance such as that shown here for D-fructose is observed but an opposite behavior was observed upon

Phenylboronate Eu3+ Complexes for Glucose Sensing

Bioconjugate Chem., Vol. 15, No. 6, 2004 1435

Figure 1. Absorption spectra of Eu(4) (5.64 × 10-5 M) as a function of pH. The changes in A271 are shown plotted as a function of pH in the absence (a) and presence (b) of 50 mM D-fructose. Table 1. pKa Values of Eu(4), Eu(10), and Eu(12) in the Absence and Presence of Excess D-Fructose As Measured by UV Spectroscopy complex

pKa (without D-fructose)

pKa (with D-fructose)

∆pKa

Eu(4) Eu(10) Eu(12)

7.81 8.03 8.03

5.47 5.71 5.50

2.34 2.32 2.53

binding of D-glucose to Eu(4). Plots of these absorbance changes as a function of added sugar are shown in Figure 2. The solid lines represent the best fit of these data to 1:1 binding model for all systems. Fitting of the Eu(4) and Eu(10) data to a 1:2 binding model yielded rather poor agreement with the experimental values. Hence, a 1:1 binding model was assumed to hold for all combinations of Eu3+ complexes and sugars (more evidence is provided below for formation of 1:1 complexes). The resulting values are summarized in Table 2. These data reveal a marked difference in stability between the three Eu3+ complexes and these sugars. The data for Eu(12) is typical of mono-phenylboronic acidsugar interactions with the stability order of D-frucose > D-galactose > D-glucose (15, 16). Eu(10) also displays the highest affinity for D-fructose but has an approximate 2-fold greater affinity for D-glucose than D-galactose. Eu(4), however, shows a completely different binding trend with these same sugars. This complex, the 1,7-trans-bisphenylboronate, displays the highest affinity for D-glucose followed by D-fructose, D-galactose, and the other sugars. This indicates that the distance and positioning of the two phenylboronates in Eu(4) is most favorable for a bridging a single glucose molecule between the two complexing centers. Binding of Eu3+ in the macrocyclic cavity of ligands such as these likely alters the geometry of the ligand side-chains and this in turn could have a profound effect upon the phenylboronate-sugar binding. To test this hypothesis, binding studies were also carried out between

the free ligands and these same sugars (Table 3). It should be noted that free ligand 4 binds to D-fructose with such high affinity that it became difficult to arrive at a true equilibrium situation at the concentrations required for the spectroscopic measurement, i.e., essentially all of the fructose is bound at even the lowest ratio of D-fructose to ligand 4. Hence, the Ka listed in Table 3 for this system (8325 ( 2192 M-1) has a rather large error. Nonetheless, the experiment does prove that free ligand 4 does have the highest binding affinity for D-fructose among the sugars examined. Interestingly, in the absence of Eu3+, 10 shows greater selectivity toward glucose than does 4, a result just opposite from that seen with the corresponding Eu3+ complexes. The stability of the complex formed between 4 and glucose is ∼8-fold lower than the stability of the complex formed between Eu(4) and glucose. This indicates that the selectivity of bis-phenylboronic acid derivatives for sugars is quite dependent on the distance separating the two phenylboronic acid moieties, consistent with other bis-phenylboronates (29, 34, 35). This also suggests that it may be possible to modulate the affinity of such cyclen-based systems for glucose by other factors that may influence the distance between the phenylboronates such as bulkiness of the other two amide sidechains or perhaps even the size of the lanthanide cation. CD Spectroscopy. Sugar binding can also be conveniently monitored by changes in the chirality as detected by CD spectroscopy. Molecules containing bis-phenylboronic acid receptors typically become CD active when sugars form cyclic 1:1 complexes such as those illustrated by Scheme 1a while 1:2 complexes are less rigid (Scheme 1b) and are typically CD-silent (36-39). This makes CD spectroscopy a very convenient tool for differentiating between these two possible structural patterns. The CD spectra of Eu(4) and Eu(10) are silent in the absence of sugars but become active in the presence of all sugars examined here except D-fructose. The ketose, D-fructose, appears to be unique in this regard for reasons that are

1436 Bioconjugate Chem., Vol. 15, No. 6, 2004

Trokowski et al.

Figure 2. Absorption spectra of Eu(4) (7.52 × 10-5 M) as a function of added D-fructose (top row) or D-glucose (bottom row) at a constant pH ) 10.2 (48 mM carbonate buffer). [D-fructose] was varied from 0; 1.8; 4.4; 11.3; 41.9; 108.3 mM while [D-glucose] was varied from 0; 0.9; 1.8; 5.6; 15.4; 24.9; 80.7 mM. Table 2. Binding Constants (Ka, M-1) for Formation of 1:1 Complexes between Eu(4), Eu(10), and Eu(12) and Various Saccharides at pH 10.2 saccharides

Eu(4)

Eu(10)

Eu(12)

glucose galactose fructose lactose

2275 ( 266 198 ( 20 1259 ( 106 110 ( 24

319 ( 48 124 ( 15 1503 ( 130 63 ( 9

73 ( 13 118 ( 14 820 ( 124 159 ( 27

Table 3. Binding Constants (Ka, M-1) between Free Ligands, 4 and 10, and Various Sugars at pH 10.2 saccharides

4

10

glucose galactose fructose lactose

243 ( 32 671 ( 54 8325 ( 2192 137 ( 47

1165 ( 106 274 ( 54 831 ( 208 25 ( 54

not entirely clear. This same behavior has been observed for many other bis-phenylboronate systems that have been studied using CD spectroscopy (3, 16, 40, 41). The CD spectra of Eu(4) and Eu(10) in the presence of excess D-glucose are shown in Figure 3. Clearly, both the cisphenylboronate complex (Eu(10)) and the trans-phenylboronate complex (Eu(4)) form bridging 1:1 complexes with glucose but with opposite chirality. As a check, spectra of Eu(4) and Eu(10) with also run in the presence of excess L-glucose (data not shown) and the CD spectra of these were the mirror images of those shown in Figure 3. This lends support to the opposite chirality of Eu(4)‚ D-glucose and Eu(10)‚D-glucose. Binding constants between Eu(4) were also measured by CD spectroscopy at pH 10.2 (to compare with the UV

results) and again at pH 7 (Figure 4). A fitting of the CD binding data at pH 10.2 gave a binding constant, Ka ) 2280 M-1, identical to the value determined in the UV titration. The titration performed at pH 7 gave a binding constant, Ka ) 339 M-1, again identical to the value determined previously in a CEST titration (383 M-1) (18). This binding constant is in a reasonable range for using Eu(4) to detect physiological levels of glucose (∼5 mM) by MRI. Verification of Eu(4)‚sugar stoichiometry by MALDI TOF Mass Spectrometry. To verify that glucose indeed binds to Eu(4) in a bridging manner as suggested by CD spectroscopy, mass spectroscopy was also performed on aqueous samples containing 10 mM Eu(4) plus either D-fructose or D-glucose at 1:1 and 1:2 ratios. For both glucose and fructose at pH 7, a mass peak at 1065 Da corresponding to a 1:1 complex was observed. For a sample recorded at pH 10 where the binding affinity is much higher, again a mass peak at 1066 Da dominated the spectrum. Only very small amounts of the 1:2, Eu(4)‚(sugar)2, complexes were detected at either pH value. This result verified the fits of the binding data and the CD active spectrum observed for Eu(4)‚sugars described above. MR Images of Phantoms Containing Eu(4) or Eu(12) Plus Simple Monosaccharides or Glycated HSA. To test whether these new sensors might be used to detect sugars by MRI based upon a CEST mechanism (20), phantoms consisting of four tubes (I.D. 4 mm) each containing 10 mM Eu(4) or Eu(12) and different amounts of monosaccharides were prepared in 100 mM PIPES

Figure 3. CD spectral changes of Eu(4) (left) and Eu(10) (right) in the presence of 50-fold excess D-glucose (24 mM carbonate buffer, pH 10.2; the concentrations of Eu(10) and Eu(4) were 7.52 × 10-5 M).

Phenylboronate Eu3+ Complexes for Glucose Sensing

Bioconjugate Chem., Vol. 15, No. 6, 2004 1437

Figure 4. CD intensity changes of Eu(4) (5.26 × 10-5 M) in the presence of increasing concentrations of D-glucose. The top row is data collected at pH 10.2 (24 mM carbonate buffer) while the bottom row is data collected at pH 7.2 (24 mM phosphate buffer).

Figure 5. CEST images of phantoms containing 10 mM Eu(4) or Eu(12) and 0, 5, 10, and 20 mM sugar, respectively. PIPES (100 mM) was used to buffer the pH at 7.0. The image parameters were as follows: TR/TE ) 3000/18 ms, FOV ) 40 × 40 mm, coronal thickness 2 mm, data matrix 256 × 256, saturation duration time of 2 s at a power of 1020 Hz at a frequency offset of 50 and 30 ppm. The CEST images were obtained by pixel by pixel subtraction of the image at 50 ppm from that at 30 ppm, respectively.

buffer at pH 7.0. Eu(4) was selected because it has the highest binding ability to glucose, while Eu(12) was imaged simply for comparison. Images were acquired after alternatively applying identical presaturation pulses at 50 ppm (bound water) versus 30 ppm (between the bound and bulk water peaks) (18). Although the intensities of these individual images were visually similar, the difference images (referred to as a CEST image) show clear intensity gradations that parallel the sugar concentrations. Typical CEST images for Eu(4) or Eu(12) in the presence of different amounts of glucose, fructose, and galactose were shown in Figure 5. For example, using a gray scale of 0-255 (from the darkest to the brightest), the image intensities were 208, 148, 112, and 96 for samples containing 10 mM Eu(4) and either 0, 5, 10 and 20 mM glucose, respectively, while they were 134, 114, 110, and 106 for Eu(12) phantom. This demonstrates that

Eu(4) may be used to quantitatively image glucose near physiological concentrations (∼5 mM). A comparison of the CEST images of Eu(4) in the presence of glucose or fructose suggests that fructose would likely interfere with detection of glucose if both sugars were presented at similar concentrations. This is consistent with the ∼2fold lower binding constant for Eu(4) and fructose versus glucose (Table 2). However, the concentration of fructose in tissues is typically about 100-fold lower than glucose so one would anticipate that fructose would not interfere with quantitative imaging of glucose. Galactose binding is weak for both Eu3+ complexes, and the corresponding CEST images are little affected by this sugar. CEST imaging experiments were also performed on samples containing glycated human serum albumin (HSA) to determine whether Eu(4) could be used as a sensor of sugars bound to proteins. Figure 6 shows images of phantoms containing 10 mM Eu(4) plus increasing amounts of glycated HSA (with an average of five hexose units per albumin). Assuming that Eu(4) binds to each of the five fructosamine moieties with equal binding affinity, a binding constant of 791 ( 301 M-1 was estimated from the limited CEST image intensity data of Figure 6. Although this binding constant has a rather large error, the value found is the same order of magnitude as the constants found for Eu(4) binding to simple sugars at this same pH. This indicates that amidation of fructose does not destroy recognition by Eu(4). However, the amounts of glycated proteins, such as albumin and hemoglobin, are proportional to the level of blood glucose with albumin glycated to an extent of about 10% at normal plasma levels (42). Thus, at a plasma concentration of 10% × 0.6 or ∼0.06 mM, the amount of glycated HSA present in plasma of a normal individual should not interfere with using Eu(4) as a sensor for glucose in tissue. On the other hand, this binding property could be useful as a rapid throughput analysis of the extent of glycation of HSA in vitro after separation of excess simple sugars. Glycation of proteins has been implicated in the development of diabetic complications and the aging process (43).

1438 Bioconjugate Chem., Vol. 15, No. 6, 2004

Trokowski et al.

Figure 6. CEST images of phantoms containing 10 mM Eu(4) and 0, 0.18, 0.36, and 0.72 mM glycated HSA, respectively. PIPES (100 mM) was used to buffer the pH at 7.0. The image parameters are identical to those shown in Figure 5. DISCUSSION

Three new cyclen-based tetraamide ligands containing either mono- and bis-phenylboronate extended arms were synthesized and their Eu3+ (europium) complexes characterized by UV, circular dichroism, mass spectrometry, and CEST imaging. The Eu3+ complexes of 4, 10, and 12 display quite different binding behavior with glucose, fructose, and galactose. The initial binding experiments were carried out at pH 10.2 (well above the physiological pH range) because it is well-known that phenylboronates have a much higher affinity for sugars at pH values above the pKa of the boronic acid. Given that the affinity of many phenylboronates for sugars is too low to detect at pH values near 7, most binding studies are carried out first at pH values near 10 to ensure adequate binding. The design of bis-phenylboronic acid molecules as sensors specific for glucose assumes that bis-phenylboronic acids will be positioned at the proper distance and orientation to bind glucose with high affinity and selectivity. Although this has been demonstrated previously for several fluorescent molecules containing two phenylboronate groups (29, 44-46), the degree of selectivity and binding affinity remains rather empirical. The goal of this project at the onset was to create a glucose sensor using MRI as the readout device. It has been shown recently that water exchange in cyclen-based Eu3+-tetraamide complexes is quite sensitive to the identity of the appended amine sidechains (17, 47) so it was reasonable to assume that one might be able to modulate water exchange in such systems by using glucose as a “capping” moiety over a Eu3+-bound water molecule. Hence, the trans-1,7-bisphenylboronate ligand (4) became the target ligand of choice while the cis-1,4-bis-phenylboronate ligand (10) was designed as a control. While simple mono-phenylboronic acids typically display a much higher affinity for fructose over glucose (for example, phenylboronic acid has a Ka of 162 M-1 for fructose and 5 M-1 for glucose (48)), bis-phenylboronic acids systems tend to become more selective for glucose. As anticipated, the mono-phenylboronic acid system, Eu(12) displayed an ∼11-fold selectivity for fructose over glucose and even had a greater affinity for galactose than glucose. Surprisingly, free ligand 4 also had an ∼34-fold binding selectivity for fructose over glucose and an unusually high affinity for fructose, comparable to some of the more favorable bisphenylboronate systems that have been reported (34), while free ligand 10 slightly favored glucose in preference to fructose (more typical of over bis-phenylboronate systems). This indicates that the exact positioning of the two phenylboronate moieties not only determines sugar selectivity but also substantially impacts binding affinity.

It is quite interesting and again surprising that formation of Eu3+ complexes with these same ligands has a much more dramatic impact in the case of Eu(4) than Eu(10). Now, Eu(4) favors glucose over fructose by ∼2-fold, a nearly 40-fold switch in binding selectivity upon going from free ligand 4 to Eu(4). This rather dramatic effect must be ascribed to a repositioning of the two phenylboronate side-chains upon entry of Eu3+ into the cyclen cavity and suggests that the ionic radius of the central metal ion might be used as an adjustable parameter for fine-tuning glucose selectivity and affinity. Conversely, a comparison of free ligand 10 with Eu(10) shows an opposite effect with free ligand 10 favoring glucose and Eu(10) favoring fructose. Here, the switch in binding affinity is less dramatic but nonetheless ∼7-fold. As predicted a priori, water exchange between the Eu3+bound water molecule in Eu(4) is indeed slower when glucose is bound, and this feature can be detected by CEST imaging (18). The images shown in Figure 5 indicate that water exchange is also slowed upon binding of fructose to Eu(4), but the effect is smaller in this system due to a combination of lower binding affinity and perhaps lower modulation of water exchange (the exchange rate has not been measured in this system). Addition of galactose to Eu(4) or addition of any monosaccharides to Eu(10) does not initiate a CEST effect. The later observation is not surprising since binding of sugars to the later cis-1,4-bis-phenylboronate complex would like not disturb the Eu3+-bound water site. Compared to other organic-based bis-phenylboronate glucose sensors, Eu(4) has a reasonably high binding affinity for glucose (2275 ( 266 M-1 at pH 10.2 and 339 ( 29 M-1 at pH 7) but only a modest selectivity for glucose over fructose. Sensor Eu(4) represents an improvement of about 455-fold in affinity and about 60-fold improvement in selectivity for glucose over fructose compared with simple phenylboronic acid. Although fructose and glycated HSA have similar binding affinities as glucose with Eu(4), it is unlikely that these sugars will interfere with imaging of glucose in tissues because the plasma concentrations of fructose and glycated HSA are much lower than that of glucose. Thus, Eu(4) is an important advance toward an optimal agent for imaging the tissue distribution of glucose by MRI. ACKNOWLEDGMENT

This work was supported in part by grants from the Robert A. Welch Foundation (AT-584), the National Institutes of Health (CA-84697 and RR-02584), and the Texas Advanced Technology Program. Authors thank Dr

Phenylboronate Eu3+ Complexes for Glucose Sensing

G. R. Dieckmann and Dr J. Sibert for access to their CD and UV spectrometers. LITERATURE CITED (1) Striegler, S. (2003) Selective carbohydrate recognition by synthetic receptors in aqueous solution. Curr. Org. Chem. 7, 81-102. (2) Freckmann, G., Kalatz, B., Pfeiffer, B., Hoss, U., and Haug, C. (2001) Recent advances in continuous glucose monitoring. Exp. Clin. Endocrinol. Diabetes 109, 347-357. (3) James, T. D., Sandanayake, K. R. A. S., and Shinkai, S. (1995) Recognition of sugars and related compounds by “reading-out”-type interfaces. Supramol. Chem. 6, 141-157. (4) James, T. D., Linnane, P., and Shinkai, S. (1996) Fluorescent saccharide receptors: a sweet solution to the design, assembly and evaluation of boronic acid derived PET sensors. Chem. Commun. 281-288. (5) James, T. D., Sandanayake, K. R. A. S., and Shinkai, S. (1996) Saccharide sensing with molecular receptors based on boronic acid. Angew. Chem., Int. Ed. Engl. 35, 1911-1922. (6) Mizuno, T., Takeuchi, M., Hamachi, I., Nakashima, K., and Shinkai, S. (1997) Chiroselective transcription of the sugar structure to D- or L-[CoIII(bpy)3]3+ using a boronic acid-sugar template interaction. Chem. Commun. 1793-1794. (7) Shinmori, H., Takeuchi, M., and Shinkai, S. (1998) A novel light-gated sugar receptor, which shows high glucose selectivity. J. Chem. Soc., Perkin Trans. 2, 847-852. (8) Wang, W., Gao, X., and Wang, B. (2002) Boronic acid-based sensors. Curr. Org. Chem. 6, 1285-1317. (9) Ross, B., and Bluml, S. (2001) Magnetic resonance spectroscopy of the human brain. Anat. Rec. 265, 54-84. (10) Gruetter, R., Rothman, D. L., Novotny, E. J., Shulman, G. I., Prichard, J. W., and Shulman, R. G. (1992) Detection and Assignment of the Glucose Signal in H1 NMR Difference Spectra of the Human Brain. Magn. Reson. Med. 27, 183188. (11) Seaquist, E. R., Damberg, G. S., Tkac, I., and Gruetter, R. (2001) The effect of insulin on in vivo cerebral glucose concentrations and rates of glucose transport/metabolism in humans. Diabetes 50, 2203-2209. (12) Gruetter, R., Novotny, E. J., Boulware, S. D., Rothman, D. L., Mason, G. F., Shulman, G. I., Shulman, R. G., and Tamborlane, W. V. (1992) Direct Measurement of Brain Glucose-Concentrations in Humans by C-13 Nmr-Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 89, 12208-12208. (13) Testa, C., Casieri, C., Canese, R., Carpinelli, G., Podo, F., and De Luca, F. (2001) In vivo detection of C-13-enriched glucose metabolites in mouse brain by T-SEDOR imaging. Magn. Reson. Imaging 19, 739-743. (14) Lorand, J. P., and Edwards, J. O. (1959) Polyol complexes and structure of the benzeneboronate ion. J. Org. Chem. 24, 769-774. (15) Murakami, H., Nagasaki, T., Hamachi, I., and Shinkai, S. (1993) Sugar sensing utilizing aggregation properties of a boronic-acid-appended porphyrin. Tetrahedron Lett. 34, 62736276. (16) Arimori, S., Takeuchi, M., and Shinkai, S. (1996) Sugarsensing by chiral orientation of dimeric boronic-acid-appended porphyrins which show selectivity for glucose and xylose. Chem. Lett. 77-78. (17) Zhang, S. R., Merritt, M., Woessner, D. E., Lenkinski, R. E., and Sherry, A. D. (2003) PARACEST agents: Modulating MRI contrast via water proton exchange. Acc. Chem. Res. 36, 783-790. (18) Zhang, S., Trokowski, R., and Sherry, A. D. (2003) A Paramagnetic CEST Agent for Imaging Glucose by MRI. J. Am. Chem. Soc. 125, 15288-15289. (19) Wu, K., Zhang, S., Kovacs, Z., and Sherry, A. D. (2000) Dota-amide derivatives with extended arms form lanthanide complexes with unusual kinetic inertness. J. Nucl. Med. 41, 1085.

Bioconjugate Chem., Vol. 15, No. 6, 2004 1439 (20) Zhang, S., Winter, P., Wu, K., and Sherry, A. D. (2001) A novel europium(III)-based MRI contrast agent. J. Am. Chem. Soc. 123, 1517-1518. (21) Kovacs, Z., and Sherry, A. D. (1995) General-Synthesis of 1,7-Disubstituted 1,4,7,10-Tetraazacyclododecanes. J. Chem. Soc., Chem. Commun. 185-186. (22) Kovacs, Z., and Sherry, A. D. (1997) pH-controlled selective protection of polyaza macrocycles. Synthesis 759-763. (23) Bellouard, F., Chuburu, F., Kervarec, N., Toupet, L., Triki, S., Le Mest, Y., and Handel, H. (1999) Cis-diprotected cyclams and cyclens: a new route to symmetrically or asymmetrically 1,4-disubstituted tetraazamacrocycles and to asymmetrically tetrasubstituted derivatives. J. Chem. Soc., Perkin Trans. 1, 3499-3505. (24) Anelli, P. L., Murru, M., Uggeri, F., and Virtuani, M. (1991) Highly regioselective synthesis of 1,7-diprotected 1,4,7,10tetraazacyclododecane derivatives. J. Chem. Soc., Chem. Commun. 1317-1318. (25) Baker, W. C., Choi, M. J., Hill, D. C., Thompson, J. L., and Petillo, P. A. (1999) Synthesis of Lipophilic Paramagnetic Contrast Agents. J. Org. Chem. 64, 2683-2689. (26) Seaman, W., and Johnson, J. R. (1931) Derivatives of phenylboric acid, their preparation and action upon bacteria. J. Am. Chem. Soc. 7, 711-723. (27) Yoon, J., and Czarnik, A. W. (1992) Fluorescent Chemosensors of Carbohydrates - a Means of Chemically Communicating the Binding of Polyols in Water Based on ChelationEnhanced Quenching. J. Am. Chem. Soc. 114, 5874-5875. (28) Valeur, B., Pouget, J., Bourson, J., Kaschke, M., and Ernsting, N. P. (1992) Tuning of Photoinduced EnergyTransfer in a Bichromophoric Coumarin Supermolecule by Cation Binding. J. Phys. Chem. 96, 6545-6549. (29) Eggert, H., Frederiksen, J., Morin, C., and Norrild, J. C. (1999) A new glucose-selective fluorescent bisboronic acid. First report of strong alpha-furanose complexation in aqueous solution at physiological pH. J. Org. Chem. 64, 3846-3852. (30) Tsukagoshi, K., and Shinkai, S. (1991) Specific complexation with mono- and disaccharides that can be detected by circular dichroism. J. Org. Chem. 56, 4089-4091. (31) Nakashima, K., and Shinkai, S. (1995) Diaza-18-crown-6based sugar receptor bearing two boronic acids. Possible communication between bound sugars and metal cations. Chem. Lett. 6, 443-444. (32) James, T. D., Sandanayake, K. R. A. S., and Shinkai S. (1994) A glucose-specific molecular fluorescence sensor. Angew. Chem., Int. Ed. Engl. 106, 2287-2289. (33) Takeuchi, M., Mizuno, T., Shinmori, H., Nakashima, M., and Shinkai, S. (1996) Fluorescence and CD spectroscopic sugar sensing by a cyanine-appended diboronic acid probe. Tetrahedron 52, 1195-1204. (34) Karnati, V. V., Gao, X., Gao, S., Yang, W., Ni, W., Sankar, S., and Wang, B. (2002) A glucose-selective fluorescence sensor based on boronic acid-diol recognition. Bioorg. Med. Chem. Lett. 12, 3373-3377. (35) Hartley, J. H., James, T. D., and Ward, C. J. Synthetic receptors. (2000) J. Chem. Soc., Perkin Trans. 1, 3155-3184. (36) Shiomi, Y., Saisho, M., Tsukagoshi, K., and Shinkai, S. (1993) Specific complexation of glucose with a diphenylmethane-3,3′-diboronic acid derivative: correlation between the absolute configuration of mono- and disaccharides and the circular dichroic-activity of the complex. J. Chem. Soc., Perkin Trans. 1, 2111-2117. (37) Suenaga, H., Mikami, M., Sandanayake, K. R. A. S., and Shinkai, S. (1995) Screening of fluorescent boronic acids for sugar sensing which show a large fluorescence change. Tetrahedron Lett. 36, 4825-4828. (38) Kondo, K., Shiomi, Y., Saisho, M., Harada, T., and Shinkai, S. (1992) Specific complexation of disaccharides with diphenyl-3,3′-diboronic acid that can be detected by circular dichroism. Tetrahedron 48, 8239-8252. (39) Sandanayake, K. R. A. S., James, T. D., and Shinkai, S. (1995) Two-dimensional photoinduced electron transfer (PET) fluorescence sensor for saccharides. Chem. Lett. 503-504.

1440 Bioconjugate Chem., Vol. 15, No. 6, 2004 (40) Takeuchi, M., Yoda, S., Imada, T., and Shinkai, S. (1997) Chiral sugar recognition by a diboronic-acid-appended binaphthyl derivative through rigidification effect. Tetrahedron 53, 8335-8348. (41) James, T. D., and Shinkai, S. (2002) Artificial receptors as chemosensors for carbohydrates. Top. Curr. Chem. 218, 159-200. (42) Garlick, R. L., and Mazer, J. S. (1983) The Principal Site of Non-Enzymatic Glycosylation of Human-Serum Albumin In vivo. J. Biol. Chem. 258, 6142-6146. (43) Sakaue, R., and Kajiyama, N. (2003) Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution. Appl. Environ. Microb. 69, 139-145. (44) Bielecki, M., Eggert, H., and Norrild, J. C. (1999) A fluorescent glucose sensor binding covalently to all five hydroxy groups of alpha-D-glucofuranose. A reinvestigation. J. Chem. Soc., Perkin Trans. 2, 449-455. (45) Norrild, J. C., and Eggert, H. (1995) Evidence for Monodentate and Bisdentate Boronate Complexes of Glucose in the Furanose Form - Application of 1JC-C-Coupling-Constants as a Structural Probe. J. Am. Chem. Soc. 117, 1479-1484.

Trokowski et al. (46) Yang, W., He, H., and Drueckhammer, D. G. (2001) Computer-guided design in molecular recognition: Design and synthesis of a glucopyranose receptor. Angew. Chem., Int. Ed. 40, 1714-1718. (47) Zhang, S., and Sherry, A. D. (2003) Physical characteristics of lanthanide complexes that act as magnetization transfer (MT) contrast agents. J. Solid State Chem. 171, 38-43. (48) Springsteen, G., and Wang, B. H. (2002) A detailed examination of boronic acid-diol complexation. Tetrahedron 58, 5291-5300. (49) Groziak, M. P., Ganguly, A. D., and Robinson, P. D. (1994) Boron Heterocycles Bearing a Peripheral Resemblance to Naturally-Occurring Purines - Design, Syntheses, Structures, and Properties. J. Am. Chem. Soc. 116, 7597-7605. (50) Bazzicalupi, C., Bianchi, A., Berni, E., Calabi, L., Giorgi, C., Mariani, P., Losi, P., and Valtancoli, B. (2002) Complexation properties of a new macrocyclic polyaminic ligand (L) containing amidic pendant arms: crystal structure of [PbL](ClO4)2. Inorg. Chim. Acta 329, 93-99.

BC0498976