Fluorescent Chelates for Monitoring Metal Binding with

Biomacromolecules 2003, 4, 114-121

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Fluorescent Chelates for Monitoring Metal Binding with Macromolecules M. Islam, M. Khanin, and O. A. Sadik* Department of Chemistry, State University of New York at Binghamton, P.O. Box 6016, Binghamton, New York 13902-6016 Received July 25, 2002

Metals and radionuclides are usually coupled with proteins together with suitable ligands for therapeutic, tumor-imaging, pharmaceuticals, and biocompatibility applications. Several ligands that can strongly coordinate a given nuclide in a specific valency are already known. However, the demand for bifunctionality has limited the applications of these ligands. We hereby report the molecular design of a receptor system based on the linkage of protein to monoazo ligands. By use of basic coordination chemistry, 4-(3quinolinoazo)hydroxybenzoic acid (QABA) and derivatives were successfully conjugated to ovalbumin, bovine serum albumin, and alkaline phsophatase at a site that was distinct from the metal binding site. The presence of carboxylic acid linkage in the QABA served as a convenient bridge for protein conjugation and may allow the generic application of these ligands for bioconjugate synthesis while ensuring a high in vivo stability. The ligand-protein conjugates were characterized using UV-vis spectroscopy, Fourier transform infrared spectroscopy, thin layer charoatography, NMR, and surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The conjugate was tested for the ability to recognize nonradioactive Ga3+ at a physiological pH, and a binding constant of 1 × 1020 was recorded. Also, the in vitro testing results indicated that the fluorescent conjugates exhibited significant selectivity for gallium compared to Pb2+, Hg2+, Zn2+, Cu2+, Fe3+, and Co2+ while no responses were obtained for alkaline and alkaline earth metals. These attributes could allow these conjugates to be used as a model for imaging sensors and for metal detection. Introduction The field of immunotargeting for in vivo diagnosis of diseases consists of several diverse strategies, based on the delivery of cytotoxic proteins, conventional chemotherapy, and radioimmunotherapy. The targeting strategy, involving the selective destruction of malignant cells using immunoconjugate, has attracted considerable interest because of the apparent simplicity of the concept. Typically, the strategy involves the transport of potentially therapeutic radionuclides to the desired “site-of-action” by target-selective vehicles. The target vehicles consist of protein reagents coupled with metals/radionuclides. Numerous radiometals have been successfully coupled to antibodies using suitable, strongly coordinating multidentate ligands or chelators. Polycarboxylic acids have also been used extensively for the chelating of metal ions due to the ease of generating intramolecular anhydrides of such compounds.1,2 Some workers have reported the preparation of bis-anhydride ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DPTA).3 DTPA was coupled to human serum albumin, IgG, and fibrinogen.4-6 Many of these reagents are commercially available and have been found to produce the highest coupling efficiency in both bicarbonate buffer (pH 8.2) and borate buffer (pH 8.6). * To whom correspondence may be addressed: fax, 607-777-4478; e-mail, [email protected].

Isothiocyanates have also been used to introduce polycarboxylic chelating agents into proteins for radiophamaceutical purposes.7 Other workers have described the preparation and application of (S)-4-{2,3-bis[biscarboxymethyl)amino]propyl}phenyl isothiocyanate (CITC) for use in labeling monoclonal antibodies.7 Typical reaction condition occurred with the ratio of CITC to IgG being about 9:1, while the reaction continued for about 2 h at 37 °C and a pH range of 9.0-9.5. It was found that the incorporation of up to three or four chelators of this type did not seriously affect immunoreactivity. The in vivo behavior of a given metal-chelate complex is not easy to predict. Hence the choice of potential multidentate ligands is usually guided by conventional chelation chemistry. Once a radionuclide or an element is selected for conjugation, the next step is to develop a bifunctional form of the “matched” multidentate ligand. This step consists of a coordination site for metal complexation and a linking site for covalent coupling of any available functional group within the protein (usually the -amino group of lysine). This however, requires that the ligand have at least two binding sites. Yet, it is often difficult to synthetically introduce a reasonable linking group without changing its coordination properties. We have previously reported the molecular design of a receptor system based on the linkage of a protein to monoazo ligands.8 By use of basic coordination chemistry, the environmentally sensitive monoazo ligand was conjugated to a protein-based receptor

10.1021/bm025622m CCC: $25.00 © 2003 American Chemical Society Published on Web 12/19/2002

Fluorscent Chelates

Biomacromolecules, Vol. 4, No. 1, 2003 115

Figure 1. Structures of PAR derivatives and the new fluorescent conjugates: (1) PAR; (2) QABA; (3) PAGPA; (4) QABA-AP.

at a site distinct from the binding site. This is a limited strategy since most bioconjugates of interest in medical and pharmaceutical communities have no corresponding chromophore or indicator. On the basis of 2-pyridylazo (PAR) literature,10-16 we speculated that a chelate-protein complex would be a viable conjugate depending on the favorable arrangement of its heterocyclic nitrogen, the azo group, and the phenolic hydroxyl group before it could bind with metal ions. The existence of several pKa values should also allow a wide pH range to be covered with the same indicator even though all azo indicators are nonfluorescent. Neither PAR (Figure 1, 1) nor any of its derivatives has been previously reported to be suitable for the preparation of biological conjugates (Figure 1). Thus there is a need to develop general strategies for achieving target responses for metal binding with radionuclides. We hereby report the preparation and characterization of a novel, fluorescently active, carboxylic acid derivative of PAR (Figure 1, 2-4). These ligands were conjugated to ovalbumin, bovine serum albumin, and alkaline phosphatase. The resulting conjugate was used to test for gallium under in vitro sandwich assay format and was found to exhibit relatively higher binding affinity for gallium as compared with the non-carboxylic derivative.8 The antitumoral and therapeutic effects of gallium nitrate have been widely demonstrated in laboratory animals.17-21 The low degree of gallium toxicity appears to indicate that it may be suitable for the treatment of various tumors.18 The substitution of PAR with carboxylic acid groups is expected to ensure a high in vivo stability of the chelate, increase the solubility as well as renal clearance of the chelate. It may provide an additional functional group for coupling of the chelate to a carrier protein. Moreover, when covalently bound, the ligand may be so close to the protein such that it may no longer function effectively as a chelator. Thus we have appended a spacer arm to the carboxylic group before attaching the chelate to the protein as in 3.

Figure 2. Signal generation strategy using PAR-protein derivatives.

binding to induce high specificity without changing the coordination properties. The metal-conjugate binding can be selectively controlled using (i) size-to-charge ratio, (ii) binding constant, (iii) stoichiometry, and (iv) optimal pH. The signal generation strategy employed involves a noncompetitive assay format that utilizes the sandwich techniques such as illustrated in Figure 2. First, a receptor linked to a captor chelator is immobilized, and a metal analyte is added that chelates with the immobilized captor chelator. There is a greater number of captor chelator sites than metal analytes to ensure complete adsorption of the analytes. After this, another detector chelator with an enzyme (or fluorescent) label is added. The reaction between the enzyme-labeled chelator and the substrate takes place after eliminating the excess-labeled chelator. Then optical signal is generated by measuring either the change in the absorption of the substrate using a wavelength scan or the fluorescence from the enzyme marker. Thus, the concentration of the labeled chelator is a direct indication of the analyte concentration. Although this technique has been used in environmental immunoassay,1 to our knowledge, this strategy has not been reported for metal binding to macromolecules. Equation 1 gives the relationship between pH change in PAR with the absorbance signal as follows:

Signal Generation Strategy The use of PAR ligands for immunological applications relies on the selective recognition of the chelate-protein

pH ) pKa′ - log

{

}

(Smax - S) (S - Smin)

(1)

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where Smax is the maximum signal of the completely dissociated form, S is the signal at any given pH, and Smin is the signal of the totally undissociated (conjugated) form (HA). For fluorescence measurements, a similar relationship is obtained as shown in eq 2 pH ) pKa′ - log

[( ) ] Fmax -1 F I-

(2)

where Fmax is the fluorescence intensity of the undissociated ligand (HI) completely in the phenolate form and FI- is the intensity of the I- form at a given pH. For living organisms, a highly selective recognition mechanism is based on the stereospecific binding of an antigenic determinant (antigen, hapten epitope) together with an antibody. Hence signal generation strategies described here may provide a unique template for fingerprinting of cancerous cells in the presence of a suitable antibody. Molecular receptors that can recognize metal chelates from the ligand have now been prepared as shown in Figure 1. Experimental Section Reagents and Instrumentation. Analytical reagent (AR) grade chemicals were used throughout the experiments. All solutions were made using Nanopure water having a resistivity of 18 MΩ/cm or higher. The following chemicals were purchased from sources as indicated: From Sigma Chemicals, we have 98% 3-aminoquinoline, N-hydroxysuccinimide (NHS), ovalbumin (OVA, 98% lot 37H7015), BSA (>97%, lot 102H9308), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), Ga(NO3)3, sodium dihydrogen phosphate (NaH2PO4 (99%). Others include alkaline phosphatase (lot 96120639) and p-nitrophenyl phosphate disodium (PNPP, 5 mg/tablet) (lot 96111570) from Pierce Chemicals (Rockford, IL), CuSO4‚5H2O, Co(NO3)3‚6H2O, and FeCl3‚6H2O from Aldrich Chemicals, and Pb(NO3)3, NH2OH‚HCl, and FeSO4‚ (NH4)2SO4‚6H2O from Baker. To remove all traces of metal ions, all glassware were soaked in 3 M HNO3 for 3 days and also in water for a day prior to use. Instrumentation. All protein conjugates were characterized using a Hewlett-Packard diode array UV/visible spectrophotometer (model 8453) equipped with a 1 cm pathlength cell. Protein concentration was determined using a E1% 280 with a molecular weight of 45 000 for ovalbumin and 66 500 for BSA. Optical densities were measured using a 100-fold dilution to give an absorbance reading of 0.10.3. In addition, Fourier transform infrared spectra of lyophilized samples in KBr pellets were obtained using a Perkin-Elmer spectrophotometer (model 1600 FT-IR). The enzyme-linked immunosorbent assay (ELISA) system used for the analysis of Ga consisted of Maxisorb I 96-well microplate and microplate reader (ELX 800 UV) from BioTek Instruments. The plate reader’s “instrument control and data analysis” software was from Biotek KC-4 Software. The characterization of all protein conjugates was performed using a Ciphergen protein chip array system equipped with a surface-enhanced laser desorption ionization time-of-flight

mass spectrometer (SELDI-TOF-MS) detector. The pipets used are capable of delivering 1 mL having different adjustable ranges between 20 and 200 µL (single-channel pipet). Some of the range includes 0-25 µL adjustable, positive displacement pipets, eight-channel 50-200 µL adjustable pipets, and borosilicate glass tubes (12 × 75 or 13 × 100 mm). Lobconco, FreeZone 12 L, model 77540, was used for freeze-drying. Stock Solutions. A stock solution of metals (1.0 × 10-2 M) was prepared by dissolving a calculated amount of the salts (99.99%) in an appropriate amount of 1 M nitric acid. This was diluted to the desired volume and then further diluted to 1 × 10-2 M before use. A buffer solution of acetate was used for pH adjustment at 0.1 ( 0.001 M. A stock solution of 0.01 M Ga(NO3)3 was prepared using 0.2557 g of Ga(NO3)3 plus 2 drops of dilute HNO3. This was made up to 1 L. The following buffers were used for the preparation of ELISA standards and enzyme tracer dilution: (i) carbonate-containing buffer having pH of 9.6, 0.32 g of Na2CO3, 0.533 g of NaHCO3, and 0.2 g of NaN3 dissolved and diluted to 1 L; (ii) phosphate-buffer saline (PBS) of pH 7.2, 8.0 g of NaCl, and 0.123 of Na2H2PO4.7H2O; (iii) 0.1 M acetate buffer of pH 5.5, 13.6 g of sodium acetate and 1.05 mL of glacial acetic acid diluted to 1 L in a volumetric flask. The substrate solution was made from 9 mL of acetate buffer and 1.0 mL of diethanolamine, 0.20 g of MgCl2‚6H2O, and two PNPP tablets. The 100 mM sodium acetate buffer, having pH 5.5, was used for both washing and incubation of standards and samples. The metal solutions used for interference tests were prepared using different amounts of Ga(III) and interference metal ions. Synthesis and Characterization of QABA (4-(3-Quinolinoazo)-3-hydroxybenzoic Acid)). QABA was synthesized according to Scheme1. A solution of 1 mL of glacial acetic acid and 1.50 g (10.40 mmol) of finely powdered 3-aminoquinoline was dissolved in a minimum amount of absolute ethanol (2 mL). Sodium nitrite (0.5 g) was dissolved in 4 mL of water, and 1 mL of concentrated sulfuric acid solution was added in a roundbottom flask. The resulting solution was stirred for 6 h at 0-5 °C. 3-Hydroxybenzoic acid (1.44 g, 10.40 mmol) was dissolved in minimum amount of absolute ethanol (3 mL) and was added to the cooled diazotized solution. Then the reaction mixture was stirred for 6 h at 0-5 °C and then overnight under room temperature. A deep red precipitate was observed during the coupling reaction. The resulting mixture was filtered, extracted with ethyl acetate, and separated by column chromatography using ethyl acetate and petroleum ether (1:4) as eluting solvent. QABA was purified by silica gel column chromatography. The purity was assessed by thin layer chromatography (TLC) and high-field (300 MHz) NMR spectroscopy. NMR signals of 4-(3-quinolinoazo)-3-hydroxybenzoic acid: 1H NMR (CDCl3, 300 MHz), δ 13.66 (s, 1H), 9.58 (d, J ) 2.31 Hz, 1H), 8.91 (s, 1H), 8.85 (d, J ) 9.51 Hz, 1H), 8.71 (d, J ) 2.16 Hz, 1H), 8.23 (d, J ) 8.4 Hz,1H), 8.14 (d, J ) 7.98 Hz, 1H), 8.05 (d, J ) 7.26 Hz, 1H) 7.70 (t, 1H, J ) 8.415 Hz) 7.86 (t, 1H, J ) 7.90 Hz).

Fluorscent Chelates


Scheme 1

Scheme 2

The Fourier transform infrared (FTIR) spectrum revealed the presence of ν(OH), ν(CdO), and ν(C-N) bands of phenolic OH, carboxylic acid, and quinoline ring at 3300, 1660, and 1310 cm-1, respectively. Yield obtained was 8%. Synthesis of PAR-Linked Ethylpentanoic Acid (4-(2Pyridylazo)(3-hydroxy-5-phenoxy)ethylpentanoic Acid, PAHPA). The synthesis of a new PAR-linked carboxylic acid (PAHPA) was carried out with a minimal number of steps. The first step in the synthesis of PAHEP (ethyl 5-(4(2-pyridylazo)-3-hydroxyphenoxy)pentanoate) involves the reaction of PAR, 4-(2-pyridylazo)resorcinol, with 5-bromovalerate in DMF as per Scheme 2. The second step involves the hydrolysis of the corresponding PAR-linked ester, PAHEP. A solution of 10 mL of DMF, 0.25 g (1.05 mmol) of finely powdered PAR, and 0.100 mL (1.05 mmol) of 5-bromopentanoic acid ester (ethyl 5-bromovalerate) were added to a round-bottom flask. The resulting solution was stirred for 8 h under a nitrogen atmosphere at 90 °C. Then the reaction mixture was stirred overnight under room temperature. A deep red precipitate was observed during the reaction. The resulting mixture was filtered and subjected for workup. NMR spectroscopy was then performed after separation of the reaction mixture by silica gel column chromatography. From the NMR spectra, the following signals were obtained for PAHEP: 1H NMR (DMSO-d6, 300 MHz) 13.92 (s, 1H), 8.39 (d, 1H, J ) 5.66 Hz), 7.68 (t, 1H, J ) 7.28 Hz), 7.63 (d, 1H, J ) 7.88 Hz), 7.39 (d, 1H, J ) 9.30 Hz), 7.10 (t, 1H, J ) 6.06 Hz), 6.35 (dd, 1H, J ) 2.83, 9.30 Hz), 6.09 (d, 1H, J ) 2.63 Hz), 3.93 (q, 2H, J ) 7.28 Hz), 3.84 (t, 2H, J ) 5.86 Hz), 2.21 (t, 2H, J ) 6.47 Hz), 1.62 (m,

4H), 1.04 (t, 4H, J ) 6.87 Hz). The yield of 78% was obtained for PAHEP. Hydrolysis of PAHEP (Ethyl 5-(4-(2-Pyridylazo)-3hydroxyphenoxy)pentanoate). PAHEP (0.5 g) was refluxed with 5 mL of 5% KOH solution using methanol as solvent for 3 h. After hydrolysis, the resulting mixture was filtered and subjected for workup. NMR spectroscopy was then performed after separation of the reaction mixture by silica gel column chromatography. From NMR results, it was confirmed that the new PAR-linked carboxylic acid is obtained. The NMR signals obtained are as follows: 1H NMR ((DMSO-d6, 300 MHz) 13.96 (s, 1H), 12.01 (s, 1H), 8.43 (d, 1H, J ) 5.25 Hz), 7.68 (t, 1H, J ) 7.28 Hz), 7.63 (d, 1H, J ) 7.88 Hz), 7.41 (d, 1H, J ) 9.30 Hz), 7.10 (t, 1H, J ) 6.06 Hz), 6.36 (dd, 1H, J ) 2.83, 9.30 Hz), 6.10 (d, 1H, J ) 2.63 Hz), 3.86 (t, 2H, J ) 5.86 Hz), 2.25 (t, 2H, J ) 6.47 Hz), 1.67 (m, 4H). The yield of 85% was recorded for this reagent. Protein Conjugation. QABA-protein conjugates were prepared using a modified procedure previously described by Tijssen et al.21,22 This procedure was carried out in two steps. In step 1, the NHS derivative of QABA was generated. This was used either directly or stored until needed. In step 2, the reaction of activated QABA with protein was achieved. Briefly, the procedure employed is as follows: 0.0318 g (108.5 µmol) of QABA was reacted with 0.020 g (173 µmol) of NHS with 0.0249 g (130 µmol) of EDC. The mixture was added to 5 mL of dry DMF at room temperature with constant stirring for 6 h. NHS-activated QABA was formed as the carbodiimide became transformed to urea. This was later isolated after dilution with water and extracted with

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ethyl acetate to remove carbodiimide, which could deactivate the protein in the subsequent step. During step 2, the NHSactivated QABA and ovalbumin were mixed together at 273 K for 1 h in 0.1 mmol/L PBS (pH 7.2). The mixture was allowed to react at 4 °C or ice-cooled and left overnight. The mixture was extensively dialyzed against phosphate buffer (pH 7.2) to remove the unreacted QABA before being freeze-dried. Similar reaction procedures were used to generate the enzyme-labeled chelator (QABA-AP). Conjugate Characterization. Mass spectrometric characterization of all protein conjugates was performed using a Ciphergen protein chip array system equipped with SELDITOF-MS detector. A hydrophobic-surface, array-protein chip was used for the experiment. A Ciphergen H-4 chip was washed with acetonitrile, and the samples were placed on the H-4 protein chip array and washed away with PBS buffer (90% PBS in acetonitrile). After sample processing, the chip was dried and energy absorbing molecule (EAM) was applied to each spot of the H-4 protein chip to facilitate desorption and ionization in the protein chip reader. The retained proteins were eluted from the protein chip array using laser desorption/ionization. Ionized proteins were detected, and their mass was accurately determined using time-of-flight mass spectrometry. Results and Discussions Protein conjugates were characterized using NMR, FTIR, TLC, UV-vis, and mass spectroscopy as described in the Experimental Section. The NMR results confirmed that new PAR-linked carboxylic acid was obtained. It also showed that both QABA and its ester hydrolysis were formed. FTIR experiments were carried out to determine the presence of major amide functional groups in the conjugates. The spectra range was within 4000-200 cm-1. The most important QABA functional groups are those having absorption bands of stretching vibration frequencies in the range 1200 and 1240 cm-1. These are assigned to the C-OH vibrational modes and NdN- functions (1130-1160 cm-1). The presence of both symmetric and asymmetric N-H stretching bands in the range 3550-3450 and 3450-3350 cm-1, respectively, was observed for the QABA-OVA, QABABSA, and QABA-AP conjugates. In addition, amide II bands with medium intensities arising from deformation modes at 1650-1580 cm-1 were also present. The presence of CdO at 1650 cm-1 and NH at 1530-1540 cm-1 showed the characteristic bands of amides derived from the conjugated protein, which were absent from the QABA spectra. The IR spectra of the protein-bonded QABA exhibited a conspicuous feature of strong, broad bands of C-H for protein (3100 cm-1), OH (3300 cm-1), and C-O (1100 cm-1) functional groups that were wider and more intense as a result of the presence of the protein. Ciphergen protein chip mass spectrometry (CP-SELDITOF-MS) was used to determine the molecular weight and fragment ions of the protein adduct and the accompanying neutral molecules formed during fragmentation. CP-TOFMS results indicated that the QABA was conjugated to the proteins. With a molecular weight of 66500 for BSA, a 1:1

Islam et al.

Figure 3. UV-vis spectra of QABA with Zn2+ and Hg2+ ions in 0.001 M PBS buffer (pH 7.2): (a) 1.0 × 10-6 M QABA; (b) 1.0 × 10-5 M Zn2+; (c) 1.0 × 10-6 M Hg2+.

QABA-BSA conjugate was estimated from the CP-SELDITOF-MS measurements. The QABA-BSA corresponded to a peak at ∼66800 amu. This also corresponded to the degree of conjugation estimated from the UV-vis measurements. Using a molecular weight of 45 000 for OVA, a 1:2 conjugate OVA (QABA)2 was estimated from the MS measurements, which corresponded to peak at ∼45600 amu. This also corresponded to the degree of conjugation estimated from UV-vis measurement. Molecular Recognition. According to the signal generation strategy described earlier, the ligand must retain its molecular recognition properties after linkage with the proteins. Hence, the conjugate must bind to the metal ion of interest without changing its coordination properties. We tested the ability of the carboxylic acid derivative (QABA) and its protein conjugate to bind to metals ions using absorbance measurements both in the solution and in solid phases. In the solution phase, the UV-vis spectra of QABA showed a characteristic peak in the region at ∼400 nm. This peak shifted to higher wavelength (∼500 nm) when it formed a chelate with several metal ions including the group 1 and II metals. Figure 3 shows the example of absorbance spectra obtained for zinc and mercury ions. The ligands were found to bind to most of the metals tested except the alkali metals. The transition of QABA and the chelate occurring at UVvis spectra can be attributed to the electronic π to π* (anti-π bond) taking place in both reagents. The absorption spectrum of QABA-chelate was due to the loss of hydrogen atom from the ortho-OH, thus resulting in ionization of the QABA molecule. In addition, the lone pair of electrons in the oxygen atom can be conjugated with the phenyl ring to lower the energy of the π* bond, thus resulting in lower energy difference of π to π* transition. In that case, the absorbance shifted to longer wavelength. UV-vis characterization using PAHPA also exhibited a similar behavior, thus confirming that the new derivatives in Figure 1, structures 2 and 3, could bind metal ions without changing the coordination properties. Sensitivity and Coordination to Gallium. To confirm the sensitivity of this protein-azo complex to different metal ions, the UV-vis spectrum was also recorded in 0.001 M phosphate buffer of pH 7.4. The spectrum of the solution was determined for several dilutions of metal ions made up in the same buffer. A typical spectrum recorded is shown in Figure 4. Two absorption maxima were obtained at 280 nm


Fluorscent Chelates Table 1. Binding Constants Recorded for QABA-Metal Complexes metal ion Ga3+ Pb2+ Hg2+ Zn2+ Cu2+ Fe3+ Co2+

metal ion concn (nM)

z/r (pm) × 1000

log βa

stoichiometry

λmax (nm)

optimum pH

IC50b

1× 1 × 106 1 × 106 1 × 106 1 × 106 1 × 106 1 × 106

4.84 1.67 1.81 2.70 2.86 4.69 2.70

20.30 4.57 5.95 5.30 4.43 7.50 4.89

1:2 1:1 1:1 1:1 1:1 1:2 1:1

505 510 512 498 520 515 510

7.0 10.0 5.0 5.2 4.5 5.0 4.5

1 × 10-6 NMc NM NM NM NM NM

106

a β ) binding constant. b The response of sandwich assay to metals was characterized with respect to gallium. This response is expressed as the concentration of the gallium required reducing, or inhibiting, the assay response by 50%. This is defined as 50% inhibition level or IC50. The data showed that none of the potential cross-reactants exhibited measurable cross reactivity relative to gallium(III). c NM ) not measurable. The responses were too low to allow any significant determination of the LC50.

Figure 5. Sensitivities of QABA-OVA conjugate for different metal ions: (a) 1 × 10-5 M QABA-OVA; (b) metal ion concentration 1 × 10-6 M in 0.001 M PBS buffer (pH 7.2).

Figure 4. UV-vis spectra of QABA conjugated to ovalbumin (1 × 10-5 M QABA-OVA) at different Ga3+ concentrations in 0.001 M PBS buffer (pH 7.2): (a) 2.5 × 10-5 M; (b) 5.0 × 10-5 M; (c) 7.5 × 10-5 M; (d) 1 × 10-4 M.

for protein and 410 nm for QABA. The UV-vis absorbance at 280 nm in Figure 4 was due to the conjugated proteins. These results indicated that QABA was successfully conjugated to the proteins. From absorbance measurements, the relative ratio of OVA to QABA was estimated to be 1:2. Ga formed a 1:2 ratio of chelate to QABA, i.e., Ga (QABA)2, by coupling to the N and O atoms. Since gallium has a coordination of 6, the geometry is octahedral. Gallium is used in tumor detection and is believed to exhibit therapeutic properties.20-22 After different metals were added to QABAOVA, the QABA signal decreased due to the formation of metal-QABA complex, which was found to be dependent on the concentration of the metal. The maximum spectra attributed to the protein at 280 nm remained unchanged. This result indicated that the gallium ions could still bind to the QABA ligand even after conjugation, while the activity of the protein was also still retained. A similar behavior was recorded for the PAHPA ligand. Coordination with Different Metals. We then studied the ability of this new chelating agent (and its protein derivatives) to bind to different metal ions. As expected (Figure 5), we found that in solution, QABA formed tridentate 1:2 and 1:1 complexes with various metal ions including Ga3+, Hg2+, Pb2+, Cu2+, Co2+, and Zn2+. As noted for PAR ligands, the formation of complex in QABA was

also believed to occur through the coordination at the pyridine nitrogen, the azo nitrogen further from the heterogeneous cyclic ring, and the ortho hydroxyl group (Table 1). The most stable form of the QABA complex is its tridentate 1:2 chelate with Ga3+. However, the reagent by itself in solution did not show any selectivity; therefore we tested the utility in a sandwich assay format. Monitoring Conjugate Binding with Gallium. To optimize the specificity for gallium, the QABA-protein conjugate was immobilized on a solid phase format (Figure 6). The sensitivities of the quantitative chelate assays for different concentrations were determined using QABAOVA and QABA-BSA, and the native QABA as the immobilized chelator. QABA-OVA and QABA-BSA conjugates were found to exhibit significant selectivity for gallium, while QABA by itself showed no significant absorbance values beyond that of the blank. The increased sensitivity of QABA-OVA conjugate relative to QABABSA could be attributed to the higher ratio of coupling during conjugation. The selectivity of the QABA-OVA conjugates originated from a stronger attachment to the plate, thus decreasing the loss of QABA-protein conjugates during the assay. This attachment helped to stabilize the ligand in the active site and the resulting protein conformational change constituted the basis for the selectivity observed. In contrast, the relatively weaker attachment of QABA:BSA or QABABSA to the plate could result in higher loss of PAR-protein molecules. The reaction was carried out under various conditions, including different metal ions, pH, ionic strength, and concentrations. Here we observed that QABA formed a

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Figure 6. Four-parameter fit calibration curve obtained for the detection of Ga3+ using the QABA-OVA conjugate in 0.001 M PBS buffer (pH 7.2): (a) 1 × 10-5 M QABA-OVA in PBS. log concentrations in ascending order were 1 × 10-6, 5 × 10-6, 1 × 10-7, 5 × 10-7, 1 × 10-8, and 5 × 10-8 M.

complex only with Ga ions. It appears that the enhanced selectivity obtained was due to the changes in the metal/ size-to-charge ratio. We expected metals, such as iron(III), exhibiting comparable r/z ratios to Ga3+ to display similar characteristics. We actually recorded low levels of interference with Fe3+, which was easily removed by reaction with hydroxylamine. The presence of the pyridine nitrogen, the azo nitrogen, and the ortho-hydroxyl group enabled strong binding with the metal, which was greatly influenced by the charge. In addition to the size-to-charge ratio, it appears that other factors might have affected the selectivity for determination of Ga(III). These factors include the binding constant, the optimum pH, and the stoichiometry (Table 1). Also, the remarkable assay selectivity observed using QABA-protein conjugate was substantiated by actual independent data obtained for the complexation of the different metals tested using the native QABA reagent. Fluorescence Measurements. Measuring either the change in absorption of the substrate or the fluorescence from the protein-chelate conjugate with the metal ions resulted in a sensitive optical signal. Therefore, the fluorescence properties of QABA ligand were investigated. We found that it was fluorescently active with excitation maxima at 345 nm and emission maxima at 460 nm. It was further observed that the fluorescence could be quenched by oxygen. The QABA was exposed to different molecules, including pure nitrogen and pure oxygen in ethanol solution at room temperature (Figure 7). The QABA was found to undergo fluorescence quenching by oxygen by ∼45%. This result may provide a way of immobilizing the chelator for solution phase binding reactions. Conclusions We have synthesized and characterized novel carboxylic acid derivatives of monoazo chelate and tested the ability of the reagent to form complex with Ga3+ at physiological pH. The ligands were conjugated to ovalbumin, bovine serum

Figure 7. Typical fluorescence spectra of 1 × 10-5 M QABA in the presence of pure nitrogen, air, and oxygen in ethanol. Excitation wavelength was 345 nm and emission maximum was 460 nm.

albumin, and enzymes. Characterization of these conjugates using NMR, FTIR, MS, TLC, and UV-vis confirmed that the conjugates have been successfully prepared. When tested for gallium using in vitro sandwich assay format, the chelate was shown to exhibit significant selectivity and detection in the nanomole per liter range was achieved. The ligand was also found to fluoresce and could be used for quantitative detection of metals and for inherent development of a selective immunoconjugate in therapeutic applications. Acknowledgment. We thank Dr. J. Schulte for helping with the NMR analysis used in this work. We acknowledge the following agencies for funding: US-EPA, Center for Advanced Technology through the IEEC, and NSF. References and Notes (1) Blake, C.; Gould, B. J. Analyst 1984, 109, 533-547. (b) Szurdoki, F.; Kido, H.; Hammock, B. D. Bioconjugate Chem. 1995, 6, 145. (2) Gansgow, O. A. Nucl. Med., Biol. 1991, 18, pp 369-381. (b) Echelman, W. C. J. Pharm. Sci. 1975, 64, 704-706. (3) Hnatowich, D. J. Science 1983, 220, 613-615. (4) Layne, W. W.; et al. J. Nucl. Med. 1982, 23, 627-630. (5) Subramanian, K. M.; Wolf, W. J. Nucl. Med. 1990, 31, 480-488. (6) Bickel, U.; et al. Bioconjugate Chem. 1994, 5, 31-39. (7) Li, M.; Meares, C. F. Bioconjugate Chem. 1993, 4, 275-283.

Fluorscent Chelates (8) Xu, H.; Lee, E.; Sadik, O. A.; Bakhtiar, R.; Drader, J.; Hendricson, C. Anal. Chem. 1999, 71, 5271. (9) Gao, J.; Guangling, H.; Kanf, J.; Bai, G. Talanta 1993, 40 (No. 2), 195. (b) Flaschka, H. A.; Barnnard, A. J., Jr. Chelates in Analytical Chemistry; Marcel Dekker: New York, 1972; Vol. 4, p 114. (10) Flaschka, H. A.; Barnard, A. J. Chelates in Analytical Chemistry; Marcel Dekker: New York, 1972; Vol. 4, 127. (11) Sarzanini, C.; Porta, V.; Mentasti, E. New J. Chem. 1989, 13, 463. (12) Gao, J., Guanglin H., Kanf, J., Bai G., Talanta 1993, 40 (No. 2), 195. (13) Rao, A.; Malik, A.; Kapoor, J. Talanta 1993 (No. 2), 201. (14) Khoo, S.; Ming, S.; Cai, Q.; Khan, M.; Guo, S. Electroanalysis 1997, 9 (No. 1), 2. (15) Ivanov, V.; Morozko, S. J. Anal. Chem. 1996, 51 (No. 10), 989. (16) Bae, Z.; Park, Y.; Lee, S.; Jeon, W.; Chang, H. Bull. Korean Chem. Soc. 1996, 17 (No. 11), 995.

Biomacromolecules, Vol. 4, No. 1, 2003 121 (17) Collery, P.; Morel, M.; Millart, H.; Perdu, D.; Lavaud, F.; Anghileri, L.; Pluot, M.; Choisy, H.; Pechery, C. Metal Ions in Biology and Medicine; Collery, P., Poirier, L., Etienne, J., Eds.; Libbey Eurotext: Paris, 1994. (18) Collery, P.; Millart, H.; Chiosy, H. Anticancer Res. 1992, 12, 1920. (19) Wong, H.; Terner, U. K.; English, D.; Noujaim, B. C.; Hill, J. R. Int. J. Nucl. Med. Biol. 1980, 7, 9. (20) Hart, M. M.; Adamson, R. H. Proc. Natl. Acad. Sci. U.S.A. 1971, 68 (No. 7), 1623. (21) Tijssen, P. Practice and Theory of Enzyme Immunoassays; Elsevier: New York, 1985. (22) Harlow, E., Lane, D., Eds. Antibodies: A laboratory Manual; Cold Spring Habor Laboratory: Cold Spring Habor, NY, 1988.

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Fluorescent Chelates for Monitoring Metal Binding with

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