Structure-Based Design of a Sulfonamide Probe for Fluorescence Anisotropy Detection of Zinc with a Carbonic Anhydrase-Based Biosensor

Abstract

Given the avid and selective metal binding properties of naturally-occurring metalloproteins, it is possible to exploit these systems in the development of novel sensors, i.e., "biosensors", for the detection of trace quantities of metal ions. Here, we exploit the high affinity of human carbonic anhydrase II (CAII) for zinc in the detection of nanomolar concentrations of this metal ion by fluorescence anisotropy using a fluorescein-derivatized arylsulfonamide probe, 4-aminosulfonyl[1-(4-N-(5-fluoresceinylthioureido)butyl)]benzamide (3). This probe was designed through an iterative, structure-based approach and was demonstrated to bind tightly only to the zinc-bound holoenzyme (K d ) 2.3 nM) and not the metal-free apoenzyme. Furthermore, the probe exhibits anisotropy that is proportional to the concentration of bound zinc, and this behavior can be exploited in the detection of zinc in the 10-1000 nM range. Strategies for the structure-based design of improved CAII-based metal ion biosensors are considered in view of these results.

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…In a study relevant to the binding of arylsulfonamides to CA II, Krishnamurthy et al have recently reported the binding of fluorinated benzenesulfonamides (207)(208)(209)(210)(211)(212) to BCA II. 182 By constructing a quantitative structure-activity relationship (QSAR), the investigators separated the influence of fluorination of the ring on electrostatic-Lewis basicity of ArSO 2 NH − toward the Zn II cofactor and toward the hydrogen-bond network (interactions that they assumed were dependent on pK a )-and hydrophobic-contacts of the phenyl ring with the enzyme (an interaction that they assumed was dependent on log P, the logarithm of the octanol/aqueous buffer partition coefficient)-interactions (eq 22; Figure 16A).…”

“…As mentioned previously, Krishnamurthy et al constructed a QSAR between the affinity (K d ArSO 2 NH − ) of fluorinated benzenesulfonamides (207)(208)(209)(210)(211)(212) for BCA II and pK a and log P of the ligands (eq 22; Figure 16A). 182 Assuming that the two hydrogen bonds between the enzyme and ligand were equal to one another in energy ( Figure 13B), their analysis allows the partitioning of affinity to the structural interactions of the Zn II -N bond, the hydrogen-bond network, and the hydrophobic contacts of the phenyl ring between the ligand and enzyme (Figure 18; section 10.2.4).…”

“…We included substituted benzenesulfonamides that we presume do not have hydrophobic contacts with CA II other than a conserved interaction of their phenyl rings. Quantitative structure-activity relationships (QSARs) between ΔG ∘ ArSO 2 NH − (A), ΔH ∘ ArSO 2 NH − (B), and −TΔS ∘ ArSO 2 NH − (C) and pK a and log P for the binding of fluorinesubstituted benzenesulfonamides (207)(208)(209)(210)(211)(212) to BCA II. Data are shown for QSARs in which those for 4-fluorobenzenesulfonamide (209, 4-FBS) were omitted; Krishnamurthy et al have suggested that this ligand interacts in a different way with the enzyme than do the other ligands.…”

Carbonic Anhydrase and Heavy Metals

“…In particular, Cu 2+ , Co 2+ , Zn 2+ , Cd 2+ , and Ni 2+ have been determined at concentration down the picomolar range (Fierke and Thompson, 2001;Thompson and Jones, 1993;Mey et al, 2011) by changes in fluorescence emission (Thompson et al, 2000) and excitation wavelength ratios (Thompson et al, 2002), lifetimes (Thompson and Patchan, 1995), and anisotropy (polarization) (Elbaum et al, 1996;Thompson et al, 2000). The sensitivity, selectivity, analyte binding, kinetics and stability of the biosensors have been improved by subtle modification of the protein structure by directed mutagenesis (Kiefer et al, 1995;Hunt et al, 1999;DiTusa et al, 2001;McCall et al, 2004;Burton et al, 2000).…”

Chemistry of zinc(II) fluorophore sensors

“…Further development from the CA inhibitors are sulfonamide-fluorescein conjugate 14 (Elbaum et al 1996) and dapoxyl sulfonamide 15 (Thompson et al 1999). The design of 14 was achieved by an iterative structure-based procedure in which the X-ray crystal structure of the CAII-arylsulfonamide complex was determined and analyzed for the optimal attachment point of fluorescein.…”