Anionic Substrates Research Articles

Synthesis and Evaluation of a New Series of Tri-, Di-, and Mono-N-alkylcarbamylphloroglucinols as Bulky Inhibitors of Acetylcholinesterase

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as a new series of bulky inhibitors of acetylcholinesterase that may block the catalytic triad, the anionic substrate binding site, and the entrance of the enzyme simultaneously. Among three series of phloroglucinol-derived carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols (1-4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8), and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12), tridentate inhibitors 1-4 are the most potent inhibitors of mouse acetylcholinesterase. When different n-alkylcarbamyl substituents in tridentate inhibitors 1-4 are compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All inhibitors 1-12 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. Thus, tridentate inhibitors 1-4 are supposed to be hydrolyzed to bidentate inhibitors 5-8 after the enzyme catalysis. Subsequently, bidentate inhibitors 5-8 and monodentate inhibitors 9-12 are supposed to yield monodentate inhibitors 9-12 and phloroglucinol, respectively, after the enzyme catalysis. This means that tridentate inhibitors 1-4 may act as long period inhibitors of the enzyme. Therefore, inhibitors 1-4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease. Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.

Modulation of cellular responses on engineered polyurethane implants

An in vivo rat cage implant system was used to study the effect of polyurethane surface chemistries on protein adsorption, macrophage adhesion, foreign-body giant cell formation (FBGCs), cellular apoptosis, and cytokine response. Polyurethanes with zwitterionic, anionic, and cationic chemistries were developed. The changes in the surface topography of the materials were determined using atomic force microscopy and the wettability by dynamic contact angle measurements. The in vitro protein adsorption studies revealed higher protein adsorption on cationic surfaces when compared with the base, while adsorption was significantly reduced on zwitterionic (**p < 0.01) and anionic (*p < 0.05) polyurethanes. Analysis of the exudates surrounding the materials revealed no differences between surfaces in the types or levels of cells present. Conversely, the proportion of adherent cells undergoing apoptosis, as determined by annexin V-FITC staining, increased significantly on anionic followed by zwitterionic surfaces (60 + 5.0 and 38 + 3.7%) when compared with the base. Additionally, zwitterionic and anionic substrates provided decreased rates of macrophage adhesion and fusion into FBGCs, whereas cationic surfaces promoted macrophage adhesion and FBGC formation. Visualization of the F-actin cytoskeleton by Alexa Fluor 488 phalloidin showed a significant delay in the cytoskeletal fusion response on zwitterionic and the anionic surfaces. The real-time polymerase chain reaction (PCR) analysis of proinflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-10) and pro-wound healing cytokines (IL-4 and TGF-β) revealed differential cytokine responses. Cationic substrates that triggered stimulation of TNF-α and IL-4 were associated with more spread cells and higher FBGCs, whereas zwitterionic and anionic substrates that suppressed these cytokines levels were associated with less spread cells and few FBGCs. These studies have revealed that zwitterionic and anionic polyurethane surface chemistries can not only reduce nonspecific adhesion, fusion, and inflammatory events but also effectively promote cellular apoptosis in vivo.

Ring-Cleaving Dioxygenases with a Cupin Fold

Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an Fe(II) center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O(2), resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O(2) is supported by model chemistry.

Cost-Effective Filter Materials Coated with Silver Nanoparticles for the Removal of Pathogenic Bacteria in Groundwater

The contamination of groundwater sources by pathogenic bacteria poses a public health concern to communities who depend totally on this water supply. In the present study, potentially low-cost filter materials coated with silver nanoparticles were developed for the disinfection of groundwater. Silver nanoparticles were deposited on zeolite, sand, fibreglass, anion and cation resin substrates in various concentrations (0.01 mM, 0.03 mM, 0.05 mM and 0.1 mM) of AgNO3. These substrates were characterised by SEM, EDS, TEM, particle size distribution and XRD analyses. In the first phase, the five substrates coated with various concentrations of AgNO3 were tested against E. coli spiked in synthetic water to determine the best loading concentration that could remove pathogenic bacteria completely from test water. The results revealed that all filters were able to decrease the concentration of E. coli from synthetic water, with a higher removal efficiency achieved at 0.1 mM (21–100%) and a lower efficiency at 0.01 mM (7–50%) concentrations. The cation resin-silver nanoparticle filter was found to remove this pathogenic bacterium at the highest rate, namely 100%. In the second phase, only the best performing concentration of 0.1 mM was considered and tested against presumptive E. coli, S. typhimurium, S. dysenteriae and V. cholerae from groundwater. The results revealed the highest bacteria removal efficiency by the Ag/cation resin filter with complete (100%) removal of all targeted bacteria and the lowest by the Ag/zeolite filter with an 8% to 67% removal rate. This study therefore suggests that the filter system with Ag/cation resin substrate can be used as a potential alternative cost-effective filter for the disinfection of groundwater and production of safe drinking water.

Glyphosate and ATP binding by mononuclear Zn(ii) complexes with non-symmetric ditopic polyamine ligands

Binding of Zn(II) by the ditopic ligands L1py, L2py and L1para, composed of a cyclam unit linked to the linear polyamines 1,4,8,11-tetraazaundecane (L1py and L2para) and 1,4,7-triazaheptane (L2py) via a 2,6-dimethylpyridinyl (L1py and L2py) or a 1,4-dimethylbenzyl spacer (L2para), has been analyzed by means of potentiometric and (1)H and (13)C NMR measurements. All ligands form stable mononuclear Zn(II) complexes in a wide pH range, featuring the metal ion bound to the macrocyclic unit. The open-chain polyamine unit can easily bind several protons in aqueous solution affording protonated metal complexes at neutral and acidic pH values. These complexes behave as bifunctional receptors for the anionic substrates N-(phosphonomethyl)glycine (glyphosate or PMG) and ATP. Potentiometric, (1)H and (31)P NMR measurements show that the Zn(II) complex with L1py is the better receptor for both substrates, thanks to the simultaneous presence of a pyridine linker functionalized at its 2,6 positions and of a flexible linear tetraamine chain. In fact, these structural features allow a stronger interaction of PMG and ATP with both the protonated tetraamine moiety and the Zn(II)-cyclam core.

Conjugated Polymer-Based Real-Time Fluorescence Caspase Assays

We have developed conjugated polyelectrolyte-based fluorescence turn-on assays for caspase 3 and 8. These assays are composed of a cationic polyphenylene ethynylene polymer PPE4+ and p-nitroaniline modified caspase peptide substrate. The fluorescence of the assay is initially turned-off because of the efficient quenching of the polymer by p-nitroaniline moiety on anionic peptide substrates. A turn-on effect is observed due to the cleavage of the peptide by the enzyme and formation of the neutral p-nitroaniline unit which has no quenching on the polymer. We validated this assay design and obtained kinetic parameters of caspase 3 and caspase 8. These assays demonstrated good sensitivity as in pmol/L (0.1 units/mL) for caspase 3 and nmol/L (0.2 units/mL) for caspase 8. This method also showed high specificity by using caspase 3 assay as a model system and the results demonstrated that other proteases including caspase 8, papain, pepsin, and trypsin did not show observable fluorescence turn-on effect. The dose-response curve of a caspase inhibitor Z-VAD-FMK was evaluated by caspase 3 assay, by which the IC(50) value was determined to be 0.73 μM and was in a good agreement with the literature reported value at 0.62 μM. This design could be applied into the in vitro screening of small molecular inhibitors for drug discovery.

Anion deposition into ferritin

When the iron core of equine spleen ferritin is reduced, anions in solution cross the protein shell and enter the ferritin interior as part of a charge balancing reaction. Anion sequestration inside ferritin during iron core reduction was monitored using ion selective electrodes, inductively coupled plasma emission, and energy-dispersive X-ray spectroscopy. The requirement for anion translocation to the ferritin interior occurs because upon iron core reduction, two OH− ions per iron are released or neutralized inside ferritin leaving a net positive charge. Halides and oxoanions were tested as anionic substrates for this reaction. A general trend for the halides showed that the smaller halides accumulated inside ferritin in greater abundance than larger halides, presumably because the protein channels restrict the transfer of the larger anionic species. In contrast, oxoanion accumulation inside ferritin did not show selectivity based on size or charge. Vanadate and molybdate accumulated to the highest concentrations and nitrate, phosphate and tungstate showed poor accumulation inside ferritin. Fe(II) remains stably sequestered inside ferritin, as shown by electron microscopy and by column chromatography. Upon oxidation of the iron core, the anions are expelled from ferritin, and OH− ions coordinate to the Fe(III) to form the original Fe(O)OH mineral. Anion transport across the ferritin protein shell represents an important mechanism by which ferritin maintains proper charge balance inside the protein cavity.

Mechanism of anion selectivity and stoichiometry of the Na + /I - symporter (NIS)

I(-) uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na(+)/I(-) symporter (NIS) with an electrogenic 2Na(+):1I(-) stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I(-) transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the K(m) for the anion substrates. Unlike WT NIS, which mediates symport of Na(+) and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 21 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na(+)/galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.

Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells

Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.

Molecular model of human heparanase with proposed binding mode of a heparan sulfate oligosaccharide and catalytic amino acids

Heparan sulfate is abundantly present in the extracellular matrix. As other glycosaminoglycans, it is synthesized in the Golgi apparatus and then exposed on the cell surface. The glucuronidase activity of human heparanase plays a major role in the structural remodeling of the extracellular matrix, which underlies cell migration, hence tumor invasion. Heparanase is therefore a major target for anti-cancer treatment. Several inhibitors of its enzymatic activity have been synthesized. However, their design is limited by the absence of experimental structure of the protein. Homology modeling is proposed based on the structure of the endoxylanase from Penicillium simplicissimum co-crystallized with a series of xylan oligosaccharide. The new heparanase model is consistent with the few experimental data suited for the validation of such work. Furthermore, the presence of natural substrates in the template structure allowed us to propose a binding model for a hydrolyzed heparin sulfate pentasaccharide. Several lysine residues have been identified to play a key role in binding to the anionic polysaccharide substrate. In addition, two phenylalanine residues are also potentially important for the interaction with the substrate. The enzymatic mechanism investigated in the light of this new model allows for the proposal of several amino acids that can influence the protonation state of the nucleophile and the proton donor.

Exploring the Binding Ability of Polyammonium Hosts for Anionic Substrates: Selective Size-Dependent Recognition of Different Phosphate Anions by Bis-macrocyclic Receptors

Binding of mono-, di-, and triphosphate, adenosine diphosphate (ADP), and adenosine triphosphatase (ATP) with receptors L1-L3, composed of two [9]aneN(3) units separated by a 2,9-dimethylene-1,10-phenanthroline (L1), a 2,6-dimethylenepyridine (L2), or a 2,3-dimethylenequinoxaline (L3) spacer, has been studied by means of potentiometric titrations, (1)H and (31)P NMR measurements in aqueous solutions, and molecular modeling calculations. In the case of inorganic phosphates, the binding properties of the receptors appear to be determined by their geometrical features, in particular the distance between the two [9]aneN(3) units imposed by the spacer separating the two macrocyclic units. While L1 is able to selectively bind triphosphate over di- and monophosphate, L3 selectively coordinates the smaller monophosphate anion. Finally, L2 shows preferential binding of diphosphate. (1)H and (31)P NMR measurements show that the complexes are essentially stabilized by charge-charge and hydrogen-bonding interactions between the anion and the protonated amine groups of the macrocyclic subunits of the receptors. Molecular dynamics simulations suggest that the larger distance between the two macrocyclic units of L1 allows this receptor to form a larger number of hydrogen-bonding contacts with triphosphate, justifying its selectivity toward this anion. Conversely, in the case of L3, the two facing [9]aneN3 units give rise to a cleft of appropriate dimensions where the small monophosphate anion can be conveniently hosted. Considering nucleotide coordination, L1 is a better receptor for ATP and ADP than L2, thanks to the higher ability of phenanthroline to establish stabilizing π stacking and hydrophobic interactions with the adenine units of the guests.

Rectification of Nanopores at Surfaces

At the nanoscale, methods to measure surface charge can prove challenging. Herein we describe a general method to report surface charge through the measurement of ion current rectification of a nanopipette brought in close proximity to a charged substrate. This method is able to discriminate between charged cationic and anionic substrates when the nanopipette is brought within distances from ten to hundreds of nanometers from the surface. Further studies of the pH dependence on the observed rectification support a surface-induced mechanism and demonstrate the ability to further discriminate between cationic and nominally uncharged surfaces. This method could find application in measurement and mapping of heterogeneous surface charges and is particularly attractive for future biological measurements, where noninvasive, noncontact probing of surface charge will prove valuable.

Cyclen-Based Lanthanide Complexes as Luminescent Anion Receptors

This review highlights the research carried out during the last decade or so in the field of anion coordination using cyclen-based lanthanide complexes as luminescent receptors (cyclen = 1,4,7,10-tetraazacyclododecane). Herein, the fundamental concepts explaining the particularly good affinity between cyclen derivatives and lanthanides and the way to sense the interaction with anionic substrates are firstly discussed. This is followed by a selection of examples from the recent literature describing these cationic receptors as powerful optical sensors of environmental, biological and pharmaceutical relevance. Keywords: Lanthanides, cyclen, anions, complexation, detection, luminescence, sensors, thiocyanate, polycarboxylates, metalloreceptors

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Cyclen-Based Lanthanide Complexes as Luminescent Anion Receptors

This review highlights the research carried out during the last decade or so in the field of anion coordination using cyclen-based lanthanide complexes as luminescent receptors (cyclen = 1,4,7,10-tetraazacyclododecane). Herein, the fundamental concepts explaining the particularly good affinity between cyclen derivatives and lanthanides and the way to sense the interaction with anionic substrates are firstly discussed. This is followed by a selection of examples from the recent literature describing these cationic receptors as powerful optical sensors of environmental, biological and pharmaceutical relevance. Keywords: Lanthanides, cyclen, anions, complexation, detection, luminescence, sensors, thiocyanate, polycarboxylates, metalloreceptors

A Study of the Kinetics of La3+-Promoted Methanolysis ofS-Aryl Methylphosphonothioates: Possible Methodology for Decontamination of EA 2192, the Toxic Byproduct of VX Hydrolysis

The kinetics of the La3+-catalyzed methanolysis of a series of S-aryl methylphosphonothioates (4a-e, phenyl substituents = 3,5-dichloro, 4-chloro, 4-fluoro, 4-H, 4-methoxy) were studied at 25 °C with s(s)pH control. The reaction involves saturation binding of the anionic substrates to dimeric La3+/methoxide catalysts formulated as La2(3+)(-OCH3)x, where x = 2-5 depending on the solution s(s)pH. Cleavage of the La3+-bound methylphosphonothioates is fast, ranging from 5 × 10(-3) s(-1) to 5.5 × 10-(5) s(-1) for substrates 4a-e at a s(s)pH of 8.4 and 1.6 × 10(-1) s(-1) to 4 × 10(-3) s(-1) at a s(s)pH of 11.7. The rate accelerations for the methanolysis of substrates 4a-e, relative to their background methoxide-promoted reactions, average 7 × 10(10) and 1.5 × 10(9), respectively, at s(s)pH's of 8.4 and 11.7. The catalytic system is predicted to cleave EA 2192 (S-2(N,N-di-iso-propylaminoethyl)methylphosphonothioate), a toxic byproduct of the hydrolysis of VX, with a t1/2 between 4 and 8 min at a s(s)pH of 8.4, and 27 min at a s(s)pH of 11.7.

Basic hydrolysis of quinolinyl N,N‐dimethylcarbamates: a two‐step mechanism

AbstractThe reactivity of 6‐quinolinyl and 8‐quinolinyl N,N‐dimethylcarbamates was examined in several aqueous basic media. A quadratic dependence was observed for the constant rates upon hydroxide concentration for both compounds, which is a typical behaviour of a mechanism involving a base‐catalysed deprotonation of the tetrahedral intermediate with the formation of a dianion at high concentrations of hydroxide ion, while at lower concentrations a specific‐base catalysed addition–elimination mechanism seems to be predominant. The reactivity of 8‐quinolinyl N,N‐dimethylcarbamate was also studied in several amine buffers, showing specific base catalysis. The reactivity of 6‐quinolinyl N,N‐dimethylcarbamate was studied in H2O and in D2O and the solvent isotope effect supports the proposal of a mechanism involving a specific‐base hydrolysis. All results confirm the existence of a mechanism with a rate determining step involving the substrate anion and a second mole of hydroxide ion. This mechanism was so far unknown for carbamate reactivity, being only known to occur with amides. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Selective photocatalytic degradation of aquatic pollutants by titania encapsulated into FAU-type zeolites

The selective photocatalytic degradation of charged pollutants in water was achieved on titania encapsulated into FAU-type zeolites. The electrostatic attraction of cationic substrates and repulsion of anionic substrates by the negatively charged zeolite framework facilitated the selective photocatalytic degradation of charged substrates. The hybrid zeolite–titania photocatalysts were prepared through the ion-exchange method. The titania clusters were mainly well distributed within the cavities of FAU-type zeolites whereas no TiO 2 nanoparticles aggregates were observed on the external surface of zeolite crystals. The hybrid zeolite–titania photocatalysts were characterized by diffuse reflectance UV–visible spectroscopy, transmission electron microscopy, energy-dispersive X-ray analysis and X-ray photoelectron spectroscopy. The selective degradation of charged pollutants was investigated by employing three pairs of oppositely charged substrates. The comparison between the cationic and anionic substrates clearly showed that the degradation rates for the cationic substrates on the hybrid photocatalysts are markedly higher than those for the anionic substrates. Among the cationic substrates, the smaller cations such as tetramethylammoniums were preferentially degraded. This enabled the selective removal of cationic substrates among the mixture. Such a selective photocatalytic degradation of water pollutants may provide a useful strategy for the development of economical photocatalytic process by targeting only the most recalcitrant pollutant.

Enantiomeric discrimination of aromatic-containing anionic substrates using cationic cyclodextrins

Cationic α-, β-, and γ-cyclodextrins were prepared by reacting the corresponding native cyclodextrin (CD) with glycidyltrimethylammonium chloride (GTAC). The reaction conditions were varied in order to alter the degree of substitution of GTAC units on the cyclodextrin. The CD-GTAC derivatives, which retain a positive charge independent of pH, were evaluated as water-soluble chiral NMR solvating agents for anionic substrates. The CD-GTAC derivatives are considerably more effective at causing enantiomeric discrimination in the 1H NMR spectra of aromatic-containing anionic substrates than the neutral native cyclodextrins. Derivatives with a degree of substitution of about 1.5 were more effective than those with lower or higher degree of substitution. Not one of the α-, β-, and γ-CD-GTAC derivatives was consistently the most effective at causing enantiomeric discrimination for all of the substrates examined herein.

Anthracene appended pyridinium amide–urea conjugate in selective fluorometric sensing of L-N-acetylvaline salt

A new anthracene labeled pyridinium amide–urea conjugate 1 has been designed and synthesized. The receptor shows a different fluorometric response with L-N-acetylvaline and L-N-acetylalanine salts in CH3CN in contrast to the other salts of L-N-acetyl α-amino acids and (S)-α-hydroxy acids studied. Upon complexation of the tetrabutylammonium salt of L-N-acetylvaline, the emission of 1 increases accompanied by the formation of a new band at higher wavelength and this characteristic change distinguishes it from other anionic substrates studied. The binding interaction has been studied by 1H NMR, fluorescence and UV titration experiments.