differential metal binding interactions of the imidazolinones revealed by nmr and uv spectroscopy

Pestic. Sci. 1997, 49, 17È28

Differential Metal Binding Interactions of theImidazolinones Revealed by NMR and UVSpectroscopyKannan Rajamoorthi,* Bijay K. Singh, Stephen Donovan, Dale L. Shaner,Srinivasan Rajan & Gerald W. StocktonAmerican Cyanamid Company, Agricultural Products Research Division, PO Box 400, Princeton,NJ 08543-0400, USA

(Received 17 December 1995 ; revised version received 2 May 1996 ; accepted 23 June 1996)

Abstract : NMR and UV spectroscopy and molecular modeling methods wereapplied to probe the interaction of the two imidazolinones, imazethapyr (5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid) and its structuralisomer CL 303,135 (5-ethyl-3-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)pico-linic acid), with metal ions. Both the imidazolinones inhibit the enzyme acetohy-droxyacid synthase (AHAS) in vitro. However, while imazethapyr is a herbicidethat is used widely in agriculture, CL303,135 does not exhibit herbicidal activity.Imazethapyr and CL303,135 exhibited considerable di†erences in their inter-actions with metals. In the metal complex of imazethapyr, the carboxyl moietybinds strongly and the pyridine nitrogen binds weakly with metals. In the case ofCL303,135, both the pyridine nitrogen and the carboxyl group that are posi-tioned ortho to each other participated strongly in the binding and were found toact together as a strong bidentate ligand to a metal ion. Both of the imid-azolinones form predominantly 2 : 1 complexes with multivalent metal ions.However, imazethapyr binds two orders-of-magnitude more weakly(1É0 ] 109 M~2) with metal ions compared to CL303,135 (1É7 ] 1011 M~2). Theinteractions of the model compounds, nicotinic acid and picolinic acid, withmetals were examined similarly. It was concluded that the strong affinity ofCL303,135 for metals compared to imazethapyr may a†ect its absorption fromsoil into plants, or its translocation in plants, thereby explaining the di†erencesin herbicidal activity of imazethapyr and CL303,135.

Key words : CL303,135, imazethapyr, metal complex, molecular modeling,NMR, UV

1 INTRODUCTION

Imidazolinones such as imazapyr (2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid), imaza-quin (2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)quinoline-3-carboxylic acid) and imazethapyr (5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinicacid) are all commercial herbicides used widely in agri-culture.1,2 They exhibit extremely low mammalian tox-icity and high efficiency, resulting in very lowapplication rates and low environmental impact.3 Theyall inhibit the enzyme acetohydroxyacid synthase

* To whom correspondence should be addressed.

(AHAS; acetolactate synthase ; acetolactate pyruvate-lyase (carboxylating) ; EC 4.1.3.18), which is present inplants and bacteria, but not in animals.4 AHAS cata-lyzes reactions involving pyruvate and 2-ketobutyratein the biosynthesis of the branched-chain amino acidsleucine, isoleucine and valine. However, not all imid-azolinones that are AHAS inhibitors are able to func-tion as herbicides. For example, two isomers of animidazolinone, CL303,135 and imazethapyr, exhibitcomparable inhibition of AHAS in vitro, but on theother hand exhibit a considerable di†erence in herbi-cidal activity.5 This is thought to arise because herbi-cidal activity also depends on rapid absorption andtranslocation of the inhibitor in the xylem and phloem,

17Pestic. Sci. 0031-613X/97/$09.00 1997 SCI. Printed in Great Britain(

18 Kannan Rajamoorthi et al.

and accumulation in the meristemic regions, as well ason the rate of metabolism of the inhibitor. While theabsorption, translocation and metabolism of imaze-thapyr in plants has been thoroughly studied as arequirement for the registration of the herbicide withgovernment regulatory agencies, there is currently nocomparable information for CL303,135 due to the highcost of such studies. However, such studies, when com-bined with the metal binding studies presented in thispaper, could provide valuable mechanistic information.

Multiple factors may a†ect the absorption and trans-location of herbicides in plants. One hypothesis is thatthe strong binding of imidazolinones with metal ionsthat are present in soils and plants may a†ect theirabsorption and translocation, and even their interactionwith AHAS. Copper is found commonly in many soilsand plants. It has been argued by McBride6 thatorganic molecules in soil act to mobilize or immobilizeCu2`, with the net e†ect depending on the nature of theadsorbing mineral as well as the type of organic. Thus,if immobilization of an inhibitor by copper occurs in thesoil, it will prevent uptake of the inhibitor by the plant.On the other hand, if the inhibitor is taken up by theplant, strong complexation by a metal may simplyprevent e†ective translocation in the plant. Earlierstudies of the interaction of imidazolinone herbicideswith soil indicated that the herbicides imazethapyr andimazaquin showed a much higher affinity for copper-saturated montmorillonite clay than for calcium- orpotassium-saturated clay (D. L. Shaner, unpublishedresults). Therefore, in the present work, copper(II) wasused in comparative studies of interactions of metal ionswith imidazolinones to explore why many AHAS inhib-itors are not e†ective herbicides.

We selected two isomers of an imidazolinone, imaze-thapyr and CL303,135 (Fig. 1), that exhibit comparable

in-vitro activity against AHAS 4 kM and 13 kM,(I50respectively), and at the same time exhibit a consider-able di†erence in their herbicidal activity (63 g ha~1versus [4000 g ha~1, respectively).5 NMR and UVspectroscopic and molecular modeling methods wereapplied to study the interactions of the two imid-azolinones with metal ions. NMR is a powerful methodto study molecular interaction as it gives information atthe atomic level on the distance of two interacting mol-ecules. Both NMR and UV methods yield informationon the binding affinity and composition of complexesformed between organic molecules and metals. Possiblestructures for the copper complexes of CL303,135 andimazethapyr are proposed using molecular modelingtools based on the NMR and UV binding data. Therole of the imidazolinone ring in these compounds isalso predicted by carrying out limited studies on theiranalogs, nicotinic and picolinic acids (Fig. 1), respec-tively. The inhibition kinetics of AHAS enzyme by theimidazolinones studied in presence of copper is alsoreported.

2 EXPERIMENTAL

2.1 Materials

As the diamagnetic Cu(I) is sensitive to light and air andreadily degrades to the Cu(II) state, Cu(II) was used forthe binding studies. Copper(II) chloride and sulfate,nicotinic and picolinic acids were purchased fromAldrich and europium(III) chloride from Fluka. Imaze-thapyr and CL303,135 were obtained from the Analyti-cal Standards Distribution at American CyanamidCompany. All the samples were dried under vacuum for

Fig. 1. Structures of imidazolinones and their analogs : Imazethapyr, CL303,135, nicotinic acid and picolinic acid.

Metal binding interactions of the imidazolinones 19

48 h before use. SigmaPlot software (Jandel ScientiÐc,CA) was used for curve Ðt analysis of the binding dataand for the linear regression analysis.

2.2 NMR Studies

The interactions of imazethapyr and CL303,135 withcopper and europium were studied by [1H] and[13C]NMR under the same conditions of pH (D5È6)and concentration (D5 mg ml~1) in deuterium oxide.The pH 5È6 was selected for these studies to maintainthe carboxyl group mostly in the anionic state (CO2~)and avoid the precipitation of copper, and also becausea small change in pH around this value due to the addi-tion of metal does not a†ect the chemical shift of theimidazolinones signiÐcantly. [1H] and [13C]NMRspectra were obtained on Bruker AM-500 and AMX-300, and AMX-500 spectrometers. Sodium 3-trimethylsilylpropionate-2,2,3,3- (TSP-deuterated)d4was used as an internal chemical shift reference for deu-terium oxide solvent. Typical conditions for(D2O)[1H]NMR were as follows : 16 K data points, 3È4 kHzsweep width, pulse width 3È5 ks (30¡) ; relaxation delay10 s. For [13C]NMR: 32 K data points, 20È30 kHzsweep width, pulse width 3 ks (30¡), relaxation delay1È3 s. To assign carbon spectra of imidazolinones withand without metal, two-dimensional heteronuclearmultiple bond correlation (HMBC) experiments7h9 werecarried out using a triple resonance gradient probe onthe Bruker AMX-500 instrument. HMBC data werecollected using 4È5 mg of imidazolinone in 0É75 ml

(pH 5È6) at room temperature using 2028 ] 512D2Odata matrix with acquisition times of 80 and 200 ms inthe F1 and F2 dimension, respectively. Forty scans wererecorded per value and the total measuring time wast1approximately 15 h.

2.3 UV Studies

UV spectra were obtained on a Perkin-Elmer Lambda4B UV/VIS spectrophotometer. Cells with a 1-cm pathlength were used. For studies of the complexes, solu-tions (3 ml) containing the imidazolinone (0É05 to0É1 mg ml~1 in 0É05 M ammonium sulfate bu†er, pH5É5) were placed in the sample and reference cells.Copper sulfate solution (0É004 M, 10È200 kl) was addedto the sample cell and an equal volume of bu†er(without copper) to the reference cell, and the di†erencespectra were recorded.

2.4 pH Measurements

For the NMR studies, pH was measured using a specialNMR pH electrode and a Corning pH meter, model

245. No correction was applied for the deuteriumisotope e†ect of Orion Research pH/millivoltD2O.meter 811 was used for the UV studies. The pH meterwas standardized at a pH of 4É0 and 7É0 at 21¡C. ThepH was adjusted using dilute hydrochloric acid andsodium hydroxide to the accuracy of ^0É1.

2.5 Molecular modeling

The structures of the imidazolinoneÈcopper complexeswere calculated using a suite of “CACheÏ molecularmodeling programs on a Macintosh Quadra 900 com-puter equipped with an internal CAChe card. First, thegeometries of the initial structures were optimized usingaugmented molecular mechanics with the MM2 forceÐeld parameters. The Ðnal geometry was found usingZINDO with INDO/1 (Intermediate Neglect of Di†er-ential Overlap) parameters. ZINDO is M. C. ZernerÏsINDO/1 semi-empirical molecular orbital calculationprogram using a valence-electron only procedure. TheINDO/1 parameter set contained spectroscopic param-eters for all of the elements of concern, includingcopper.

2.6 Enzyme extraction

AHAS was extracted from Black Mexican Sweet (BMS)corn cells. The growing conditions for BMS corn cellshave been described previously.10 Cells were powderedin liquid nitrogen and then homogenized in 100 mM

potassium phosphate bu†er (pH 7É0) containing pyru-vate 10 mM, magnesium chloride 5 mM, EDTA 5 mM,Ñavin adenine dinucleotide (FAD) 100 mM, valine 1 mM,leucine 1 mM, glycerol 100 ml litre~1 and dithiothreitol1 mM. The homogenate was Ðltered through a nyloncloth and centrifuged at 25 000g for 20 min. The super-natant was desalted on a Bio-Rad Econo-Pac 10 DGcolumn (Bio-Rad, Richmond, CA) that had been pre-equilibrated with 100 mM potassium phosphate bu†er(pH 7É0) containing sodium pyruvate 100 mM, magne-sium chloride 10 mM, thiamine pyrophosphate 1 mM

and FAD 10 mM. The desalted enzyme extract was usedfor the assay procedure.

2.7 AHAS assay

AHAS activity was measured by estimation of the con-version of pyruvate to acetolactate, after conversion ofacetolactate to acetoin by decarboxylation in the pres-ence of acid.11 Standard reaction mixtures containedthe enzyme in 50 mM potassium phosphate bu†er(pH 7É0) containing pyruvate 100 mM, magnesium chlo-ride 10 mM, thiamine pyrophosphate 1 mM and FAD10 mM. This mixture was incubated at 37¡C for 1 h. The


reaction was stopped by adding sulfuric acid to a Ðnalconcentration of 0É85%, or by adding sodium hydroxidesolution (4 M) to a Ðnal concentration of 0É67 M. Thereaction product was allowed to decarboxylate at 60¡Cfor 15 min. The acetoin formed was determined afterincubating with creatine (0É17%) and 1-naphthol (1É7%)by the method of Westerfeld.12

3 RESULTS AND DISCUSSION

3.1 Interaction of imazethapyr and CL303,135 withcopper by [1H ]NMR

In deuterium oxide at pH 5É5 the paramagnetic Cu(II)broadens the resonances for protons 6, 5a, 2d, 2e and2f/2g of both the pyridine and imidazolinone rings ofimazethapyr (Fig. 2), suggesting the participation ofboth rings in the interaction with copper. In the case ofCL303,135, it is only the pyridine ring that participatesin the binding with copper as only the proton reso-nances 6 and 5a of pyridine ring were broadened bycopper (Fig. 3). Similar results were obtained for com-parable studies carried out in acetonitrile (data notshown).

The extent of line broadening caused by copper canbe related to the distance between copper and thespeciÐc proton. The Solomon and Bloembergen

Fig. 2. [1H]NMR spectra of imazethapyr (1É61 ] 10~5 M)and its complex at di†erent mole ratio (f) of copper to imaze-

thapyr (given in parenthesis) in deuterium oxide at pH 5É0.

Fig. 3. [1H]NMR spectra of CL303,135 (1É48 ] 10~5 M) atdi†erent mole ratio (f) of copper to CL303,135 (given in

parenthesis) in deuterium oxide at pH 4É8.

equation13,14 for the electronic contribution to the spin-spin relaxation time, can be related to the observedT2 ,linewidth and simpliÐed to :

1T2p

\ 1T2(obs)

[ 1T2(0)

\ !fr6 (1)

This equation is based on assumptions such as theextreme narrowing condition, negligible scalar contribu-tion and fast chemical exchange.15 and areT2(obs) T2(0)the observed relaxation times in the presence andabsence of paramagnetic ion, respectively. The trans-verse relaxation times, were estimated from the line-T2 ,widths (*l) at half peak height f is the(T 2~1 \n*l).ratio of paramagnetic copper ion concentration to imid-azolinone concentration, r is the metal ion-proton inter-nuclear distance, and ! is a constant. Qualitatively, theabove equation means that the protons closest to thecopper ion will be preferentially broadened and this ishighly sensitive because of the sixth-power dependence.Distances can be compared quantitatively by taking theslopes for two protons from the plots of against fT 2p~1(Fig. 4). Such NMR-derived relative distance informa-tion is used to determine the site(s) of the interactionand also to model the structure of the complex usingstandard molecular modeling methods (see below). Inthe part (b) of Fig. 4, the slope of the curve for theproton 6 is greater than that of the protons 5a, indicat-


Fig. 4. Plot of the measured values of for the protons of1/T2p(a) imazethapyr and (b) CL303,135 as a function of the moleratio (f) of copper to imazethapyr and CL303,135, respectively.The solid line represents the Ðtted Ðrst-order linear regressionline. In (a), the slopes of the curves for the protons 6, 2d, 5aand 4 are 6É76, 3É12, 1É76 and 0É84, respectively. In (b), theslopes of the curves for the protons 6 and 5a are 21É16 and

0É75, respectively.

ing that the proton 6 is closer to copper than are the 5amethylene protons in the copper complex of CL303,135.Similarly, in the case of the imazethapyr complex, theslope for the proton 6 is greater than that of 2d, 4 and5a protons (part (a) of Fig. 4), indicating that the proton6 is again closer to copper than are protons 2d, 4 and5a. The Ðnding that the proton 6 is closer to copperthan any other protons in the complexes of imazethapyrand CL303,135 indicates that the pyridine ring partici-pates in the binding in both cases.

3.2 Binding of imazethapyr and CL303,135 witheuropium by [1H ]NMR

Copper was not useful for determining the compositionand binding constant of the complex by NMR becauseit caused severe NMR line-broadening upon addition offar less than one equivalent (Figs 2 and 3). Therefore,one of the lanthanides, europium, was used in addi-tional NMR studies to determine the composition andbinding affinity of the complex.

Lanthanides are widely used to study the structureand conformation of small organic molecules, peptidesand proteins by NMR.16h18 When the lanthanidecation is bound to a ligand, it can induce changes inboth chemical shift and nuclear spin relaxation. Lantha-nides can be classiÐed into three groups : (i) cations suchas Eu3` and Nd3`, which have very short electronrelaxation times, causing chemical shift perturbationbut negligible line broadening ; (ii) cations such as Gd3`and Eu2`, which have long electron relaxation times,causing only a large isotropic broadening e†ect ; (iii)cations such as Ho3` with intermediate relaxationtimes that cause changes in both line width and chemi-cal shift. For the studies of imidazolinone complexes,Eu3` was used. Both Cu2` and Eu3` are paramagnetic

cations. Both can form four- and six-coordinate com-plexes. Unlike d-block transition metals like copper, thelanthanides, for which 4f electrons are responsible fortheir properties, can also form higher than six-coordinate complexes.19 The NMR data obtained forthe lanthanide complexes are easier to interpret thanNMR data for transition metals. The 4f electrons inlanthanides are well screened and their orbitals havelittle overlap with those of ligand electrons. Therefore,all perturbations by lanthanides can be attributed to thepseudocontact (though-space) interaction, and thecontact (though-bond) interaction may be safelyneglected.

Eu3` a†ects only NMR chemical shifts and does notbroaden the lines signiÐcantly. Therefore, stoichiometricamounts of europium can be added. In the europiumbinding studies, the Eu3` was titrated against the imid-azolinone by following the changes in the chemical shiftof the proton NMR resonances. In the case ofCL303,135, large chemical shift changes were observedfor proton 6 upon the addition of europium, and amaximum was reached with the addition of two equiva-lents (part (b) of Fig. 5). In contrast, a relatively smallchemical shift change was observed for proton 6 of ima-zethapyr for the same amount of europium, and it con-tinued to increase asymptotically even after the additionof four equivalents of europium (part (a) of Fig. 5).Qualitatively, these observations reveal that the bindingaffinity of CL303,135 for europium is higher than thatof imazethapyr.

A non-linear least-squares curve Ðtting routine in theSigmaPlot scientiÐc graphing software was used toanalyze the binding data for a simple 1 : 1 complexaccording to the equilibrium:

M ] L% ML (2)

where M is a metal and L is an imidazolinone. Accord-ing to Stockton and Martin,20 the observed population

Fig. 5. Plot of the measured NMR chemical shift changes (*)for the proton 6 of (a) imazethapyr (0É027 M) and (b)CL303,135 (0É026 M) as a function of the mole ratio (f) of euro-

pium to imazethapyr and CL303,135, respectively.


average shielding, p, is given by

p \ [pf pf ] pcpc],

where and are the populations, and and thepf pc pf pcNMR shieldings, of the free and complexed ligand,respectively. The shifts * and are then deÐned as*c

and Thus, the NMR shift **\ p[ pf *c \ pc[ pf .can be related to the concentration of the complex[ML ] by the formula :

*\ [ML ]L 0

*c

(3)

where is the total amount of ligand (free] bound).L 0In the titration of imidazolinones against metal, the

metal ion concentration, [M], is varied while the totalimidazolinone concentration, remains constant. ForL 0 ,every [M], the corresponding * value was measured.The * value was also calculated theoretically by esti-mating the value of [ML ] as shown below from themetal and imidazolinone concentrations and assumedequilibrium constant K and values.*c[ML ]\

(L 0] M0] 1/K) [ J(L 0] M0] 1/K)2[ 4L 0M02

(4)

The NMR binding data for both imazethapyr andCL303,135 do not Ðt the equation for a simple 1 : 1complex (ML ).

Since the NMR binding data do not Ðt the 1 : 1complex equation, JobÏs method, also known as thecontinuous variations method,21 was used to determinethe stoichiometry of the complex. The overall concen-tration of the two species, [metal]] [imidazolinone],was kept constant and the mole fraction x \ [metal]/([metal]] [imidazolinone]) was varied from 0 to 1.The quantity * multiplied by [imidazolinone] wasplotted against x. * is the di†erence between the chemi-cal shift of free imidazolinone and the observed valuefor a given mole fraction x. For both imazethapyr andCL303,135, the maximum of the plot occurred around0É3 (Fig. 6), indicating the predominant existence of a2 : 1 (imidazolinone : metal) complex.

The only one experimentally observed value ofchemical shift, which is also the average of both the freeand bound imidazolinone, and many unknown param-eters preclude the curve-Ðt analysis of the NMR bindingdata for the 2 : 1 complex. Therefore, the reverse titra-tion procedure22 was used in which metal concentration

was kept constant and at least 10 times lower than(M0)the imidazolinone concentration thus guaranteeing(L0),

The imidazolinone concentration wasL 0? ML 2 .varied. Under this reverse condition, the following

Fig. 6. JobÏs plot of the quantity *] [imidazolinone] versusmole fraction (x) for the interaction of (a) imazethapyr and (b)

CL303,135 with europium.

equation for binding constant K was obtained for thecomplex, ML 2 .

L 02\ M0*c(L 0/*) [ 1/K (5)

* is the induced chemical shift change for a given con-centration and is the chemical shift change for(L 0) *cthe complex The intercept of the plot of versusML 2 . L 02

(Fig. 7) gives the binding constant. The bindingL 0/*constant for imazethapyr (K \ 1É5 ] 105 M~2) wasfound to be much less than that of the CL303,135(K\ 7É4 ] 107 M~2).

3.3 Interaction of imazethapyr and CL303,135 witheuropium by [13C ]NMR

As the [1H]NMR data alone cannot be used to conÐrmthe involvement of the carboxyl in binding, [13C]NMRdata were also collected. In [13C]NMR studies, copperhas not a†ected the chemical shifts of the 13C reso-nances but caused severe line-broadening. Therefore,europium was used again as it a†ects predominantly

Fig. 7. The plot of versus for the interaction of (a)L 02 L 0/*imazethapyr and (b) CL303,135 with europium. The twodotted lines around the solid Ðrst-order regression linedescribe the 95% conÐdence intervals. The intercepts are

[0É655 and [0É001 36 for (a) and (b), respectively.


NMR chemical shifts with little or no broadening of thelines.

For [13C]NMR studies of interaction of imid-azolinones with europium, one equivalent of metal forimazethapyr and a half-equivalent for CL303,135 wasadded and pH was maintained around 5È6. When anequivalent amount of europium was added toCL303,135, a few carbons were not detectable becauseof severe line-broadening, perhaps due to their stronginteraction with europium. The [13C]NMR spectra ofimidazolinones and their complexes with europiumwere assigned using a carbonÈproton multiple bondcorrelation experiment (HMBC). In the HMBC experi-ment, less sensitive nuclei carbons are detected indirect-ly via the more sensitive nuclei protons which are twoor three bonds away from the connected carbons. Theexperiments with direct observation of carbon signalsare not useful for assigning the [13C]NMR spectra ofimidazolinones and their complexes because of their low

concentration, which is due to their poor aqueous solu-bility. The HMBC results including the assignments arelisted in Table 1 for imazethapyr and CL303,135.

The quaternary carbons, 3 and 2a, that do not haveany long-range correlation with protons were assignedbased on comparison with similar compounds andchemical shift prediction. When the metal is bonded tothe carboxyl oxygen, the carboxyl carbon is deshieldedand shifts downÐeld (Table 1). At the same time, thecarbon attached to carboxyl group is predicted to beshielded and, therefore, expected to be shifted upÐeld.Molecular orbital theory23 predicts this charge transferto be propagated along the carbon backbone, produc-ing alternating shifts which decrease with the inversethird power of the distance. For imazethapyr, it wasfound that the carbon of the carboxyl group shifteddownÐeld by 12 ppm and the carbon attached to thecarboxyl group shifted upÐeld by 16 ppm (Table 1). Allother pyridine ring carbons were a†ected to only a

TABLE 113C Chemical Shifts for Imazethapyr and CL303,135 with and without Europium

Imazethapyr Imazethapyr] Europium (1 : 1)

Chemical Shift HMBC Results Chemical Shift HMBC ResultsCarbon (d) (Protons) (d) (Protons)

2b 196É9 197É3 2d3a 176Æ1 4 188Æ7 42aa 171É0 172É06 151É9 4, 5a 152É9 4, 5a5 146É5 6, 5a 147É1 6, 5a, 5b2 144É3 4, 6 143É6 4, 64 139É4 6, 5a 140É7 63a 138Æ3 122Æ02c 76É8 2d, 2f/2g 76É3 2d, 2f/2g2e 37É2 2d, 2f/2g 37É7 2d, 2f/2g5a 28É3 4, 6 28É7 4, 6, 5b2d 22É6 23É0 2e2f/2g 19É2, 19É0 19É5, 19É4 2e5b 17É0 5a 17É3 5a

CL 303,135 CL 303,135 ] Europium (1 : 0É5)

3b 197É1 3e 195É6 3d2aa 174Æ9 217Æ63a 172Æ7 4 165Æ36 153Æ4 4 167Æ5 4, 5a5 144Æ2 5b, 5a 130Æ2 5a, 5b2 154Æ5 4 139Æ2 44 140Æ5 5a 152Æ0 5a3a 125Æ8 97Æ03c 76É5 3d, 3e, 3f/3g 76É3 3d, 3f/3g3e 37É2 3d, 3f/3g 36É7 3d, 3f/3g5a 28É1 4, 5b 29É1 5b, 43d 22É6 3e 21É83f/3g 19É1, 19É0 3e 19É1, 19É0 3e5b 17É1 5a 17É9 5b

a See text for assignment details.


small extent (around 1 ppm), and the imidazolinonering carbons remained una†ected (Table 1), suggestingthe strong participation of the carboxyl group. A weakparticipation of the pyridine nitrogen in the binding isalso possible. However, in the case of CL303,135, thecarbons of the pyridine ring and the carboxyl werea†ected strongly by the europium addition (Table 1),indicating the strong participation of the carboxylgroup and the nitrogen of the pyridine ring in binding.Again, the carbons of the imidazolinone ring remaineduna†ected except the carbon 3a. The change in chemicalshift of 3a could be due to charge transfer e†ect becauseof the binding of europium to the carboxyl and nitrogenof the pyridine ring. This is in agreement with the[1H]NMR results that none of the protons in the imid-azolinone ring was a†ected by the addition of copper.

From the [13C]NMR experiments, it becomes clearthat only the pyridine ring and the carboxyl areinvolved in binding for both the imazethapyr andCL303,135. This is in agreement with [1H]NMR resultsfor CL303,135 only. In the case of imazethapyr, it wasfound in [1H]NMR studies that 2d and 2e protons ofthe imidazolinone ring were broadened by the additionof copper. This could still be possible without the directinvolvement of the imidazolinone ring in the binding ifthe geometry of the copper complex is such that copperis close to those protons of the imidazolinone ring (seeSection 4).

3.4 Binding of nicotinic and picolinic acids witheuropium by [1H ]NMR

Similar titration studies with europium were carried outfor nicotinic and picolinic acids. These reference com-pounds lack the imidazolinone ring and serve asanalogs of imazethapyr and CL303,135, respectively.These model compounds were chosen to avoid the pos-sible binding sites from the imidazolinone ring. Nicotin-ic and picolinic acids showed a similar di†erence in theinteraction behavior with europium to that of imaze-thapyr and CL303,135, respectively. The picolinic acidprotons showed a large chemical shift change andreached a maximum change within the addition of oneequivalent of europium (part (b) of Fig. 8) similar toCL303,135 (part (b) of Fig. 5). The chemical shifts ofnicotinic acid protons changed gradually in smallincrements and kept changing asymptotically even afterthe addition of two equivalents of europium (part (a) ofFig. 8) similar to imazethapyr (part (a) of Fig. 5). Thistype of interaction behavior with europium indicatesqualitatively that the nicotinic acid binds weakly witheuropium compared to picolinic acid.

As found with imazethapyr and CL303,135, the NMRtitration data of europium against nicotinic and pico-linic acids also do not Ðt a simple 1 : 1 complex equa-tion, suggesting similar mechanisms of interaction to

Fig. 8. Plot of the measured NMR chemical shift changes (*)for (a) proton 2 of nicotinic acid (0É025 M) and (b) proton 6 ofpicolinic acid (0É025 M) as a function of the mole ratio (f) of

europium to nicotinic acid and picolinic acid, respectively.

those of imazethapyr and CL303,135, respectively.Perhaps the imidazolinone ring of the imidazolinonesdoes not interact directly with the metal ion, which is inagreement with the [13C]NMR Ðndings. When thesame reverse titration procedure was applied to nicotin-ic and picolinic acids, no measurable chemical shiftchange was observed for nicotinic acid because of a veryweak complex formation. In the case of picolinic acid,the binding constant K was estimated to be1É0 ] 104 M~2, which is 1000 times less than that ofCL303,135. Therefore, it can be concluded that theimidazolinone ring in imazethapyr and CL303,135improves the binding affinity for metal but does notparticipate directly in the binding.

3.5 Binding copper to imazethapyr and CL303,135probed by UV spectroscopy

As an alternative method, UV spectroscopy wasapplied, not only to check the NMR results but alsobecause it permitted the use of copper directly in thebinding studies. UV titration experiments were carriedout, varying the copper concentration and keeping theimidazolinone concentration constant. A single isobesticpoint around 200 nm was observed for the imazethapyrcomplex (part (a) of Fig. 9), whereas two isobestic pointsaround 200 and 270 nm were found for the CL303,135complex (part (b) of Fig. 9). At higher concentrations ofcopper sulfate, a few spectra do not intersect at 200 nm(part (b) of Fig. 9), which could be due to absorption ofcopper sulfate itself. Since the copper solution absorbsat lower wavelengths, 200È250 nm, a wavelengtharound 290È300 nm was chosen for complexationstudies. As found for the NMR binding data, the UVbinding data also do not Ðt the simple 1 : 1 complexequation.

A modiÐed JobÏs plot24 was used to extract thebinding constant in addition to the stoichiometry of thecomplex. The modiÐed JobÏs plot uses a normalizedy-scale instead of the absorbance scale. This y-scale is


Fig. 9. UV spectra of the complexes of (a) imazethapyr (1É35 ] 10~4 M) and (b) CL303,135 (1É72 ] 10~4 M) with copper at variousconcentrations of Cu2` solution ranging from 1É37 ] 10~5 M to 1É59 ] 10~4 M. The arrow indicates the direction of increasing

concentration of Cu2`.

based on the quotients obtained when the experimentalabsorbances (A) are divided by the maximum absorb-ance, The latter is obtained by measuring theA' .absorbance of a solution containing an excess of imid-azolinone over the metal concentration against a(xmax)reference solution containing an identical imidazolinoneconcentration. is the mole fraction of metal of theX'stoichiometric composition of the complex. Using thisnormalization concept, the following equation wasderived for the binding constant of the complex Mm L n :

K \ [(m ] n)/T ](m`n~1)m~mn~ny'(1[ y')~(m`n)

(6)

where T is the total concentration of the metal andimidazolinone. In the modiÐed JobÏs plot of y versusmole fraction of metal (x), the occurred around 0É3x'for both imazethapyr and CL303,135 (Fig. 10), indicat-ing the predominant existence of a 2 : 1 complex. In the

Fig. 10. ModiÐed JobÏs plot of UV data of y against molefraction (x) for the interaction of (a) imazethapyr and (b)

CL303,135 with copper.

case of CL303,135, the plot is almost a perfect triangle,indicating very strong complex formation. For imaze-thapyr, the plot is slightly curved, indicating weakercomplex formation than that of CL303,135.

For m : n\ 1 : 2, the above binding constant equa-tion simpliÐes to :

log K \ 0É3522 [ 2 log T ] log y'[ 3 log(1 [ y')

(7)

Using this binding constant equation and the valuey'from the plots (Fig. 10), the binding constants were esti-mated.

The binding constant for the CL303,135-coppercomplex (1É7 ] 1011 M~2) is two orders of magnitudegreater than that of the imazethapyrÈcopper complex(1É0 ] 109 M~2). This conclusion is consistent with theresults from earlier NMR studies of europium binding,though the binding constants were lower (D105 toD107). The similar results obtained using europiumjustify the use of this lanthanide as a surrogate forcopper in the NMR studies.

4 MOLECULAR MODELING

NMR and UV spectroscopy data indicated thatCL303,135 forms a predominantly 2 : 1 complex withcopper and only the pyridine ring nitrogen and the car-boxyl group of CL303,135 are involved in the binding.Based on these data, only one possible structure for thecopper complex of CL303,135 can be proposed, asshown in Fig. 11. The geometry was Ðrst optimized by amolecular mechanics energy minimization (augmentedMM2), followed by a semi-empirical molecular orbitalcalculation using INDO/1 parameters optimized for


Fig. 11. A proposed structure for a 2 : 1 complex of CL303,135 with copper, involving only the pyridine ring.

Fig. 12. Three possible structures for a 2 : 1 complex of imazethapyr with copper, involving both pyridine and imidazolinone rings.


Fig. 13. Inhibition of AHAS by (a) imazethapyr and (b)CL303,135 at varying concentrations of cupric chloride solu-

tion : 0, 6É25, 12É5 and 25É0 kM.(È) (|) (K) (L)

spectroscopy (ZINDO). The proposed structure satisÐesthe NMR data including the relative distances (see part(b) of Fig. 4), i.e. proton 6 is closer to copper than the 5amethylene protons. None of the imidazolinone ringprotons is closer to copper in the model structure, asindicated by [13C]NMR data, suggesting that the imid-azolinone ring of CL303,135 is not involved in metalbinding.

In the case of imazethapyr, the imidazolinone ring ispositioned between the carboxyl group and the nitrogenof the pyridine ring. In [1H]NMR studies with copper,the methyl group of the imidazolinone ring showed asigniÐcant broadening. If the methyl broadening is dueto direct involvement of the imidazolinone ring in thebinding, at least three models can be proposed for the2 : 1 complex of imazethapyr with copper (Fig. 12) : (1)The carboxyl group and the imine nitrogen of the imid-azolinone coordinated to copper ; (2) The nitrogen ofthe pyridine ring and the imine nitrogen of the imid-azolinone ring coordinated to copper ; (3) The nitrogenof the pyridine and the imine nitrogen of the imid-azolinone ring from one molecule and the carboxylgroup and the imine nitrogen of the imidazolinone ringfrom another molecule coordinated to copper.However, no single model can satisfy all the NMR data,including the relative distances. In model (1), proton 6 isnot the closest to copper as indicated by the NMR

results (Fig. 4) ; instead, the 2d-methyl group is theclosest. In model (2), although proton 6 is closer tocopper as indicated by [1H]NMR data, the carboxylgroup cannot be involved in the binding, which doesnot agree with the [13C]NMR results. Model (3) is thecombination of models (1) and (2). A proof for model (3)may not be possible with the fast-exchange limit for theNMR data. Since [13C]NMR results also indicate thatthe imidazolinone ring may not directly participate inthe binding, the copper complex of imazethapyr mightcontain copper coordinating to only the carboxyl groupand pyridine ring nitrogen. A complex with coppercoordinating to the pyridine ring nitrogen and the car-boxyl group oxygen to form a chain of staggered four-or six-coordination complexes can be proposed for theimazethapyr complex, as proposed by Kleinstein andWebb25 for metal complexes of nicotinic acid. Sincesimilar metal interactions were observed for imaze-thapyr and CL303,135 compared to nicotinic and pico-linic acid, respectively, it can be concluded that thestructure of the imidazolinone complexes could besimilar to those proposed for nicotinic acid and pico-linic acids.25 Although the imidazolinone ring in theimidazolinones appears to improve the binding, it doesnot seem to participate directly in the binding.

5 AHAS ENZYME ASSAY

As compared to CL303,135, imazethapyr is a slightlybetter inhibitor of wild-type AHAS isolated from BMScells (Fig. 13). The for imazethapyr is 4 kM, andI5013 kM for CL303,135. Since the CL303,135 binds morestrongly with copper than imazethapyr, it is interestingto determine the relative AHAS inhibition by the imid-azolinones in the presence of copper. Cupric chloridealone at concentrations up to 25 kM did not inhibitAHAS. Therefore, a copper concentration less than25 kM was used for studies of AHAS inhibition by imid-azolinones. In the presence of copper, signiÐcantlyhigher concentrations of the imidazolinones wererequired to achieve the same degree of inhibition (Fig.13). However, the e†ect of copper on the kinetics ofinhibition was similar for both of the imidazolinones.This implies that di†erential interaction of copper withimidazolinones cannot be invoked to explain their dif-ferent AHAS inhibition in vitro.

6 CONCLUSIONS

The binding studies of imazethapyr and CL303,135 withcopper by UV & NMR and with europium by NMRclearly indicate that both of these imidazolinones form2 : 1 imidazolinone : metal complexes, and thatCL303,135 interacts more strongly with metals thanimazethapyr. The strong affinity of CL303,135 formetals is due to the ability of CL303,135 to form chelate


bonds through the pyridine nitrogen and a carboxyloxygen forming a stable Ðve-membered ring. Imaze-thapyr cannot form such a chelate ring owing to stericfactors caused by the relative positions of the carboxylgroup and imidazolinone ring. The mechanisms ofinteraction of imazethapyr and CL303,135 are similarto those of their analogs nicotinic and picolinic acids,respectively. Although the imidazolinone ring in imaze-thapyr and CL303,135 does not participate directly inthe binding, its presence does improve the binding affin-ity. Copper increases the for AHAS enzyme for bothI50of these imidazolinones. Although the di†erent affinitiesof imazethapyr and CL303,135 for metals did not causea signiÐcant di†erence in their AHAS inhibition in vitro,this may still be a factor that leads to di†erential uptakeand translocation in plants.

ACKNOWLEDGEMENT

We thank Dr Karl-Heinz Ott for his helpful commentson the manuscript.

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differential metal binding interactions of the imidazolinones revealed by nmr and uv spectroscopy

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