esr study of the radical cations 1,1'-dimethylene and 1,1'-trimethylene...

ESR Study of the Radical Cations 1,1'-Dimethylene and 1,1'-Trimethylene 2,2'-Bipyridinium: Charge-Transfer Complexes between Their Dications and the Donor Cysteine

A N G E L A S A N C H E Z - P A L A C I O S * a n d R U T D E L G A D O Departamento de Qufmica Ffsica Aplicada, Facultad de Ciencias, Universidad Aut6noma, 28049 Madrid, Spain

In order to compare the acceptor properties of l , l '-dimethylene and 1,1'- trimethylene 2,2'-bipyridinium dications opposite to a donor cysteine and the stabilities of their cation radicals, UV-visible spectrophotometric and ESR studies were carried out. The diquat dication (DQ 2+) forms a red charge-transfer (CT) complex with cysteine (Cys z-) of the ion-pair type. The complex presents two CT bands at 348 and 494 nm, respectively. By use of the Benesi-Hildebrand and other treatments (Scott, Foster, and Scatchard), the association constant for the ion association and ~x were determined. The results obtained by all the procedures are in fair agreement. The triquat dication (TQ 2+) does not form a CT complex with cysteine (CysZ-). The triquat radical (TQ. +) was generated by its reduction with alkaline sodium dithionite, and it was detected by electron spin resonance spectroscopy at room temperature. The ESR spectrum of the diquat radical (DQ. +) was reinvestigated. The corre- sponding hyperfine coupling and g factors of the radicals DQ ~. and TQ7 were given.

Index Headings: 2,2'-Bipyridinium derivatives; Charge-transfer complex; Cysteine; ESR spectroscopy.

I N T R O D U C T I O N

The viologens are diquaternary salts of 1,10-phenantrolinium (I), 4,4'-bipyridinium (II), and 2,2'-bipyridinium (III) (see Fig. 1). In aqueous solution, the viologens are colorless. They undergo one-electron reduction to give strongly colored radical cations.1 Early on, they were used as oxidation-reduction indicators in biology. 2 Viologens have been extensively studied in recent years, in their role as electron relays in photochemical reactions used for solar energy conversion (e.g., the photolytic decomposi- tion of water into hydrogen)? Methyl viologen (II, R = Me), also known as paraquat and diquat (III, n = 2), is a potent herbicide as well. Paraquat and diquat function by transferring the electron from the primary acceptor of photosystem I and forming the radical cations. The dications are then regenerated by reaction of DQ. + and PQ-+ with molecular oxygen. The other product of the reaction is the superoxide radical. 4

The diquat molecule has not been as widely studied as paraquat. It is a very potent herbicide and extremely toxic to human beings? Consequently, the diquat molecule is encountered in cases of accidental or intentional inges- tion. Its study has presented a double interest, both to identify appropriate analytical methods for further re- search and to find an explanation of its biological activity. The herbicidal mode of action appears to depend on the

Received 12 November 1993; accepted 29 April 1994. * Author to whom correspondence should be sent.

facile and reversible reduction to the radical cation. The structure of the dication and radical cation and the formation of charge-transfer (CT) complexes also play an important role in determining its herbicidal activity. 6

1,1'-Trimethylene-2,2'-bipyridinium (III, n = 3), triquat (TQ 2÷ ) is reduced, like the DQ 2÷, by two single- electron reversible steps. 7 Both steps are favored by the coplanafity of the pyridine rings.

With regard to dications (DQ 2÷ and TQ 2÷ ), the dihedral angle between both aromatic rings has been related to the ultraviolet spectra of their diquaternized salts. Pre- vious work by Homer and Tomlinson 8 correlated the dihedral angle with the intensity and shift of a given absorption maximum of some derivatives of 2,2'-bipyridinium.

A resolved spectrum of the diquat cation radical has been reported. 9-1~ The spectrum of the triquat radical cation has still not been presented.

It is known that the pyridinium cation or bipyridinium cation derivatives with planar structures form charge- transfer complexes with an extensive range of donors.12-14 Paraquat and diquat are derivatives of the bipyridinium cation. The charge-transfer complexes of paraquat, with a wide range of neutral and anionic electron donors, have been published. 15.16 The CT complexes of diquat have not been extensively studied.

Since the formation of free radicals and their CT complexes plays a significant role in the biological activity of the cations, the aim of this work is twofold. First, we wish to compare the acceptor capacity of dications DQ 2+ and TQ 2+ opposite a common donor. We have chosen cysteine in aqueous solution as an electron donor. The amine, sulfhydryl, and carboxilic groups are expected to contribute to CT complexation. UV-visible absorption spec- trometry was employed for detection and study of the CT complex that was formed. Second, an analysis of the ESR spectra of diquat and triquat cation radicals will be presented; we will assign the hyperfine coupling constants (hccs) obtained and compare the results with previously reported values. 9,1°

EXPERIMENTAL

S p e c t r o c o p i c D a t a . A Varian E-12 spectrometer oper- ating a field modulation of 100 kHz was employed. Mea- surements were carried out with low microwave power (2-5 mW). An optimum modulation width of 0.05 or 0.5 Gauss was used. The g factor was determined by means of a high-precision frequency meter and a gaussmeter.

926 Volume 48, Number 8, 1994 0003-7028/94/4808-092652.00/0 APPLIED SPECTROSCOPY © 1994 Society for Applied Spectroscopy

R-- R 2

5 6 (CH2)n (CHz)n

1 11 III

FIG. 1. Chemical structures of 1,10-phenantrolinium (I), 4,4'-bipyfidinium (ll), and 2,2'-bipyfidinium (III).

The relative intensities of ESR signals were calculated from the following equation9 ~

YAH~p Icx (1)

S.M

where I is proportional to the spin concentration of the paramagnetic species, Y is half the peak-to-peak amplitude of the first derivative line, ~Hpp is the peak-to-peak linewidth, S is the gain of the amplifier, and M is the modulation amplitude in Gauss.

The central lines of the spectra of the diquat and triquat radicals (denoted lines 10 and 14) were used for the quan- tification.

Absorption measurements in the ultraviolet and visible region were performed on a Hitachi U-2000 spectrometer. One-centimeter quartz cells were used for all measurements. A Durrum 110 stopped-flow spectrometer was used for measurements in the initial seconds of the reaction.

Reagents. All chemicals utilized were of analytical re- agent grade. Doubly distilled water was used throughout. Aqueous 400 ug mL- ' diquat and triquat solutions were prepared from pure chemicals, and less concentrated solutions were prepared by suitable dilution. Sodium dithionite solutions were prepared in 0.2 M buffer just be- fore use. Diquat was supplied by Dr. S. Ehrenstorfer. Triquat was obtained according to the procedure of Ho- mer and Tomlinson. 8 1, l '-Trimethylene 2,2'-bipyridinium dibromide was characterized by its C, H, N com- bustion analysis, 200-MHz ~H NMR spectrum, and chemical ionization mass spectrum (see Table I).

Procedure. Formation of tke CT Complex. When a diquat solution (4 × 10 -2 mM) in NaOH (20 mM) was added to solid L-cysteine (30 raM), a red color appeared. Then the optical absorption was measured with a UV- visible spectrophotometer at different time intervals.

Generation of Radicals. Equal volumes of diquat or triquat and sodium dithionite solutions were mixed in 0.1 M buffer at pH 8 and 10, respectively. The final concentrations of the reactives were 0.5 mM ofdiquaternary salts and 11.5 mM or 5.74 mM for the reductor in each case. The mixed solution was transferred to an ESR thin, flat cell, and the spectrum was recorded at room temperature. The formation of the radical cations was mon-

1,000 -

AB5-

0.500

I I I I 300 4 0 0 5 0 0 6(30 700

Wavelength (nm)

FIG, 2. Absorption spectrum o fd iquat (4,34 x 10 .2 raM) in NaOH (0.24 M) solution with cysteine (40 raM): (curve 1) 1 min after mixing; (curve 2) 3 min after; (curve 3) 10 min later; (curve 4) 20 min later; (curve 5) 60 min later.

itored. In the case of the triquat sample, sodium dithionite solution was previously deoxygenated with nitrogen.

RESULTS AND DISCUSSION

Absorption Studies of Charge-Transfer Complexes. Figure 2 shows the absorption spectra at different time intervals carried out by mixing diquat in NaOH solution and the amino acid cysteine. One minute after mixing (curve 1), two bands are seen at 494 and 348 nm (and traces of DQ 2÷, UV peak); 3 min later, the intensities of the two bands suddenly increase (curve 2), and the UV peak vanishes. When the reaction time is 10 min, the maximum absorbance is attained (curve 3).

The new absorption bands in the visible region could be attributed to intermolecuar CT interaction between the acceptor cation diquat (DQ 2+ ) and the donor anion cysteine (Cys-2).

To prove the significance of the hydroxide ions, we mixed diquat in a tris(hydroxy methyl)aminomethane solution along with cysteine. The spectrum is the same as that without cysteine; only the UV peak of DQ 2+ was observed.

Since the donor undergoes acid-base equilibration [pK, (Cys): 1.9; 8.2; 10.317], it is not surprising that the complexation of this species with DQ 2+ is a function of the [OH-]/[cysteine] ratio. In fact, it was observed that the red-colored compound was not formed if [OH-]/[cysteine] was less than 2. Moreover, the [Cys2-]/[DQ 2÷] ratio is also important; the higher concentrations of the donor can be useful for rapidly reaching equilibrium and thus minimizing the time of complex formation.

In order to analyze the donor capability of amine, car- boxylate, or sulfhydryl groups, the cystine (dimer of cysteine) was used as a donor. The red color is not formed but, in the visible range, a very weak wide band (~ 415

TABLE I. Analytical and spectroscopic data for the triquat.

Anal. calcd, for C~3H~4NBr2H20 Anal. found

]H NMR (200 MHz, D20)

MS (C.I.) m / z (relative intensity)

C = 41.52; H = 4.26; N = 7.45 C = 42.71; H = 4.13; N = 7.32

(in ppm) 2.9 (m, -CH2- central), 4.5 (m, 2H axial), 5.0 (m, 2H equato- rial), 8.3 (m, Hs), 8.45 (d, H3), 8.8 (m, H4) and 9.2 (d, H6) a

199 (100, M + 1), 157 (35)

"Note: m = multiplet; d = doublet.

APPLIED SPECTROSCOPY 927

0.800

0.600

A494

0.400

0.2 O0

0.000 0

(a)

I I I 10 20 30

t ( r n i n )

I 40

0 . 8 0 0 - ( b )

0.600

A494

0.400

0.200

0 . 0 0 0 I I 0.00 O.02 0,04

Cysteine (M)

FIO. 3. (a) Effect o f NaOH concentration on the stabil i ty o f the red CT complex DQz+/Cys 2-. [DQ 2+] = 3.1 x 10 -2 raM, [H2Cys] = 30 raM; symbols • , [], and • indicate 0.18, 0.25, and 0.43 M NaOH, respectively. (b) Var iat ion o f absorbance at 494 nm as a function of [H2Cys]. [DQ 2÷] = 3.1 x 10 -2 raM; symbols [ ] and • indicate 0.18 and 0.25 M NaOH, respectively.

nm) appears together with the UV peak of DQ 2÷. This visible band is unstable with time (bathochromic shift). The visible band might be a charge transfer between the acceptor diquat and the donors hydroxide ~8 or amine.

In conclusion, the carboxilate anion or the amine group does not contribute significantly to CT complexation, although the negative charge of its deprotonated form (CO0-) is an important factor in the ionic interaction with DQ 2+. Donor capability is provided by the sulfhydryl group.

To establish the optimum condition for the formation of the red complex, we studied the magnitude of the absorption as a function of the main variables.

The influence of hydroxide concentration on the stability of the complex at different NaOH concentrations was examined over the range 0.18 M to 0.43 M (Fig. 3a).

The curves present two clearly differentiated parts; the former, of positive slope, corresponds to the formation of the CT complex, and the latter, practically parallel to the time axis, indicates its stability. The curves obtained in the other cysteine concentrations are similar. The compound produced with cysteine was the most stable in 0.18 M NaOH solution. In NaOH solutions more dilute than 0.18 M, the CT complex was formed, but very slowly. For instance, when [NaOH] is equal to 0.08 M and [cys-

TABLE II. Equilibrium constant and molar absorption coefficients for the diquat/cysteine CT complex at room temperature.

Method

KDA c348 x 10 -4 ~494 x 10 -4 x 10 -2 KDA ~348 KDA ~494 (M- ' cm ') (M -I c m - ' ) (M I)a X 10 -7 X 10 -7

Benesi 1.43 2.32 5.05 1.07 1.17 Hildebrand Scott 1.45 2.31 5.47 1.00 1.26 Foster 1.44 2.32 5.00 1.04 1.16 Scatchard 1.44 2.33 4.94 1.03 1.15

° Measured at 494 nm, ± 44 M - ' .

teine] is equal to 4 mM, the time required to attain the maximum absorbance (Amax) at CT is 48 min. On the other hand, it was observed that the value of Amax is slightly lower when the ratio [OH-]/[cysteine] is less than 5.

Figure 3b shows the dependence OfAma x at 494 nm on [cysteine] at fixed [DQ 2÷] and [NaOH]. The absorbance increases with the cysteine concentration until a limit concentration is reached; if the cysteine solution is more concentrated, the absorption decreases. Furthermore, the absorption is greater with less alkaline solution. Here, the cysteine ranged from 8 to 40 mM, and the time to attain maximum absorbance varied between 30 to 15 min.

If the diquat/cysteine charge-transfer complex is 1:1, the equilibrium reaction could be represented as:

KDA

DQ 2÷ + Cys 2- = DQCys (2)

where KDA symbolizes the equilibrium constant for the 1:1 CT complex. This could also be expressed by the well- known Benesi-Hildebrand equation ~9 as:

[DQ2+]o = 1 1 + - ( 3 )

Ax KDAex[Cys2-]0 E x

where [DQZ÷]0 and [CysZ-]0 are the concentrations of diquat and cysteine, respectively; Ax is the maximum absorbance of the complex at wavelength X for a 1-cm pathlength; and ~x is the molar absorptivity of the CT bands. (The formula is valid only if [Cys 2- ]o is much more than [DQ 2+ ]0 and if the donor and acceptor do not absorb in the region studied.)

A plot of [DQ2÷]o/Ax against 1/[Cys2-]o is expected to be linear; ex = 1/intercept and KDA = intercept/slope.

Other methods of analysis of these absorbance data, based upon algebraic rearrangement of the fundamental equation in order to minimize extrapolation errors and identify multiple equilibria, are available from the literature (see Scott, Foster, and Scatchard in Ref. 19).

A series of solutions were made with constant [DQZ÷]o (3.1 x 10 -2 mM at 494 nm and 4.34 x 10 -z mM at 348 nm) in NaOH solution and [Cys2-]o varying from 50 to 2 raM. Reasonably straight lines were obtained by the different methods employed. The results are summarized in Table II.

The high value of%94 (2.32 x 104 M -1 cm -1) will provide a suitable method for the determination of the herbicide diquat. In effect, there was a linear relationship between [DQZ+]o and Amax at Xcr over the range 1 to 8 gg mL -~. The slope (at 494 nm) was 2.12 x 104 M - ' cm- ' .

The 308-nm UV absorption of DQ ÷2 and the 378-nm visible absorption ofdiquat radical cation D Q t (see Table

928 Volume 48, Number 8, 1994

TABLE IlL Spectral data. Species Xma, (nm) ~ X 10 -3 (M -~ cm -~)

DQ 2+ 308 20.3 a 308-311 19.0 b 308 20.3 c

TQ 2+ 287 16.0 a 287 15.6 b • 289 15.4 c

DQ. + 378, 427 (s), 443, 460 32.1, 4.72, 4.36, 3.18" 370 32 d 366 38 °

TQT 386, 494 8.86, 2.4 a • .., 493 ..., 2.0 d 364, 465 15, 3.4 e

" This work. b With 10 -4 solutions in HzO, see Ref. 8.

With 10 -4 solutions in H20, see Ref. 7. Reduction with dithionite, see Ref. 25. Pulse radiolysis, see J. A. Farrington, M. Ebert, and E. J. Land, J. Chem. Faraday Trans. I, 74, 665 (1978).

Ca)

2.5G

III) have been used for the determinat ion o f the herbicide. The 494-nm visible absorption o f the DQ2+/Cys 2- system will provide a selective method for the analysis ofdiquat . The presence o f paraquat does not interfere because it does not form a CT complex with cysteine. The difference between E494 (Table II) and ~308 (Table III) is 3.0 x 103, and, moreover , the interferences are smaller in the visible region than in the UV region; the radical gives a more intense band than at 494 nm, but it is unstable.

The high value o f the equil ibrium constant KoA may be related to the ionic character of the complex. Weaker complexes are formed as a result o f dipole interactions or dispersion forces (< 10). We also observed that the red CT complex diquat/cysteine in alkaline solvent turned a stable purple color when it was extracted in a less polar solvent, 1-butanol. The absorption spectrum is similar to the one given in Fig. 2, but a ba thochromic shift was produced in both CT bands. Their new positions are at 378 and at 518 rim. The solvent sensitivity o f the CT bands in ion-pair complexes (pyridinium iodides) was studied by Kosower. 2°

Verhoeven et alJ 4 studied the CT complexes between 4-cyano pyr idinium and neutral aromatic organic donors. They explained the apparent dependence Of KDA on wavelength, because the position o f the CT band depends on the donor concentration, which modifies the polarity o f the medium. The variat ion in the concentrat ion o f cysteine or alkali modifies its polarity. In 0.16 M N a O H solution, a decrease o f cysteine from 50 m M to 2 m M results in a shift o f the first CT band from 347 nm towards 348.5 nm and the second CT band from 495 n m towards 492.5 nm. The value o f KoA was est imated at ~kma x where the absorbance is the same in a greater wavelength interval. Therefore, we have not found an explanation o f the variat ion in the value o f KDA with the CT bands.

The presence o f two CT bands is due to an electron transition from two energy levels in the donor, or f rom the acceptance at two energy levels in the acceptor; 2~ the energy difference between both levels will be the sepa- rat ion o f the CT bands.

Cysteine was added to triquat in N a O H solution, and the absorption spectrum vs. t ime was recorded. There were no CT bands present, and only a UV band at 287

(b) 11

14

17

FIG. 4. ESR spectrum of triquat radical in buffer pH 10; triquat 0.5 raM, sodium dithionite 5.74 mM. (a) Modulation width 0.05 G; (b) modulation width 0.5 G.

n m was observed (cation TQ 2+, Table III), but it was more unstable than in an acetate buffer.

ES R of Diquat and Triquat Cation Radicals. The ESR spectrum o f TQ .+ is shown in Fig. 4. At a small modulation width (0.05 G), the spectrum consists o f approximately 43 lines (Fig. 4a). Increasing the modulat ion width f rom 0.05 to 0.5 yields a spectrum of 27 lines with a linewidth f rom 0.75 G (Fig. 4b). The total width is 38.8 G. In Fig. 3b lines 11 and 17 have different heights because sodium dithionite generates the SO27 radical anion which is always present in equil ibrium with dithionite. 22 The ESR spectrum of diquat cation radical, presented in a prel iminary work, ~ is more complex (~ 155 lines, AHpp = 0.15 G and a total width o f 41.5 G) and has a hyperfine structure that is well-resolved. When the modulat ion width was 0.5 G, the spectrum consisted o f only 19 lines; line 8 was higher than line 12 because o f the singlet o f SO27. TQ .+ and D Q . + have a g factor o f 2.0027.

To establish the op t imum condit ions for the ESR signal deve lopment o f the tr iquat radical, we studied the relative intensity o f line 14 as a function o f the main variables.

The influence o f pH on the stability o f the reduced tr iquat with the use o f buffer solutions was examined over the range o f 9 to 12. The radical was the most stable at p H 10.

The effect o f sodium dithionite concentrat ion on the stability o f the tr iquat radical was considered. There is a


0.600

0.4 O0

¢,, 0.2 O0

32

24 ~.

16

8

I 0 50

(3.000 I I ~ I I I i I 0 10 20 30 40

t ( m i n )

FiG. 5. Stability o f triquat and diquat radicals by visible and electron spin resonance measurements: (1) absorbance at 378 nm, [DQ 2+] = 2.2 x 10 _2 mM, [$2042-] = 2.8 mM, pH 8; (2) absorbance at 386 nm, [TQ z+] = 5.04 x 10 2 mM, [$2042 ] = 1.15 mM, pH 10; (3) I~0, [DQ 2+] = 0.5 mM, [$2042-] = 11.5 mM, pH 8; (4) 1,4, [DQ 2+] = 0.5 mM, [$2042 ] = 5.74 mM, pH 10.

great dependence of [dithionite] on [TQ.+]. Thus maximum and more stable values of 1,4 were reached when the [dithionite]/[TQ 2+ ] ratio was ~ 10-15; the [SO2-] decreased with the reaction time since the I,,/I,7 ratio changed from 2 to 1.2 (in an interval from 3 to 40 min- utes). Also, the ESR signal of the triquat radical showed a decrease of 29% after 3 h; 4 h later only the SOz- singlet was detected. In contrast, in the case of the diquat radical, the ESR signal reached a maximum and was stable within a larger range of reductor concentrations; [SOz-] was practically constant during the course of the experiment since the ratio 18/1,2 increased from 1.5 to 2. The DQ +- signal dropped to around 10% of its original value 24 h after its generation together with the dithionite radical. Finally, the singlet of SO2- was the last registered.

The dissolved oxygen in the solution of DQ 2+ (or TQ 2+ ) with dithionite attacks the newly formed free radicals; furthermore, the presence of a reductor regenerates the radicals, leading to the reduced viologens:

$2042- 52042-

DQ 2+(orTQ 2+) = DQ ± ( o r T Q ±) ~- D Q ( o r T Q )

colorless o2 green (or red) o2 yellow (4)

The [DQ +. ] radical was not affected in a degassed solution, but the 18/1,2 ratio was decreased (1.2). On the other hand, [TQ+.] suddenly increased within 20-25 min, and sub- sequently decreased slowly [Fig. 5 (4)]. At the same time, the [SO27] radical decreased sharply in an early step of the reaction; in about 15 min 111 was nearly equal to 117. When the dithionite solution was not deoxygenated, a smaller amount of triquat radical was formed (measured by the intensity of line 14). For a reaction time of 22 min, 1,4 was approximately half that for degassed solution. On the other hand, 25 min after the triquat had been mixed,

II III

t IV V

L,

FIG. 6. Canonical structures of the diquat radical.

the ESR signal was kept nearly constant and then decreased more rapidly.

These results prove that the dications DQ 2+ and T Q 2+ will be reduced by dithionite (in which SO2 v is the re- active species) to radical cations. 2a Also the SO2 v radical anion may react with the dissolved oxygen. The second electron transference could be due to only SO27 or to both reductant SO27 and $2042-. DQ +. is more stable than TQ .+, which is continuously generated by the SO2 ~ present in the alkaline medium. Their stability is related to the dihedral angle between the two pyddinium rings in the dications TQ 2+ and DQ 2+.

The absorbance peak positions and molar absorption coefficient of the dications and their radicals are taken from the literature and reexamined in this work. They are listed in Table III. We have verified that the UV absorption maxima of DQ 2+ and TQ 2+ in acetate buffer pH 4 show greater stability and reproducibility than in water. The hypsochromic shift (21 nm) and a decrease in the intensity of the T Q 2+ band indicate a minor conjugation w~hen the chain length is increased by a CH2 group. The difference in chemical shift (A~) between the H-3 and H-5 ring protons is an indirect measure of the dihedral angle between aromatic tings of 2,2'-bipyridines. 24 The A6 values of 0.62 ppm and 0.12 ppm for DQ 2+ and T Q 2+ denote a larger dihedral angle in the TQ z+; thus the system loses conjugation. In the DQ 2+ both aromatic rings are nearly coplanar. The dihedral angle N(1)--C(2)--C(2')-N(1 ') according to X-ray analysis is 20.40, 9 which is in agreement with the theoretical calculations of 21.6°. 25 Calcu- lations for the TQ 2+ gave a value of 38.70. 25 Thus, the diquat radical cation will be the most stable, as was shown by ESR spectroscopy.

Figure 5 shows the stability of diquat and triquat radicals obtained by reduction with dithionite with the use of visible absorption or ESR spectrum intensity. The [DQ -+] decreases exponentially with reaction time (curves 1 and 3). A plot of absorbance at 378 nm (or I,o) against [DQ 2+] gave a straight line. In Table III the values of ~x are given (reference time, 10 rain). The absorbance (at 386 nm) or 1,4 of the triquat radical increased until a maximum was reached, and then both began to decrease (curves 2 and 4). There is a relationship between dication concentration and maximum absorbance; the values of Ex are given in Table III.

In a stopped-flow apparatus, the maximum concentration of the diquat radical was quickly reached (in 20 s); for the triquat radical, after 4 s the absorbance produced a ~55% increase, then 8 s later a - 6 3 % increase, and

930 Votume 48, Number 8, 1994

TABLE IV. ESR hyperfine coupling constants (G).

Cation Position

radical N,N' 5,5' 4,4' 6,6' 3,3' 2,2' CH3 CH2

Paraquat" 4.23 1.57 -.- 1.33 1.57 1.33 3.99 ..- Diquat b 4.06 2.94 2.38 0.56 0.28 . . . . . . 6.44 Diquat c 4.08 2.54 2.90 0.56 0.36 . . . . . . 6.98 Diquat d 4.10 3.00 2.48 0.26 0.58 . . . . . . 3.46 Triquat o 4.5 2.7 1.7 . . . . . . . . . . . . 2.9

" Irradiation in ethanol solution, see Ref. 26. b Sodium dithionite in buffer pH 8, this work. c Zinc in trifluoracetic acid, see Ref. 9.

Zinc in ethanol, see Ref, 10. ° Sodium dithionite in buffer pH 10, this work.

after 3 min only a ~69% increase. An estimation of~37 s for DQ -+ and E386 for TQ .+ gives a higher value for DQ. + than that obtained with a UV-visible spectrometer.

Hyperfine Coupling Constants. The hyperfine structure of DQ .+ and TQ .+ is due to coupling of the unpaired electron with two equivalent nitrogens of spin 1 and the protons in no equivalent positions of spin 1/2. Conse- quently a maximum number of 52 × 34 or 52 × 35 lines should be expected. The splitting constants of DQ +. are listed in Table IV. The results show that the unpaired electron will be delocalized over the molecule. These values are in the algebraic ratio of 2:4:17:21:29:46, which explains the uniform spacing of the lines in the spectrum. The assignment is justified by consideration of the canonical structures (see Fig. 6) and the pattern of the paraquat radical, PQ.+. This assignment was first reported by Johnson and Gutowsky. 26

In the -CH2-CH 2- bridge, only two methylene protons are split. This phenomenon was discussed in detail by Sullivan and Williams. 9 In the --CH2-CH2- bridge with pseudoaxial and pseudoequatorial protons, presumably the arrangement of the pseudoaxial protons would be more favorable for couplings with the unpaired electron in the nitrogen 2pz orbital.

The DQ. + hyperfine coupling constants off ing protons given in Table IV are in satisfactory numerical agreement with those presented by other authors, although the assignments were in disagreement. Sullivan and Williams based their assignments on an HMO calculation, and Rie- ger and Rieger ~° assigned the hccs by the change in the NMR line broadening of DQ 2÷ with traces of DQ .+. The methylene proton coupling of 6.44 is comparable with the value of Sullivan and Williams of 6.98, and it is in disagreement with the value of 3.46 given by Rieger and Rieger.

The ESR spectrum of triquat was analyzed to provide the parameters listed in Table IV. The assignment was justified by the analogy of the diquat radical. The smallest splitting in the 6 and 3 positions was not resolved because of the low signal-to-noise ratio and the large number of lines. The methylene proton coupling of 2.9 G could be assigned to the four neighboring methylene protons coupling to the nitrogen atom; the protons of the CH2 central group would have a negligible splitting. A computer sim- ulation using these splitting constants is in good agreement with the experimental spectrum. The nitrogen couplings (Table IV) are similar in the three radicals PQ+., DQ. ~, and TQ. +.

CONCLUSIONS

This work clearly demonstrates the formation of two CT bands of DQ 2+ with cysteine (Cys2-). The complex was found to be stable, and the results obtained indicate that the analytical method is sensitive to micrograms of diquat.

The ESR intensity data showed that DQ .+ is more stable than TQ. + in alkaline solutions. Moreover, the singlet of the SO2r radical was detected together with both radical cations. A well-resolved ESR spectrum of DQ -+ was obtained to permit unambiguous analysis of hccs. The ESR spectrum ofTQ ~. was interpreted, but the signal-to-noise ratio was not good enough to allow a complete determination of hccs.

ACKNOWLEDGMENT

The authors express their gratitude to the DGICYT for financial support, Project 84-392.

1. L. A. Summers, Tetrahedron 24, 5433 (1968). 2. L. Michaelis, Chem. Rev. 16, 243 (1968). 3. M. Gr~itzel, Biochim. Byophys. Acta 683, 221 (1982). 4. L. A. Summers, TheBirpyridilim Herbicides (Academic Press, Lon-

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932 Volume 48, Number 8, 1994

esr study of the radical cations 1,1'-dimethylene and 1,1'-trimethylene...

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