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1H and 13C NMR relaxation investigation of the copper(II) complex of amitraz, a formamidine pesticide
Polyhedron Vol. 12, No. 19, pp. 2355-2358, Printed in Great Britain
1993 0
‘H AND 13C NMR RELAXATION INVESTIGATION THE COPPER COMPLEX OF AMITRAZ, A FORMAMIDINE PESTICIDE ELENA GAGGELLI,
NICOLA GAGGELLI
and
0277-5387/93 $6.00+ .W 1993 Pergamon Press Ltd
OF
GIANNI VALENSIN*
Department of Chemistry, University of Siena, Siena 53 100, Italy and ALESSANDRO
FRANCHI
USL 30, Siena, Siena 53100, Italy (Received 23 March 1993 ; accepted 2 June 1993)
Abstract-The copper(I1) complex of amitraz, a formamidine pesticide, was investigated by measuring ‘H NMR spin-lattice relaxation rate enhancements (PRRE) in [2H6]-DMS0 solution. The occurrence of fast exchange conditions was demonstrated by the temperature dependence of the PRRE. The PRRE was interpreted by the Solomon-BloembergenMorgan approach ; the number of solvent molecules in the coordination shell was calculated at q = 2 ; the reorientational correlation time in the 1: 1 metal complex was assumed to be the same as in the free ligand and calculated at z, = 69 ps. The metal ion was shown to stabilize a cisoid conformation of the ligand.
Arnitraz, {N,N’[(methylimino)dimethylidyne]di2,4-xylidine} (Fig. l), is a formamidine acaricide and insecticide effective against a wide variety of phytophagous mites and insects. It is synthesized from 2,4_dimethylaniline and it reverts to several products on mammalian metabolism and environmental degradation. I-3 Extensive use in agriculture has raised the question of its toxicity, especially in light of its interaction with the hepatic mixed-function oxidase systems’ and also with components of the biogenic amine system.4 In this communication we present NMR evidence that amitraz can act as an effective ligand for metal ions such as Cu*+, forming stable complexes that mai play a significant role in eliciting the biochemical activity. EXPERIMENTAL Materials
used without further purification. Solutions were made in [2H6]-DMS0 or in H20 and carefully deoxygenated by bubbling nitrogen gas through. The desired copper concentration was obtained using stock solutions of Cu(ClO4)2 in [2H6]-DMS0 or in H20. Methods
NMR experiments were performed on a Varian VXR-200 spectrometer at controlled temperature ( f 1 K). Chemical shifts were referenced to internal TMS. Spin-lattice relaxation rates were measured with inversion recovery pulse sequences. Spin-spin relaxation rates were measured with the Carr-Purcell-Meiboom-Gill pulse sequence. The rate values were calculated, in both cases, by linear regression analysis of the recovery or decay curves of longitudinal or transverse magnetization components. 13C{‘H} nuclear Overhauser effects were measured with gated decoupling techniques.
Amitraz (standard analysis reagent) was obtained from Ehrenstorfer Lab. (Augsburg) and
RESULTS
AND DISCUSSION
Different proton spin-lattice relaxation rate * Author to whom corrcspondcnce should be addressed. enhancements (PRRE) were measured in the pres2355
E. GAGGELLI et al.
2356
Fig. 1. Molecular formula of amitraz.
ence of copper(I1) ions (Table 1). The experimental paramagnetic contributions to the relaxation rates, R,, were calculated by : Rip = Riobs- Rif
(i = 1) 2),
(1)
where Riobs and Rif are the rate values measured in the presence and in the absence, respectively, of relatively small concentrations of the paramagnetic ion. Consideration of the temperature dependence of such PRRE (Fig. 2) suggests that fast exchange conditions apply, such that : R, = qfR.+,
(i = 1,2),
(2)
where q is the number of coordinated ligand molecules and f is the fraction of bound ligand, the relaxation rate of which is given by R,u. In such situations the PRRE can be interpreted in terms of the Solomon-Bloembergen-Morgan theory ;5-7 since Rzp >> R,, was measured (Table 1) it was concluded that Rzp is contributed by the scalar interaction, whereas the paramagnetic contribution to the spin-lattice relaxation rate, R,,,, is mainly determined by the dipolar electron spin-nuclear spin interaction given by :‘v9
where r is the ion-proton distance measured in 8, ; OH = 1.257 x 10’ rad s- ’ is the proton Larmor frequency ; z, is the correlation time modulating the nuclear spin-electron spin dipoledipole interaction ; yH = 2.6753 x 10’ rad s- ’ G- ’ is the proton magnetogyric ratio ; ge = 2.002322 is the electronic g factor ; Be = 0.9273 1 x lo- *’ erg G- ’ is the Bohr magneton. The motional correlation time, in the case of copper(I1) complexes with small ligands, is a rotational correlation time8*9which can be reasonably assumed to be the same as for the free ligand. The validity of this assumption might be questioned but only at a superior level of accuracy, which is not required in this case (a large error in r, in fact only yields small uncertainties in evaluating distances). The motional correlation time was, therefore, calculated by measuring the i3C spin-lattice relaxation rates of the free ligand, summarized in Table 2. Since the 13C-‘H dipolar interaction may not provide the unique relaxation mechanism, especially for methyls, the spin-lattice relaxation rates were scaled down with the measured nuclear Overhauser effect (n.0.e.) through : (4)
(3) where qmax(the maximum observable n.0.e. when
Table 1. Paramagnetic proton relaxation rates for amitraz (0.1 mol dmp3) in [‘HJ-DMSO at T = 295 K in the presence of [Cu’+] = 4.3 x lo-’ mol dmm3 and calculated ion-proton distances r&u-H)o (A)
6
Proton
@Pm)
N--CHS H3 CH 3(6’) cHH,;s?
3.31 8.29 2.21 2.18 6.97
1.10 1.98 0.75 1.25 -
6.87 9.44 4.86 2.21 7.31
4.0 3.6 4.2 3.9
Hg HI0
6.90 6.82
0.22 1.76
7.11 9.04
5.2 3.7
nCalculated from eq. (3) with z, = 69 ps. ‘Calculated from eq. (3) with t, = 8 ps.
~(CU-H)~ (A) 2.8 2.7
2357
‘H and 13CNMR relaxation
Table 3. Proton spin-lattice relaxation rates (R ,) of water and DMSO in the free solvent and in the presence of amitraz (0.1 mol dmp3) or Cu(ClO.&* 6H20 (4.30 x 10m4 mol dm-3) or both at T = 295 K n!
,P -
I
l
0
0
I
‘
-J ; ; ; , , l
2.9
3.0
3.1
3.2
3.3
Amitraz (mol dm- ‘)
Solvent
.
0.1
Fig. 2. Temperature dependence of PRRE for selected protons of amitraz (0.1 mol dm- ‘) in [*H,]-DMSO.
the relaxation rate is dominated by the dipolar interaction) = 1.98. From the calculated dipolar relaxation rates, R Idip, an effective reorientational correlation time of 69.3 + 15.3 ps for modulation of the internuclear vectors of the four methynes was calculated.” As for the methyls the same calculations yield correlation times in the range 6.1-8.5 ps, suggesting “unlocked” free rotation. * ’ The number of ligands in the coordination sphere was evaluated by measuring the solvent proton relaxation rates, shown in Table 3. A large PRRE (R,, = 3.88 s- ‘) is measured for DMSO protons in the presence of [Cu”] = 0.43 mmol dmm3, which is reduced (R,, = 1.64 s- ‘) by the presence of ligand (0.1 mol dm- 3). The reduction is due to a decreased number of solvent molecules in the metal coordination sphere. Application of eq. (3) would yield q = 2.5, as compared with q = 6 for the free solvated ion, suggesting that speciation may occur in DMSO. However, repetition of the measurements in H20, where the presence of the ligand (0.1 mol dm- ‘) lowered the solvent PRRE from its free hexaaquo ion value (R,, = 20.6 s- ‘) to RIP = 13.7 s- ‘, yields q = 4.0 for the complex. It is conTable 2. ’3C NMR parameters of amitraz (0.1 mol dm- ‘) in [‘HJ-DMSO at T = 295 K
Cl c3
C5 C8 C7 Cg Cl0 CS C*
G
28.49 130.63 145.93 132.56 1so.74 126.90 118.37 20.34 17.53
(s-l)
n.0.e. (q units)
Tc (ps)
0.59 1.14
1.75 1.89
8.5 53.9
0.11
1.12
0.22 1.49 1.53 1.78 0.64 0.46
1.19 1.93 1.95 1.91 1.62 1.59
4.3 x 10-4 4.3 x 10-4
0.41 0.75 21.06 14.47
-
H@
0.1 0.1
R,
4.3 x 10-4 4.3 x 10-4
0.1
3.4
71.6 74.6 85.0 8.5 6.1
(5) 0.04 0.08 3.92 1.72
DMSO
103/T (K-1)
Carbon
CU2+ (mol dm- 3,
eluded that the major species existing in water solution is a metal complex, ML(H20)4, having four solvent molecules in the first coordination shell and that preferential solvation by water molecules gives, in the partially hydrated DMSO solvent, metal complexes having mixed solvation shells, ML(DMSO),(H,O),_,. Since q = 2 for the amitraz molecule that contains pairs of symmetric protons, we can conclude that the main species is a chelated ML complex in which the two imino nitrogens of one ligand molecule coordinate the copper ion. The effective formation of a predominant ML, species, in which the coordinating atom is the methylamino nitrogen, can be excluded with the aid of computer graphics molecular modelling. If coordination were occurring via the sp3 nitrogen there would be no possibility of bringing the aromatic protons close enough to the metal to account for the observed PRRE. These observations were ratified by calculating ion-proton distances (Table 1). Agreement with the proposed structure becomes very good, especially if the calculations are performed taking into account the correlation time for methyls as in the free state. It is concluded that the ligand retains the same motional flexibility in the bound and in the free state. Metal binding evidently stabilizes a cisoid conformation around the N4--C5 bond, thus bringing H3 and H,. quite close to each other. In order to ascertain whether this conformation was also somehow stabilized in the free state selective and double-selective proton spin-lattice relaxation rates were measured for H3 and H 1o in order to obtain the dipolar cross-relaxation rate (e3,, o) between the two.12,13
g3.10
=
010.3
-
R;*‘O-RT$’
=
R:$O-R$
(5)
2358
E. GAGGELLI
where RF’ is the spin-lattice relaxation rate after selective excitation of the i resonance and R,‘j is the rate measured for proton i after double-selective excitation of i andj resonances. The H J-H1 o internuclear distance was calculated at 2.54 8, for amitraz in the free state using the measured value of cr3,1,, (a3, I o = 0.07 s- ‘) in the following equation : l4
’
et al.
2. C. 0. Knowles and H. J. Benezet, J. Environ. Sci. Health 1981, B16, 547. 3. R. E. Homish, J. Agric. Food. Chem. 1984,32, 114. 4. H. J. Benezet, K. M. Chang and C. 0. Knowles, Pesticides and Venom Neurotoxicity (Edited by D. L.
Shankland, R. M. Hollingworth and T. Smyth), p. 189. Plenum Publishing Corporation, New York (1978). I. Solomon, Phys. Rev. 1955,99, 559. N. Bloembergen, J. Chem. Phys. 1957,27, 572. A. W. Nolle and L. 0. Morgan, J. Chem. Phys. 1957,
(6)
26, 642.
where r, = 69.2 ps was assumed. From the molecular model it is calculated that, at values of 0” and 90” for the C3-N4-C5-C 1o torsional angle, the H3-HI o distance is found at 2.1 and 3.5 A, respectively, whereas a distance of 2.5 A corresponds to a torsional angle of about 45”. It is concluded that the free ligand in solution exists in some “preferential” conformation, which is further stabilized by coordination with divalent copper ions.
9.
10. 11.
12.
I. Bertini and C. Luchinat, NMR of Paramagnetic Species in Biological Systems. Benjamin Cummings, Menlo Park (1986). G. Valensin and G. Navon, in Metal Ions in Biological Systems (Edited by H. Sigel), Vol. 21, p. 1. Marcel Dekker, New York and Base1 (1987). A. Allerhand, D. Doddrell and R. Komoroski, J. Chem. Phys. 1971,55, 189. F. W. Wehrli and T. Wirthlin, Interpretation of Carbon-13 NMR Spectra, p. 255. Heyden, London (1980). L. D. Hall and H. D. W. Hill, J. Am. Chem. Sot. 1976,98,
1269.
REFERENCES
13. E. Gaggelli and G. Valensin, J. Chem. Sot., Perkin
1. E. C. Kimmel, J. E. Casida and L. 0. Ruzo, J. Agric.
14. J. H. Noggle and R. E. Schirmer, The Nuclear Overhauser @@ct. Academic Press, New York (1971).
Trans. 2 1990,401. Food. Chem. 1986,34,157.
1993 0
‘H AND 13C NMR RELAXATION INVESTIGATION THE COPPER COMPLEX OF AMITRAZ, A FORMAMIDINE PESTICIDE ELENA GAGGELLI,
NICOLA GAGGELLI
and
0277-5387/93 $6.00+ .W 1993 Pergamon Press Ltd
OF
GIANNI VALENSIN*
Department of Chemistry, University of Siena, Siena 53 100, Italy and ALESSANDRO
FRANCHI
USL 30, Siena, Siena 53100, Italy (Received 23 March 1993 ; accepted 2 June 1993)
Abstract-The copper(I1) complex of amitraz, a formamidine pesticide, was investigated by measuring ‘H NMR spin-lattice relaxation rate enhancements (PRRE) in [2H6]-DMS0 solution. The occurrence of fast exchange conditions was demonstrated by the temperature dependence of the PRRE. The PRRE was interpreted by the Solomon-BloembergenMorgan approach ; the number of solvent molecules in the coordination shell was calculated at q = 2 ; the reorientational correlation time in the 1: 1 metal complex was assumed to be the same as in the free ligand and calculated at z, = 69 ps. The metal ion was shown to stabilize a cisoid conformation of the ligand.
Arnitraz, {N,N’[(methylimino)dimethylidyne]di2,4-xylidine} (Fig. l), is a formamidine acaricide and insecticide effective against a wide variety of phytophagous mites and insects. It is synthesized from 2,4_dimethylaniline and it reverts to several products on mammalian metabolism and environmental degradation. I-3 Extensive use in agriculture has raised the question of its toxicity, especially in light of its interaction with the hepatic mixed-function oxidase systems’ and also with components of the biogenic amine system.4 In this communication we present NMR evidence that amitraz can act as an effective ligand for metal ions such as Cu*+, forming stable complexes that mai play a significant role in eliciting the biochemical activity. EXPERIMENTAL Materials
used without further purification. Solutions were made in [2H6]-DMS0 or in H20 and carefully deoxygenated by bubbling nitrogen gas through. The desired copper concentration was obtained using stock solutions of Cu(ClO4)2 in [2H6]-DMS0 or in H20. Methods
NMR experiments were performed on a Varian VXR-200 spectrometer at controlled temperature ( f 1 K). Chemical shifts were referenced to internal TMS. Spin-lattice relaxation rates were measured with inversion recovery pulse sequences. Spin-spin relaxation rates were measured with the Carr-Purcell-Meiboom-Gill pulse sequence. The rate values were calculated, in both cases, by linear regression analysis of the recovery or decay curves of longitudinal or transverse magnetization components. 13C{‘H} nuclear Overhauser effects were measured with gated decoupling techniques.
Amitraz (standard analysis reagent) was obtained from Ehrenstorfer Lab. (Augsburg) and
RESULTS
AND DISCUSSION
Different proton spin-lattice relaxation rate * Author to whom corrcspondcnce should be addressed. enhancements (PRRE) were measured in the pres2355
E. GAGGELLI et al.
2356
Fig. 1. Molecular formula of amitraz.
ence of copper(I1) ions (Table 1). The experimental paramagnetic contributions to the relaxation rates, R,, were calculated by : Rip = Riobs- Rif
(i = 1) 2),
(1)
where Riobs and Rif are the rate values measured in the presence and in the absence, respectively, of relatively small concentrations of the paramagnetic ion. Consideration of the temperature dependence of such PRRE (Fig. 2) suggests that fast exchange conditions apply, such that : R, = qfR.+,
(i = 1,2),
(2)
where q is the number of coordinated ligand molecules and f is the fraction of bound ligand, the relaxation rate of which is given by R,u. In such situations the PRRE can be interpreted in terms of the Solomon-Bloembergen-Morgan theory ;5-7 since Rzp >> R,, was measured (Table 1) it was concluded that Rzp is contributed by the scalar interaction, whereas the paramagnetic contribution to the spin-lattice relaxation rate, R,,,, is mainly determined by the dipolar electron spin-nuclear spin interaction given by :‘v9
where r is the ion-proton distance measured in 8, ; OH = 1.257 x 10’ rad s- ’ is the proton Larmor frequency ; z, is the correlation time modulating the nuclear spin-electron spin dipoledipole interaction ; yH = 2.6753 x 10’ rad s- ’ G- ’ is the proton magnetogyric ratio ; ge = 2.002322 is the electronic g factor ; Be = 0.9273 1 x lo- *’ erg G- ’ is the Bohr magneton. The motional correlation time, in the case of copper(I1) complexes with small ligands, is a rotational correlation time8*9which can be reasonably assumed to be the same as for the free ligand. The validity of this assumption might be questioned but only at a superior level of accuracy, which is not required in this case (a large error in r, in fact only yields small uncertainties in evaluating distances). The motional correlation time was, therefore, calculated by measuring the i3C spin-lattice relaxation rates of the free ligand, summarized in Table 2. Since the 13C-‘H dipolar interaction may not provide the unique relaxation mechanism, especially for methyls, the spin-lattice relaxation rates were scaled down with the measured nuclear Overhauser effect (n.0.e.) through : (4)
(3) where qmax(the maximum observable n.0.e. when
Table 1. Paramagnetic proton relaxation rates for amitraz (0.1 mol dmp3) in [‘HJ-DMSO at T = 295 K in the presence of [Cu’+] = 4.3 x lo-’ mol dmm3 and calculated ion-proton distances r&u-H)o (A)
6
Proton
@Pm)
N--CHS H3 CH 3(6’) cHH,;s?
3.31 8.29 2.21 2.18 6.97
1.10 1.98 0.75 1.25 -
6.87 9.44 4.86 2.21 7.31
4.0 3.6 4.2 3.9
Hg HI0
6.90 6.82
0.22 1.76
7.11 9.04
5.2 3.7
nCalculated from eq. (3) with z, = 69 ps. ‘Calculated from eq. (3) with t, = 8 ps.
~(CU-H)~ (A) 2.8 2.7
2357
‘H and 13CNMR relaxation
Table 3. Proton spin-lattice relaxation rates (R ,) of water and DMSO in the free solvent and in the presence of amitraz (0.1 mol dmp3) or Cu(ClO.&* 6H20 (4.30 x 10m4 mol dm-3) or both at T = 295 K n!
,P -
I
l
0
0
I
‘
-J ; ; ; , , l
2.9
3.0
3.1
3.2
3.3
Amitraz (mol dm- ‘)
Solvent
.
0.1
Fig. 2. Temperature dependence of PRRE for selected protons of amitraz (0.1 mol dm- ‘) in [*H,]-DMSO.
the relaxation rate is dominated by the dipolar interaction) = 1.98. From the calculated dipolar relaxation rates, R Idip, an effective reorientational correlation time of 69.3 + 15.3 ps for modulation of the internuclear vectors of the four methynes was calculated.” As for the methyls the same calculations yield correlation times in the range 6.1-8.5 ps, suggesting “unlocked” free rotation. * ’ The number of ligands in the coordination sphere was evaluated by measuring the solvent proton relaxation rates, shown in Table 3. A large PRRE (R,, = 3.88 s- ‘) is measured for DMSO protons in the presence of [Cu”] = 0.43 mmol dmm3, which is reduced (R,, = 1.64 s- ‘) by the presence of ligand (0.1 mol dm- 3). The reduction is due to a decreased number of solvent molecules in the metal coordination sphere. Application of eq. (3) would yield q = 2.5, as compared with q = 6 for the free solvated ion, suggesting that speciation may occur in DMSO. However, repetition of the measurements in H20, where the presence of the ligand (0.1 mol dm- ‘) lowered the solvent PRRE from its free hexaaquo ion value (R,, = 20.6 s- ‘) to RIP = 13.7 s- ‘, yields q = 4.0 for the complex. It is conTable 2. ’3C NMR parameters of amitraz (0.1 mol dm- ‘) in [‘HJ-DMSO at T = 295 K
Cl c3
C5 C8 C7 Cg Cl0 CS C*
G
28.49 130.63 145.93 132.56 1so.74 126.90 118.37 20.34 17.53
(s-l)
n.0.e. (q units)
Tc (ps)
0.59 1.14
1.75 1.89
8.5 53.9
0.11
1.12
0.22 1.49 1.53 1.78 0.64 0.46
1.19 1.93 1.95 1.91 1.62 1.59
4.3 x 10-4 4.3 x 10-4
0.41 0.75 21.06 14.47
-
H@
0.1 0.1
R,
4.3 x 10-4 4.3 x 10-4
0.1
3.4
71.6 74.6 85.0 8.5 6.1
(5) 0.04 0.08 3.92 1.72
DMSO
103/T (K-1)
Carbon
CU2+ (mol dm- 3,
eluded that the major species existing in water solution is a metal complex, ML(H20)4, having four solvent molecules in the first coordination shell and that preferential solvation by water molecules gives, in the partially hydrated DMSO solvent, metal complexes having mixed solvation shells, ML(DMSO),(H,O),_,. Since q = 2 for the amitraz molecule that contains pairs of symmetric protons, we can conclude that the main species is a chelated ML complex in which the two imino nitrogens of one ligand molecule coordinate the copper ion. The effective formation of a predominant ML, species, in which the coordinating atom is the methylamino nitrogen, can be excluded with the aid of computer graphics molecular modelling. If coordination were occurring via the sp3 nitrogen there would be no possibility of bringing the aromatic protons close enough to the metal to account for the observed PRRE. These observations were ratified by calculating ion-proton distances (Table 1). Agreement with the proposed structure becomes very good, especially if the calculations are performed taking into account the correlation time for methyls as in the free state. It is concluded that the ligand retains the same motional flexibility in the bound and in the free state. Metal binding evidently stabilizes a cisoid conformation around the N4--C5 bond, thus bringing H3 and H,. quite close to each other. In order to ascertain whether this conformation was also somehow stabilized in the free state selective and double-selective proton spin-lattice relaxation rates were measured for H3 and H 1o in order to obtain the dipolar cross-relaxation rate (e3,, o) between the two.12,13
g3.10
=
010.3
-
R;*‘O-RT$’
=
R:$O-R$
(5)
2358
E. GAGGELLI
where RF’ is the spin-lattice relaxation rate after selective excitation of the i resonance and R,‘j is the rate measured for proton i after double-selective excitation of i andj resonances. The H J-H1 o internuclear distance was calculated at 2.54 8, for amitraz in the free state using the measured value of cr3,1,, (a3, I o = 0.07 s- ‘) in the following equation : l4
’
et al.
2. C. 0. Knowles and H. J. Benezet, J. Environ. Sci. Health 1981, B16, 547. 3. R. E. Homish, J. Agric. Food. Chem. 1984,32, 114. 4. H. J. Benezet, K. M. Chang and C. 0. Knowles, Pesticides and Venom Neurotoxicity (Edited by D. L.
Shankland, R. M. Hollingworth and T. Smyth), p. 189. Plenum Publishing Corporation, New York (1978). I. Solomon, Phys. Rev. 1955,99, 559. N. Bloembergen, J. Chem. Phys. 1957,27, 572. A. W. Nolle and L. 0. Morgan, J. Chem. Phys. 1957,
(6)
26, 642.
where r, = 69.2 ps was assumed. From the molecular model it is calculated that, at values of 0” and 90” for the C3-N4-C5-C 1o torsional angle, the H3-HI o distance is found at 2.1 and 3.5 A, respectively, whereas a distance of 2.5 A corresponds to a torsional angle of about 45”. It is concluded that the free ligand in solution exists in some “preferential” conformation, which is further stabilized by coordination with divalent copper ions.
9.
10. 11.
12.
I. Bertini and C. Luchinat, NMR of Paramagnetic Species in Biological Systems. Benjamin Cummings, Menlo Park (1986). G. Valensin and G. Navon, in Metal Ions in Biological Systems (Edited by H. Sigel), Vol. 21, p. 1. Marcel Dekker, New York and Base1 (1987). A. Allerhand, D. Doddrell and R. Komoroski, J. Chem. Phys. 1971,55, 189. F. W. Wehrli and T. Wirthlin, Interpretation of Carbon-13 NMR Spectra, p. 255. Heyden, London (1980). L. D. Hall and H. D. W. Hill, J. Am. Chem. Sot. 1976,98,
1269.
REFERENCES
13. E. Gaggelli and G. Valensin, J. Chem. Sot., Perkin
1. E. C. Kimmel, J. E. Casida and L. 0. Ruzo, J. Agric.
14. J. H. Noggle and R. E. Schirmer, The Nuclear Overhauser @@ct. Academic Press, New York (1971).
Trans. 2 1990,401. Food. Chem. 1986,34,157.