J. Phys. Chem. 1982, 86, 4011-4016
counting system. A Spectra-Physics Ar+/Kr laser was employed and the power of the exciting line, at 20 490 cm-', was ca. 30 mW measured a t the sample. Polycrystalline samples were sealed in capillary tubes and stored for several weeks a t a temperature of 45 "C prior to use. During the period of measurement samples were maintained a t 45 "C by the use of a modified microscope hotstage arrangement. Confirmation that specimens were indeed in phase I11 was achieved by reference to the lattice-mode region of the Raman spectrum.
4011
AnalaR-grade ammonium nitrate (Hopkin and Williams) was used in this study. Maximum deuteration (95%+) was achieved by repeated recrystallization of samples from D,O (99 at.%) in a sealed system under reduced pressure. NH26N03(97 at. %) and NH4N1803were purchased from Prochem B.O.C. Ltd. and deuterated by the procedure given above.
Acknowledgment. This work was supported by U.S.Air Force Grant AFOSR-81-0207.
Electron Nuclear Double Resonance Study of the Spin-Label Tanol (Tempol) Oriented in the Inclusion Compound 2'-Hydroxy-2,4,4,7,4'-pentamethylfla~an~ F. Ohzekl,t L. D. Kispert;
C. Arroyo, and M. Steffans
Chemistry Department, University of Alabema. Tuscehwsa, Alabama 35486 (Receh4: March 26, 7962; I n Final Form: May 27, 1982)
The nitrogen quadrupole tensor has been determined by ENDOR measurements at -20 "C for the spin-label tanol (tempol) oriented in the inclusion compound 2'-hydroxy-2,4,4,7,4'-pentamethylflavan. The principal values are equal to +1.46, +0.11, and -1.57 MHz, with the direction of the 1.46-MHz quadrupole coupling lying parallel to the direction of the largest nitrogen hyperfine coupling. The other two quadrupole tensor components do not lie parallel to the remaining nitrogen hyperfine coupling directions nor are the couplings cylindrically symmetric. Two exchangeableprotons are observed to be strongly dipolar coupled to the electron. Coupling constants and direction cosines for nine weakly coupled protons were deduced and attempts were made to assign them.
Introduction Nitroxide radicals have been used extensively as reporter groups for biological macromolecules.' The ability to deduce structural features of macromolecules has depended in part on how accurately the g and A tensors are known for a rigid spin-label. Ideally single-crystal EPR studies of oriented spin-labels can yield such information; however, few have been carried out.' Estimates based on frozen solution or powder EPR samples are insensitive to in-plane variations and weakly coupled protons are usually not resolved. In an attempt to accurately measure the nitrogen hyperfine and g tensors as well as the principal directions, single crystals were grown of the spin-label tanol (also known as tempol) included in the clathrate 2'-hydroxy2,4,4,7,4'-pentamethylflavan(I). Using the precursor to (1)
(11)
/OH
51
- 2'
5'-
3'
'5-1 H5-3
tanol as a diluent, we grew a 1:lOOO molar mixture of tanol *Currentaddress: 1-11-3, Hongo, Bunkyo-ku, Tokyo 113, Japan. f NSF Undergraduate Research Participant, summer 1981. 0022-3654/82/2086-4011$01.25/0
and precursor as a one-to-one inclusion compound in clathrate (I). Resolved EPR spectra were obtained and the hyperfine and g tensors were ,reported.2 However, it was noted at selected crystal orientations that the 1:l:l EPR spectrum was split by a proton doublet from at least two different protons. It is the purpose of this paper to examine the origin of the resolved doublets and to compare the principal nitrogen Q tensor directions to those of the nitrogen hyperfine couplings. Previous low-temperature NMR studies of polycrystalline nitroxide radical^^-^ have revealed that the spinlabel tempol (tanol) exists in the solid state in nearly a W-type conformation4 as evidenced by the appearance of a large positive isotropic coupling of 7.84 MHz for the 4-1 methyl proton and a positive 0.64-MHz coupling for the 5-3 proton. All other proton couplings were negative and I2.13 MHz. Initially, it was assumed that the resolved proton coupling in the EPR spectrum of tanol included in I was due to this methyl proton. However, our ENDOR and deuteration study reveals that this is not the case and that no large isotropic proton couplings are present. Instead, the EPR doublets are due to two dipolar coupled exchangeable protons that are located approximately 2.1 A from the unpaired electron density. It is also noted that ENDOR spectra can be easily obtained near room temperature for a 5% molar concentration of spin-label; however, only one branch of the ENDOR doublet is ob(1) L. J. Berliner, Ed., "Spin Labeling, Theory and Applications", Academic Press, New York, 1976, and references cited therein. (2) W. Smith and L. D. Kispert, J. Chem. Soc., Faraday Tram. 2,73, 152 (1977). (3) D.Ondercin, Ph.D. Thesis, University of Rochester, Rochester, NY. _ _ , 1978. -. . _. (4) D. Ondercin, T. Sandreczki, and R. W. Kreilick, J. Magn. Reson.,
--.(5) F..----,Ferrieu and M. Nichtachien, Chem. Phys. Lett., 11, 46 (1971).
34. 151 - - - (1979).
0 1982 American Chemical Society
4012
The Journal of Physical Chemistry, Vol. 86, No. 20, 7982
Ohzeki et
served for each strongly coupled proton, presumably as a result of strong cross relaxation. On the other hand, both branches of the weakly coupled protons are detected. When the temperature is lowered below -50 "C, the ENDOR spectrum of the weakly coupled protons disappears. Poor ENDOR resolution of the weakly coupled protons prevents an accurate analysis to be carried out but estimates of the coupling tensors are made nevertheless. Experimental Section Single crystals of the inclusion compound 2'-hydroxy2,4,4,7,4'-pentamethylflavan (I) containing 5 mol ?& of the spin-label tanol (11)(2,2,6,6-tetramethyl-4-piperidinol-loxyl) and 45 mol % of the spin-label precursor 2,2,6,6tetramethyl-4-piperidinol(III)were grown by slow evaporation of an ethanol solution as previously reported.2 Preparation of purified I has been describede6 A single crystal of I containing 5 mol % I1 and 45 mol % I11 was also grown from deuterioethanol where the exchangeable hydroxyl protons in I1 and I11 as well as the N-H proton were substituted for deuterium. To prepare deuterated 11,we first purified tanol by recrystallization from isopropyl ether until the melting point (69-71 "C) checked with the literature and IR indicated absence of impurities. Selective deuteration of the exchangeable protons in I1 and I11 was carried out by dissolving each separately in D20under an N2 atmosphere, letting them stand, and then distilling off the D20 under vacuum. This procedure was repeated 3 times. IR indicated >90% OD present. I is insoluble in D20, so little exchange results; 50 mol % I was dissolved in ETOD and to this solution was added the deuterated 5 mol % I1 and the 45 mol % deuterated 111. To the solution was added -2 mL of D20 to reduce the solubility of I in solution. Under optimum conditions of temperature and concentration crystals were grown in a few days by evaporation at 29 "C by flowing prepurified dry N2 over the solution. However, it took nearly 4 months to find optimum conditions. Since the crystal structure is not known, an arbitrary orthogonal X 'YZ' reference axis system was used where the X'axis is parallel to the needle axis of the crystal and the Y' axis is perpendicular to the crystal plate. The X Y Z ' axis system corresponds respectively to the YZX reference system used in ref 2. ENDOR spectra were recorded between 5 and 42 MHz in each of the three orthogonal planes X'Y', YZ', Z'X'with a Varian E-1700 spectrometer located at the Biomedical ESR Center in Milwaukee, WI. The angular ENDOR data were used to determine the principal proton hyperfine coupling and were further checked by using a homemade ENDOR spectrometer previously described.' ENDOR spectra were recorded at -20 "C. This temperature was selected to prevent any excessive heating of the crystal by the rf source and yet high enough to obtain ENDOR spectra. It was found that the proton ENDOR spectral intensity decreased markedly below -50 "C and was absent at 77 K. The nitrogen ENDOR intensity increases upon cooling to -50 "C and is absent at 77 K. The nitrogen hyperfine coupling has been previously determined by EPR measuremenh2 Results The ENDOR spectrum recorded at -20 "C with the observing field set on the MI = 0 EPR line of the pro(6)W. Baker and D. M. Besly, J. Chem. Soc., 1103 (1940);W.Baker, R.F. Curtis, and J. F. McOmir, ibid., 76 (1951);W.Baker, R. F. Curtis, and M. G. Edwards, ibid., 83 (1951). (7) J. S.Hwang, A. C. Dickinson, and L. D. Kispert, J.Phys. Chem., 83,3381 (1979).
I
I
I
2 9 MHZ
18
;j" \."\ I
I
I
33
al.
I
37
ENDOR TANoL
n
20
\ Figure 1. ENDOR spectrum of m o l incorporated in clathrate I obsewed between 8 and 37 MHz when H 11 (cos 76", sln 76", 0) and theobseWgReklissetattheM,=OESRUne. NoENWRlineswere obsewed over those frequencies not shown. The four lines between 29 and 37 MHz are due to the interactlon with a slngle nltrogen while the lines at approximately 19 and 8.5 MHz are due to strongty coupled protons.
TABLE I: Nitrogen Hyperfine and Quadrupole Couplings and Direction Cosines direction cosinesu
couplings, AN
QN
MHz
X'
Y'
2'
103.0 22.1 17.5 1.4, 0.1, -1.5,
-0.5971 0.4146 0.6868 -0.5961 0.6386 0.4867
0.7967 0.2068 0.5679 0.7973 0.3990 0.4530
-02934 -0.8862 t0.4538 -0.0951 -0.6581 0.7469
Since the triclinic crystal structure of I is not known, the X , Y ,and 2' directions are arbitrary. For instance, redefining e = - e in the Y ' Z ' plane only changes the sign of each element of the last column of direction cosines.
tonated sample and H II (cos 76", sin 76", 0) is given in Figure 1. The peaks centered about 13.6 MHz are due to weakly coupled protons. No additional proton peaks are observed as the temperature is decreased below -20 "C. Resolved peaks at approximately 19.3 and 8.5 MHz are due to two protons which are more strongly coupled to the electron. The four lines between 29 and 37 MHz are due to the electron coupled to a single nitrogen. Upon rotation of the crystal the four-line spectrum due to nitrogen collapses to a two-line spectrum centered at AN/2 which changes back to a four-line spectrum when AN/2 is a minimum. As the crystal is rotated, the four-line to two-line pattern is resolved at each angle except in the region between 12 and 14 MHz. Techniques for generating nitrogen hyperfine (A) and quadrupole (Q)tensors to second order without assuming that A and Q are coaxial have been given by Thuomas and Lund8 while a secondorder treatment in an arbitrary coordinate system has been given by I w a ~ a k i . The ~ four frequencies vl, v2, v3, and v4 due to nitrogen are fitted to the equation k2/4)[(vl + ~2 + ~3 + ~ 4 ) 16b2] = I.gA2.gI = T" (1) where b = g,p,H. To determine the undiagonalized hyperfine tensor T",we least-squares fit the values of eq 1 at each angle 0 to eq 2. The value P calculated from eq Ta = CY
+ p cos 20 + y sin 28
(2)
3 is used to construct the tensor Tq according to 1.Tq.I
6P =
(VI
-~
+ (US - ~ 4 )
2 )
(3)
= PI.Ta.Iby least-squares fitting PA2 to eq 2 from which (8)K. A. Thoumas and A. Lund, J. Magn. Reson., 18, 12 (1975). (9)M.Iwasaki, J.Magn. Reson., 16, 417 (1974).
ENDOR Study of the Spin-Label Tanoi
21 23
The Journal of Physical Chemistry, Vol. 86, No. 20, 7982 4013
11
1
//A
I
TABLE 11: ENDOR-Derived Proton Couplings, Direction Cosines, and Effective Distances for the Two Largest Proton Couplings direction cosines
X'
couplings, MHz
-11.85' -7.58 16.80 A h -0.88
A,, 0
50
100
x' +Plane
150'
0
50 I00 Y'F-Plane
150'
0
50 100 150' z'X'-Plane
2. Experimentalangular dependence of proton 1 (0)and proton 2 (A)ENDOR spectwn in the three orthogonal planes (X'Y', Y'Z', and Z'X'). The solid lines are the frequency positions calculated by using the couplings and direction cosines given in Table I1 (set 0 = -0 in the Z'X' plane to obtain the phase listed in Table 11).
the diagonalized Q tensor can be deduced from Q = A-l.g-1. T%g-1.A-1. The nitrogen hyperfine (A) and quadrupole (Q) tensors for protonated I1 deduced from this analysis are given in Table I. The experimental angular dependence of the proton ENDOR peaks at 19 and 8.5 MHz of Figure 1 are given in Figure 2. Careful measurement shows that these two peaks are not centered about the free-proton frequency which varies from 13.55 to 13.65 MHz as the crystal is rotated. This small variation in free-proton frequency occurs because the observing ENDOR position is always the central EPR line (mIN= 0). Instead the center of the two proton lines varies from 13.6 to 15.7 MHz. Thus,these two lines in Figure 1 are assigned as the upper branch of proton 2 and the lower branch of proton 1 and are indicated by open triangles and open circles in Figure 2, respectively. An extensive search was carried out for the corresponding lower and upper branch lines. They were found (although with low intensity) at some orientations in the ENDOR spectra by using a very large crystal. Presumably this intensity variation occurs because the Txl relaxation path is dominant for the strongly coupled protons. The presence of electron spin exchange arising from the high concentration of spin-label could also be responsible. No such variation was observed for the weakly coupled protons. A least-squares fit to the experimental ENDOR data for protons 1 and 2 produced an undiagonalized tensor which upon diagonalizing gave rise to the principal values and direction cosines listed in Table 11. Using these principal values, we calculated the solid curves given in Figure 2. An EPR study of the deuterated crystal shows the complete absence of the doublet coupling resolved at various angles for the protonated sample. ENDOR measurements of the deuterated crystal show that the ENDOR lines for protons 1 and 2 have disappeared. However, the ENDOR spectrum for the weakly coupled protons remained the same upon deuteration. It is noted that, when the deuterated spin-label sample is cooled to 77 K, the line width at -20 "C increases 3.2 G. Typically the line width of the deuterated sample at -20 "C varies between 4.5 and 5.3 G depending on angle. The same variation with angle still occws at 77 K, however, with a line width on the order of 8 G . Thus, no change occurs in the large proton couplings (1 and 2). Previously a 1.2-MHz increase in the largest nitrogen coupling was observed over this change
12.64b 11.09 -17.65' 2.03
Y
2'
Proton 1 0.0969 0.9929 0.5811 -0.1161 2.07 0.8078 -0.0412
-0.0662 -0.8062 0.5880
Proton 2 -0.4564 0.3522 2.09 -0.8171
0.6259 0.5256 -0.5762
-0.6324 0.7744 0.0194
' Couplings deduced from the v + branch. Couplings deduced from the v - branch. ' If the sign of the largest dipolar coupling is assigned a t value, then the isotropic coupling is negative. d (r) is in angstroms. /
15
I
1
14
z V W
213 W CY LL
12
I1
I
50 100 150'
x' V - P l a n e
0
.
50 IO0 Y'f-Plane
150'
.
0
.
50 IO0 150'Z'x'-Plane
Flgure 3. Angular dependence of four weakly coupled protons. The solid lines are those calculated by using the couplings and direction cosines in Table 111, v+ branch, with 0 = -0 in the Z'X plane.
in temperature.2 The reason for the line-width increase is unknown; however, it is possible that crystallographic disorder occurs at 77 K giving rise to more sites than the triclinic site observed at -20 "C. This would be consistent with the observation that the clathrate crystals always crack upon cooling to 77 K. An alternative explanation would be to assume that the motion of the methyl protons has slowed, giving rise to nonequivalent proton couplings. An analysis of the weakly coupled proton ENDOR spectra proved to be quite difficult. In Figure 3 are given the angular variation of the v+ branches of four of the nine weakly coupled protons deduced from the ENDOR spectra. Differences between calculated (solid line) and experimental frequencies are due in part to the poor ENDOR resolution observed. This problem is particularly evident when the angular dependence for all nine proton couplings is plotted (Figure 4). Despite the poor resolution, hyperfine couplings and direction cosines were deduced for nine protons and are listed in Table 111. A comparison of the calculated curves for the v+ branch (solid lines in each case) based on these couplings and the experimental frequencies are given in Figure 4. Because of the complexity of the ENDOR spectrum over the frequency range of 12-14 MHz, it was found impossible to follow the ENDOR peaks for those protons exhibiting a coupling tensor with the largest component less than 1 MHz. In such w e s the peaks were overlapped at all angles. It should be
4014
1
i 98,"
3
0
I
I ] " . :1 !
I ,
,
50' 100' 150'
0
X' Y - P l a n e
I
,
50' I& 150' YZ- Plane
0
C
.A
.
I
50'
.
loOD 150'
2 x'-Ptane
Flgure 4. Angular dependence of the Y+ ENDOR frequencies for nine weakly coupled protons. The sdid lines are the Y+ ENDOR frequencies calculated by using the direction cosines and couplings given in Table I11 with 6 = -6 in the Z'X'plane.
TABLE 111: Dipolar ( A d ) and Isotropic (Abo)Couplings (MHz) and Direction Cosines for the Weakly Coupled Protons protona
A(-
Ohzeki et at.
The Journal of Physical Chemistry, Vol. 86, No. 20, 1982
direction cosines
A h
1
Ad
4.85 -0.99 - 3.86 B(-1 4.43 -0.85 -1.92 - 2.51 C(- 1 4.27 -0.82 -1.35 - 2.92 D(-1 3.80 -0.73 -0.95 -2.86 E(+ 1 -2.37 -1.20 -0.56 2.93 F(+ 1 -3.42 +0.69 0.46 2.95 G(+ 1 -3.36 0.31 -0.08 3.44 H(- ) 3.76 -0.99 -0.56 - 3.20 I ( + ) -0.55 -3.48 0.40 3.08
(r)b
X'
Y
Z'
3.08
0.7430 -0.6693 0.0439 0.7640 0.4278 0.4813 0.7968 0.6030 -0.0397 0.9827 -0.1422 -0.1184 0.9880 -0.1374 -0.0708 0.8341 -0.5131 -0.2026 0.9529 0.3024 -0.0224 0.5858 -0.7702 0.2521 0.6959 -0.7055 0.1342
-0.2720 -0.2959 0.9157 0.0388 0.7140 -0.6991 0.0204 0.0390 0.9990 0.1725 0.4729 0.8641 0.0916 0.1519 0.9841 0.5516 0.7692 0.3226 0.2222 -0.6461 0.7302 0.6897 0.3105 -0.6541 0.1136 0.2927 0.9494
0.6115 0.6816 0.4019 -0.6441 0.5528 0.5288 -0.6039 0.7968 -0.0188 0.0669 0.8696 -0.4893 0.1245 0.9788 -0.1627 -0.0097 -0.3808 0.9246 -0.2064 0.7008 0.6829 0.4256 0.5571 0.7131 0.7091 0.6455 -0.2838
-0.49
3.18 3.22 3.34
3.91 3.64 3.46 3.35
3.59
a The letter refers t o the particular curve in Figure 3 or 4 while the t or - refers to the v + or V - branch. A change of signs of the coupling constant changes branches. (r) is the effective dipolar distance in angstroms.
pointed out that, for the proton couplings listed in Table 111, at least one hyperfine component for each coupling is greater than 3 MHz which permits spectral resolution at some angles. Small but nonzero isotropic couplings are observed for all couplings in Table 111. If the isotropic component is removed, the average distance from each proton to the unpaired electron can be estimated from the largest component of the dipolar tensor, and they are given in Table 111. Unfortunately the crystal structure for I containing I1 is not known, so a comparison between the direction cosines deduced for the hyperfine couplings in Tables I1 and
I11 and the corresponding crystal structure vector directions cannot be made. However, it is instructive to make a few assumptions concerning the crystal structure and the tanol conformation. First, assume that the conformation of the tanol radical observed in single crystals of tanol is retained in the clathrate and calculate the directions and distances expected for I containing 11. Secondly, consider the possibility that the clathrate has forced the tanol radical into another conformation. Since the crystal structure of tanol single crystals is known,1° we arbitrarily projected the vector direction connecting the 5-2 proton and the center of the N-O bond along the (0,0,1) direction of an XYZ axis system. This same transformation was also applied to all other intervector directions between the center of the N-0 bond and the remaining protons in tanol. These directions and distances were then compared to those deduced from the ENDOR measurement. However, since tanol in the clathrate I crystallizes in a triclinic space group,2 some point of reference was needed so that the distances and directions derived from the ENDOR couplings could be assigned to the various protons. It was assumed that the direction of proton 1 should be along the (0,0,1) direction. This was originally based on a wrong assumption that the large proton coupling direction was due to a nearby ring or methyl proton. Fortunately, the direction chosen seems to be approximately correct. The transformation necessary to project the direction of proton 1 along the (0,0,1) direction was also applied to all the ENDOR lines. In Table IV are given the crystal structure distances from each proton to the center of the NO group for a c\
N, C
-0 IIa
out-of-plane angle of -9". As the negative angle increases, the distance from the NO group to the 5-2 proton decreases. To correct for the fact that the unpaired electron density is distributed in the p orbitals on nitrogen and oxygen, one replaces" the point dipole expression by [1/($ + d2)]3/2where d = 1.4 A. The best matches between the vector directions deduced from ENDOR with those from the crystal structure are listed in Table IV for an out-ofplane angle of -9" and -31". The angular differences in the vector directions in most instances are small (if the angular difference for the 1-1 proton is ignored); however, an out-of-plane of -9" appears to be given an overall closer fit. The distances calculated from ENDOR give less than a perfect fit although they are similar to the distances calculated from the crystal structure. None of the proton coupling directions corresponded to the N-O to 1-1 proton direction although the distance derived from coupling E (3.91 A) closely matches. This less than perfect match suggests that tanol may exist in another conformation in the clathrate than that found in the pure crystal. A referee has made a clever suggestion that allows the presence of rotating methyl protons at 250 K. He pointed out that the absence of coincident principal axes for both the quadrupole and hyperfine coupling tensors suggests that absence of reflection symmetry about a plane perpendicular to the CNC plane containing the N-0 bond and thus there must be (10) L. J. Berliner, Acta Crystallogr. Sect. B , 26, 1198 (1970);P.J. Lajzerowicz-Bonneteau,ibid., 24, 196 (1968). (11)A. Pullman and E. Kochanski, Int. J. Quantum Chem., 15, 251 (1967);A. Calder, A. R. Forrester, P. G. James, and G. R. Luckhurst, J. Am. Chem. SOC.,91, 3724 (1969).
ENDOR Study of the Spin-Label Tanol
The Journal of Physical Chemistry, Vol. 86, No. 20, 1982 4015
TABLE IV: Effective Proton to Electron Density Distances ( R ) Derived from Crvstal Structure and ENDOR Data crystal data
angular differences: deg ENDOR R d
Droton 1-1
2-1 2-2 4-1 4-2 4-3 5-1 5-2 5-3
3.58 3.12 3.68 3.30 2.50 3.01 2.48 2.13 3.12
3.84 3.42 3.94 3.58 2.86 3.32 2.85 2.55 3.42
l3.91, 3.59, 3.46, 3.22, 3.64, 3.18, 3.35, 3.08, 3.34,
E(+)]
I(+) G(+ )
C(-) F(+ )
B(-) H(-) A(-) D(-)
- 9"
- 31"
[68.4] 24.4 1.3 12.4 15.1 30.7 10.9 12.2 10.7
[69.3] 26.4 12.3 21.0 17.4 26.4 5.5 18.4 7.8
Assuming a -9" "out-of-plane angle" for substructure IIa where the minus sign designates a bend toward the C-5 carbon. Corrected for electron distribution. Angular differences between ENDOR and crystallographic directions assuming - 9" and - 31" out-of-plane angle. In angstroms.
more than nine nonequivalent protons. It was further suggested that the clathrate may have forced the spin-label tanol into a croiilee conformation (proposed for tanone12) rather than the chair conformation proposed for tanol.12 If tanol exists in the croiilee structure, all 17 protons become nonequivalent. If the methyl protons are allowed to rotate, then only nine weakly coupled protons are observed, the same number as reported in Table 111. Since the methyl protons are close to the high spin density on the N-O bond, a measurable anisotropy would be observed, even though they are y protons. When the temperature was lowered, the methyl proton rotation would slow, causing an increse in the EPR line width and a loss of ENDOR intensity below 250 K, as observed experimentally. Providing the increase in line width with decreasing temperature is not due to site disorder and the loss of ENDOR intensity not due to electron-electron exchange effects, this type of conformation would be preferred over that found in tanol crystals. It is then unfortunate that the ENDOR resolution for the weakly coupled protons is so marginal; otherwise, a calculation of the distances and directions for the croiilee conformation would be justified as accurate distances and directions from ENDOR measurements would be possible. Given such a situation, it would be possible to assign a best model Conformation in the absence of crystallographic data for I containing II. The calculations that have been carried out serve only to show that the nine proton couplings given in Table 111are probably due to the methyl or ring protons on II. The ENDOR data are not good enough to determine the conformation of I1 in I.
Discussion The ENDOR-derived nitrogen hyperfine couplings listed in Table I are in close agreement with the couplings derived from EPR measurements reported in ref 2. It is interesting to note that, although the AN tensor is nearly cylindrically symmetric, the deduced nitrogen quadrupole tensor (Table I) is not. Even though it is difficult to assess the accuracy of the quadrupole couplings reported in Table I because the absolute value of each component seems to be quite sensitive to small errors in the input data, the quadrupole couplings in Table I are believed to be accurate to *O.l MHz. The axis corresponding to the +la46 MHz coupling lies parallel to the axis corresponding to the largest positive nitrogen coupling. The axes corresponding to the other two quadrupole couplings (0.l1 and -1.5, MHz) lie approximately 21.5O from the direction of the 22.1- and 17.5-MHz nitrogen couplings, respectively. This would (12)R.Briere, H. Lemaire, A. Rassat, P. Rey, and A. Rousseau, Bull. SOC.Chim. Fr., 4479 (1967).
place the largest negative quadrupole coupling near to the direction of the nitrogen lone pair. There appear to be only a few examples where the quadruple coupling has been reported for a nitrogen-centered radical. Analysis of the nitrogen quadrupole tensor for NO2 showssJ3 that the +1.6-MHz coupling direction lies parallel to largest nitrogen hyperfine coupling; however, the directions of the other two tensor components of -0.2 and -1.4 MHz are not parallel to the remaining two axis directions of the A tensor. Thus, the charge distribution around nitrogen in NO2 is similar to that in 11. Noncylindrical symmetry and similar values have also been observed for the nitrogen quadrupole tensor in magnetically dilute single crystals of Ag(I1) and Cu(I1) tetraphenylp~rphyrins'~ where the largest coupling was positive and located perpendicular to the M-N direction but parallel to the heterocycle plane. Similar values for the Q tensor and the lack of cylindrical symmetry were also observed for Cu(g1~)~ complexes in a-glycine cry~tals,'~ and Cu(I1) complexes in L-alanine crystals.16 However, in Cu(I1)-alanine complexes the axis corresponding to the largest positive value was found to lie parallel to the direction of the largest A value coupling while the largest and negative coupling for C u ( g 1 ~was ) ~ found to be located parallel to the largest positive A value. In copper 8hydroxyquinolinate," the axis of the largest negative coupling is oriented along the nitrogen lone pair in the plane of the metal complex, not so different from that in tanol. Since the quadrupole couplings give estimates of total charge distribution at the nitrogen, obviously the charge distribution varies from system to system. It also appears that nuclear quadrupole couplings are only qualitatively understood since ab initio attempts to calculate field gradients have not been particularly successful.14 Thus, no attempt was made to calculate field gradients for nitrogen in I. Theoretical studiesl8 predict that large proton coupling constants will be observed if the nitroxide radicals form the W configuration. The W configuration occurs when the dihedral angles Or = *180° and 8, = Oo where Br and 8, are the dihedral angles between the y or /3 protons and the orbital containing the unpaired electron in a radical like a propyl radical. The appearance of positive couplings (13)S. N.Rustgi and H. C. Box, J. Chem. Phys., 59, 4763 (1973). (14)T. G. Brown and B. W. Hoffman, Mol. Phys. 39, 1073 (1980). (15)M. Fujimoto, C. A. McDowell, and T. Takui, J. Chem. Phys., 70, 3694 (1979). (16)R. Calvo, S. B. Oseroff, and H. C. Abache, J. Chem. Phys., 72, 760 (1980). (17)G.H.Rist and J. S.Hyde, J. Chem. Phys., 50, 4532 (1969). (18)Y.Ellinger and R. Subra, J. Chem. Phys., 62, 10 (1975).
4010
The Journal of Physical Chemistry, Vol. 86,No. 20, 1982
was given as proof that the structure of tanol in the pure crystalline form exists in nearly a W c o n f i r g ~ r a t i o n . ~ ~ ~ In the present clathrate inclusion compound, it will be difficult for the spin-label tanol to exist in a W configuration as the orientation of just the methyl group would be a property of the clathrate and not that of the spinlabel. Theoretically it is predicted that, as By decreases or 8, increases, the magnitude of the proton coupling constants is predicted to decrease and change spin when 8, I120° and 8, I40". On the basis of these predictions, the absence of large positive isotropic proton couplings for tanol oriented in I suggests that the clathrate has prevented the W configuration from forming. Although the absolute sign of the isotropic proton coupling constants cannot be determined from the current ENDOR study, the sign of the largest anisotropic component was taken as positive, thus defining the sign of the isotropic component. Using this convention, most of the isotropic components are negative and small (
