1752
Anal. Chem. 1986, 58, 1752-1756
(24) Lenkinski, R . E.; Elgavish, G. A.; Reuben, J. J . Magn. Reson. 1978, 32, 367. (25) Krasutskii, P. A.; Yurchenko. A. G.: Rodionov, V. N.; Antipin, M. Y.; Struchkov. Y. T. Teor. Eksp. Khim. 1983, 19, 685; Chem. Abstr. 100 .- 94795 - .. - - w .. (26) Krasutskii, P. A,; Yurchenko, A. G.; Jones, M., Jr.; Kornilov. M. Y.; Degtyarev. L. S.: Zamkovoi, V. I. Teor. EXP. Khim. 1982, 18, 189; Chem. Abstr. 97 92457h. (27) Krasutskii, P. A,; Yurchenko, A. G.; Rodionov, V. N.: Jones, M.,Jr. Tetrahedron Lett. 1982, 23, 37 19. (28) Krasutskii. P. A.; Yurchenko, A. G.;Rodionov, V. N.;Kulik, N. I . Teor.
Eksp. Khim. 1984, 20, 54; Chem. Abstr. 101 321699. (29) Peters, J. A.; Nieuwenhuizen, M. S.;Raber, D. J. J. Magn. Reson. 1985, 65, 417.
RECEIVED for review July 15, 1985. Resubmitted February 10,1986. Accepted February 27,1986. This work was supported by the International Research and Exchanges Board (IREX).
Solvent Suppression in High-Resolution Proton Nuclear Magnetic Resonance Based on Control of Transverse Relaxation Rate Thomas M. Eads,' Scott D, Kennedy, and Robert G. Bryant* Departments of Radiology, Biophysics, and Chemistry, University of Rochester Medical Center, Rochester, New York 14642
A general method Is described for the acGu11yl(dtkn of nudear magnetic resonance proton spectra In water and other proton-containlng solvents, whkh mkrlmlzes the spectral distortions usually associated with the Intense solvent resonance. The technique Involves the addltlon of a relaxatlon agent, whkh dMferentlaUy Increases the sdvenl transverse relexatkn rate while leaving the solute resonances largely unaffected, combhred with a data acquldtlon strategy that exploits the Induced relaxation rate difference. I n the present examples we have used the common pulse sequence associated wlth the Melboom-Glll modlfkatbn of the Carr-Purcell spln-echo train experiment wlth the pulses closely spaced to suppress phase modulatlon In the accumulated spectra due to scalar couplings. Alternatlve schemes that spln lock the magnetization dlrectly work equally well, though they may be more dlffkult to implement on commerclal Instruments. Examples of inorganic chemicals and biochemicals are provlded, and the features that control the solvent suppresslon by this method are presented quantltatlvely.
The problem of suppressing the solvent peak in high-resolution 'H NMR has been approached in several ways: substitute D20 for H20; arrange for selective nonexcitation of the solvent spectral region (I);or arrange for solvent magnetization to be small at the time of acquisition by presaturation, by spectral selection based on longitudinal relaxation time, T , (recently reviewed in ref 1and 2), by spin-echo methods based on transverse relaxation time, T2 (3-6); or suppress detection of single quantum transitions, including HzO, or apply multiple-quantum techniques (7). The basis of spin-echo Fourier transform (SEFT) methods is to exploit the difference, when it exists, between the transverse relaxation times of water protons and solute protons. The water proton T2 may be fortuitously short in concentrated or heterogeneous systems (e.g., packed cells, intact tissues, foods, and slurries), and thus, SElV spectra may show resolved low-molecular-weight solute 'Present address: Basic Food Science Laboratory, Kraft, Inc., 801
Waukegan Rd., Glenview, IL 60025.
0003-2700/86/0358-1752$01.50/0
resonances. However, control of solvent T2is desirable for applications where the water proton T, is naturally long, as in dilute or compartmented systems. This paper presents a general method for solvent suppression based on control of Tzusing paramagnetic reagents. In preliminary reports we demonstrated some applications of the method ( 4 9 ) . Here, we review the relevent principles and extend the results to inorganic and organic solutes in simple aqueous systems, to metabolites in complex natural body fluids, and to metabolites in living cells.
EXPERIMENTAL SECTION Ethanol was 95% U.S.P. 3-(Trimethykilyl)-l-propaneaulfonic acid sodium salt (TSS),deuterium oxide (DzO, 99.9 atom % ,H), and cobalt(II1)sepulchrate chloride were obtained from Aldrich Chemical Co. All other chemicals were reagent grade (ACS) or better. Cobalt(II1) sepulchrate and lactose solutions were measured without adjustment of pH. Human cerebrospinal fluid was collected in biopsy and frozen without treatment. Human blood plasma was obtained frozen from the hospital blood bank. Human urine was collected and used immediately. Rat brain was dissected from laboratory Wistar rata and placed in 0.1 M sodium phosphate buffer, pH 7.4, at room temperature. The cortex was minced, then soaked and rinsed 4 times, each soak lasting 1 min with a buffer containing 0.1 M sodium phosphate, 22 vol % DzO, 0.3 mM MnClZ;the sample was then centrifuged into an NMR tube. NMR samples were made to contain 10% DzO for field frequency, lock, 0.1-1% of a saturated solution of TSS in water (depending on the relative concentration of the main solutes of interest) as internal primary chemical shift reference, and about 0.2 mM MnClZ,added as a 10 mM solution prepared freshly from refrigerated 1 M stock solution. TSS was not added to minced brain tissue. The TSS, DzO, MnCl,, and test fluids were pipetted directly into a 5-mm NMR tube (Wilmad 507PP), final volume 0.500 mL. 'H NMR spectra were obtained at ambient temperature (28 f 2 "C) on an IBM WP270 SY spectrometer, operating frequency 270 MHz (lH), and using the standard 5-mm probe. External field homogeneity was obtained by adjusting magnet shim coils while observing the free induction decay (FID)of residual protons in a sample of DzO spinning at 30 d= 1Hz. The line width obtained was typically 0.50 f 0.05 Hz; subsequently,a resolution of 0.15-0.2 Hz could be observed in spectra of organic solutes. Single-pulse spectra were obtained by attenuating both the transmitted radio 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986 frequency pulse power into the probe and the rf signal coming from the probe by 8-12 dB. The result is a pulse angle much Iess than 90" and a signal that does not saturate the receiver. Spin-echo spectra were obtained with attenuators removed, by executing the spin-echo pulse sequence (D-90",-( ~-180'~-~)~acquisition)where T is typically 0.6ms and n is adjusted to reduce the water peak; n is typically 1 ~ 0 in0the experiments reported here. The pulse sequence is the Meiboom-Gill modification (IO) of the Carr-Purcell spin-echo sequence ( I l ) , usually used to generate a series of echoes for determination of T,, but used here t o effectively spin lock the magnetization during the relaxation period; 90"and 180" pulse widths were 4.5 and 9.8 /.a, respectively. The usual phase cycling with quadrature detection was employed. A relaxation delay of 5 s was sufficient to avoid resonance saturation. Sweep widths were 2000 or 2500 Hz, and 8K or 16K data points were collected. An exponential multiplication was applied to the FID, corresponding to 0.4-1.0-Hz line broadening. All samples were spun at 30 Hz. For lines much broader than the residual instrumental inhomogeneity (about 0.1 Hz), proton T2values could be estimated from the line width T2*= 1/7r Av,where Av is the full line width at half-height. 'H T, values were also measured by the spin-echo technique, using a series of spectra corresponding to different values of n in the CPMG pulse train. T z was determined by least-squares fit to the relation Z ( t ) = Io exp(-t/Tz) where Z is the integrated intensity in arbitrary units and t = 21r7 in seconds.
RESULTS AND DISCUSSION The relaxation rates l/Tl and 1/T2of a nucleus in chemical exchange with a Paramagnetic center in solution are given by (la) 1/Tl,obsd = P m / ( T l M + 7ex) + 1 / T l D + 1 / T l , O S
do,
-
IU)
'
"""OI
" " "10 ' ' " " " ' 10 FREQUENCY, MHz
'
'
"
'
1753
'*.1
241
20
..
. . a
5
..
4-
(Ib) = pm/(TZM + 7ex) + l/TZD + l/TZ,OS where 1/TD is the diamagnetic contribution (e.g., due to bulk water in a diamagnetic aqueous solution), 1OTi,os is due to l//TZ,obsd
so-called outer sphere relaxation, which is small in our experiments, TiM is the relaxation time of the nucleus in the paramagnetic center, T,, is the mean residence time of the observed species in the paramagnetic environment, and P,,, is the probability of the observed spin sampling the paramagnetic site. The relaxation rates in the paramagnetic environment are given by the Solomon-Bloembergen equations, (12, 13) 1/T1~ = 2C[3~,/[1 W?T,2] 77,/[1 + W?T>]] -k 2CTTe/[1 + W ~ T ~ (2a) ] ]
+
+
1 / T 2 ~= C[47,
+ ( 3 ~ , / [ 1+ UI%,~]) + 1 3 ~ , / [ 1+ w?7>]]
+ C'ITe + (Te/[l + U,27,2])]
+
(2b)
+
where C = (1/15)y2g2p2S(S l)/r6,C'= (1/3)(A/h)'S(S I),w, and WI are the electron and nuclear Larmor precession frequencies, y is the gyromagnetic ratio of the observed nucleus, p is the Bohr magneton, A / h is the electron nuclear hyperfine coupling constant, r is the distance between the paramagnetic ion and the nucleus, g is the Lande g factor for the electron, S is the electron spin quantum number, and 7c and 7 , are the correlation times for the dipolar and scalar interactions, respectively. The ~ / T , M contain terms that disperse near the charac1. Note in addition that l/TzM teristic frequencies UT' contains terms linear in T , and 7,, that may also be field dependent, as shown by
-
1/7e =
-k I / T , ~
1/7, = 1/7, -k
l/Tex
+1
(3)
/ ~ ~
(4)
where T , is the electron longitudinal relaxation time and T , is the rotational correlation time of the ion-ligand complex. The electron spin relaxation rate is given to a good approximation by 1/TS
= B7,[1/(1 -k
WS27,2)
+ 4/(1 + 4U,2TV2)]
(5)
Figure 1. Longitudinal relaxation rate for solvent water protons obtained in the presence of manganese(I1)ion as a function of magnetic field strength using a nuclear magnetic dispersion spectrometer constructed in this laboratory: (top)a 0.146 mM MnCi, solution at 286 K; the inflection marked E is from the scalar term in the relaxation equation, that marked C is from the dipolar contribution and occurs when w,7, 1; (bottom) a 0.4 mM MnCI, solution also containing 2.5 mM bovine serum albumin at 286 K. The maximum is due to the
-
relaxatbn tlme for the elctron dominating correlation time for the dipolar
contrlbution to the relaxation rate. where T , is a correlation time for the fluctuations in the zero-field splitting and B contains the zero-field splitting parameter; 1, or 7 , becomes field dependent only when T , is short relative to T~. and 7., The field dependence of the correlation time may lead to a maximum in the longitudinal relaxation rate as shown in Figure 1. At high field, however, the longitudinal relaxation rate falls, but the terms linear in correlation time maintain high transverse relaxation rates. In summary, a t high fields longitudinal relaxation may be inefficient, but transverse relaxation rates may remain high. Efficient paramagnetically induced solvent relaxation depends on rapid chemical exchange of the relaxed nucleus between the paramagnetic environment and the bulk solution. Thus, a solute species not sampling the first coordination sphere of the ion is affected only weakly by the presence of a paramagnetic center that may dramatically alter the solvent relaxation rates. Thus, we may employ a T,-selective detection scheme to exploit the difference between solvent and solute T2induced by a paramagnetic relaxation agent. As we have previously indicated (5), n in the CPMG spin echo sequence may be adjusted to allow magnetization components with small T2values to decay in the transverse plane before detection of those with larger values; and by spacing the 180' pulses closely, the magnetization is practically spin locked during the echo train, which has the effect of suppressing phase modulation in coupled multiplets that may complicate
1754
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986 SINGLE WLSE
CPMQ SPIN ECHO
Table I. Solvent Suppression Parameters for Solutions of MnC12"
exponential constant calcdc obsdd
preexponential factor* for [MnC12] 0.0 0.2 0.5 1.0 2.0 5.0
,I:.,
-
-s_i
L
1.00 1.14 0.766 1.38 1.55 e
15.8 29.4 138 300 637
16.1 35.0 148 294 566
a Refer to eq 8 in the text. *Integrated intensities for water (w) and acetone (s) resonances, measured on single-pulse spectra either containing paramagnetic reagent MnC12 (P) or not (D). 'Calculated as [Thp- TzwP]/TBPT2wP from relaxation times determined in CPMG spin-echo experiments. Negative of the slope of a semilog plot of measured suppression ratio [Zwp(t)/Z:(t)]/ [I:/ ZwD]vs. CPMG echo time 2nr. e Water line too broad for reliable estimate of intensitv.
I1
LA
A
Figure 2. Water peak suppression in solutions of manganese(I1) ion. Single-pulse and CPMG spin-echo proton NMR spectra were acquired at 270 MHz on aqueous solutions containing ethanol (5 % v/v), acetone (2.5%). D20(lo%), 0.4% of saturated TSS, and varying concentrations of MnCI,: (A) 0.0 mM; (B), 0.2 mM, with 2177 = 768 ms, suppression ratio R , = 5.3 X lo-? (C) 0.5 mM, 2n7 = 192 ms, R , = 7.7 X (D) 1.0 mM, 2nr = 96 ms, R , = 1.9 X 1 0 5 (E) 2.0 mM, 2n7 = 48 ms, R , = 3.7 X (F) 5.0 mM, 2n7 = 12 ms, R , = 3.1 x 10-4
spectra obtained using a two-pulse sequence. Equations 1-5 predict that the paramagnetic contribution to the line width will be proportional to the concentration of the ion. The broader the water resonance, the shorter the pulse train required for the H 2 0 transverse magnetization to decay. The paramagnetic center may also affect solute resonances if there is significant first coordination sphere interaction between the paramagnetic ion and the solute. Thus, in Figure 2 for aqueous solutions of ethanol plus acetone at low manganese ion concentrations, the water resonance is slightly broadened, solvent suppression is achieved in the spin-echo spectrum with fairly long pulse trains (n = 320,2n7 = 768 ms), and solute resolution is maintained as shown in the ethanol methylene quadruplet. As the concentration of manganese(I1) ion increases, smaller values of n are sufficient for suppression; however, solute resolution suffers. The decay of a resonance in the transverse plane is given by I ( t ) / I o = exp(-t/TJ (6) where I k the intensity of the peak and we assume the effective H1is small, eliminating the need to consider Tlp. The experimental suppression ratio is the ratio of water peak intensities before and after application of the suppression sequence. However, often absolute intensities are not easily available or comparable. To avoid these problems, we measure the intensity ratio of water to a convenient solute singlet (acetone) from the spin-echo experiment and normalize that to the single-pulse experiment to obtain
(7) where we have represented the different experiments by the superscripts D (diamagnetic solution, single-pulse experiment) and P (paramagnetic, CPMG spin-echo experiment), and
l i . ' i I '
5.5
5.0
'
I " '
4.5
'
4.0
' I ' " ' 1
3.5
3.0
" ' I
2.5
CHEMICAL SHIFT (PPM)
Figure 3. Water peak suppression in 0.32 M aqueous cobait(II1) sepulchrate: (A) single-pulse spectrum, (e) 128X vertical expansion of spectrum in A, (C) CPMG spin-echo spectrum of a sample also containing 0.2 mM MnCI,, 2n 7 = 480 ms. Thirty-two transients were acquired in each spectrum.
R,(t) is the water suppression ratio. Substitution and rearrangement give
where Iwp(0) and IF(0)represent the intensities observed for n = 0 in a CPMG experiment (equivalent to a single-pulse experiment), and the entire preexponential factor is a constant. The intensities in eq 8 are proportional to the mole fractions of protons of components. If the efficiency of excitation and detection is the same for water as it is for solute, and if this is true whether or not a paramagnetic reagent is present, then the preexponential factor will be unity. In any case it will be close to unity as shown by the data summarized in Table I. Equation 8 takes into account the fact that solute resonances also decay somewhat during the echo train. The greatest suppression of H20 intensity relative to a solute clearly occurs when the difference between their T2values is the greatest. In experiments separate from those in Figure 2, we measured the water, ethanol, and acetone T, values by the CPMG
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
4.5
5.0
4.0
1755
3.5
C
1
"
"
1
"
"
1
"
"
/
"
"
1
"
"
4.0
4.5
5.0
3.5 1
CHEMICAL SHIFT (PPM)
I
Flgm 4. Water peak suppression in aqueous solution of lactose: (A) , ; (E) single-puise spectrum single spectrum of lactose, 0.20 M in HO of lactose, 0.20 M In D,O, (C) CPMG spin-echo spectrum of sample in A also containing 0.2 mM MnCi,, 2n T = 480 ms. Thirty-two transients were acquired in each spectrum.
b
7
6
5
4
3
2
1
I
/
I
l
I
I
I
7 6 5 4 3 2 1 CHEMICAL SHIFT (PPM)
8
0
0
\
A 0.0
7.5
\
7.0 I
I
I
,
I
,
,
I
1
0
7
6
5
4
3
2
1
0
CHEMICAL SHIFT (PPM) 4.5 4.0 3.5 3.0 CHEMICAL SHIFT (PPM)
Figure 5. Water peak suppression in human vine-CPMG spin+cho spectrum of urine containing 0.2 mM MnCi,, 2177 = 480 ms: (A) spin-echo spectrum, (E) expansion of downfield region, (C) expansion of "carbohydrate" region.
method. The exponential constants in eq 8 were calculated from the T2values for water and acetone and compared well with those obtained from the slope of a semilog plot of experimental suppression ratio vs. t, for the entire range of manganese ion concentration (Table I). Thus, eq 8 provides a sufficient description of the solvent suppression. Several examples are shown below where this technique is applied to different classes of compounds. Obviously, the approach suffers when first coordination sphere interactions with the solute of interest are strong, a problem that may be solved by judicious choice of ligands coordinated to the relaxation agent. An interesting consequence of the technique is that the severe broadening of resonances a t high field associated with large solvent magnetization is eliminated. The broadening from so-called radiation damping (14) results only from the solute magnetization and is, therefore, much less
Flgurs 8. Water peak suppression in human blood plasma: (A) singlepuise spectrum of plasma without MnCi,; (E) 128X vertical expansion of spectrum A (C) CPMG spin-echo spectrum of plasma containing 0.3 mM MnCi,, 2n7 = 240 ms; (D) expansion of "carbohydrate" region of spectrum C. One hundred twenty eight transients were acquired in each spectrum.
severe. An important point is that in many natural systems the water or solvent resonance may be broad, e.g., tissues, foods, rocks, and microporous media. In such systems the and either a pulse train or spin relaxation agent is unneces8~~1y locking experiment by itself may yield excellent spectra (15). As an example of an application to inorganic chemistry, we chose as a solute the octaazabicycloeicosane cage compound cobalt(II1) sepulchrate (16). At the concentration used (0.32 M), the spectrum is only observable (Figure 3B) in the presence of the water peak,with sufficient vertical expansion; however, the spectral distortions associated with the presence of the intense water peak are relieved in the spin-echo spectrum of the solution with 0.2 mM MnC12 added (Figure 3C), and resolution is virtually unaffected. Proton NMR spectra of lactose are shown in Figure 4. From comparison of the spectrum of lactose dissolved in D2O with the spin-echo spectrum of the sample with MnC12 (Figure
1756
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
T ' " ' 1 ' '" '" '
4
1 ' '
' I
1-
" ' ~ " ' " ' '
2
3
1
B
/
A
ll
'
l " " l " " ~ " " I " " I
5
4
3
2
1
CHEMICAL SHIFT (PPM) Flgure 7. Water peak suppression in human cerebrospinal fluidCPMG spin-echo spectrum, 2n 7 = 480 ms, of a sample containing 0.2 mM MnCI,: (A) full spectrum,(B) expansion of carbohydrate reglon. Two hundred fifty six transients were acquired.
4B,C) the manganese-induced broadening was found to be about 0.4 Hz.The solvent-suppressed spectrum shows that resonances otherwise obscured by the water peak (e.g., the doublet at 4.68 ppm) can be clearly observed by this method. Sugars seem not to interact appreciably with this particular paramagnetic reagent (9). The spin-echo spectrum of human urine to which Mn(I1) ion has been added is shown in Figure 5. Resonances due to creatinine (sharp peaks near 3.05 and 4.1 ppm) are immediately suggested by comparison with spectra of the pure compound. Resonances typical of carbohydrates appear in the region from 3 to 4 ppm. Very little information is gained by using a much shorter value o f t (180 ms) than that used for Figure 5 (480 ms), and little is lost at t = 720 ms; thus, solute T2values are long, and the solvent-suppressed spectrum provides a faithful representation of the principal solute profile. The water suppression ratio in the urine spectrum but the concentration of the is still of the order of 5 X most abundant solute (creatinine) is lower by at least 20-fold than that of solutes in the previous samples; thus, the residual water peak is pronounced, but resonances within 0.15 ppm of the water peak at 4.8 ppm are readily discerned. The single-pulse spectrum of human blood plasma without MnC1, (Figure 6A,B) shows a strong multiplet centered at 2.6 ppm that disappears upon addition of MnClz and application of the CPMG pulse sequence. This may be due to interaction with manganese ion, or the chemical species may have a naturally short T P In the spin-echo spectrum of plasma containing 0.3 mM MnC12 (Figure 6C,D), resonances due to glucose (3-4 ppm) are immediately identifiable. Resonances in the 0.7-2.1 ppm range may contain contributions from lactic acid and lipids, although we have not attempted assignment. The residual water peak again seems large, despite efficient suppression, since the principal solute, glucose, is only present at about 5-6 mM in plasma. Urine and plasma spectra have been reported previously (I 7-19). The spin-echo spectrum of human cerebrospinal fluid containing 0.2 mM MnClz (Figure 7) is similar to that of blood plasma (Figure 6), with glucose dominating. The water peak could be further reduced by using echo times up to 720 ms without affecting the appearance of the spectrum.
1
1
1
1
1
1
1
1
:
8
7
6
5
4
3
2
1
0
CHEMICAL SHIFT (PPM) Flgun 8. Water peak suppression in rat brain. Minced tissue was washed in 80 mM phosphate buffer (pH 7.4) containing 20% D,O and 0.2 mM MnCi,: (A) CPMG spin-echo spectrum, 2n 7 = 600 ms;(B) expansion of spectrum in A.
The natural line width of water in intact rodent brain is fairly broad (about 16 Hz), and a spin-echo experiment reduced the water peak without addition of a relaxation agent. However, in many preparations a residual narrow water signal (4-6 Hz) was found presumably due tin part to the few percent of normal saline buffer used to bathe the brain pieces in the NMR tube. Nonetheless, carbohydrate and lipid resonances were clearly observable in eight scans, and none of these resonances changed in its relative intensity when the echo time was increased from 300 to 1000 ms. The spectrum was improved significantly by using D20 buffer as the bathing medium as shown in Figure 8. Similar results have been obtained from muscle (15).
LITERATURE CITED (1) Hore, P. J. J. Magn. Reson. 1983, 55, 283-300. (2) Redfleld, A. 0.NMR: Bask frinciples and Progress; Plntar, M. M., Ed.; Springer-Veriag: Berlin, 1976; Vol. 13, p 137. (3) Rabenstein, D. L.; Isab, A. A. J. Magn. Reson. 1979, 36, 281-286. (4) Rabenstein, D. L.; Fan, S.: Nakashima. T. T. J. Magn. Reson. 1985, 64, 541-546. (5) Rabenstein, D. L. J. Biochem. Biophys. Methods 1984, 9 , 277-306. (6) Bryant, R. G.; Eads, T. M. J. Magn. Reson. 1985, 6 4 , 312-315. (7) Dumouiin. C. L. J. M g n . Reson. 1985, 64. 36-46. (8) Bryant, R. G.; Eads, T. M. J. M g n . Reson. 1985, 6 4 , 312-315. (9) Eads, T. M.; Bryant. R. G., submitted for publication. (10) Meiboom, S.; (3111. D. Rev. Sci. Instrum. 1958, 29, 688. (11) Carr, H. Y.; Purcell, E. M. fhys. Rev. 1954, 94, 630. (12) Solomon, I . fhys. Rev. 1955, 99, 559-565. (13) Solomon, I.; Bloembergen. N. J. Chem. fhys. 1956, 25, 261-266. (14) Abragam. A. The Principles of Nuclear Magnetism; Oxford University: London, 1961; Chapter 111. (15) Wllllams, S. R.; Gadlan, D. G.; Proctor, E.: Sprague, D. B.; Talbot, D. F. J. Magn. Reson. 1985, 63.406-412. (16) Creaser, I. E.; Harrowfleld, J. M.; Herlt, A. J.; Sargeson, A. M.; Spring borg, J.; Geue, R. J.; Snow, M. R. J. Am. Chem. SOC. 1977, 99, 3181. (17) Nicholson, J. K.; O'Flynn. M. P.; Sadler. P. J.; Macleod, A. F.; Juul, S. M.; Sijnksen, P. H. Blochem. J . 1984, 217(2), 365-375. (18) Nicholson, J. K.; Buckingham, M. J.; Sadler, P. J. Biochem. J. 1983, 277, 605-615. (19) Nicholson, J. K.; Tlmbrell, J. A,; Sadler, P. J. Mol. fharmacol. 1985, 27(6), 644-651.
RECEIVED for review December 30,1985. Accepted March 27, 1986. This work was supported by the National Science Foundation (PCM-8408620) and the National Institutes of Health (GM-34541).
