JOURNAL OF MAGNETIC RESONANCE 64, 54 l-546 ( 1985)

Attenuation of the Water Resonance in Fourier Transform ‘H NMR

Spectra of Aqueous Solutions by Spin-Spin Relaxation

DALLAS L. RABENSTEIN,* SHIYAN FAN,? AND THOMAS T. NAKASHIMA

Department of Chemistry, University ofAlberta, Edmonton, Alberta T6G 2G2, Canada

Received June 4, 1985

The study of molecules in dilute aqueous solution by ‘H NMR has been hampered
by the strong water resonance. Not only does the water resonance obscure a large part
of the spectrum, it also makes it difficult to detect the much weaker resonances from
the solute molecules by pulse Fourier transform NMR because of the limited dynamic
range of analog-todigital converters. Several methods have been developed in attempts
to solve the dynamic range problem, including selective saturation of the water res-
onance (1, 2), the inversion recovery pulse sequence (WEFT) (3, 4) and selective
excitation pulse sequences which excite only the spectral region of interest (5-9). While
the water resonance can be considerably reduced in intensity with the saturation and
selective excitation methods, they have the disadvantage that resonances near that of
water cannot be observed. Nuclei whose resonances are in this region can be observed
with the inversion-recovery method if their spin-lattice relaxation times are different
from that of the water protons.
We report here a new and effective method with which the water resonance can be
completely eliminated and resonances near the water resonance can be observed. In
the water attenuation by T2 relaxation (WATR) experiment described here, the spin-
spin relaxation time of the water protons is selectively reduced by chemical exchange,
and the ‘H NMR spectrum is measured by the Car-r-Pmcell-Meiboom-Gill (CPMG)
pulse sequence (90”(x)-(T- 180”( y)-r),-acquisition) (IO). By making the time between
the 90” pulse and acquisition of the free induction decay long enough, the water
resonance is completely eliminated. Phase modulation of homonuclear coupled mul-
tiplet patterns is suppressed by using a sufficiently short time between successive 180”
pulses. The development of the WATR experiment follows from our finding that the
water resonance can be selectively eliminated from ‘H NMR spectra of aqueous sus-
pensions of red blood cells and from aqueous protein solutions with the spin-echo
pulse sequence (I I).
Several classes of compounds which have labile protons can be used as reagents to
selectively decrease the T2 of the water protons by chemical exchange; we describe
here the use of NH&l. The rate of exchange of protons between Hz0 and NHd+ (12)

* To whom correspondence should be addressed at Department of Chemistry, University of California,

Riverside, California 9252 I.
t On leave from Zhongshan University, People’s Republic of China.

541 0022-2364185 $3.00

Copyright 0 1985 by Academic f’res. Inc.
All rights of reproduction in any form reserved.
542 COMMUNICATIONS

is slow on the NMR time scale at low pH, intermediate at intermediate pH, and fast
at high pH. This is reflected in the T2of the water protons, as illustrated by the results
in Fig. 1. The rate of exchange, and thus T2, is also dependent on the concentration
of NH&l. At pH 6.5, the T2of the water protons is reduced by factors of 93 and 160
by adding 0.25 and 0.50 M NH&I.
The spectra in Figs. 2 and 3 illustrate the application of the results in Fig. I to the
measurement of ‘H NMR spectra of dilute aqueous solutions. The spectra in Fig. 2
are for a pH 6.5 solution containing 1 m&I alanine, histidine, threonine, and methionine
and 0.5 M NH4Cl in 99% HzO/ 1% D20. Spectrum A was obtained by the single-pulse
method, while spectrum B was measured by the CPMG pulse sequence. Details are
given in the figure legend. The height of the water resonance in (A) is 3300 times that
of the methionine methyl resonance at 2.123 ppm, while the water resonance is com-
pletely eliminated from (B). Resonances which were completely obscured by the water
resonance in (A) are easily observed in (B).
The spectra in Fig. 3 are for a 10 rmI4 solution of the octapeptide angiotensin II in
99% HzO/ 1% D20. The pH is 6.5 and the solution also contains 0.5 M NH&l. Again,
the water resonance is completely eliminated in (A), revealing resonances for the
protons on the alpha carbons of the amino acid residues, including a resonance at
4.72 ppm. Spectrum A was measured with a total T2 relaxation period of 0.40 s

1.0
3420

-2.01

1 I 1 1 I # 1 I

3.0 5.0 7.0 9.0

PH
FIG. I. The pH dependence of the spin-spin relaxation time of the water protons in distilled water, 0.25
M NH,CI solution and 0.50 M NH&l solution. Tz values were determined at 25°C by the CPMG pulse
sequence with ‘T = 0.0005 s on a Bruker WM-360 spectrometer. One scan was collected.
 COMMUNICATIONS 543

FIG. 2. 360 MHz ‘H NMR spectra measured by the single-pulse method (A) and CPMG pulse sequence
(B) for a pH 6.5 solution containing 1 mkf alanine, histidine, threonine, and methionine and 0.5 M NH&l
in 99% H20/I% D20. Measurement conditions: (A) -4’ flip angle, 560 scans, (B) 7 = 0.0003 s, 670 180”
pulses, 96 scans. The phase of the 180” pulses was alternated to give a +90” phase relation relative to the
90” pulse. Both FIDs were digitized with a 16 bit ADC and were exponentially multiplied with a line
broadening of 0.3 Hz.

10 9 8 7 4 3 2 1 01
8 5
pm

Fffi. 3.360 MHz ‘H NMR spectra measured by the CPMG pulse sequence for a pH 6.5 solution of 10 mM
angiotensin Ii and 0.5 M NH&l in 99% HzO/l% &O. Measurement conditions: (A) 7 = 0.0003 s, 670 180”
pulses, 100 scans,(B) and (C) Q = 0.0003 s, 330 180” pulses, 100 scans. The height of the water resonance in
the spectrum from which (B, C) were taken is 13 times the height of the resonance at 3.117 ppm.
544 COMMUNICATIONS

between the 90” pulse and acquisition of the FID. With a delay this long, some other
resonances are also reduced in intensity, including those from protons on the nitrogens
of the peptide linkages and the broad resonances from carbon-bonded protons of the
proline residue. These both can be observed by decreasing the T2relaxation period to
0.20 s, as shown by (B) and (C) in Fig. 3. Not only are resonances from protons on
the peptide nitrogens observed, but coupling to these protons is also observed in res-
onances for the protons on the alpha carbons of the amino acid residues, as shown
by the spectra in Fig. 4. Spectrum A is the same as (C) in Fig. 3, while (B) is for a
D20 solution of angiotensin II at pD 6.5. As a result of coupling to the protons on
the peptide nitrogens, the doublets at 3.96 and 4.04 ppm in (B) are triplets in (A),
while the triplets at 4.39 and 4.53 ppm in (B) are quartets in (A). The fidelity of the
spectrum measured by the CPMG pulse sequence can also be judged by comparison
of the two spectra in Fig. 4.
The results in Fig. 5 provide an example of the application of the WATR method
to the measurement of two-dimensional COSY spectra of dilute aqueous solutions.
The sample was a pH 6.5 solution containing 10 mM alanine, histidine, threonine,
and methionine and 0.5 M NH&l in 99% H20/ 1% D20. The pulse sequence used
for the COSY experiment (90(o)-tl-90(o)-D2- 180(B)-D2-acquisition@)) is a slight
modification of the “delayed’ COSY (13) and SUPER COSY (14) experiments, both
of which are used to enhance cross peaks due to small couplings. In the application
shown here, the spin-echo part of the pulse sequence is used to selectively attenuate
the resonance from the water protons by T2 relaxation. The phases of all three pulses
and the receiver were cycled to enhance the N-type responses and effect quadrature
detection in both domains.
The examples presented here demonstrate that WATR is a simple and efficient
method for solving the dynamic range problem encountered in measuring FT ‘H
NMR spectra of dilute aqueous solutions. As compared to other methods, a distinct

I I ,
3.5 3.0 2.5
fvm
FIG. 4. Portions of the 360 MHz ‘H NMR spectra of (A) a pH 6.5 solution of angiotensin II and 0.5 M
NH&l in 99% H20/1% D20 and (B) a pD 6.5 solution of angiotensin II in DzO. (A) is from the same
spectrum as(B) and (C) in Fig. 3.
 COMMUNICATIONS 545

FIG. 5. A portion of the COSY spectrum for a pH 6.5 solution of 10 mM alanine, histidine, threonine,
and methionine and 0.5 M NH&I in 99% HrO/l% 40. The pulse sequence is given in the text. A D2 of
0.10 s was used to attenuate the Hz0 resonance. The Hz0 resonance and other diagonal resonances were
further suppressed by processing the data with shifted Gaussian wexghting functions in t, and fr (13, IS).
The frequency range in both domains was 2700 Hz and 2D data matrix was IK by 128 t, values with zero
filling twice on the Fr axis.

advantage of the WATR method is that resonances can be observed in the 3.5-6 ppm
region. Also, spinning side bands from the water resonance are eliminated. The spectra
presented here were measured at 360 MHz; since the :selective reduction in the T2 of
the water protons is by chemical exchange, the WATR method will be even more
efficient at higher frequencies. The elimination of the water resonance by T2 relaxation
in spin-echo ‘H NMR spectra of aqueous suspensions of red blood cells and aqueous
protein solutions is also more efficient the higher the spectrometer frequency (II),
546 COMMUNICATIONS

suggesting that the short T2 value of the water protons and the frequency dependence
of the water elimination in these samples is also due to chemical exchange. A disad-
vantage of the WATR method is that there also is some attenuation of the resonances
of interest by T2 relaxation. As a result, application of the method is limited to the
observation of resonances from protons whose T2’s are longer than the chemical ex-
change shortened T2 of the water protons. Since T2’s decrease as molecular size in-
creases, the WATR method will be of limited use in measuring ‘H NMR spectra of
aqueous solutions of macromolecules. However, the results in Figs. 3 and 4 indicate
it works well with molecules as large as octapeptides.
The WATR method with NH&l as the water proton exchange reagent is most
efficient in the pH region 6-7, depending on the NH&l concentration. We are studying
other compounds with labile protons for use as chemical exchange reagents at other
pH values; e.g., we have found that the log T2 vs pH curve for ammonium phosphate
solutions goes through a minimum around pH 5.2.

ACKNOWLEDGMENTS
This research was supported in part by the University of Alberta and by an operating grant from the
Natural Sciences and Engineering Research Council of Canada to D.L.R. The Bruker WM-360 spectrometer
was purchased with an equipment grant from the Alberta Heritage Foundation for Medical Research.

REFERENCES
1. J. P. JESSON, P. MEAKIN,AND G. KNIESSEL, J. Am. Chem. Sot. 95,6 I8 (1973).
2.1. D. CAMPBELL, C. M. DOSSON, G. JEMINET, AND R. J. P. WILLIAMS, FEBS Left. 49, 1 I5 (1974).
3.S. L. PATT AND B. D. SYKES, J. Chem. Phys. 56,3182 (1972).
4.F. W. BENZ, J. FEENEY, ANDG. C. K. ROBERTS, J. Magn. Reson. 8, I14 (1972).
5.A. G. REDRELD, S. D. KUNZ, AND E. K. RALPH, J. Mugn. Reson. 19, I 14 (1975).
6.P. PLATEAU AND M. G&RON, J. Am. Chem. Sot. 104,731O (1982).
7.
G. M. CLORE, B. J. KIMBER, AND A. M. GRONENBORN, J. Mugn. Reson. 54, 170 (1983).
8.P. J. HORE, J. Magn. Reson. 54, 539 (1983).
P. J. HORE, J. Mugn. Reson. 55, 283 (1983).
9.
10. S. MEIBO~M AND D. GILL, Rev. Sci. Insrum. 29,688 (1958).
Il. D. L. RABENSTEIN AND A. A. ISAB, J. Mugn. Reson. 36,281 (1979).
12. M. T. EMERSON, E. GRUNWALD, AND R. A. KROMHOUT, .I. Chem. Phys. 33,547 (1960).
13. A. BAX AND R. FREEMAN, J. Mugn. Reson. 44, 542 (198 I).
14. A. KUMAR, R. V. HOSUR AND K. CHANDRASEKHAR, J. Mugn. Reson. 60, 143 (1984).
IS. A. BAX, R. A. BYRD, AND A. ASZALOS, J. Am. Chem. Sot. 106,7632 (1984).