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Solvent peak suppression in high resolution NMR
JOURNAL OF MAGNETIC RESONANCE 64,3
12-3 15 ( 1985)
Solvent Peak Suppression in High Resolution NMR ROBERTG.BRYANTANDTHOMASM.EADS
.. _ ...
Departments of Radiology, Biophysics, and Chemistry, University of Rochester Medical Center, Box 648, 601 Elmwood Avenue, Rochester, New York 14642 Received April 4, 1985 The solvent proton nuclear magnetic resonance peak may be. suppressedto arbitrarily low levels (~10~‘) by exploiting the effects of paramagnetic relaxation reagents on the solvent transverse relaxation rates. Specifically, we demonstrate successfulelimination of the water peak in aqueous solutions with retention of solute resolution by application of the Meiboom-Gill modification of the Catr-Purcell spin-echosequenceusedin combination with severalclassesof paramagnetic relaxation agentsthat are added at low concentrations (~0.2 mM). We show that the method is quite general and that its use permits observation of solute resonancesotherwise obscured by the water resonance. 0 1985 Academic PIES, I~C.
A number of important chemical and biochemical problems involve the necessity of accumulating ‘H NMR spectra in solvents rich in protons such as water. The dynamic range problem associatedwith observing resonancesin such solvents is well known and there are several methods involving rf pulse strategiesfor minimizing the effects of strong solvent peaks (1-3). In most cases,these methods leave at least some region of the spectrum unobservable becauseof selective excitation at one or several frequencies.The elimination or reduction of water proton resonancesbasedon relaxation time selectivity is another approach. Early methods exploited the generally longer T, of solvent protons, while more recently T2 effects have been used by Rabenstein and Isab (4). We report here a very general method for solvent suppressionbased on control of solvent transverse relaxation rate by using several distinct classesof paramagnetic reagents,and we show how to obtain high-resolution proton spectra from aqueous solutions without interference from the water resonance. The effects of paramagnetic ions on solvent relaxation rates are well known and have been widely applied in biophysical studies (5.6). The paramagnetic contributions to solvent proton T;’ and T;’ are similar at low values of the magnetic field strength. At high fields, the longitudinal and transverserates may diverge becauseterms linear in the correlation time, which may increasewith field strength, dominate. Thus, solvent T2 may become short while T, becomes long. Efficient paramagnetically induced solvent relaxation depends on rapid chemical exchangeof the relaxed nucleus between the paramagnetic environment and the bulk solution. Thus, a solute speciesnot sampling the first coordination sphere of the ion is affected only weakly by the presenceof a paramagnetic center that may dramatically alter the solvent relaxation rates. The essenceof the method is to exploit the large difference between solvent and solute T2. 0022-2364185 $3.00 Copyriat 8 1985 by Academic ReJs, Inc. All rights of reproduction in any form reserved.
312
SOLVENT PEAK SUPPRESSION
313
Although several pulse and acquisition schemesare possible for T+elective observation of solutes, a simple one is to use the Meiboom-Gill modification of the CarrPurcell spin-echo sequence(7,8). This choice has two advantagescompared to a Hahn spin-echo sequence:(1) By spacing the ?rpulses closely, the magnetization is practically spin locked during the echo train, which has the effect of suppressingthe echo phase modulation causedby scalar (J) coupling; peak amplitude distortions in the spectrum are thus m inimized. (2) Phase problems associatedwith acquiring data after a long delay following excitation are m inimized. The solvent suppression scheme is to add a low concentration of a paramagnetic reagent to the solution, and to execute a spin-echo train pulse sequence(D-90,“-(r180,“-r),-acquisition) where r is typically 1 ms or less and n is adjusted to be large enough to reduce or eliminate the solvent peak. The value of II may be 40 to 400 for solvent suppression of the order of lop4 for the types and concentrations of relaxation reagentswe have used.
I
I
I
I
I
5 4 3 2 I CkEMCAL SHIFT, ppm FIG. I. Water peak suppression using several types of paramagnetic reagents.Proton spectra were obtained either with a single (very weak) pulse (D-pulse-acquisition) (A) or with a CPMG pulse sequence (D-90”(r-180”-r),-acquisition) (B-D), where D = 10 s, T = 0.6 ms, and the weak pulse or the 90” pulse is phase cycled, consistent with quadrature detection. Bach sample contains 5% (vol) of 95% ethanol (USP) 0.86 M, 2.5% acetone, 0.34 M; 1% of a saturated solution of 3-(trimethylsilyl)-I-propanesulfonic acid, sodium salt (Aldrich); HZ0 at 81.5% (A, B), 78.5% (C), or 67.6% (D); > 10% *Hz0 (lock); and an appropriate paramagnetic reagent. (A) No reagent; one-pulse. (B) 200 N MnClz ; CPMG sequence,n = 240. (C) 10 m M CuS04, 1.6 M NH,OH; CPMG, n = 40. (D) 200 a 1:l complex of bovine serum albumin: Mn (BSA from Sigma, No. A4378); CPMG, n = 160. Insets show expansion of the ethanol methylene multiplet: horizontal scale 0.2 ppm. Spectra were obtained on 0.5 ml samples in 5 m m NMR tubes (Wilmad No. 507PP) spinning at 30 + 1 Hz, at 28 + 1°C on an IBM WP 270 SY spectrometer; 90 and 180” pulse widths are 4.5 and 9.8 PCS, respectively; sweep width 2000 Hz, 8K data points; chemical-shit? scale is relative to the high-field peak of internal TSS.
314
BRYANT
AND EADS
Any reagent that induces highly efficient solvent transverse relaxation in the high fields now common for most proton spectroscopy may be used for this purpose. We demonstrate two fundamentally different types of reagent: (1) those that have large electron-nuclear hype&e coupling constants and rapid rotational diffusion such as manganese(I1) or square planar copper complexes, and (2) a paramagnetic species bound to a macromolecule whose slow tumbing allows the strong field dependence of the electron relaxation times to dominate terms in T;’ that are linear in these times. In either case, the paramagnetic center must be accessible to water or solvent protons and have a relatively low affinity for the solute species of interest. Examples of the first type are hexaaquomanganese(I1) ion and tetraamminecopper(I1) ion. In the copper case, the solvent protons couple to the paramagnetic center by rapid proton exchange with ammonia protons in the first coordination sphere of the complex. In the hexaaquomanganese(I1)complex, water molecuk exchange is very rapid. The spinecho spectra shown in Figs. 1B and C demonstrate that both of these reagents are very
A
\,i A!!!!!& L i,
B
I
1 L, A I
6
’
I
,
r
I
I
5 4 3 2-T-z CHEMICAL SHIFT. pDm
FIG. 2. Water peak suppression in milk. Single pulse (A) and CPMG pulse (B) spectra obtained as described in the legend to Fig. I, except D = 5 s. (A) Control: pasteurized, homogenized whole milk, 8096 (vol); 1.46 mM BSA; 1.2% saturated TSS; 20% ‘Hz0 (lock). Single-pulse sequence, 32 scans. The spectrum and its I50 X vertical expansion are shown. (B) Milk plusparamagnetic reagent: milk, 8096, 1.46 m&f BSA - Mn; 1.2% saturated TS!$20% ‘Hz0 (lock). CPMG pulse sequence, n = 320,32 scans. An exponential multiplication (EM) of the FID corresponding to 0.4 Hz line broadening was applied. No baseline corrections were applied to either spectrum. Arrow indicates residual ‘I&O peak; its suppression ratio is approximately 1.5 X 10’.
SOLVENT PEAK SUPPRESSION
315
effective for eliminating the solvent peak by Tz selectivity, with little sacrifice in solute resolution. A second class of relaxation reagentsis represented by manganese(I1)ion bound to a protein molecule. The spin-echo spectrum shown in Fig. 1D demonstrates that the relaxation efficiency of the manganese-serumalbumin complex is comparable to that ofthe low molecular weight reagents.Either may have advantagesin particular applications. Of course, these relaxation reagentsare by no means unique. Use of this method allows direct observation of solute peaks at or near the resonance frequency of water protons. This is shown in Fig. 2 for a sample of whole m ilk, which is 88% water or 97 M in water protons. The severe distortions in the single pulse spectrum of m ilk (Fig. 2A) are relieved in the spin-echo spectrum of m ilk to which 200 ,uM BSA Mn is added. Peakspreviously obscuredby water are now clearly visible. Sensitivity and resolution are excellent, as shown in the region from 3.2 to 5 ppm, in which lactose, the most abundant low molecular weight solute in m ilk (about 4.6% by weight (IO), or 135 ti) contributes. Some resonance (0.5 to 2.5 ppm) due to aliphatic protons of m ilk fat show reduction in amplitude due to their shorter T2's (determined on the control data not shown). Virtues of this method are that it is very easy to use, it eliminates the water protoninduced radiation damping (9), and it may be executed with relaxation agents that differ widely in their chemical properties. The clear disadvantageis that the technique is not well suited for solutes with short T2 values such as proteins. Nevertheless, this solvent peak elimination strategy appears to have considerable potential for wide ap plication in chemistry, biochemistry, and medicine. l
ACKNOWLEDGMENTS This work was supported by researchgrants from the National ScienceFoundation (PCM-8 106054, PCM8408620) and the National Institutes of Health (GM-29428, GM-34541). Very helpful discussions with Scott Kennedy, V. P. Chacko, and S. Ganapathy and assistancefrom Melanie Nath and Scott Swanson, are gratefully acknowledged. We thank Professor Richard F. Botch for accessto his NMR spectrometer. Note added in prooJ An alternate and complementary method of solvent suppression has recently heen reported, C. L. Dumoulin, J. Mugn. Reson. 64,38 (1985). REFERENCES 1. A. G. REDFIELD,“NMR: Basic Principles and Progress”(M. M. Pintar, Ed.), Vol. 13, p. 137, SpringerVerla8 Berlin, 1976. 2. A. G. REDFIELD,in “Methods in Enzymology” (C. H. W. Hits and S. N. Timasheff, eds.), Vol. 49, p. 253, Academic Press, New York, 1978. 3. P. J. HORE, J. Magn. Reson. 55, 283 (1983). 4. D. L. RABENSTEINAND A. A. ISAB,J. Magrz. Reson. 36,281 (1979). 5. R. A. DWEK, “Nuclear Magnetic Resonancein Biochemistry: Applications to Enzyme Systems,”Chaps. 9-l 1, Oxford Univ. Press (Clarendon), Oxford, 1973. 6. D. R. BURTON,S. FORSEN,G. KARLSTROM,AND R. A. DWEK, Progr. NMR Spectrosc. 13, 1 (1979). 7. H. Y. CARR AND E. M. PURCELL,Phys. Rev. 94,630 (1954). 8. S. MEIEZOOM AND D. GILL, Rev. Sci. Instrum. 29,688 (1958). 9. A. ABRAGAM,“The Principles of Nuclear Magnetism,” Chap. III, Oxford Univ. Press, London, 1961. 10. M. WINDHOLZ (Ed.), “Merck Index,” 9th ed., p. 806, Merck, Rahway, N.J., 1976.
12-3 15 ( 1985)
Solvent Peak Suppression in High Resolution NMR ROBERTG.BRYANTANDTHOMASM.EADS
.. _ ...
Departments of Radiology, Biophysics, and Chemistry, University of Rochester Medical Center, Box 648, 601 Elmwood Avenue, Rochester, New York 14642 Received April 4, 1985 The solvent proton nuclear magnetic resonance peak may be. suppressedto arbitrarily low levels (~10~‘) by exploiting the effects of paramagnetic relaxation reagents on the solvent transverse relaxation rates. Specifically, we demonstrate successfulelimination of the water peak in aqueous solutions with retention of solute resolution by application of the Meiboom-Gill modification of the Catr-Purcell spin-echosequenceusedin combination with severalclassesof paramagnetic relaxation agentsthat are added at low concentrations (~0.2 mM). We show that the method is quite general and that its use permits observation of solute resonancesotherwise obscured by the water resonance. 0 1985 Academic PIES, I~C.
A number of important chemical and biochemical problems involve the necessity of accumulating ‘H NMR spectra in solvents rich in protons such as water. The dynamic range problem associatedwith observing resonancesin such solvents is well known and there are several methods involving rf pulse strategiesfor minimizing the effects of strong solvent peaks (1-3). In most cases,these methods leave at least some region of the spectrum unobservable becauseof selective excitation at one or several frequencies.The elimination or reduction of water proton resonancesbasedon relaxation time selectivity is another approach. Early methods exploited the generally longer T, of solvent protons, while more recently T2 effects have been used by Rabenstein and Isab (4). We report here a very general method for solvent suppressionbased on control of solvent transverse relaxation rate by using several distinct classesof paramagnetic reagents,and we show how to obtain high-resolution proton spectra from aqueous solutions without interference from the water resonance. The effects of paramagnetic ions on solvent relaxation rates are well known and have been widely applied in biophysical studies (5.6). The paramagnetic contributions to solvent proton T;’ and T;’ are similar at low values of the magnetic field strength. At high fields, the longitudinal and transverserates may diverge becauseterms linear in the correlation time, which may increasewith field strength, dominate. Thus, solvent T2 may become short while T, becomes long. Efficient paramagnetically induced solvent relaxation depends on rapid chemical exchangeof the relaxed nucleus between the paramagnetic environment and the bulk solution. Thus, a solute speciesnot sampling the first coordination sphere of the ion is affected only weakly by the presenceof a paramagnetic center that may dramatically alter the solvent relaxation rates. The essenceof the method is to exploit the large difference between solvent and solute T2. 0022-2364185 $3.00 Copyriat 8 1985 by Academic ReJs, Inc. All rights of reproduction in any form reserved.
312
SOLVENT PEAK SUPPRESSION
313
Although several pulse and acquisition schemesare possible for T+elective observation of solutes, a simple one is to use the Meiboom-Gill modification of the CarrPurcell spin-echo sequence(7,8). This choice has two advantagescompared to a Hahn spin-echo sequence:(1) By spacing the ?rpulses closely, the magnetization is practically spin locked during the echo train, which has the effect of suppressingthe echo phase modulation causedby scalar (J) coupling; peak amplitude distortions in the spectrum are thus m inimized. (2) Phase problems associatedwith acquiring data after a long delay following excitation are m inimized. The solvent suppression scheme is to add a low concentration of a paramagnetic reagent to the solution, and to execute a spin-echo train pulse sequence(D-90,“-(r180,“-r),-acquisition) where r is typically 1 ms or less and n is adjusted to be large enough to reduce or eliminate the solvent peak. The value of II may be 40 to 400 for solvent suppression of the order of lop4 for the types and concentrations of relaxation reagentswe have used.
I
I
I
I
I
5 4 3 2 I CkEMCAL SHIFT, ppm FIG. I. Water peak suppression using several types of paramagnetic reagents.Proton spectra were obtained either with a single (very weak) pulse (D-pulse-acquisition) (A) or with a CPMG pulse sequence (D-90”(r-180”-r),-acquisition) (B-D), where D = 10 s, T = 0.6 ms, and the weak pulse or the 90” pulse is phase cycled, consistent with quadrature detection. Bach sample contains 5% (vol) of 95% ethanol (USP) 0.86 M, 2.5% acetone, 0.34 M; 1% of a saturated solution of 3-(trimethylsilyl)-I-propanesulfonic acid, sodium salt (Aldrich); HZ0 at 81.5% (A, B), 78.5% (C), or 67.6% (D); > 10% *Hz0 (lock); and an appropriate paramagnetic reagent. (A) No reagent; one-pulse. (B) 200 N MnClz ; CPMG sequence,n = 240. (C) 10 m M CuS04, 1.6 M NH,OH; CPMG, n = 40. (D) 200 a 1:l complex of bovine serum albumin: Mn (BSA from Sigma, No. A4378); CPMG, n = 160. Insets show expansion of the ethanol methylene multiplet: horizontal scale 0.2 ppm. Spectra were obtained on 0.5 ml samples in 5 m m NMR tubes (Wilmad No. 507PP) spinning at 30 + 1 Hz, at 28 + 1°C on an IBM WP 270 SY spectrometer; 90 and 180” pulse widths are 4.5 and 9.8 PCS, respectively; sweep width 2000 Hz, 8K data points; chemical-shit? scale is relative to the high-field peak of internal TSS.
314
BRYANT
AND EADS
Any reagent that induces highly efficient solvent transverse relaxation in the high fields now common for most proton spectroscopy may be used for this purpose. We demonstrate two fundamentally different types of reagent: (1) those that have large electron-nuclear hype&e coupling constants and rapid rotational diffusion such as manganese(I1) or square planar copper complexes, and (2) a paramagnetic species bound to a macromolecule whose slow tumbing allows the strong field dependence of the electron relaxation times to dominate terms in T;’ that are linear in these times. In either case, the paramagnetic center must be accessible to water or solvent protons and have a relatively low affinity for the solute species of interest. Examples of the first type are hexaaquomanganese(I1) ion and tetraamminecopper(I1) ion. In the copper case, the solvent protons couple to the paramagnetic center by rapid proton exchange with ammonia protons in the first coordination sphere of the complex. In the hexaaquomanganese(I1)complex, water molecuk exchange is very rapid. The spinecho spectra shown in Figs. 1B and C demonstrate that both of these reagents are very
A
\,i A!!!!!& L i,
B
I
1 L, A I
6
’
I
,
r
I
I
5 4 3 2-T-z CHEMICAL SHIFT. pDm
FIG. 2. Water peak suppression in milk. Single pulse (A) and CPMG pulse (B) spectra obtained as described in the legend to Fig. I, except D = 5 s. (A) Control: pasteurized, homogenized whole milk, 8096 (vol); 1.46 mM BSA; 1.2% saturated TSS; 20% ‘Hz0 (lock). Single-pulse sequence, 32 scans. The spectrum and its I50 X vertical expansion are shown. (B) Milk plusparamagnetic reagent: milk, 8096, 1.46 m&f BSA - Mn; 1.2% saturated TS!$20% ‘Hz0 (lock). CPMG pulse sequence, n = 320,32 scans. An exponential multiplication (EM) of the FID corresponding to 0.4 Hz line broadening was applied. No baseline corrections were applied to either spectrum. Arrow indicates residual ‘I&O peak; its suppression ratio is approximately 1.5 X 10’.
SOLVENT PEAK SUPPRESSION
315
effective for eliminating the solvent peak by Tz selectivity, with little sacrifice in solute resolution. A second class of relaxation reagentsis represented by manganese(I1)ion bound to a protein molecule. The spin-echo spectrum shown in Fig. 1D demonstrates that the relaxation efficiency of the manganese-serumalbumin complex is comparable to that ofthe low molecular weight reagents.Either may have advantagesin particular applications. Of course, these relaxation reagentsare by no means unique. Use of this method allows direct observation of solute peaks at or near the resonance frequency of water protons. This is shown in Fig. 2 for a sample of whole m ilk, which is 88% water or 97 M in water protons. The severe distortions in the single pulse spectrum of m ilk (Fig. 2A) are relieved in the spin-echo spectrum of m ilk to which 200 ,uM BSA Mn is added. Peakspreviously obscuredby water are now clearly visible. Sensitivity and resolution are excellent, as shown in the region from 3.2 to 5 ppm, in which lactose, the most abundant low molecular weight solute in m ilk (about 4.6% by weight (IO), or 135 ti) contributes. Some resonance (0.5 to 2.5 ppm) due to aliphatic protons of m ilk fat show reduction in amplitude due to their shorter T2's (determined on the control data not shown). Virtues of this method are that it is very easy to use, it eliminates the water protoninduced radiation damping (9), and it may be executed with relaxation agents that differ widely in their chemical properties. The clear disadvantageis that the technique is not well suited for solutes with short T2 values such as proteins. Nevertheless, this solvent peak elimination strategy appears to have considerable potential for wide ap plication in chemistry, biochemistry, and medicine. l
ACKNOWLEDGMENTS This work was supported by researchgrants from the National ScienceFoundation (PCM-8 106054, PCM8408620) and the National Institutes of Health (GM-29428, GM-34541). Very helpful discussions with Scott Kennedy, V. P. Chacko, and S. Ganapathy and assistancefrom Melanie Nath and Scott Swanson, are gratefully acknowledged. We thank Professor Richard F. Botch for accessto his NMR spectrometer. Note added in prooJ An alternate and complementary method of solvent suppression has recently heen reported, C. L. Dumoulin, J. Mugn. Reson. 64,38 (1985). REFERENCES 1. A. G. REDFIELD,“NMR: Basic Principles and Progress”(M. M. Pintar, Ed.), Vol. 13, p. 137, SpringerVerla8 Berlin, 1976. 2. A. G. REDFIELD,in “Methods in Enzymology” (C. H. W. Hits and S. N. Timasheff, eds.), Vol. 49, p. 253, Academic Press, New York, 1978. 3. P. J. HORE, J. Magn. Reson. 55, 283 (1983). 4. D. L. RABENSTEINAND A. A. ISAB,J. Magrz. Reson. 36,281 (1979). 5. R. A. DWEK, “Nuclear Magnetic Resonancein Biochemistry: Applications to Enzyme Systems,”Chaps. 9-l 1, Oxford Univ. Press (Clarendon), Oxford, 1973. 6. D. R. BURTON,S. FORSEN,G. KARLSTROM,AND R. A. DWEK, Progr. NMR Spectrosc. 13, 1 (1979). 7. H. Y. CARR AND E. M. PURCELL,Phys. Rev. 94,630 (1954). 8. S. MEIEZOOM AND D. GILL, Rev. Sci. Instrum. 29,688 (1958). 9. A. ABRAGAM,“The Principles of Nuclear Magnetism,” Chap. III, Oxford Univ. Press, London, 1961. 10. M. WINDHOLZ (Ed.), “Merck Index,” 9th ed., p. 806, Merck, Rahway, N.J., 1976.