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Proton Fourier transform nmr spectroscopy in H2O solutions on a JEOL PFT-100. The WEFT, SWEFT, and VASE pulse sequences
JOURNAL
OF MAGNETIC
RESONANCE
19,99-107
(1975)
Proton Fourier Transform NMR Spectroscopyin Hz0 Solutions on a JEOL PFT.-100.The WEFT, SWEFT, and VASE Pulse Sequences THOMAS R. KRUGH* Department
of Chemistry,
AND WILLIAM
University
of Rochester,
C. SCHAEFER Rochester,
N. Y. 14627
Received February lo,1975 An inexpensive pulse generating circuit which adds a pulse to the pulse train generated by the JEOL DP-1 pulse programmer is described. This circuit allows for the generation of a 180”-~-90” pulse sequence with a continuously variable interval (7). The 180”-r-90” pulse sequence has been used to record the LOO-MHz proton Fourier transform NMR spectrum of 5 x lob4 M pdC-dG in an Hz0 solution. The use of a 180”-71-1800-72-90” (SWEFT) pulse sequence for the simultaneous elimination of any two resonances is illustrated. A variable angle signal elimination (VASE) pulse sequence that allows for the use of a minimum interval between the pulses is discussed. An inexpensivecircuit for the generation of a homogeneity spoiling pulse is also presented. INTRODUCTION
In the last few years the use of Hz0 as a solvent in proton NMR studies has provided valuable information on the structure and dynamics of proteins and nucleic acids (Z-5). Until recently, continuous wave techniques have been most frequently used to record the proton NMR spectra of samples dissolved in an Hz0 solvent because the presence of the large solvent peak complicates the Fourier transform (FT) experiments. In 1971 Redfield and Gupta (6-7) reported the use of a long observation pulse which acts as a 45” to 90” pulse for resonances of interest while not flipping the solvent resonance. Redfield et al. (personal communication) have also recently proposed a modification of the long pulse method. Several other approaches have also been proposed for either minimizing the residual HDO resonance or recording spectra in HZ0 solutions, including saturation of the solvent resonance (&IO), synthesized excitation (II), correlation spectroscopy (12), and the WEFT pulse sequence (13-24). In a previous communication (15) we demonstrated that the 180”~r-90” (WEFT) pulse sequence may be used to record the proton spectra dissolved in H,O solutions down to the millimolar concentration range. The value of r in the WEFT pulse sequence must be carefully chosen in order to minimize the residual H,O resonance. On our JEOL PFT100 this presents a problem because the DP-I pulse programmer provides only two digit selection of the interval. In the following sections we present an inexpensive circuit that provides a continuously variable interval for the WEFT pulse sequence as well as a homogeneity spoiling pulse (HSP) to eliminate the transverse components of the magnetization after the 180” pulse. The addition of the pulse adding circuit (PA-l) * To whom correspondence should be addressed. Copyri!#t 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
99
100
KRUGH
AND
SCHAEFER
to the JEOL DP-I pulse programmer provides the capability of generating a ( I X0’---z,1SO”-r,--90”--T), pulse sequence. We show below that this pulse sequence may be used to eliminate any two resonances with different T, values; because this corresponds ro a second WEFT pulse sequence we will refer to the (180”-r,-180”-T,--90”--T), pulse sequence as the SWEFT pulse sequence. In addition, we discuss the use of a variable angle signal elimination (VASE) pulse sequence, &z-90”, in which B is a variable angle between 90 and 180 degrees. INSTRUMENTAL
The JEOL DP-1 pulse programmer provides a choice of single pulses (O-T),,, a T, pulse sequence (28-2-6-T),, and a DEFT pulse sequence, where 8 is the pulse angle, z is the interval between the pulses, and T is the time between the last pulse in a given pulse sequence and the initiation of a new pulse sequence. In the T, pulse mode the value of the pulse width is usually adjusted so that 8 is 90”. to provide the inversion recovery pulse sequence (16). The value of 0 may be arbitrarily chosen by adjusting the continuously variable pulse width on the JEOL DP-I . On the other hand, the two digit selection of the interval is especially inconvenient for recording the spectra of samples dissolved in H,O solutions when T is greater than 1 set because the possible values of the interval are 1.O, 1.1, I .2, . . ., seconds. The 0.1 set steps are much too large for convenient minimization of the residual water resonance in the WEFT pulse sequence. Tn order to circumvent this problem and to simplify the process of obtaining spectra in H,O solutions when z is less than 1 set (at low temperatures), we have developed the pulse adding circuit (PA-l) shown in Fig. 1. This circuit is designed as an adjunct to the JEOL DP-1 pulse programmer and is used to generate and add a single pulse to the pulse sequence generated by the DP-I . The PA-l circuit (Fig. 1) is triggered by either the positive or negative trigger from the DP-1; the choice of the trigger depends upon the pulse sequence desired, as discussed below, A delay between the trigger and the pulse generation is provided by ICI and IC2 in an additive fashion. Two delay circuits were used in order to provide a maximum delay of 2.3 sec. A tenturn potentiometer was used for R, to provide convenient resolution in setting the delay time, while a one-turn potentiometer was used for RI to provide a coarse selection of the delay time. The PA-I pulse width is determined by the value of R5 and, in the present configuration, is adjustable between 13 and 52 psec (corresponding to pulse angles of between 65” and 260” on our spectrometer). The pulse width is generally set to provide either a PO@or 180” irradiation pulse. This pulse is then added to the pulse train generated by the DP- I and the new pulse train is returned to the DP-1 pulse amplifier circuit. The only required modification of the DP-I unit is to break the connection between terminal C and pin 1 on the input to the pulse amplifier card (PCB-7) and to insert a switch that either completes the normal circuit or feeds the composite pulse output of the PA-I into the pulse amplifier, as indicated in Fig. 2. This switch and the BNC connector were mounted on the rear of the DP-1. For convenience, the DP-1 “monitor” output was also changed, as indicated in Fig. 2. With this configuration it is not actually necessary to install the switch, because the external pulse generator circuit has been designed so that the DP-I pulse programmer functions normally when the PA-1 is turned off. For convenience we provided a switch that sends either the DP-I positive
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101
PULSEADDER 4 MONITOR
0
POSITIVE TRIGGER
FIG. 1. Schematic diagram of the pulse generator and adding circuit (PA-l) and the homogeneity spoiling circuit (HSP-1). The buffers are all 7404 hex inverters. The component values of the control time constant circuits are RX--500 k.Q pot; &--100 kS2 trimpot for calibration; R,--100 kQ helipot; R4-100 kQ trimpot for calibration; R5--25 kS2 pot; R,-20 k.Q trimpot for calibration; R,-5 MQ pot; Cl---2.0 mfd; C,-10.0 mfd; C,-2400 pfd; G-O.33 mfd. The timing capacitors are metahzed Mylar, the others are ceramic. All fixed resistors are l/4 W, 5 % composition. The relay is a Magnacraft reed relay, type W-104 MPCK, S.P.D.T., 12 V, 300 Q, but it operates off 5 V. V,, and returns to the I.C.‘s are not shown.
--
__----__
*
43-
L-PJB-7 PULSE AMPLIFIER ------_-C----. FIG. 2. Modifications
of the JEOL- DP-1 Pulse Programmer.
102
KRUGH
AND
SCHAEFER
trigger, the DP-1 negative trigger, or the PA-l delay trigger to the computer to initiate the accumulation of the FID. The “positive” and “negative” triggers of the DP-1 are actually misnomers because both triggers consist of a momentary drop from a +5 V output. When the DP-I is in the single pulse mode both the positive and negative triggers are generated just before each pulse, while in the Tl mode the DP-1 generates the negative trigger just before the 180” (28) pulse, whereas the positive trigger is generated just before the 90” (0) pulse. The positive trigger is used to trigger the EC-100 computer when the DP-1 and PFT-1Oq are in the standard configuration. The positive trigger from the DP-I is used to trigger the computer in the 180”-2,-180”-2,-90” pulse sequence, while the PA-l delay trigger is used in the 180”-r-90” pulse sequence (when the PA-I is used to generate the 90” pulse). The negative trigger allows for observation of the free induction decay after the initial pulse, or, with a sufficiently long accumulation time, the response of the nuclei to the entire pulse train may be monitored. This is a convenient option in setting up the homogeneity spoiling pulse and the SWEFT pulse sequence. The homogeneity spoiling pulse circuit (HSP-1) was designed in order to eliminate the need for the HP 8013A pulse generator that was used in our initial experiments (1.5). In this circuit (Fig. 1) IC4 provides a variable delay (0.01 to 2 set) after receipt of the trigger pulse, IC5 provides a variable pulse width (0.01 to 2 set), and 1C6 drives the relay. The relay mode is switch selectable between the normally open and normally closed positions. We usually use the normally open position and then short the Ycoarse resistor in the homogeneity control circuit to generate the HSP (15). The advantage of this approach for generating the HSP is that it does not require any modilication of the PFT-100. The controls for both the PA-I and the HSP-1 were mounted on the blank panel that replaces the Spin Decoupler Unit of the PS-100. The circuits were mounted on a chassis attached to this panel. RESULTS
The design of the PA-l and HSP-1 circuits includes a switch-selectable monitor output (Fig. 1) so that the pulse widths and the intervals between the pulses may be measured on an oscilloscope or a frequency counter, The stability of the delay in the pulse generating circuit is critical since fluctuations or a drift in the interval between pulses will affect the water elimination experiments. The drift of the delay in our pulse generating circuit is less than _+0.0002 set over a few hour time period. This stability is more than adequate for our experiments. The WEFT pulse sequence with a continuously variable interval is produced by setting the DP-1 in the single pulse mode with a pulse width corresponding to a pulse of 180”. Turning on the PA-I adds a second pulse whose width is set to provide a 90” pulse. The delay time is then adjusted until a minimum FID is observed. The advantage of including a homogeneity spoiling pulse is indicated in Fig. 3. Note that without the HSP a significant amount of transverse magnetization remains at the end of even this relatively long interval of 1.5744 sec. The inclusion of an HSP in the WEFT pulse sequence generally reduces the amplitude of the FID (after the 90” pulse) due to the residual water magnetization by a factor of 3-6 under normal operating conditions. The spectrum of 5 x 10M4 M pdC-dG in an H,O solution (Fig. 4) illustrates the sensitivity limits of these experiments. This spectrum was recorded after 45 minutes
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1 -----------------180” I HSP 03 set)
SPECTRA
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103
I ---- T(,,5744~)---------90”(ACCUMULATE) I
FIG. 3. Free induction decayafter the initial pulse in a 180”~2-W” pulse sequencefor a sample of 0.03 M puke +0.03 M sodium acetatein H,O, pH 6.9,25”C. A homogeneity spoiling pulseof 0.3 sec. duration was applied after a 0.08 se-cdelay in the bottom spectrum. Both spectrawere recordedwith a 5.1 set repetition time. The inset on the bottom spectrumhasan I-fold gain increase.
of accumulation. The proton spectra of samples in the millimolar concentration range in Hz0 solutions are relatively easy to record. However, at these low concentrations we generally observe a distortion in the baseline that is especially evident in the region upfield of the residual solvent resonance. We have not located the exact cause of this baseline distortion (e.g., see (17)) but it is generally not a problem because we are especially interested in the NH and NH2 protons that have resonances downfield of the water resonance. The S WEFT pulse sequence. The 180”~r,-1 SO”-r,-90” pulse sequence may be used to eliminate any two resonances with different spin-lattice relaxation times in much the same manner as the 180”-r-90” pulse sequence is used to eliminate a single resonance. In the SWEFT pulse sequence the value of z1 is adjusted so that the magnetizations of the two resonances to be eliminated have already passed through their null values before the application of the second 180” pulse. The second 180” pulse inverts all the magnetization vectors and, if the values of r1 and z2 are adjusted properly, the magnetization vectors of the two resonances to be eliminated will simultaneously reach a null value after an interval of t2 seconds from the second 180” pulse. An example of this double elimination is shown in Fig. 5. In this case the spinlattice relaxation times of the guanine-Zamino protons and the G-H8 proton are sufficiently short so that they are easily observed, while the Hz0 solvent signal and the remaining nucleotide proton resonances are eliminated. The SWEFT pulse sequence will be useful in assigning resonances or in determining the chemical shifts of overlapping resonances, while simultaneously eliminating a large solvent resonance. The
104
KRUGH
3
AND
SCHAEFER
2
1
PPm FIG. 4. IOO-MHz proton FT spectrum of 0.5 m&f pdC-dG at 3°C in H@, pH 6.9,25”C, using the 180”-7-90” pulse sequence. T = 0.85 set; repetition = 3.0 set; HSP = 0.6 set; 863 accumulations. The chemical shifts are given with respect to the solvent resonance.
SWEFT pulse sequence may also be used to determine the relaxation times of protons with spin-lattice relaxation times that are much shorter than the solvent reraxation time (13). To produce the SWEFT pulse sequence the DP-1 is set in the T1 mode. The pulse width and pulse delay of the PA-l are set to provide a 180” pulse at the appropriate time between the 180” and 90” pulses generated by the DP-1. The first interval tl is somewhat longer than that which is required to minimize the water resonance m&g the WEFT pulse sequence, while the value of 72 is principally determined by the relative values of the spin-lattice relaxation times of the two signals to be eliminated. A homogeneity spoiling pulse is applied after the initial 180” inversion pulse. The use of the PA-l to add the second 180” pulse to the 180*-90” pulse sequence generated by the DP-I means that the two intervals TVand r2 may not be independently adjusted since 51 + T* = zap-l. However, the SWEFT pulse sequence is relatively easy to set up if the FID is recorded for the entire pulse sequence, by selecting the negative trigger of the DP-1 as the computer trigger. DISCUSSION
The major advantage of using the WEFT sequence for recording spectra in II20 solutions is that it may be performed on commercial Fourier transform NMR spectrometers. Recording proton NMR spectra in Hz0 solutions is almost as routine as in D,O solutions with the availability of a continuously variable interval and homogeneity
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105
0.02M pdG-dC WI
SWEFT
FIG. 5.100 MHz proton FT spectra of 0.02 M pdG-dC in HzO, pH 7.0,2”C. The bottom spectrum was recorded with the SWEFT pulse sequence (rl = 0.83 set, r2 = 0.20 set) while the top spectrum was recorded with the WEFT pulse sequence (T = 0.63 set). A homogeneity spoiling pulse was used in both pulse sequences. The pulse intervals in the SWEFT spectrum were selected so that the nucleotide ribose Hl’ protons were eliminated along with the solvent resonance.
spoiling pulse. It should be noted, however, that the relative intensities of the lines may be significantly distorted by the use of the WEFT, the SWEFT, or other multiple pulse techniques. The Tl of Hz0 at 25°C is on the order of 2-3 seconds and thus the intensity of any proton with a Tl greater than 4.5 set will be perturbed. Chemical exchange or cross-relaxation with the solvent protons will also affect the intensities of proton resonances. The magnetization of protons undergoing chemical exchange with the solvent will be reduced by an amount that will depend upon the relative spin lattice relaxation times of the protons in the two states and the exchange rate. This effect is somewhat analogous to the “transfer of saturation” that occurs for protons exchanging with a solvent whose resonance has been saturated (17-19). Chemical exchange (transfer of saturation) effects will generally be the most troublesome because the prime motivation for performing experiments in Hz0 solutions is to be able to observe resonances that do exchange with DzO solvent. Lowering the temperature will help to minimize chemical exchange effects, but it should be clear that the intensities of proton resonances (as opposed to their chemical shifts) should be cautiously interpreted in spectra obtained by the use of these multiple pulse techniques. The addition of the PA-l circuit to the DP-1 unit provides a great deal of flexibility in selecting the appropriate pulse sequence. One very useful example, the, SWEFT sequence, has already been discussed. Any pulse sequence of the form (&t-$-T) may
106
KRUGH AND SCHAEFER
be generated because of the independence in setting the pulse widths of the PA-1 and the DP-I. One example of this sequence is the (90”~z(HSP)-90”-T), pulse sequence (20), which is especially useful for measuring long Tl's. A second possibility is the use of a pulse sequence in which the original pulse is in the range 90” < 8 < 180” and the second pulse is a 90” pulse. This pulse sequence may be used in much the same manner as the 180”-2-90” pulse sequence to eliminate the solvent resonance. The homogeneity spoiling pulse is used to eliminate the large transverse components of the magnetization between the pulses. This sequence has the advantage of significantly reducing the time required (T) for the longitudinal component of the water magnitization to reach a null value, which may be an important consideration if chemical exchange during the WEFT pulse sequence results in diminished intensities of the exchangeable protons. The miaimum interval between the pulses is essentially limited to the time required to eliminate the transverse component of the water magnetization. The initial pulse angle is determined (or limited by) the relaxation times of thesolvent signal and the protons of interest. In preliminary experiments with this variable angle signal elimination (VASE) pulse sequence we found that the HSP required 0.2-0.3 set to destroy the large transverse component of the water magnetization after an initial pulse of I IO degrees. A much more effective HSP is required (eg. by simultaneously shorting out several of the coarse homogeneity potentiometers) in order to take full advantage of using this sequence to minimize chemical exchange effects. It should be noted that the intensities of the proton resonances may be significantly distorted with the use of the VASE pulse sequence. The distortion in the intensities may be used to advantage if the prime interest of recording the spectrum in an H,O solution is to observe the NH and NH2 protons, because these protons generally have the shortest relaxation times and will thus suffer the smallest perturbation in their intensities. The potential of this new approach for observing the resonances of chemically exchanging protons increases as the temperature of the experiment increases. This work was partially National Cancer Institute.
ACKNOWLEDGMENT supported by Public Health Service Research Grant CA-14103 from the REFERENCES
1. 2. 3. 4.
5. 6. 7. 8. 9. 10. II. 12. 13. 14. IS.
J. D. GLICKSON,
C. C. MCDONALD,
AND W. D. PHILLIPS,
Biochem. Bigphys. Res. Comm. 35, 491.
498(1969). D. R. KEARNS, D. J. PATEL, AND R. G. SHLJLMAN, Nature 229,338 (1971). D. M. CROTHERS, C. W. HILBERS, AND R. G. SHULMAN, Proc. Nat. Acad. Sci. U.S. 7D,2899 M. RASZKA AND N. 0. KAPLAN, Proc. Nat. Acad. Sci. U.S. ($2025 (1972). T. R. KRUGH AND M. A. YOUNG, Biochem. Biophys. Res. Comw., 62, 1025 (1975). A. G. REDFIELD AND R. K. GUPTA, Ado. Magn. Rewnance 5,81 (1971). A. G. REDFIELD AND R. K. GUPTA, J. Chem. Phys. 54,1418 (1971). J. P. JESSON, P. MEAKIN, ANDG. KNEISSEL,J. Amer. Chem. Sac. 95,618 (1973). J. SCHAEFER, J. Magn. Resonance 6,670 (1972). H. E. BLEICH AND J. A. GLASEL, J. Magn. Resonance, in press. B. L. TOMLINS~N AND H. D. W. HILL,J. Chem. Phys. 59,1775 (1973). J. D. GLICKSON, J. DADOK, AND G. R. MARSHALL, Biochemistry 13, 11 (1974). S. L. PAN AND B. D. SYKES, J. Chem. Phys. 563182 (1972). F. W. BENZ, J. FEENEY, AND G. C. K. ROBERTS, J. Magn. Resonance 8,114 (1972). E. S. M~EERRY AND T. R. KRUGH, J. Magn. Reunance, 17, 128 (1975).
(1973).
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M. P. KLEIN, AND D. E. PHELPS, J. Chem. Phys. 48,3831(1968). 17. J. SCHAEFER AND E. 0. STWSKAL, J. Magn. Resonance 15,173 (1974). 18. S. FOR&N AND R. A. HOFFMAN, J. Chem. Phys. 39,2892 (1963). 19. P. H. VON DREELE, A. i. BREWSTER, J. DADOK, H. A. SCHERAGA, F. A. BOVEY, M. F. FERGER, V. Du VIONEAUD, Proc. Nat. Acad. Sci. U.S. 69,2169 (1972). 20. G. G. MCDONALD AND J. S. LEIGH, JR., J. Mugn. Resonance 9,358 (1973).
107
16. R. L. VOLD, J. S. WAUGH,
AND
OF MAGNETIC
RESONANCE
19,99-107
(1975)
Proton Fourier Transform NMR Spectroscopyin Hz0 Solutions on a JEOL PFT.-100.The WEFT, SWEFT, and VASE Pulse Sequences THOMAS R. KRUGH* Department
of Chemistry,
AND WILLIAM
University
of Rochester,
C. SCHAEFER Rochester,
N. Y. 14627
Received February lo,1975 An inexpensive pulse generating circuit which adds a pulse to the pulse train generated by the JEOL DP-1 pulse programmer is described. This circuit allows for the generation of a 180”-~-90” pulse sequence with a continuously variable interval (7). The 180”-r-90” pulse sequence has been used to record the LOO-MHz proton Fourier transform NMR spectrum of 5 x lob4 M pdC-dG in an Hz0 solution. The use of a 180”-71-1800-72-90” (SWEFT) pulse sequence for the simultaneous elimination of any two resonances is illustrated. A variable angle signal elimination (VASE) pulse sequence that allows for the use of a minimum interval between the pulses is discussed. An inexpensivecircuit for the generation of a homogeneity spoiling pulse is also presented. INTRODUCTION
In the last few years the use of Hz0 as a solvent in proton NMR studies has provided valuable information on the structure and dynamics of proteins and nucleic acids (Z-5). Until recently, continuous wave techniques have been most frequently used to record the proton NMR spectra of samples dissolved in an Hz0 solvent because the presence of the large solvent peak complicates the Fourier transform (FT) experiments. In 1971 Redfield and Gupta (6-7) reported the use of a long observation pulse which acts as a 45” to 90” pulse for resonances of interest while not flipping the solvent resonance. Redfield et al. (personal communication) have also recently proposed a modification of the long pulse method. Several other approaches have also been proposed for either minimizing the residual HDO resonance or recording spectra in HZ0 solutions, including saturation of the solvent resonance (&IO), synthesized excitation (II), correlation spectroscopy (12), and the WEFT pulse sequence (13-24). In a previous communication (15) we demonstrated that the 180”~r-90” (WEFT) pulse sequence may be used to record the proton spectra dissolved in H,O solutions down to the millimolar concentration range. The value of r in the WEFT pulse sequence must be carefully chosen in order to minimize the residual H,O resonance. On our JEOL PFT100 this presents a problem because the DP-I pulse programmer provides only two digit selection of the interval. In the following sections we present an inexpensive circuit that provides a continuously variable interval for the WEFT pulse sequence as well as a homogeneity spoiling pulse (HSP) to eliminate the transverse components of the magnetization after the 180” pulse. The addition of the pulse adding circuit (PA-l) * To whom correspondence should be addressed. Copyri!#t 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
99
100
KRUGH
AND
SCHAEFER
to the JEOL DP-I pulse programmer provides the capability of generating a ( I X0’---z,1SO”-r,--90”--T), pulse sequence. We show below that this pulse sequence may be used to eliminate any two resonances with different T, values; because this corresponds ro a second WEFT pulse sequence we will refer to the (180”-r,-180”-T,--90”--T), pulse sequence as the SWEFT pulse sequence. In addition, we discuss the use of a variable angle signal elimination (VASE) pulse sequence, &z-90”, in which B is a variable angle between 90 and 180 degrees. INSTRUMENTAL
The JEOL DP-1 pulse programmer provides a choice of single pulses (O-T),,, a T, pulse sequence (28-2-6-T),, and a DEFT pulse sequence, where 8 is the pulse angle, z is the interval between the pulses, and T is the time between the last pulse in a given pulse sequence and the initiation of a new pulse sequence. In the T, pulse mode the value of the pulse width is usually adjusted so that 8 is 90”. to provide the inversion recovery pulse sequence (16). The value of 0 may be arbitrarily chosen by adjusting the continuously variable pulse width on the JEOL DP-I . On the other hand, the two digit selection of the interval is especially inconvenient for recording the spectra of samples dissolved in H,O solutions when T is greater than 1 set because the possible values of the interval are 1.O, 1.1, I .2, . . ., seconds. The 0.1 set steps are much too large for convenient minimization of the residual water resonance in the WEFT pulse sequence. Tn order to circumvent this problem and to simplify the process of obtaining spectra in H,O solutions when z is less than 1 set (at low temperatures), we have developed the pulse adding circuit (PA-l) shown in Fig. 1. This circuit is designed as an adjunct to the JEOL DP-1 pulse programmer and is used to generate and add a single pulse to the pulse sequence generated by the DP-I . The PA-l circuit (Fig. 1) is triggered by either the positive or negative trigger from the DP-1; the choice of the trigger depends upon the pulse sequence desired, as discussed below, A delay between the trigger and the pulse generation is provided by ICI and IC2 in an additive fashion. Two delay circuits were used in order to provide a maximum delay of 2.3 sec. A tenturn potentiometer was used for R, to provide convenient resolution in setting the delay time, while a one-turn potentiometer was used for RI to provide a coarse selection of the delay time. The PA-I pulse width is determined by the value of R5 and, in the present configuration, is adjustable between 13 and 52 psec (corresponding to pulse angles of between 65” and 260” on our spectrometer). The pulse width is generally set to provide either a PO@or 180” irradiation pulse. This pulse is then added to the pulse train generated by the DP- I and the new pulse train is returned to the DP-1 pulse amplifier circuit. The only required modification of the DP-I unit is to break the connection between terminal C and pin 1 on the input to the pulse amplifier card (PCB-7) and to insert a switch that either completes the normal circuit or feeds the composite pulse output of the PA-I into the pulse amplifier, as indicated in Fig. 2. This switch and the BNC connector were mounted on the rear of the DP-1. For convenience, the DP-1 “monitor” output was also changed, as indicated in Fig. 2. With this configuration it is not actually necessary to install the switch, because the external pulse generator circuit has been designed so that the DP-I pulse programmer functions normally when the PA-1 is turned off. For convenience we provided a switch that sends either the DP-I positive
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PULSEADDER 4 MONITOR
0
POSITIVE TRIGGER
FIG. 1. Schematic diagram of the pulse generator and adding circuit (PA-l) and the homogeneity spoiling circuit (HSP-1). The buffers are all 7404 hex inverters. The component values of the control time constant circuits are RX--500 k.Q pot; &--100 kS2 trimpot for calibration; R,--100 kQ helipot; R4-100 kQ trimpot for calibration; R5--25 kS2 pot; R,-20 k.Q trimpot for calibration; R,-5 MQ pot; Cl---2.0 mfd; C,-10.0 mfd; C,-2400 pfd; G-O.33 mfd. The timing capacitors are metahzed Mylar, the others are ceramic. All fixed resistors are l/4 W, 5 % composition. The relay is a Magnacraft reed relay, type W-104 MPCK, S.P.D.T., 12 V, 300 Q, but it operates off 5 V. V,, and returns to the I.C.‘s are not shown.
--
__----__
*
43-
L-PJB-7 PULSE AMPLIFIER ------_-C----. FIG. 2. Modifications
of the JEOL- DP-1 Pulse Programmer.
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SCHAEFER
trigger, the DP-1 negative trigger, or the PA-l delay trigger to the computer to initiate the accumulation of the FID. The “positive” and “negative” triggers of the DP-1 are actually misnomers because both triggers consist of a momentary drop from a +5 V output. When the DP-I is in the single pulse mode both the positive and negative triggers are generated just before each pulse, while in the Tl mode the DP-1 generates the negative trigger just before the 180” (28) pulse, whereas the positive trigger is generated just before the 90” (0) pulse. The positive trigger is used to trigger the EC-100 computer when the DP-1 and PFT-1Oq are in the standard configuration. The positive trigger from the DP-I is used to trigger the computer in the 180”-2,-180”-2,-90” pulse sequence, while the PA-l delay trigger is used in the 180”-r-90” pulse sequence (when the PA-I is used to generate the 90” pulse). The negative trigger allows for observation of the free induction decay after the initial pulse, or, with a sufficiently long accumulation time, the response of the nuclei to the entire pulse train may be monitored. This is a convenient option in setting up the homogeneity spoiling pulse and the SWEFT pulse sequence. The homogeneity spoiling pulse circuit (HSP-1) was designed in order to eliminate the need for the HP 8013A pulse generator that was used in our initial experiments (1.5). In this circuit (Fig. 1) IC4 provides a variable delay (0.01 to 2 set) after receipt of the trigger pulse, IC5 provides a variable pulse width (0.01 to 2 set), and 1C6 drives the relay. The relay mode is switch selectable between the normally open and normally closed positions. We usually use the normally open position and then short the Ycoarse resistor in the homogeneity control circuit to generate the HSP (15). The advantage of this approach for generating the HSP is that it does not require any modilication of the PFT-100. The controls for both the PA-I and the HSP-1 were mounted on the blank panel that replaces the Spin Decoupler Unit of the PS-100. The circuits were mounted on a chassis attached to this panel. RESULTS
The design of the PA-l and HSP-1 circuits includes a switch-selectable monitor output (Fig. 1) so that the pulse widths and the intervals between the pulses may be measured on an oscilloscope or a frequency counter, The stability of the delay in the pulse generating circuit is critical since fluctuations or a drift in the interval between pulses will affect the water elimination experiments. The drift of the delay in our pulse generating circuit is less than _+0.0002 set over a few hour time period. This stability is more than adequate for our experiments. The WEFT pulse sequence with a continuously variable interval is produced by setting the DP-1 in the single pulse mode with a pulse width corresponding to a pulse of 180”. Turning on the PA-I adds a second pulse whose width is set to provide a 90” pulse. The delay time is then adjusted until a minimum FID is observed. The advantage of including a homogeneity spoiling pulse is indicated in Fig. 3. Note that without the HSP a significant amount of transverse magnetization remains at the end of even this relatively long interval of 1.5744 sec. The inclusion of an HSP in the WEFT pulse sequence generally reduces the amplitude of the FID (after the 90” pulse) due to the residual water magnetization by a factor of 3-6 under normal operating conditions. The spectrum of 5 x 10M4 M pdC-dG in an H,O solution (Fig. 4) illustrates the sensitivity limits of these experiments. This spectrum was recorded after 45 minutes
FOURIER
TRANSFORM
1 -----------------180” I HSP 03 set)
SPECTRA
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103
I ---- T(,,5744~)---------90”(ACCUMULATE) I
FIG. 3. Free induction decayafter the initial pulse in a 180”~2-W” pulse sequencefor a sample of 0.03 M puke +0.03 M sodium acetatein H,O, pH 6.9,25”C. A homogeneity spoiling pulseof 0.3 sec. duration was applied after a 0.08 se-cdelay in the bottom spectrum. Both spectrawere recordedwith a 5.1 set repetition time. The inset on the bottom spectrumhasan I-fold gain increase.
of accumulation. The proton spectra of samples in the millimolar concentration range in Hz0 solutions are relatively easy to record. However, at these low concentrations we generally observe a distortion in the baseline that is especially evident in the region upfield of the residual solvent resonance. We have not located the exact cause of this baseline distortion (e.g., see (17)) but it is generally not a problem because we are especially interested in the NH and NH2 protons that have resonances downfield of the water resonance. The S WEFT pulse sequence. The 180”~r,-1 SO”-r,-90” pulse sequence may be used to eliminate any two resonances with different spin-lattice relaxation times in much the same manner as the 180”-r-90” pulse sequence is used to eliminate a single resonance. In the SWEFT pulse sequence the value of z1 is adjusted so that the magnetizations of the two resonances to be eliminated have already passed through their null values before the application of the second 180” pulse. The second 180” pulse inverts all the magnetization vectors and, if the values of r1 and z2 are adjusted properly, the magnetization vectors of the two resonances to be eliminated will simultaneously reach a null value after an interval of t2 seconds from the second 180” pulse. An example of this double elimination is shown in Fig. 5. In this case the spinlattice relaxation times of the guanine-Zamino protons and the G-H8 proton are sufficiently short so that they are easily observed, while the Hz0 solvent signal and the remaining nucleotide proton resonances are eliminated. The SWEFT pulse sequence will be useful in assigning resonances or in determining the chemical shifts of overlapping resonances, while simultaneously eliminating a large solvent resonance. The
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PPm FIG. 4. IOO-MHz proton FT spectrum of 0.5 m&f pdC-dG at 3°C in H@, pH 6.9,25”C, using the 180”-7-90” pulse sequence. T = 0.85 set; repetition = 3.0 set; HSP = 0.6 set; 863 accumulations. The chemical shifts are given with respect to the solvent resonance.
SWEFT pulse sequence may also be used to determine the relaxation times of protons with spin-lattice relaxation times that are much shorter than the solvent reraxation time (13). To produce the SWEFT pulse sequence the DP-1 is set in the T1 mode. The pulse width and pulse delay of the PA-l are set to provide a 180” pulse at the appropriate time between the 180” and 90” pulses generated by the DP-1. The first interval tl is somewhat longer than that which is required to minimize the water resonance m&g the WEFT pulse sequence, while the value of 72 is principally determined by the relative values of the spin-lattice relaxation times of the two signals to be eliminated. A homogeneity spoiling pulse is applied after the initial 180” inversion pulse. The use of the PA-l to add the second 180” pulse to the 180*-90” pulse sequence generated by the DP-I means that the two intervals TVand r2 may not be independently adjusted since 51 + T* = zap-l. However, the SWEFT pulse sequence is relatively easy to set up if the FID is recorded for the entire pulse sequence, by selecting the negative trigger of the DP-1 as the computer trigger. DISCUSSION
The major advantage of using the WEFT sequence for recording spectra in II20 solutions is that it may be performed on commercial Fourier transform NMR spectrometers. Recording proton NMR spectra in Hz0 solutions is almost as routine as in D,O solutions with the availability of a continuously variable interval and homogeneity
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105
0.02M pdG-dC WI
SWEFT
FIG. 5.100 MHz proton FT spectra of 0.02 M pdG-dC in HzO, pH 7.0,2”C. The bottom spectrum was recorded with the SWEFT pulse sequence (rl = 0.83 set, r2 = 0.20 set) while the top spectrum was recorded with the WEFT pulse sequence (T = 0.63 set). A homogeneity spoiling pulse was used in both pulse sequences. The pulse intervals in the SWEFT spectrum were selected so that the nucleotide ribose Hl’ protons were eliminated along with the solvent resonance.
spoiling pulse. It should be noted, however, that the relative intensities of the lines may be significantly distorted by the use of the WEFT, the SWEFT, or other multiple pulse techniques. The Tl of Hz0 at 25°C is on the order of 2-3 seconds and thus the intensity of any proton with a Tl greater than 4.5 set will be perturbed. Chemical exchange or cross-relaxation with the solvent protons will also affect the intensities of proton resonances. The magnetization of protons undergoing chemical exchange with the solvent will be reduced by an amount that will depend upon the relative spin lattice relaxation times of the protons in the two states and the exchange rate. This effect is somewhat analogous to the “transfer of saturation” that occurs for protons exchanging with a solvent whose resonance has been saturated (17-19). Chemical exchange (transfer of saturation) effects will generally be the most troublesome because the prime motivation for performing experiments in Hz0 solutions is to be able to observe resonances that do exchange with DzO solvent. Lowering the temperature will help to minimize chemical exchange effects, but it should be clear that the intensities of proton resonances (as opposed to their chemical shifts) should be cautiously interpreted in spectra obtained by the use of these multiple pulse techniques. The addition of the PA-l circuit to the DP-1 unit provides a great deal of flexibility in selecting the appropriate pulse sequence. One very useful example, the, SWEFT sequence, has already been discussed. Any pulse sequence of the form (&t-$-T) may
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be generated because of the independence in setting the pulse widths of the PA-1 and the DP-I. One example of this sequence is the (90”~z(HSP)-90”-T), pulse sequence (20), which is especially useful for measuring long Tl's. A second possibility is the use of a pulse sequence in which the original pulse is in the range 90” < 8 < 180” and the second pulse is a 90” pulse. This pulse sequence may be used in much the same manner as the 180”-2-90” pulse sequence to eliminate the solvent resonance. The homogeneity spoiling pulse is used to eliminate the large transverse components of the magnetization between the pulses. This sequence has the advantage of significantly reducing the time required (T) for the longitudinal component of the water magnitization to reach a null value, which may be an important consideration if chemical exchange during the WEFT pulse sequence results in diminished intensities of the exchangeable protons. The miaimum interval between the pulses is essentially limited to the time required to eliminate the transverse component of the water magnetization. The initial pulse angle is determined (or limited by) the relaxation times of thesolvent signal and the protons of interest. In preliminary experiments with this variable angle signal elimination (VASE) pulse sequence we found that the HSP required 0.2-0.3 set to destroy the large transverse component of the water magnetization after an initial pulse of I IO degrees. A much more effective HSP is required (eg. by simultaneously shorting out several of the coarse homogeneity potentiometers) in order to take full advantage of using this sequence to minimize chemical exchange effects. It should be noted that the intensities of the proton resonances may be significantly distorted with the use of the VASE pulse sequence. The distortion in the intensities may be used to advantage if the prime interest of recording the spectrum in an H,O solution is to observe the NH and NH2 protons, because these protons generally have the shortest relaxation times and will thus suffer the smallest perturbation in their intensities. The potential of this new approach for observing the resonances of chemically exchanging protons increases as the temperature of the experiment increases. This work was partially National Cancer Institute.
ACKNOWLEDGMENT supported by Public Health Service Research Grant CA-14103 from the REFERENCES
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