- Email: [email protected]
Chapter 6 The DANTE-Z experiment
chapter 6 The DANTE-Z Experiment
D. Canet Laboratoire de Methodologie RMN (URA CNRS 4 0 6 - LESOC; FU CNRS EO08- INCM) Universite Henri Poincare, Nancy 1, B.R 239 54506 Vandoeuvre les Nancy Cedex France
C. Roumestand Centre de Biochimie Structurale (UMR CNRS 9955- U 414 INSERM/Universite de Montpellier I) 15 Avenue Charles Flahault 34060 Montpellier Cedex France
Methods for Structure Elucidation by High-Resolution NMR Edited by Gy. Batta, K.E. KSver and Cs. Szantay, Jr. @ 1997 Elsevier Science B.V. All rights reserved 121
Selectivity has become one of the experimental procedures routinely used by the NMR spectroscopist [1, 2]. Its objective is to reduce the measuring time with 1D counterparts of 2D (or nD) experiments or to improve the spectral resolution in these latter experiments. Selectivity is usually achieved by a soft pulse or alternatively by a train of hard pulses of small flip angle (the DANTE experiment [3]). Both approaches are equivalent provided that the amplitude modulation of the soft pulse is implemented in the DANTE train through a modulation of pulse durations [4]. However, the simplest soft pulse, i.e., rectangularly shaped (or the equivalent DANTE train with pulses of identical durations) exhibits unwanted sinc oscillations on each side of the selected region with in addition a strong dispersive component. Both these drawbacks can be circumvented by the DANTE-Z variant [5], this experiment relying on the selective profile of the z magnetization component. As compared with the profiles of the transverse components, the one of longitudinal magnetization possesses the unique feature of much reduced sidelobes and, obviously, does not involve any disturbance from the other components of magnetization (fig. 1). In order to take full advantage of the z component profile (shown at the bottom of fig. 1), one must substract it from a profile corresponding to the equilibrium magnetization and perfectly flat over the frequency zone of interest. This is achieved by the following basic phase cycle
(1)
[(O)x-T-(O)+x-~-]n (Tr/2) (Acq)•
where 0 stands for a pulse of small flip angle with 2nO = 7r, r being the classical precession interval of the DANTE sequence, and where the (7r/2) pulse converts the longitudinal magnetization into transverse magnetization. The first step is just a standard DANTE inverting train whereas the second step leaves essentially the longitudinal magnetization at its equilibrium value over the frequency range of interest. Substraction of these two results yields consequently a transverse magnetization whose profile reflects the one shown in the bottom of fig. 1. However, the DANTE trains generate unwanted dispersive components which can be eliminated by two further phase steps. The complete phase cycling is given in table 1, in accordance with the notations of the general DANTE-Z sequence given below
[(O)~l--T--(O)~2--T]n (7r/2)~3 (Acq)~4. 123
(2)
124
D. Canet and C. Roumestand
i00 8O 60 4O 20 0 -20 -40 -60 i
-80
-i00 i00 80 60 40 20 0 -20 -40 -60 -80 -i00
0 -20
40
60
80
-i00 -i00
-80
-60
-40
-20
0
20
40
60
80
I00
Itz
Fig. 1. A comparison of the profiles relative to the three components of magnetization after application of an inverting DANTE pulse train.
The D A N T E - Z E x p e r i m e n t
125
TABLE 1 The four-step phase cycle of the D A N T E - Z sequence. r
(/92
qr
r +
x
x
x
X
--X
X
X
X
--X
--
-x
+
x
-x
-
-
First of all, a four-step phase cycle may appear excessive. Fortunately, the two additional steps can be omitted by means of the application of a B0 gradient pulse prior to the 7r/2 pulse [6], which has the virtue of defocusing unwanted dispersive components. Secondly, it remains the problem of unwanted (negative) excitations at frequencies equal to (2k + 1)/2r (k being an integer); those are specific of the DANTE-Z pulse train, since the classical DANTE train involves only positive sidebands at frequencies equal to k/7-. Again, these negative sidebands can be canceled by a simple modification of the DANTE-Z sequence [6] which becomes [(O)x
(O)+x--7"]n(90)(Tr/2)x (Acq)+,
(3)
where (9o) stands for a B0 gradient pulse, according to the above proposed procedure for limiting the number of phase cycling steps. As a consequence, eq. (3) represents the most efficient and simple version of the DANTE-Z sequence. However, because selectivity is ultimately governed by the total duration of the pulse train, and because the r value is dictated by the frequency of the first positive side-band (inherent to the classical DANTE pulse train), n must be multiplied by two and concomitantly each pulse length must be divided by two. This has to be done in order to obtain a selectivity identical to that of the original sequence. The DANTE-Z sequence has been employed successfully as a 1D substitute in pseudo 3D experiments [7] and also as a "band-selective" technique in multidimensional experiments [8] in order to improve the spectral resolution. The efficiency of the DANTE-Z procedure over the simple DANTE sequence is illustrated by the spectra shown in fig. 2. The quality of the profile pertaining to the selected region can be improved by a modulation of the pulse lengths within the pulse train, which mimics the amplitude modulation of a simple soft pulse [8]. Simple modulation schemes can be devised for attenuating or even suppressing the side-lobes in the vicinity of the selected region (as shown in fig. 3). Alternatively, more elaborated modulation schemes as those of the B URP family [9] can be run in the DANTE-Z mode [10]. This mode actually offers a
126
A
r
A
D. Canet and C. Roumestand
m
p.. ,-.-.o
r,,9
S
.......................
m ~
,,,.q,
q
q
.4
.,in
, l .--.---.,a --..,,a
-,--..u
,o 4-
.4
-o -.I
.---.,o 9. 4 . . . . .
The DANTE-Z Experiment
127
Fig. 3. Experimental profiles (same experimental setting as in fig. 2) (a) of the basic DANTEZ sequence, (b) of DAZ 363, (c) of DAZ 22622 (the actual modulation schemes of DAZ 363 and DAZ 22622 can be found in ref. [7]). +..._
Fig. 2. Left: Experimental profiles of the conventional DANTE sequence (top) and of the DANTE-Z sequence (bottom). The sample used was 5% H20 in D20 with a tiny amount of copper sulfate added (leading to a T~ of approximately 3 s). The different traces were obtained by shifting the carrier frequency in 50 Hz steps without readjustment of the spectrometer phase. For each experiment, four scans were acquired in order to perform the complete phase cycling of DANTE-Z. Right: (a) The conventional ~H spectrum of a small protein (toxin "7:60 residues) in D20 at 318 K; (b) selection of the aromatic region by the conventional DANTE sequence; (c) same as (b) using the DANTE-Z procedure. Experiments were performed at 200 MHz using a "routine" AC200 Bruker spectrometer.
128
D. Canet and C. Roumestand
QD 00
-3.5 ..
~
+dl
00
~176
"4.0
~.. Ot
o,
Q'e
eo
IIII
o+ oh
O0
I0
'
, o
-4.5
+
00
I~O 9o +
oO+ ~,
ee
.o.,~o
.~,
9. OD O0
"5.0 08
.
-5.5 j~l
I0
Oo
"6.0 |
|
!
i
i
9.5
9.0
8.5
8.o
7.5
ppm
"3.5
ii*
i* -4.0
'%.,
"
++
rj. "4.5
"5.0
"5.5
"6.0 I
I
I
I
i
9.5
9.0
8.5
8.0
7.5
ppm
Fig. 4. Bottom: the Double-Band-Filtered COSY spectrum obtained by selection through DANTE-Z of the Ha region (prior to the evolution interval) and by the selection through SPIN-PINGING [11] of the amide region (before the acquisition interval) of toxin % Top: the corresponding region of a standard COSY spectrum. Note, in the bottom diagram, the considerable increase in spectral resolution as well as the occurrence of additional crosspeaks (indicated with asterisks). Experiments were performed at 360 MHz (Bruker AMX360) in H20 at 318 K. The 50 W "class C" amplifier of the proton channel was used as transmitter.
The DANTE-Z Experiment
129
m u c h more convenient way of setting the instrumental parameters of the sequence; the tedious calibration of B U R P soft pulses reduces to the calibration of a 90 ~ hard pulse [10]. The benefit of D A N T E - Z is exemplified by the two-dimensional diagrams of fig. 4. Finally, it can be m e n t i o n e d that the ideas underlying the D A N T E - Z m e t h o d o l o g y are closely related to the S P I N - P I N G I N G sequence [11], in which the starting configuration is transverse magnetization. Both methods yield roughly the same results and present the same advantages. However, as far as the effects of relaxation p h e n o m e n a are concerned, the D A N T E - Z sequence appears to be slightly in favor [10].
References [1] R. Freeman, Chem. Rev. 91 (1991) 1397. [2] H. Kessler, S. Mronga and G. Gemmeker, Magn. Reson. Chem. 29 (1991) 527. [3] G.A. Morris and R. Freeman, J. Magn. Reson. 29 (1978) 433. [4] J. Friedrich, S. Davies and R. Freeman, J. Magn. Reson. 75 (1987) 390. [5] D. Boudot, D. Canet, J. Brondeau and J.-C. Boubel, J. Magn. Reson. 83 (1989) 428. [6] C. Roumestand and D. Canet, J. Magn. Reson. B 106 (1995) 68.
[7] D. Boudot, C. Roumestand, E Toma and D. Canet, J. Magn. Reson. 90 (1990) 221. [8] C. Roumestand, D. Canet, N. Mahieu and E Toma, J. Magn. Reson. A 106 (1994) 168. [9] H. Geen and R. Freeman, J. Magn. Reson. 93 (1991) 93. [10] C. Roumestand, J. Mispelter, C. Austruy and D. Canet, J. Magn. Reson. B 109 (1995) 153. [ 11] P. Xu, X.-L. Wu and R. Freeman, J. Magn. Reson. 83 (1989) 404.
D. Canet Laboratoire de Methodologie RMN (URA CNRS 4 0 6 - LESOC; FU CNRS EO08- INCM) Universite Henri Poincare, Nancy 1, B.R 239 54506 Vandoeuvre les Nancy Cedex France
C. Roumestand Centre de Biochimie Structurale (UMR CNRS 9955- U 414 INSERM/Universite de Montpellier I) 15 Avenue Charles Flahault 34060 Montpellier Cedex France
Methods for Structure Elucidation by High-Resolution NMR Edited by Gy. Batta, K.E. KSver and Cs. Szantay, Jr. @ 1997 Elsevier Science B.V. All rights reserved 121
Selectivity has become one of the experimental procedures routinely used by the NMR spectroscopist [1, 2]. Its objective is to reduce the measuring time with 1D counterparts of 2D (or nD) experiments or to improve the spectral resolution in these latter experiments. Selectivity is usually achieved by a soft pulse or alternatively by a train of hard pulses of small flip angle (the DANTE experiment [3]). Both approaches are equivalent provided that the amplitude modulation of the soft pulse is implemented in the DANTE train through a modulation of pulse durations [4]. However, the simplest soft pulse, i.e., rectangularly shaped (or the equivalent DANTE train with pulses of identical durations) exhibits unwanted sinc oscillations on each side of the selected region with in addition a strong dispersive component. Both these drawbacks can be circumvented by the DANTE-Z variant [5], this experiment relying on the selective profile of the z magnetization component. As compared with the profiles of the transverse components, the one of longitudinal magnetization possesses the unique feature of much reduced sidelobes and, obviously, does not involve any disturbance from the other components of magnetization (fig. 1). In order to take full advantage of the z component profile (shown at the bottom of fig. 1), one must substract it from a profile corresponding to the equilibrium magnetization and perfectly flat over the frequency zone of interest. This is achieved by the following basic phase cycle
(1)
[(O)x-T-(O)+x-~-]n (Tr/2) (Acq)•
where 0 stands for a pulse of small flip angle with 2nO = 7r, r being the classical precession interval of the DANTE sequence, and where the (7r/2) pulse converts the longitudinal magnetization into transverse magnetization. The first step is just a standard DANTE inverting train whereas the second step leaves essentially the longitudinal magnetization at its equilibrium value over the frequency range of interest. Substraction of these two results yields consequently a transverse magnetization whose profile reflects the one shown in the bottom of fig. 1. However, the DANTE trains generate unwanted dispersive components which can be eliminated by two further phase steps. The complete phase cycling is given in table 1, in accordance with the notations of the general DANTE-Z sequence given below
[(O)~l--T--(O)~2--T]n (7r/2)~3 (Acq)~4. 123
(2)
124
D. Canet and C. Roumestand
i00 8O 60 4O 20 0 -20 -40 -60 i
-80
-i00 i00 80 60 40 20 0 -20 -40 -60 -80 -i00
0 -20
40
60
80
-i00 -i00
-80
-60
-40
-20
0
20
40
60
80
I00
Itz
Fig. 1. A comparison of the profiles relative to the three components of magnetization after application of an inverting DANTE pulse train.
The D A N T E - Z E x p e r i m e n t
125
TABLE 1 The four-step phase cycle of the D A N T E - Z sequence. r
(/92
qr
r +
x
x
x
X
--X
X
X
X
--X
--
-x
+
x
-x
-
-
First of all, a four-step phase cycle may appear excessive. Fortunately, the two additional steps can be omitted by means of the application of a B0 gradient pulse prior to the 7r/2 pulse [6], which has the virtue of defocusing unwanted dispersive components. Secondly, it remains the problem of unwanted (negative) excitations at frequencies equal to (2k + 1)/2r (k being an integer); those are specific of the DANTE-Z pulse train, since the classical DANTE train involves only positive sidebands at frequencies equal to k/7-. Again, these negative sidebands can be canceled by a simple modification of the DANTE-Z sequence [6] which becomes [(O)x
(O)+x--7"]n(90)(Tr/2)x (Acq)+,
(3)
where (9o) stands for a B0 gradient pulse, according to the above proposed procedure for limiting the number of phase cycling steps. As a consequence, eq. (3) represents the most efficient and simple version of the DANTE-Z sequence. However, because selectivity is ultimately governed by the total duration of the pulse train, and because the r value is dictated by the frequency of the first positive side-band (inherent to the classical DANTE pulse train), n must be multiplied by two and concomitantly each pulse length must be divided by two. This has to be done in order to obtain a selectivity identical to that of the original sequence. The DANTE-Z sequence has been employed successfully as a 1D substitute in pseudo 3D experiments [7] and also as a "band-selective" technique in multidimensional experiments [8] in order to improve the spectral resolution. The efficiency of the DANTE-Z procedure over the simple DANTE sequence is illustrated by the spectra shown in fig. 2. The quality of the profile pertaining to the selected region can be improved by a modulation of the pulse lengths within the pulse train, which mimics the amplitude modulation of a simple soft pulse [8]. Simple modulation schemes can be devised for attenuating or even suppressing the side-lobes in the vicinity of the selected region (as shown in fig. 3). Alternatively, more elaborated modulation schemes as those of the B URP family [9] can be run in the DANTE-Z mode [10]. This mode actually offers a
126
A
r
A
D. Canet and C. Roumestand
m
p.. ,-.-.o
r,,9
S
.......................
m ~
,,,.q,
q
q
.4
.,in
, l .--.---.,a --..,,a
-,--..u
,o 4-
.4
-o -.I
.---.,o 9. 4 . . . . .
The DANTE-Z Experiment
127
Fig. 3. Experimental profiles (same experimental setting as in fig. 2) (a) of the basic DANTEZ sequence, (b) of DAZ 363, (c) of DAZ 22622 (the actual modulation schemes of DAZ 363 and DAZ 22622 can be found in ref. [7]). +..._
Fig. 2. Left: Experimental profiles of the conventional DANTE sequence (top) and of the DANTE-Z sequence (bottom). The sample used was 5% H20 in D20 with a tiny amount of copper sulfate added (leading to a T~ of approximately 3 s). The different traces were obtained by shifting the carrier frequency in 50 Hz steps without readjustment of the spectrometer phase. For each experiment, four scans were acquired in order to perform the complete phase cycling of DANTE-Z. Right: (a) The conventional ~H spectrum of a small protein (toxin "7:60 residues) in D20 at 318 K; (b) selection of the aromatic region by the conventional DANTE sequence; (c) same as (b) using the DANTE-Z procedure. Experiments were performed at 200 MHz using a "routine" AC200 Bruker spectrometer.
128
D. Canet and C. Roumestand
QD 00
-3.5 ..
~
+dl
00
~176
"4.0
~.. Ot
o,
Q'e
eo
IIII
o+ oh
O0
I0
'
, o
-4.5
+
00
I~O 9o +
oO+ ~,
ee
.o.,~o
.~,
9. OD O0
"5.0 08
.
-5.5 j~l
I0
Oo
"6.0 |
|
!
i
i
9.5
9.0
8.5
8.o
7.5
ppm
"3.5
ii*
i* -4.0
'%.,
"
++
rj. "4.5
"5.0
"5.5
"6.0 I
I
I
I
i
9.5
9.0
8.5
8.0
7.5
ppm
Fig. 4. Bottom: the Double-Band-Filtered COSY spectrum obtained by selection through DANTE-Z of the Ha region (prior to the evolution interval) and by the selection through SPIN-PINGING [11] of the amide region (before the acquisition interval) of toxin % Top: the corresponding region of a standard COSY spectrum. Note, in the bottom diagram, the considerable increase in spectral resolution as well as the occurrence of additional crosspeaks (indicated with asterisks). Experiments were performed at 360 MHz (Bruker AMX360) in H20 at 318 K. The 50 W "class C" amplifier of the proton channel was used as transmitter.
The DANTE-Z Experiment
129
m u c h more convenient way of setting the instrumental parameters of the sequence; the tedious calibration of B U R P soft pulses reduces to the calibration of a 90 ~ hard pulse [10]. The benefit of D A N T E - Z is exemplified by the two-dimensional diagrams of fig. 4. Finally, it can be m e n t i o n e d that the ideas underlying the D A N T E - Z m e t h o d o l o g y are closely related to the S P I N - P I N G I N G sequence [11], in which the starting configuration is transverse magnetization. Both methods yield roughly the same results and present the same advantages. However, as far as the effects of relaxation p h e n o m e n a are concerned, the D A N T E - Z sequence appears to be slightly in favor [10].
References [1] R. Freeman, Chem. Rev. 91 (1991) 1397. [2] H. Kessler, S. Mronga and G. Gemmeker, Magn. Reson. Chem. 29 (1991) 527. [3] G.A. Morris and R. Freeman, J. Magn. Reson. 29 (1978) 433. [4] J. Friedrich, S. Davies and R. Freeman, J. Magn. Reson. 75 (1987) 390. [5] D. Boudot, D. Canet, J. Brondeau and J.-C. Boubel, J. Magn. Reson. 83 (1989) 428. [6] C. Roumestand and D. Canet, J. Magn. Reson. B 106 (1995) 68.
[7] D. Boudot, C. Roumestand, E Toma and D. Canet, J. Magn. Reson. 90 (1990) 221. [8] C. Roumestand, D. Canet, N. Mahieu and E Toma, J. Magn. Reson. A 106 (1994) 168. [9] H. Geen and R. Freeman, J. Magn. Reson. 93 (1991) 93. [10] C. Roumestand, J. Mispelter, C. Austruy and D. Canet, J. Magn. Reson. B 109 (1995) 153. [ 11] P. Xu, X.-L. Wu and R. Freeman, J. Magn. Reson. 83 (1989) 404.