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Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses
Author’s Accepted Manuscript Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses Ming Shen, Qun Chen, Jean-Paul Amoureux, Bingwen Hu www.elsevier.com/locate/ssnmr
PII: DOI: Reference:
S0926-2040(16)30030-3 http://dx.doi.org/10.1016/j.ssnmr.2016.05.001 YSNMR734
To appear in: Solid State Nuclear Magnetic Resonance Received date: 23 March 2016 Revised date: 6 May 2016 Accepted date: 19 May 2016 Cite this article as: Ming Shen, Qun Chen, Jean-Paul Amoureux and Bingwen Hu, Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses, Solid State Nuclear Magnetic Resonance, http://dx.doi.org/10.1016/j.ssnmr.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses Ming Shen, Qun Chen, Jean-Paul Amoureux, Bingwen Hu Shanghai Key Laboratory of Magnetic Resonance, School of Physics and Materials Science, East China Normal University, Shanghai, 200062 [email protected] [email protected]
Abstract We show here that composite pulses allow broad-band excitation of nitrogen-14 overtone frequencies through proton detected D-HMQC experiment (referred to 1H-{
} D-HMQC).
Experimental verifications have been performed on glycine, L-histidine and N-acetyl-valine (NAV) samples. Composite pulses enable symmetric excitations of
14
N sites with large shift differences.
Therefore, this approach is promising for recording high resolution 1H-{
} D-HMQC spectra
of most amino-acids, pharmaceutical samples and peptides.
Key words Solid-state NMR, 14N overtone spectroscopy, proton-detected D-HMQC
Introduction Solid-State NMR (SS-NMR) is a powerful tool for probing structural and dynamic information in biomolecules.
13
C and
15
N enrichments are often utilized to provide local binding and distance
constrains with atomic resolution. SS-NMR study of
14
N nucleus is less common due to its spin-1
value and its large quadrupole interaction that broadens the signal. However,
14
N SS-NMR can
provide unique information on electric field gradients, which can provide detailed information on structure and dynamics at the molecular level. Although experimentally demanding, the direct 1D detection of nitrogen-14 single-quantum transitions, 14NSQ, is nevertheless feasible under both static [1,2] and magic angle spinning (MAS) [3] conditions. However, due to the overlap of the broad line-shapes from different sites,
14
NSQ directly-detected 1D spectra with many
14
N sites are quite
difficult to analyse. As a result, the dipolar-assisted heteronuclear multiple quantum coherence (DHMQC) MAS method is often used to obtain high-resolution signals of
14
N nuclei.[4,5] In D-
2
HMQC 2D experiments, the
14
NSQ signal is indirectly detected via a more sensitive spy nucleus.
The rotor axis must be precisely at the magic-angle and the two
14
N pulses must be rotor-
synchronized for the complete averaging of the 1st-order quadrupolar coupling. Such setting yields 2D spectra with along F1 only the chemical plus
14
NSQ 2nd-order quadrupolar-induced shifts,
leading to high-resolution 14N spectra along the indirect dimension. Furthermore, the sensitivity of 14 1
NSQ NMR is largely enhanced by indirect detection, especially with the proton spy nucleus; the
H-{14NSQ} D-HMQC MAS experiment.[4,5] Unlike the single-quantum transitions, the nitrogen-14 double quantum transition, 14NDQ, is
not broadened by the 1st-order quadrupole interaction (HQ1), but only by the 2nd-order one (HQ2). This enables the direct and indirect high resolution observation of 14NDQ transition, without the high experimental demand required for 14
14
NSQ: perfect MAS angle and very stable spinning speed. This
NDQ indirect observation can be performed with excitation and detection that are performed either
at once or twice the 14N Larmor frequency. The second way is called the overtone method. The
overtone spectroscopy relies on the mixing of Zeeman and HQ1 energy-states.[6-
8] Direct detection of
signal is quite difficult due to its low sensitivity, and thus dynamic
nuclear polarization (DNP)[9] or cross-polarization [10] must be employed to save experimental time. To enhance the {
sensitivity, its indirect detection was recently realized through 1H-
D-HMQC experiments [11,12]. A detailed comparison of different schemes for indirect
detection of
14
N signal can be found elsewhere.[13] However, as a matter of fact,
spectroscopy is very sensitive to frequency offset, because the effective nutation frequency is very week for
transition, and the maximum rf-field is usually less than
attenuate the offset effects, a composite pulse (̅̅̅̅ been applied for broadband excitation in
14N
= 75 kHz. To
, referred to COM-V in this work) has first
spectroscopy under static condition.[14] Here, we
investigate the performance of several composite pulses [15] for indirect detection through 1H{
} D-HMQC experiments under MAS.
Pulse sequence and experiments The performances of broadband composite pulses in 1H-{
} D-HMQC experiment were tested
on a Bruker AVANCE-III 600 MHz spectrometer. Experiments were performed using samples of glycine, L-[U-13C]-histidine, and N-acetyl-valine (NAV), which were purchased from CortecNet and used without further purification. Samples were spun at resonance probe.
R
= 62.5 kHz with a 1.3 mm triple-
3
Fig.1. Pulse sequence for 2D 1H-{ } D-HMQC experiment. Dipolar recoupling (SR421) was used for 1 14 coherence transfer between H and N. Our study makes use of composite pulses (listed in Table 1) to excite and reconvert the transition. These composite pulses are submitted to a two-step phase cycling as a whole to allow the selection of coherences. The excitation and detection of these coherences were performed at 86.8 MHz, twice the 14N Larmor frequency at 14.1 T.
The pulse sequence of 1H-{
} D-HMQC is depicted in Fig.1. Our experiment differs
from previous works by replacing on
14
N channel the single-pulse (SP) with composite pulses as
listed in Table 1. The first three composite pulses were studied in our previous work [15] of 2H quadrupolar echo spectroscopy, while the last one has already been employed for direct excitation of
signal under static condition.[14] In our work, these composite pulses were modified
because the definition of flip angle is not valid for
14
N overtone transition, for which the nutation
depends on (i) the rf field, (ii) the quadrupolar coupling constant, and (iii) for each crystallite on the orientation of the quadrupole tensor relative to the externally applied static [8] [13]. As shown in Table 1, we simply retain the relative time scales of individual pulses for each composite pulse. The rf field was set at its maximum value depending on the probe that is used. We have first optimized on glycine the on-resonance
signal with a long single pulse. Then we have used the same
total length (tp) for modified composite pulses, which allowed and easy comparison. It must be noted that an optimization of the total length of each composite pulse could presumably still increase the signal, but we have not done such an individual optimization. In this paper, only modified composite pulses are employed, and we have used two identical modified composite pulses for excitation and reconversion of
transition. WURST pulse has been shown to be
effective for both direct excitation [16] and indirect detection of
transition [11] under MAS.
Therefore, WURST shape pulses, generated by using Bruker Shape Tool, were also employed for comparison. All these pulses were sent on resonance at the +2 were applied with the strength of
14N
R
‘overtone spinning sideband’ and
= 70 kHz, which is the maximum rf field that can be
delivered on 14N channel of our probe. The duration of composite pulses or WURST shape pulses, tp, and the sweep width of WURST shape pulses were optimized and are indicated in figure captions.
4
Table 1. Original and modified composite pulses studied in our work. Since the concept of flip angle is no longer valid for 14N overtone transition, we have modified these composite pulses so that only the relative time scales of individual pulses for each composite pulse train are kept. In this paper, only modified composite pulses are employed. These modified pulses, together with WURST scheme were applied on 14N channel at twice the Larmor frequency to replace single pulse irradiation in 1H-{ } D-HMQC experiments.
Original Composite Pulses
COM-I:
̅̅̅̅
COM-II:
̅̅̅̅̅
COM-IV:
̅̅̅̅̅
Modified Composite Pulses ̅̅̅̅
COM-Im: ̅̅̅̅̅ ̅̅̅̅̅
COM-V: ̅̅̅̅
COM-IIm:
̅̅̅̅
̅̅̅̅
COM-IVm:
̅̅̅̅
̅̅̅̅
COM-Vm: ̅
Typical 90° and 180° pulses on 1H channel were applied with an rf field strength of 150 kHz. recoupling sequence [17] with rf field strength of 2 14
R
= 125 kHz was applied to reintroduce the 1H-
N dipolar couplings and suppress most of the 1H-1H interactions. The dipolar recoupling time, τD,
was optimized for each experimental set. All the 2D spectra were recorded with t1 increment equal to one rotor period. States-TPPI was applied for hyper-complex data acquisition. The two WURST pulses in the HMQC pulse sequence were applied with opposite sweep directions. Other parameters are indicated in the figure captions.
Results and discussion In this work, we mainly focus on the sensitivity of 1H-{ to
14
} D-HMQC experiments with respect
N offset. This issue was addressed firstly by performing experiments on glycine. This
compound has a single
14
N site, thus allowing to record within reasonable time a series of 2D 1H-
} D-HMQC spectra using different excitation schemes and various offsets of the 14N carrier
{
frequency. It must be noted that with 14NDQ experiments all frequencies are doubled with respect to their values with 14NSQ. This means that the separations and linewidths of the 14NDQ resonances are twice their values with 14NSQ experiments. Therefore, when given in Hz, the robustness to offset in 14
NDQ should be divided by two to be compared with 14NSQ experiments. Fig.2 presents the response
of
signal extracted from those 2D spectra to
14
N offset at twice the Larmor frequency. It is
5
evident that the use of two long single pulses (Fig.2a) gives very intense on-resonance signal. However, the peak amplitude decays rapidly with increasing offset, resulting in a coverage bandwidth (in yellow) of 8 kHz. Such narrow excitation is attributed to the use of a long rf irradiation of tp = 220
s to achieve sufficient sensitivity. For comparison, we have fixed the total
length of WURST and modified composite pulses to the same value of 220
s. It should be noted
here that the definition of flip angle in original composite pulses is not valid as the nutation of transition cannot be described as with spin 1/2 nuclei. Nevertheless, the relative scale of individual pulses for each modified composite pulse is retained to make them quasi-adiabatic for transition.
Fig.2. Experimental signal of glycine with various excitation schemes, extracted from 2D 1H{ } D-HMQC spectra as function of offset applied at 2014N. 14N offset was varied from -14 to +14 kHz with an increment of 1 kHz. Each 2D spectrum was recorded by co-adding 16 transients with 50 t1 increments. The recycle delay was 1.5 s. SP, modified composite and WURST pulses were applied with total pulse length, tp = 220 s. For WURST pulse, the sweep range was 40 kHz.
Interestingly, the offset profiles obtained by using modified composite pulses are quasianalogous to those with a 2H quadrupolar echo spectrum with a pair of horns ‘resonating' away from the centre. Although the excitation of composite pulses is not as uniform as that of WURST shape pulse, it is still quite sufficient to cover a quite wide frequency range. For example, COM-Im leads to a broad excitation with coverage of 20 kHz, and COM-IIm and COM-IVm lead to an even broader excitation with a slightly weaker signal at certain offsets. We have found that COM-Vm gives a much narrower bandwidth as compared to a previous work on the same composite pulse.[14] This is due to the fact that longer pulses as employed in our work (220
s instead of 70
6
s) are more adequate for 14N site with smaller CQ value (1.18 MHz for glycine [18]), thus leading to much narrower excitation bandwidth. Nevertheless, the coverage of the excitation profile resulting from COM-IVm is twice larger than that resulting from single pulse in both works.
Fig.3. 2D 1H-{ } D-HMQC spectra of L-histidine recorded by using WURST and COM-IIm schemes. Skyline projections are shown on the left for comparison. Spectra obtained with other composite pulses are not shown for clarity. COM-IIm and WURST pulse lengths were tp = 400 s and WURST sweep range was 40 kHz. 2D spectra were recorded by co-adding 32 transients with 100 t1 increments. The recycle delay was 1.5 s. Projections on the left are plotted on the same
amplitude scale for comparison. It should be reminded that for most 2D experiments, the carrier frequency of the indirect dimension is preferably set in the center of spectra in order to avoid the interference of artefacts arising from the imbalance of quadrature detectors. Therefore, such symmetric excitation at the horns could be employed for recording signals with distinct chemical shifts. This is particularly helpful for indirect detection of
14
N species in biomolecules because amide and pyridine nitrogen species with well-
defined chemical shifts often co-existed in these samples. Such advantage is demonstrated on the 1
H-{
} D-HMQC spectrum of L-[U-13C]-histidine shown in Fig.3, where the two large cross
peaks corresponding to amide and pyridine nitrogen species are present. These two nitrogen sites exhibit a shift difference of 14.7 kHz. As a consequence, a symmetric excitation profile at ±7.4 kHz should be optimum as long as the carrier frequency is set in the center of
14
N dimension. This
optimum excitation is realized by using COM-IIm or COM-IVm, but is impossible to obtain with two single pulses that lead to much narrower excitation bandwidth. Shortening the long pulse lengths may help in this case, but the signal to noise ratio (S/R) would become another issue. It must be noted that WURST scheme also excites both sites, but the signal intensity is twice as small as that obtained by using COM-IIm or COM-IVm.
7
Fig.4. 2D 1H-{ } D-HMQC spectra of mixed glycine and NAV sample. Skyline projections taken from the spectra recorded with WURST pulse (a) or composite pulses (b,c,d) are shown on the left for comparison. Modified composite and WURST pulses were applied with tp = 100 s, and the WURST sweep range was 60 kHz. 2D spectra were recorded by co-adding 128 transients with 100 t1 increments. The recycle delay was 4 s. Projections on the left are plotted on the same amplitude scale for comparison.
Finally, we recorded the 2D spectra on a mixed sample of glycine and NAV with the various excitation schemes mentioned above. By using modified composite or WURST pulses, the two cross peaks arising from the
14
N site in each sample are clearly identified on the spectra (Fig.4),
which is impossible with single pulse excitation. It is verified again that composite pulses enable the improvement of the robustness with respect to 14N offset. As a result of shorter pulse block (tp = 100 s), signals with very large chemical shift difference were observed. Therefore, we anticipate that the resulting excitation bandwidth is inverse promotional to the total length of composite pulses, although further work could be performed by using spin dynamic simulations with advanced simulation software [11,19]. Furthermore, modified composite pulses are better than WURST shape pulses in this case, because of their horn-like excitation profiles.
Conclusions We have shown that in 1H-{
} D-HMQC experiments composite pulses are more robust with
respect to 14N offset than single-pulses and more efficient than WURST pulses. It has recently been shown that 1H-{14NSQ} D-HMQC experiments with selective 14NSQ excitation are very efficient and robust [20]. The present overtone strategy with composite pulses is thus an alternative approach for recording high resolution 14N signals. This method can easily be generalized and the shaped-pulses can be tailored to only excite the useful
frequency ranges. These complementary indirect-
8
detection methods, with either
14
NSQ or
, represent two promising tools to get insights into
more complex chemical environments of 14N species in biomolecules.
Acknowledgements This work was supported by the Shanghai Committee of Science and Technology (11JC1403600), the large instruments Open Foundation of East China Normal University, National Key Basic Research Program of China (2013CB921800), National Natural Science Foundation of China (21103050, 21373086) and National Natural Science Foundation of China for Excellent Young Scholars (21522303). References: [1]
L.A. O'Dell, R.W. Schurko, Fast and Simple Acquisition of Solid-State 14N NMR Spectra with Signal Enhancement via Population Transfer, J. Am. Chem. Soc. 131 (2009) 6658– 6659. doi:10.1021/ja901278q.
[2]
E.A.H. and, J.P. Yesinowski, Wide-Line 14N NMR in Solids and Reorientation-Induced Redistribution of Isochromats, in: American Chemical Society, 1996: pp. 6798–6799. doi:10.1021/ja960757j.
[3]
H.J. Jakobsen, H. Bildsøe, J. Skibsted, T. Giavani, 14N MAS NMR Spectroscopy: The Nitrate Ion, J. Am. Chem. Soc. 123 (2001) 5098–5099. doi:10.1021/ja0100118.
[4]
S. Cavadini, S. Antonijevic, A. Lupulescu, G. Bodenhausen, Indirect detection of nitrogen14 in solids via protons by nuclear magnetic resonance spectroscopy, Journal of Magnetic Resonance. 182 (2006) 168–172. doi:10.1016/j.jmr.2006.06.003.
[5]
Z. Gan, 13C/14N heteronuclear multiple-quantum correlation with rotary resonance and REDOR dipolar recoupling, Journal of Magnetic Resonance. 184 (2007) 39–43. doi:10.1016/j.jmr.2006.09.016.
[6]
M. Bloom, M.A. LeGros, Direct detection of two-quantum coherence, Can. J. Phys. 64 (1986) 1522–1528. doi:10.1139/p86-271.
[7]
R. Tycko, S.J. Opella, High-resolution nitrogen-14 overtone spectroscopy: an approach to natural abundance nitrogen NMR of oriented and polycrystalline systems, J. Am. Chem. Soc. 108 (1986) 3531–3532. doi:10.1021/ja00272a071.
[8]
R. Tycko, S.J. Opella, Overtone NMR spectroscopy, J. Chem. Phys. 86 (1987) 1761–15. doi:10.1063/1.452176.
[9]
A.J. Rossini, L. Emsley, L.A. O'Dell, Dynamic nuclear polarisation enhanced 14N overtone MAS NMR spectroscopy, Phys. Chem. Chem. Phys. 16 (2014) 12890–12899. doi:10.1039/C4CP00590B.
9
[10]
I. Haies, J. Jarvis, H. Bentley, I. Heinmaa, I. Kuprov, P. Williamson, et al., 14N Overtone NMR under MAS: signal enhancement using symmetry-based sequences and novel simulation strategies, Phys. Chem. Chem. Phys. (2015). doi:10.1039/C4CP03994G.
[11]
L.A. O'Dell, A. Brinkmann, 14N overtone NMR spectra under magic angle spinning: Experiments and numerically exact simulations, J. Chem. Phys. 138 (2013) 064201. doi:10.1063/1.4775592.
[12]
Y. Nishiyama, M. Malon, Z. Gan, Y. Endo, T. Nemoto, Proton–nitrogen-14 overtone twodimensional correlation NMR spectroscopy of solid-sample at very fast magic angle sample spinning,
Journal
of
Magnetic
Resonance.
230
(2013)
160–164.
doi:10.1016/j.jmr.2013.02.015. [13]
M. Shen, J. Trébosc, L.A. O'Dell, O. Lafon, F. Pourpoint, B. Hu, et al., Comparison of various NMR methods for the indirect detection of nitrogen-14 nuclei via protons in solids, Journal of Magnetic Resonance. 258 (2015) 86–95. doi:10.1016/j.jmr.2015.06.008.
[14]
D.K. Lee, A. Ramamoorthy, A composite pulse sequence for the broadband excitation in overtone 14 N NMR spectroscopy, Chemical Physics Letters. 280 (1997) 501–506.
[15]
M. Shen, R. Roopchand, E.S. Mananga, J.-P. Amoureux, Q. Chen, G.S. Boutis, et al., Revisiting NMR composite pulses for broadband 2H excitation, Solid State Nuclear Magnetic Resonance. 66-67 (2015) 45–48. doi:10.1016/j.ssnmr.2014.12.004.
[16]
L.A. O'Dell, C.I. Ratcliffe, 14N magic angle spinning overtone NMR spectra, Chemical Physics Letters. 514 (2011) 168–173. doi:10.1016/j.cplett.2011.08.030.
[17]
A. Brinkmann, A.P.M. Kentgens, Proton-Selective 17O− 1H Distance Measurements in Fast Magic-Angle-Spinning Solid-State NMR Spectroscopy for the Determination of Hydrogen
Bond
Lengths,
J.
Am.
Chem.
Soc.
128
(2006)
14758–14759.
doi:10.1021/ja065415k. [18]
R.E. Stark, H. Van Willigen, R.G. Griffin, Determination of bond distances and bond angles by solid-state nuclear magnetic resonance. Carbon-13 and nitrogen-14 NMR study of glycine, J. Am. Chem. Soc. 103 (1981) 2534–2539. doi:10.1021/ja00400a008.
[19]
I. Haies, J. Jarvis, L.J. Brown, I. Kuprov, P. Williamson, M. Carravetta, 14N overtone transition in double rotation solid-state NMR, Phys. Chem. Chem. Phys. (2015) 1–19. doi:10.1039/C5CP03266K.
[20]
M. Shen, J. Trébosc, O. Lafon, Z. Gan, F. Pourpoint, B. Hu, et al., Solid-state NMR indirect detection of nuclei experiencing large anisotropic interactions using spinning sideband-selective pulses, Solid State Nuclear Magnetic Resonance. 72 (2015) 104–117. doi:10.1016/j.ssnmr.2015.09.003.
10
Highlights
Composite pulses enhance robustness with respect to offset in 1H-{ Composite pulses lead to horn-like excitation profile
}D-HMQC
11
Graphical abstract
Composite pulses allow broad-band excitation in 2D 1H-{
} D-HMQC
PII: DOI: Reference:
S0926-2040(16)30030-3 http://dx.doi.org/10.1016/j.ssnmr.2016.05.001 YSNMR734
To appear in: Solid State Nuclear Magnetic Resonance Received date: 23 March 2016 Revised date: 6 May 2016 Accepted date: 19 May 2016 Cite this article as: Ming Shen, Qun Chen, Jean-Paul Amoureux and Bingwen Hu, Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses, Solid State Nuclear Magnetic Resonance, http://dx.doi.org/10.1016/j.ssnmr.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Broad-band excitation in indirectly detected 14N overtone spectroscopy with composite pulses Ming Shen, Qun Chen, Jean-Paul Amoureux, Bingwen Hu Shanghai Key Laboratory of Magnetic Resonance, School of Physics and Materials Science, East China Normal University, Shanghai, 200062 [email protected] [email protected]
Abstract We show here that composite pulses allow broad-band excitation of nitrogen-14 overtone frequencies through proton detected D-HMQC experiment (referred to 1H-{
} D-HMQC).
Experimental verifications have been performed on glycine, L-histidine and N-acetyl-valine (NAV) samples. Composite pulses enable symmetric excitations of
14
N sites with large shift differences.
Therefore, this approach is promising for recording high resolution 1H-{
} D-HMQC spectra
of most amino-acids, pharmaceutical samples and peptides.
Key words Solid-state NMR, 14N overtone spectroscopy, proton-detected D-HMQC
Introduction Solid-State NMR (SS-NMR) is a powerful tool for probing structural and dynamic information in biomolecules.
13
C and
15
N enrichments are often utilized to provide local binding and distance
constrains with atomic resolution. SS-NMR study of
14
N nucleus is less common due to its spin-1
value and its large quadrupole interaction that broadens the signal. However,
14
N SS-NMR can
provide unique information on electric field gradients, which can provide detailed information on structure and dynamics at the molecular level. Although experimentally demanding, the direct 1D detection of nitrogen-14 single-quantum transitions, 14NSQ, is nevertheless feasible under both static [1,2] and magic angle spinning (MAS) [3] conditions. However, due to the overlap of the broad line-shapes from different sites,
14
NSQ directly-detected 1D spectra with many
14
N sites are quite
difficult to analyse. As a result, the dipolar-assisted heteronuclear multiple quantum coherence (DHMQC) MAS method is often used to obtain high-resolution signals of
14
N nuclei.[4,5] In D-
2
HMQC 2D experiments, the
14
NSQ signal is indirectly detected via a more sensitive spy nucleus.
The rotor axis must be precisely at the magic-angle and the two
14
N pulses must be rotor-
synchronized for the complete averaging of the 1st-order quadrupolar coupling. Such setting yields 2D spectra with along F1 only the chemical plus
14
NSQ 2nd-order quadrupolar-induced shifts,
leading to high-resolution 14N spectra along the indirect dimension. Furthermore, the sensitivity of 14 1
NSQ NMR is largely enhanced by indirect detection, especially with the proton spy nucleus; the
H-{14NSQ} D-HMQC MAS experiment.[4,5] Unlike the single-quantum transitions, the nitrogen-14 double quantum transition, 14NDQ, is
not broadened by the 1st-order quadrupole interaction (HQ1), but only by the 2nd-order one (HQ2). This enables the direct and indirect high resolution observation of 14NDQ transition, without the high experimental demand required for 14
14
NSQ: perfect MAS angle and very stable spinning speed. This
NDQ indirect observation can be performed with excitation and detection that are performed either
at once or twice the 14N Larmor frequency. The second way is called the overtone method. The
overtone spectroscopy relies on the mixing of Zeeman and HQ1 energy-states.[6-
8] Direct detection of
signal is quite difficult due to its low sensitivity, and thus dynamic
nuclear polarization (DNP)[9] or cross-polarization [10] must be employed to save experimental time. To enhance the {
sensitivity, its indirect detection was recently realized through 1H-
D-HMQC experiments [11,12]. A detailed comparison of different schemes for indirect
detection of
14
N signal can be found elsewhere.[13] However, as a matter of fact,
spectroscopy is very sensitive to frequency offset, because the effective nutation frequency is very week for
transition, and the maximum rf-field is usually less than
attenuate the offset effects, a composite pulse (̅̅̅̅ been applied for broadband excitation in
14N
= 75 kHz. To
, referred to COM-V in this work) has first
spectroscopy under static condition.[14] Here, we
investigate the performance of several composite pulses [15] for indirect detection through 1H{
} D-HMQC experiments under MAS.
Pulse sequence and experiments The performances of broadband composite pulses in 1H-{
} D-HMQC experiment were tested
on a Bruker AVANCE-III 600 MHz spectrometer. Experiments were performed using samples of glycine, L-[U-13C]-histidine, and N-acetyl-valine (NAV), which were purchased from CortecNet and used without further purification. Samples were spun at resonance probe.
R
= 62.5 kHz with a 1.3 mm triple-
3
Fig.1. Pulse sequence for 2D 1H-{ } D-HMQC experiment. Dipolar recoupling (SR421) was used for 1 14 coherence transfer between H and N. Our study makes use of composite pulses (listed in Table 1) to excite and reconvert the transition. These composite pulses are submitted to a two-step phase cycling as a whole to allow the selection of coherences. The excitation and detection of these coherences were performed at 86.8 MHz, twice the 14N Larmor frequency at 14.1 T.
The pulse sequence of 1H-{
} D-HMQC is depicted in Fig.1. Our experiment differs
from previous works by replacing on
14
N channel the single-pulse (SP) with composite pulses as
listed in Table 1. The first three composite pulses were studied in our previous work [15] of 2H quadrupolar echo spectroscopy, while the last one has already been employed for direct excitation of
signal under static condition.[14] In our work, these composite pulses were modified
because the definition of flip angle is not valid for
14
N overtone transition, for which the nutation
depends on (i) the rf field, (ii) the quadrupolar coupling constant, and (iii) for each crystallite on the orientation of the quadrupole tensor relative to the externally applied static [8] [13]. As shown in Table 1, we simply retain the relative time scales of individual pulses for each composite pulse. The rf field was set at its maximum value depending on the probe that is used. We have first optimized on glycine the on-resonance
signal with a long single pulse. Then we have used the same
total length (tp) for modified composite pulses, which allowed and easy comparison. It must be noted that an optimization of the total length of each composite pulse could presumably still increase the signal, but we have not done such an individual optimization. In this paper, only modified composite pulses are employed, and we have used two identical modified composite pulses for excitation and reconversion of
transition. WURST pulse has been shown to be
effective for both direct excitation [16] and indirect detection of
transition [11] under MAS.
Therefore, WURST shape pulses, generated by using Bruker Shape Tool, were also employed for comparison. All these pulses were sent on resonance at the +2 were applied with the strength of
14N
R
‘overtone spinning sideband’ and
= 70 kHz, which is the maximum rf field that can be
delivered on 14N channel of our probe. The duration of composite pulses or WURST shape pulses, tp, and the sweep width of WURST shape pulses were optimized and are indicated in figure captions.
4
Table 1. Original and modified composite pulses studied in our work. Since the concept of flip angle is no longer valid for 14N overtone transition, we have modified these composite pulses so that only the relative time scales of individual pulses for each composite pulse train are kept. In this paper, only modified composite pulses are employed. These modified pulses, together with WURST scheme were applied on 14N channel at twice the Larmor frequency to replace single pulse irradiation in 1H-{ } D-HMQC experiments.
Original Composite Pulses
COM-I:
̅̅̅̅
COM-II:
̅̅̅̅̅
COM-IV:
̅̅̅̅̅
Modified Composite Pulses ̅̅̅̅
COM-Im: ̅̅̅̅̅ ̅̅̅̅̅
COM-V: ̅̅̅̅
COM-IIm:
̅̅̅̅
̅̅̅̅
COM-IVm:
̅̅̅̅
̅̅̅̅
COM-Vm: ̅
Typical 90° and 180° pulses on 1H channel were applied with an rf field strength of 150 kHz. recoupling sequence [17] with rf field strength of 2 14
R
= 125 kHz was applied to reintroduce the 1H-
N dipolar couplings and suppress most of the 1H-1H interactions. The dipolar recoupling time, τD,
was optimized for each experimental set. All the 2D spectra were recorded with t1 increment equal to one rotor period. States-TPPI was applied for hyper-complex data acquisition. The two WURST pulses in the HMQC pulse sequence were applied with opposite sweep directions. Other parameters are indicated in the figure captions.
Results and discussion In this work, we mainly focus on the sensitivity of 1H-{ to
14
} D-HMQC experiments with respect
N offset. This issue was addressed firstly by performing experiments on glycine. This
compound has a single
14
N site, thus allowing to record within reasonable time a series of 2D 1H-
} D-HMQC spectra using different excitation schemes and various offsets of the 14N carrier
{
frequency. It must be noted that with 14NDQ experiments all frequencies are doubled with respect to their values with 14NSQ. This means that the separations and linewidths of the 14NDQ resonances are twice their values with 14NSQ experiments. Therefore, when given in Hz, the robustness to offset in 14
NDQ should be divided by two to be compared with 14NSQ experiments. Fig.2 presents the response
of
signal extracted from those 2D spectra to
14
N offset at twice the Larmor frequency. It is
5
evident that the use of two long single pulses (Fig.2a) gives very intense on-resonance signal. However, the peak amplitude decays rapidly with increasing offset, resulting in a coverage bandwidth (in yellow) of 8 kHz. Such narrow excitation is attributed to the use of a long rf irradiation of tp = 220
s to achieve sufficient sensitivity. For comparison, we have fixed the total
length of WURST and modified composite pulses to the same value of 220
s. It should be noted
here that the definition of flip angle in original composite pulses is not valid as the nutation of transition cannot be described as with spin 1/2 nuclei. Nevertheless, the relative scale of individual pulses for each modified composite pulse is retained to make them quasi-adiabatic for transition.
Fig.2. Experimental signal of glycine with various excitation schemes, extracted from 2D 1H{ } D-HMQC spectra as function of offset applied at 2014N. 14N offset was varied from -14 to +14 kHz with an increment of 1 kHz. Each 2D spectrum was recorded by co-adding 16 transients with 50 t1 increments. The recycle delay was 1.5 s. SP, modified composite and WURST pulses were applied with total pulse length, tp = 220 s. For WURST pulse, the sweep range was 40 kHz.
Interestingly, the offset profiles obtained by using modified composite pulses are quasianalogous to those with a 2H quadrupolar echo spectrum with a pair of horns ‘resonating' away from the centre. Although the excitation of composite pulses is not as uniform as that of WURST shape pulse, it is still quite sufficient to cover a quite wide frequency range. For example, COM-Im leads to a broad excitation with coverage of 20 kHz, and COM-IIm and COM-IVm lead to an even broader excitation with a slightly weaker signal at certain offsets. We have found that COM-Vm gives a much narrower bandwidth as compared to a previous work on the same composite pulse.[14] This is due to the fact that longer pulses as employed in our work (220
s instead of 70
6
s) are more adequate for 14N site with smaller CQ value (1.18 MHz for glycine [18]), thus leading to much narrower excitation bandwidth. Nevertheless, the coverage of the excitation profile resulting from COM-IVm is twice larger than that resulting from single pulse in both works.
Fig.3. 2D 1H-{ } D-HMQC spectra of L-histidine recorded by using WURST and COM-IIm schemes. Skyline projections are shown on the left for comparison. Spectra obtained with other composite pulses are not shown for clarity. COM-IIm and WURST pulse lengths were tp = 400 s and WURST sweep range was 40 kHz. 2D spectra were recorded by co-adding 32 transients with 100 t1 increments. The recycle delay was 1.5 s. Projections on the left are plotted on the same
amplitude scale for comparison. It should be reminded that for most 2D experiments, the carrier frequency of the indirect dimension is preferably set in the center of spectra in order to avoid the interference of artefacts arising from the imbalance of quadrature detectors. Therefore, such symmetric excitation at the horns could be employed for recording signals with distinct chemical shifts. This is particularly helpful for indirect detection of
14
N species in biomolecules because amide and pyridine nitrogen species with well-
defined chemical shifts often co-existed in these samples. Such advantage is demonstrated on the 1
H-{
} D-HMQC spectrum of L-[U-13C]-histidine shown in Fig.3, where the two large cross
peaks corresponding to amide and pyridine nitrogen species are present. These two nitrogen sites exhibit a shift difference of 14.7 kHz. As a consequence, a symmetric excitation profile at ±7.4 kHz should be optimum as long as the carrier frequency is set in the center of
14
N dimension. This
optimum excitation is realized by using COM-IIm or COM-IVm, but is impossible to obtain with two single pulses that lead to much narrower excitation bandwidth. Shortening the long pulse lengths may help in this case, but the signal to noise ratio (S/R) would become another issue. It must be noted that WURST scheme also excites both sites, but the signal intensity is twice as small as that obtained by using COM-IIm or COM-IVm.
7
Fig.4. 2D 1H-{ } D-HMQC spectra of mixed glycine and NAV sample. Skyline projections taken from the spectra recorded with WURST pulse (a) or composite pulses (b,c,d) are shown on the left for comparison. Modified composite and WURST pulses were applied with tp = 100 s, and the WURST sweep range was 60 kHz. 2D spectra were recorded by co-adding 128 transients with 100 t1 increments. The recycle delay was 4 s. Projections on the left are plotted on the same amplitude scale for comparison.
Finally, we recorded the 2D spectra on a mixed sample of glycine and NAV with the various excitation schemes mentioned above. By using modified composite or WURST pulses, the two cross peaks arising from the
14
N site in each sample are clearly identified on the spectra (Fig.4),
which is impossible with single pulse excitation. It is verified again that composite pulses enable the improvement of the robustness with respect to 14N offset. As a result of shorter pulse block (tp = 100 s), signals with very large chemical shift difference were observed. Therefore, we anticipate that the resulting excitation bandwidth is inverse promotional to the total length of composite pulses, although further work could be performed by using spin dynamic simulations with advanced simulation software [11,19]. Furthermore, modified composite pulses are better than WURST shape pulses in this case, because of their horn-like excitation profiles.
Conclusions We have shown that in 1H-{
} D-HMQC experiments composite pulses are more robust with
respect to 14N offset than single-pulses and more efficient than WURST pulses. It has recently been shown that 1H-{14NSQ} D-HMQC experiments with selective 14NSQ excitation are very efficient and robust [20]. The present overtone strategy with composite pulses is thus an alternative approach for recording high resolution 14N signals. This method can easily be generalized and the shaped-pulses can be tailored to only excite the useful
frequency ranges. These complementary indirect-
8
detection methods, with either
14
NSQ or
, represent two promising tools to get insights into
more complex chemical environments of 14N species in biomolecules.
Acknowledgements This work was supported by the Shanghai Committee of Science and Technology (11JC1403600), the large instruments Open Foundation of East China Normal University, National Key Basic Research Program of China (2013CB921800), National Natural Science Foundation of China (21103050, 21373086) and National Natural Science Foundation of China for Excellent Young Scholars (21522303). References: [1]
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10
Highlights
Composite pulses enhance robustness with respect to offset in 1H-{ Composite pulses lead to horn-like excitation profile
}D-HMQC
11
Graphical abstract
Composite pulses allow broad-band excitation in 2D 1H-{
} D-HMQC