- Email: [email protected]
Optimized decoupling schemes in ultrafast HSQC experiments
Accepted Manuscript Optimized decoupling schemes in ultrafast HSQC experiments Laetitia Rouger, Serge Akoka, Patrick Giraudeau PII: DOI: Reference:
S1090-7807(17)30223-9 http://dx.doi.org/10.1016/j.jmr.2017.08.015 YJMRE 6154
To appear in:
Journal of Magnetic Resonance
Received Date: Revised Date: Accepted Date:
18 May 2017 25 August 2017 29 August 2017
Please cite this article as: L. Rouger, S. Akoka, P. Giraudeau, Optimized decoupling schemes in ultrafast HSQC experiments, Journal of Magnetic Resonance (2017), doi: http://dx.doi.org/10.1016/j.jmr.2017.08.015
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 proof before it is published in its final 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.
Optimized decoupling schemes in ultrafast HSQC experiments
Laetitia Rouger,a Serge Akoka,a and Patrick Giraudeau*ab
a
CNRS UMR 6230 CEISAM, Université de Nantes, Nantes, France
e-mail: [email protected] b
Institut Universitaire de France, Paris, France
1
Abstract Ultrafast (UF) 2D NMR, which enables the acquisition of 2D spectra within a single scan, is nowadays used in a large array of applications. However, the use of UF-HSQC experiments is still limited by the need to compromise between spectral widths and resolution. Interleaved acquisitions can overcome this limitation, albeit at the cost of strong artifacts. We show that the occurrence of these artifacts is mainly related to the discontinuous decoupling scheme which is generally used in UF-HSQC. In this paper, four continuous decoupling schemes using adiabatic pulses are optimized for this kind of experiments, giving access to full-range UF-HSQC spectra.
Keywords: ultrafast NMR, adiabatic decoupling, HSQC, interleaving artifacts
2
Introduction Multidimensional NMR spectroscopy is a major tool for the elucidation of chemical or biochemical compounds, as well as for the analysis of complex mixtures. Although homonuclear experiments are characterized by a high sensitivity, such spectra are commonly crowded, thus complicating structural elucidation and/or quantification. 1 H-13C experiments, at the cost of a lower sensitivity, benefit from a larger spectral width leading to a lower spectral density and subsequently to a better peak separation. Inverse-detected experiments such as HSQC suffer from particularly long acquisition durations, due to the need to record numerous t1 increments to sample the indirect dimension. Several strategies have been proposed to overcome this speed limitation [1]. A first family of approaches consists in optimizing pulse angles to minimize the inter-scan delay, such as in the SOFAST or ASAP approaches [2,3]. Other strategies are based on the reduction of the number of time increments by spectral aliasing [4,5] or by a non-uniform sampling of the indirect dimension, in the latter case associated with nonlinear reconstruction methods [6–8]. Alternatives to Fourier transform (FT) have also been developed, such as Hadamard spectroscopy [9,10]. Fifteen years ago, a generic multidimensional approach was designed by Frydman and coworkers, making it possible to acquire 2D NMR spectra within a single scan, thus dramatically reducing the acquisition duration [11,12]. In this “ultrafast” (UF) method, a spatial encoding scheme replaces the conventional incremented evolution period t1. This encoding is based on a combination of bipolar gradients and linearly frequency-swept pulses [13]. The acquisition is performed thanks to an echo-planar spectroscopic imaging scheme [14]. The detailed principles of UF 2D NMR have been described in recent reviews [15–17]. Applications of UF NMR have been reported in a variety of fields such as reaction monitoring, metabolomics, or hyphenated techniques like chromatography or dissolution dynamic nuclear polarization [15,16]. UF-HSQC experiments were introduced from the very 3
beginning of this method [12]. However, their usage is still restricted by spectral width limitations intrinsic to UF experiments (see below). Indeed, although interleaving strategies are used to successfully overcome this limitation in homonuclear experiments [18], their use for heteronuclear UF experiments induces a significant signal deterioration. In this study, after describing the specificities of UF in terms of spectral width and resolution, we show that the spectrum quality losses in interleaved UF-HSQC experiments are significantly attributable to the discontinuous decoupling scheme used during detection. We suggest four continuous decoupling schemes optimized for such experiments, giving access to wide 1H and 13C spectral widths.
Spectral width issues in UF 2D NMR In UF 2D NMR experiments, the resolution is mainly limited in the spatially-encoded dimension (called “spen dimension” in the following, the FT EPSI dimension being called “conventional dimension”). For a given resolution in the spen dimension, the spectral widths in both dimensions are related to each other and limited by the maximal gradient amplitude available [19]: ∙ ∙ = 2 ∙
∙ ∆
Equation 1
In this equation, is the gyromagnetic ratio of the detected nucleus, is the amplitude of the gradients applied during acquisition, is the sample encoded length, and are the spectral widths in the spen and conventional dimensions respectively, and ∆ is the peak width in the spen dimension. For a typical commercial configuration (400 MHz spectrometer with maximum gradient power of 65 G/cm), the achievable spectral widths (13C x 1 H) in single-scan UF-HSQC are only 40 x 4 ppm, with a full-width at mid-height of 50 Hz in the spen dimension [15].
4
Several strategies have been proposed to circumvent this limitation. A first solution consists in classical peak aliasing along the conventional dimension to alleviate the compromise between the spectral widths in the two dimensions of UF spectra [20]. Although this strategy is not applicable in the spen dimension – since no Fourier Transform is performed – several efficient methods have been developed to observe signals lying out of the observed range by tuning the peak positions in the k-space. Indeed, a band-selective refocusing pulse flanked by a bipolar gradient pair can be added prior to the mixing step [20]. We also proposed a gradient-controlled aliasing [21], based on the use of suitably chosen gradients placed on each side of the mixing period – no selective pulse is required. Finally, a spatial/spectral encoding approach has been described by Shrot et al. [19,22], using selective pulses before the spatial encoding so that each peak undergoes a specific spectral encoding. All the previously described approaches give access to more observable peaks within a single scan, but require a priori knowledge of the spectral regions to be folded, moreover the folded peaks do not appear at their real chemical shifts. The latter aspects can be a limiting factor for the general use of UF NMR. If the experiment timescale allows acquiring a few scans, another approach can be used, consisting in interleaving several ultrafast acquisitions spanning different k-space trajectories. This interleaving procedure has been proposed since the early days of ultrafast NMR [12], and is directly inspired from interleaved EPSI [23,24]. Interleaved experiments rely on the repetition of the pulse sequence, while incrementing a pre-acquisition delay ( ) by
∙ ! , "
where # is the acquisition gradient duration (see UF-HSQC
pulse sequences, Fig. 1 a), and $ the number of interleaved scans. The $ acquired scans are then processed in an interleaved manner, reducing the effective dwell time in the conventional dimension by a factor $ . Thus, becomes = 5
" ∙ !
Equation 2
Using this interleaving procedure, can also be increased without reducing : since =
%! ∙&! ∙ ! ∙' ∙
Equation 3
when # is doubled to increase , $ needs to be doubled too in order to keep constant. Finally, this procedure can also be used to reduce the demand on the gradient coil during the acquisition. However, artifacts arise from this interleaving procedure, occurring at a distance
"
for each peak in the conventional dimension, where $ is the number of
interleaved scans [18]. The intensity of those artifacts is usually low compared to the peak they are arising from, although it strongly depends on the spectrometer. Nevertheless, the superposition of these artifacts with peaks of interest strongly degrades the spectrum readability, particularly in the cases of high dynamic ranges where artifacts arising from strong peaks can have a higher intensity than small peaks of interest. In UF-HSQC experiments, the relative intensity of such artifacts is magnified by the use of discontinuous decoupling, usually used in these experiments (pulse sequence shown in Fig. 1a). This limitation is illustrated in the two first columns of Figure 2. UF-HSQC spectra of an ethanol sample were recorded using discontinuous decoupling with different values of # . Even though long acquisition gradients lead to a very short spectral width resulting in aliasing in the conventional dimension, no artifacts are observed when no interleaving is used (first column). In the second column, for each value of # , the level of interleaving ($ ) was set to avoid aliasing and restore a full spectral width in the conventional dimension. In this case, strong artifacts are observed, occurring at
"
.
The relative intensity of the artifacts
increases with the level of interleaving. Projections of the peaks along the conventional dimension show that for 8 interleaved scans and # = 2 ms, the CH3 peak is indistinguishable from its related artifacts. However, this value of # is necessary to achieve a spectral width in 6
the spen dimension up to 250 ppm using near-maximum gradient strengths. No artifacts are observable in non-decoupled spectra acquired with similar acquisition gradient durations and levels of interleaving (see Figure S1 in Supplementary Information), so that the occurrence of such interleaving artifacts can be attributed to the decoupling scheme used. Post-processing methods have been developed to reduce the artifacts arising from interleaving.[18] The first method, based on the symmetrization of the spectrum, is unsuitable to heteronuclear spectra. The second one, consisting in the subtraction of the artifacts, requires careful peak picking and is restricted to spectra with artifacts of low relative intensity and small dynamic ranges. Moreover, this approach is difficult to implement automatically and requires time-consuming and manual analysis to identify the relevant peaks versus artifacts. This post-processing method cannot be applied in the case of the spectrum acquired with 8 interleaved scans and # = 2 ms, as the original CH3 peak is indistinguishable from its artifacts.
Results and discussion Decoupling during the incremented delay A first hypothesis to explain the high relative intensity of interleaving artifacts is the lack of 13
C decoupling during the delay incremented between interleaved scans ( ). Indeed, the
evolution of the system under JC-H couplings during this delay introduces inconsistencies between the different interleaved scans. One solution to alleviate these inconsistencies could be the use of a continuous decoupling during (Fig. 1 b). The GARP4 decoupling scheme was shown to be a large-bandwidth decoupling scheme and is typically used in conventional HSQC experiments [25]. Therefore, this scheme was chosen here, as no gradient-induced frequency dispersion occurs during . As shown in the third column of Figure 2, the addition
7
of this GARP4 decoupling during leads to a decrease of interleaving artifacts, particularly significant in the case of 8 interleaved scans and # = 2 ms. The evaluation of UF-HSQC using this decoupling scheme to record full-range spectra was performed on a sample offering a greater chemical shift range than ethanol, such as concentrated ibuprofen. To record the spectrum in Figure 3a, 32 increments were interleaved, and 2 scans accumulated for phase cycling, leading to an acquisition duration of 320 s. Acquisition gradients were set to 80% of the maximal amplitude available, in order to ease the hardware demand. Such acquisition conditions led to effective spectral widths of 250 ppm (13C) and 15 ppm (1H). Despite of the artifact decrease observed in ethanol spectra, the fullrange spectrum on ibuprofen reveals a disappointing sensitivity: only one signal is above the limit of detection. This poor performance may be attributed to a lack of homogeneity of the discontinuous decoupling used during the acquisition along the entire bandwidth. Continuous decoupling schemes Here, we propose the use of continuous decoupling schemes (as illustrated in Fig. 1c) to circumvent the homogeneity and sensitivity limitations of the above-mentioned decoupling scheme (Fig. 1b) and to record full-range UF-HSQC spectra with no artefacts and a reasonable sensitivity. However, conventional decoupling schemes of HSQC pulse sequences are not suitable since decoupling in the presence of strong magnetic field gradients requires a large decoupling bandwidth. The frequency dispersion induced by the acquisition gradients is: Δ) =
%*∙&! ∙' +
Equation 4
In this equation, - is the gyromagnetic ratio of the decoupled nuclei. For a conventional gradient system such as the one used in this study (up to 105.7 G/cm), the higher frequency dispersion expected is 144 kHz for 13C, corresponding to acquisition gradients limited to 80% of their maximal amplitude in order to limit the risk of hardware damage. This high frequency dispersion dictates the use of a broadband decoupling scheme. The average RF-power of the 8
different decoupling schemes is set to 5 W (./ = 6.45 456), in order to limit sample heating effects and the risk of hardware damage. In order to evaluate the bandwidth of decoupling efficiency for the different decoupling schemes, series of 1D-HSQC were recorded on a methanol sample, while varying the decoupling offset. The methanol molecule was chosen for its single resonance in 1D-HSQC, allowing to follow its intensity depending on the decoupling offset (Fig 4b-f). As mentioned above, the GARP4 decoupling scheme was shown to be a large-bandwidth decoupling scheme and is typically used in conventional HSQC experiments [25]. Nevertheless, even when calibrated with a relatively high RF-power (5 W, ./ = 6.45 456), its bandwidth efficiency is only 44 kHz: still more than 3 times smaller than the higher
13
C
frequency dispersion arising from our gradient system (144 kHz). Larger decoupling efficiency bandwidths can be obtained by the use of adiabatic pulses [26]. We chose to optimize a few of them to design optimized continuous decoupling schemes in UF-HSQC. Independently of the adiabatic pulse used, the phase cycling is M4P5: a nested combination of the MLEV phase cycle [27] and that proposed by Tycko et al. [28]. Indeed, this phase cycle was shown to be optimal to ensure an acceptable homogeneity of the decoupling efficiency through the entire bandwidth [29]. Four different types of adiabatic pulse shapes have been optimized: constant-adiabaticity WURST [30], hyperbolic secant [31], constant-adiabaticity cosine [32] and constantadiabaticity power hyperbolic secant [33]. The maximal amplitude of each kind of decoupling pulse is calculated to guaranty an average RF-power of 5 W (./ = 6.45 456). A preliminary optimization has been done by the use of simulations of the effective bandwidth via NMRsim, for each pulse. The evaluation of the experimental bandwidth of decoupling efficiency is performed by 1D-HSQC as above for the GARP4 decoupling scheme. The series of 1D-HSQC obtained (Fig. 4c-f) shows a similar effective bandwidth for every optimized
9
pulse: 116 kHz, close to the higher frequency dispersion expected for our gradient system (144 kHz).
Application to UF-HSQC experiments The use of one of the optimized continuous decoupling schemes (caWURST) in ultrafast HSQC experiments is compared to the discontinuous decoupling on an ethanol sample, in the same conditions as in Figure 2. The projections of the peaks along the conventional dimension clearly bring out the disappearance of the artifacts due to interleaving when optimized continuous decoupling schemes are used, as shown in Figure 5. This feature significantly improves the spectrum readability, and the sensitivity of UF-HSQC experiments in cases where long acquisition gradients have to be used in order to reach higher spectral widths. Similar results (in Supplementary Information) were obtained with the three other optimized decoupling schemes, highlighting the prevalence of the effective decoupling bandwidth criterion against the decoupling pulse shape used. To demonstrate the ability of UF-HSQC with optimized adiabatic decoupling to access large spectral widths, UF-HSQC spectra have been recorded on an ibuprofen sample (Figure 3b) in the same conditions as Figure 3a, except the decoupling which was replaced by an adiabatic scheme. Contrary to the spectrum recorded with GARP4 decoupling during and discontinuous decoupling during the acquisition, all the peaks are observable in this case. Indeed, projections of the only peak observable in Figure 3a show a clear increase of the signal-to-noise ratio when the optimized adiabatic decoupling is used. Similar results (in Supplementary Information) were obtained with the three other optimized decoupling schemes. In addition, a similar spectrum acquired using a continuous GARP4 decoupling is
10
presented in Figure 3c. Although the peaks of interest are present, the spectrum appears less clean, particularly due to an artifact line at 148 ppm, with an intensity similar to the one of the less intense peak of ibuprofen. This observation is coherent with the reduced decoupling bandwidth observed in Figure 4 a. Considering that the amount of energy deposited with the GARP4 decoupling is the same than with our optimized adiabatic decoupling schemes, the latter should be preferably used.
Conclusion The results described above demonstrate the efficiency of optimized continuous decoupling schemes for UF-HSQC interleaved experiments, thus pushing the boundaries of the latter in terms of spectral widths. Indeed, the use of continuous decoupling schemes makes it possible to acquire a full-range HSQC spectrum in a few minutes. While this approach suffers from the intrinsic sensitivity limitations of UF NMR, it could be used as an alternative to conventional HSQC for many structure elucidation applications where the amount of available material is not limited, or also for reaction monitoring purposes.
Experimental Sample preparation The ethanol and methanol sample was prepared by dissolving neat compounds in D2O (10% v/v). The ibuprofen sample was prepared by weighing 300 mg of ibuprofen and dissolving it in 600 µL of aceton-d6. NMR experiments All the NMR spectra are recorded at 298 K on a Bruker Avance III spectrometer equipped with a cryogenic probe 1H/13C and z-axis gradients, at a 1H frequency of 500.13 MHz. The /
INEPT delay was optimized for each sample, to be 7∙8 11
9:;
, using the average value of <=>? .
Decoupling schemes In the discontinuous decoupling scheme used, illustrated in Fig. 1a, 180°
13
C pulses are
applied between each acquisition gradients. In Figures 2 (third column) and 3a, the
13
C pulses used in the GARP4 decoupling during
were calibrated on a 1D 13C spectrum, with a duration of 40 µs. For all continuous adiabatic decoupling schemes, the phase cycling used is M4P5: @@@A@A where @ = 0° − 150° − 60° − 150° − 0° and @A is the same sequence with all radiofrequency phases reversed. All pulse shapes are calculated in Topspin, and defined by 250 points. The constant adiabaticity WURST pulse is defined by a total sweep-width of 123 kHz, a duration of 1024 µs, an amplitude power index of 2 and ./FG = 12.9 kHz. The hyperbolic secant pulse is defined by a spectral width of 130 Hz (based on a 1 s pulse), a truncation level of 32%, a duration of 1024 µs and ./FG = 9.36 kHz. The constant-adiabaticity cosine pulse is defined by a total sweep-width of 127 kHz, a duration of 1024 µs and ./FG = 12.9 kHz. Finally, the constant-adiabaticity power hyperbolic secant pulse is defined by a total sweepwidth of 123 kHz, a duration of 2000 µs, a β parameter of 1.5, an amplitude power index equal to 1 and ./FG = 8.55 kHz. 1D-HSQC experiments The pulse sequence used to record 1D-HSQC spectra shown in Fig. 3 corresponds to the first increment of the conventional 2D-HSQC pulse sequence [34]. The INEPT delay is set to 1.760 ms. UF experiments For all the ultrafast experiments, the spatial encoding is performed using a constant-time spatial encoding scheme [35] (Fig. 1) with two pairs of 5 ms smoothed chirp pulses on both sides of the central 1 H 180° pulse. The sweep range for this encoding pulses is 75 kHz, and 12
the amplitude of the encoding gradients is adapted to obtain a
13
C frequency dispersion
equivalent to the frequency range of the pulses (42.81 G/cm). The duration of the acquisition gradients is 2018 µs, and their amplitude 84.55 G/cm. The recovery delay is set to 5 s. For the ethanol sample, 8 scans are interleaved, leading to an experiment duration of 170 s. The INEPT delay is set to 1.866 ms. For the ibuprofen sample, 16 scans are interleaved, leading an experiment duration of 320 s. The INEPT delay is set to 1.724 ms. Processing The specific processing for the ultrafast spectra is performed using home-written routines in the Bruker program Topspin. It includes zero-filling in both dimensions, a Gaussian spatial apodization in the spen dimension [36], and a sinusoidal apodization in the conventional dimension.
Bibliography [1] L. Rouger, B. Gouilleux, P. Giraudeau, Fast n-Dimensional Data Acquisition Methods, in: J.C. Lindon, G.E. Tranter, D.W. Koppenaal (Eds.), Encyclopedia of Spectroscopy and Spectrometry (Third Edition), Academic Press, Oxford, 2017: pp. 588–596. doi:10.1016/B978-0-12-409547-2.12107-9. [2] D. Schulze-Sünninghausen, J. Becker, B. Luy, Rapid Heteronuclear Single Quantum Correlation NMR Spectra at Natural Abundance, J. Am. Chem. Soc. 136 (2014) 1242– 1245. doi:10.1021/ja411588d. [3] P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015. [4] D. Jeannerat, Computer optimized spectral aliasing in the indirect dimension of 1H-13C heteronuclear 2D NMR experiments. A new algorithm and examples of applications to small molecules., J. Magn. Reson. 186 (2007) 112–122. [5] M. Foroozandeh, D. Jeannerat, Deciphered Chemical Shifts in Aliased Spectra Recorded with two Slightly Different Narrow Windows or Differential Chemical Shift Evolution, ChemPhysChem. 11 (2010) 2503–2505. doi:10.1002/cphc.201000421. [6] A.S. Stern, K.-B. Li, J.C. Hoch, Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear-prediction extrapolation and maximum-entropy reconstruction, J. Am. Chem. Soc. 124 (2002) 1982–1993. [7] H. Barkhuijsen, R. De Beer, W.M.M.J. Bovée, D. Van Ormondt, Retrieval of frequencies, amplitudes, damping factors, and phases from time-domain signals using a linear least-squares procedure, J. Magn. Reson. 61 (1985) 465–481. 13
[8] J.C. Hoch, Maximum entropy signal processing of two-dimensional NMR data, J. Magn. Reson. 64 (1985) 436–440. [9] E. Kupce, R. Freeman, Frequency-domain Hadamard spectroscopy, J. Magn. Reson. 162 (2003) 158–165. [10] E. Kupce, R. Freeman, Two-dimensional Hadamard spectroscopy, J. Magn. Reson. 162 (2003) 300–310. [11] L. Frydman, T. Scherf, A. Lupulescu, The acquisition of multidimensional NMR spectra within a single scan, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15858–15862. [12] L. Frydman, A. Lupulescu, T. Scherf, Principles and features of single-scan twodimensional NMR spectroscopy, J. Am. Chem. Soc. 125 (2003) 9204–9217. [13] Y. Shrot, L. Frydman, Spatial encoding strategies for ultrafast multidimensional nuclear magnetic resonance, J. Chem. Phys. 128 (2008) 052209. [14] P. Mansfield, Multi-planar image formation using NMR spin echoes, J. Phys. C: Solid State Phys. 10 (1977) 55–58. [15] B. Gouilleux, L. Rouger, P. Giraudeau, Ultrafast Multi-dimensional NMR: Principles and Recent Applications, in: EMagRes, John Wiley & Sons, Ltd, 2007. http://dx.doi.org/10.1002/9780470034590.emrstm1494. [16] P. Giraudeau, L. Frydman, Ultrafast 2D NMR: An Emerging Tool in Analytical Spectroscopy, Annu. Rev. Anal. Chem. 7 (2014) 129–161. [17] A. Tal, L. Frydman, Single-scan multidimensional magnetic resonance, Prog. Nucl. Magn. Reson. Spectrosc. 57 (2010) 241–292. [18] L. Rouger, B. Charrier, M. Pathan, S. Akoka, P. Giraudeau, Processing strategies to obtain clean interleaved ultrafast 2D NMR spectra, J. Magn. Reson. 238 (2014) 87–93. doi:10.1016/j.jmr.2013.11.008. [19] Y. Shrot, L. Frydman, Spatial/spectral encoding of the spin interactions in ultrafast multidimensional NMR, J. Chem. Phys. 131 (2009) 224516. [20] P. Pelupessy, L. Duma, G. Bodenhausen, Improving resolution in single-scan 2D spectroscopy, J. Magn. Reson. 194 (2008) 169–174. [21] P. Giraudeau, S. Akoka, A new gradient-controlled method for improving the spectral width of ultrafast 2D NMR experiments, J. Magn. Reson. 205 (2010) 171–176. [22] Z.D. Pardo, G.L. Olsen, M.E. Fernández-Valle, L. Frydman, R. Martínez-Álvarez, A. Herrera, Monitoring Mechanistic Details in the Synthesis of Pyrimidines via Real-Time, Ultrafast Multidimensional NMR Spectroscopy, J. Am. Chem. Soc. 134 (2012) 2706– 2715. [23] F. Hennel, Image-Based Reduction of Artifacts in Multishot Echo-Planar Imaging, J. Magn. Reson. 134 (1998) 206–213. doi:10.1006/jmre.1998.1502. [24] S.B. Reeder, E. Atalar, A.Z. Faranesh, E.R. McVeigh, Multi-Echo Segmented k-space Imaging: An Optimized Hybrid Sequence for Ultrafast Cardiac Imaging, Magn. Reson. Med. 41 (1999) 375–385. [25] A.J. Shaka, P.B. Barker, R. Freeman, Computer-Optimized Decoupling Scheme for Wideband Applications and Low-Level Operation, J. Magn. Reson. 64 (1985) 547–552. [26] M. Garwood, L. DelaBarre, The Return of the Frequency Sweep: Designing Adiabatic Pulses for Contemporary NMR, J. Magn. Reson. 153 (2001) 155–177. doi:10.1006/jmre.2001.2340. [27] M.H. Levitt, R. Freeman, Composite pulse decoupling, J. Magn. Reson. (1969). 43 (1981) 502–507. doi:10.1016/0022-2364(81)90066-4. [28] R. Tycko, A. Pines, Iterative schemes for broad-band and narrow-band population inversion in NMR, Chem. Phys. Lett. 111 (1984) 462–467. doi:10.1016/00092614(84)85541-4.
14
[29] M.R. Bendall, Broadband and Narrowband Spin Decoupling Using Adiabatic Spin Flips, J. Magn. Reson. A. 112 (1995) 126–129. doi:10.1006/jmra.1995.1021. [30] E. Kupce, R. Freeman, Adiabatic pulses for wideband inversion and broadband decoupling, J. Magn. Reson. A. 115 (1995) 273–276. [31] M.S. Silver, R.I. Joseph, D.I. Hoult, Highly selective p/2 and p pulse generation, J. Magn. Reson. 1984 (1984) 347–351. [32] J.M. Böhlen, G. Bodenhausen, Experimental aspects of chirp NMR spectroscopy, J. Magn. Reson. A. 102 (1992) 293–301. [33] A. Tannús, M. Garwood, Improved Performance of Frequency-Swept Pulses Using Offset-Independent Adiabaticity, J. Magn. Reson. A. 120 (1996) 133–137. doi:10.1006/jmra.1996.0110. [34] N. Merchak, V. Silvestre, L. Rouger, P. Giraudeau, T. Rizk, J. Bejjani, S. Akoka, Precise and rapid isotopomic analysis by 1H–13C 2D NMR: Application to triacylglycerol matrices, Talanta. 156–157 (2016) 239–244. doi:10.1016/j.talanta.2016.05.031. [35] P. Pelupessy, Adiabatic single scan two-dimensional NMR spectroscopy, J. Am. Chem. Soc. 125 (2003) 12345–12350. [36] P. Giraudeau, S. Akoka, Sensitivity and lineshape improvement in ultrafast 2D NMR by optimized apodization in the spatially encoded dimension, Magn. Reson. Chem. 49 (2011) 307–313. doi:10.1002/mrc.2746.
15
Figures OP
I · KL
a 1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
ML - ML
L3
Incrementation of OP
b
I · KL
1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
GARP4
ML - ML
L3
Incrementation of OP
c
I · KL
1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
adiabatic decoupling
ML - ML
L3
Incrementation of Figure 1. UF-HSQC pulse sequence in which decoupling is performed by (a) 180° 13C pulses between each acquisition gradient (discontinuous decoupling scheme), (b) a GARP4 decoupling during the interleaving delay additionally to discontinuous decoupling during acquisition, (c) adiabatic decoupling during both and acquisition. Narrow and wide black rectangles correspond to hard 90° and 180° pulses, respectively. Unnamed gradient shapes correspond to coherence selection gradients. Interleaving is performed by incrementing the delay as indicated.
16
Discontinuous decoupling
60
1.5
1H
(ppm)
2.0
2.4
2.2
13C
40
60
60
80
80 3
4
2 1H
(ppm)
20
(ppm)
80 4
0
(ppm)
50
50
120
120
(ppm)
1
(ppm)
0
100
2
1
-50
100
(ppm)
2 1H
0
1H
3
(ppm)
-50
3
40 60
1
-50
4
20
13C
(ppm)
-20 0
1.8
2.1
(ppm)
0
(ppm)
2.3 1H
1
-20
50
2.5
2 1H
(ppm)
13C
1H
2.2
3
0
40
2.4
60 4
1
-20
20
2.6
2
(ppm)
2.0
3
13C
2.5
4
40
(ppm)
60
30
50
50
50
1H
40
13C
13C
40
13C
20
30
(ppm)
10
20
13C
10
20
(ppm)
10
30
3.0
Discontinuous decoupling + GARP4
(ppm)
Discontinuous decoupling
Interleaved experiments
13C
Non-interleaved experiments
100 120 4
2
3 1H
1
(ppm)
Figure 2. UF-HSQC spectra of an ethanol sample, acquired with increasing acquisition gradient durations. The spectra in the left column were acquired with a discontinuous decoupling, without interleaving (which results in aliasing in the 1H dimension). The spectra in the central column were acquired with the same decoupling, but the level of interleaving was set to avoid aliasing in the conventional dimension. The spectra in the right column were also interleaved, but a GARP4 decoupling was used during additionally to the discontinuous decoupling used during the acquisition. Projections of both signals show strong interleaving artifacts, reduced by the use of GARP4 decoupling during .
17
c
b
100
150
8
6 1H
4
2
(ppm)
0
-2
100 150
200 10
8
6 1H
4
2
(ppm)
0
-2
(ppm)
(ppm)
50
150
200 10
0
13C
100
50
13C
50
0
(ppm)
0
13C
a
200 10
8
6 1H
4
2
0
-2
(ppm)
Figure 3. UF-HSQC spectrum of a concentrated ibubrofen sample acquired with 32 interleaved scans, and 2 scans accumulated, leading an acquisition duration of 5 min 30 s. (a) spectrum recorded using GARP4 decoupling during and discontinuous decoupling during acquisition. (b) spectrum recorded using an optimized caWURST decoupling scheme. (c) spectrum recorded using a continuous GARP4 decoupling.
18
44 kHz
a
-50
0
50
kHz
50
kHz
50
kHz
50
kHz
50
kHz
116 kHz
b
-50
0 116 kHz
c
-50
0
116 kHz
d
-50
0
116 kHz
e
-50
0
Figure 4. Series of 1D-HSQC spectra are recorded while varying the decoupling offset, for several decoupling schemes: GARP4 (a), optimized caWURST (b), optimized HSEC (c), optimized caCOS (d) and optimized caPowHSEC (e). The average RF-power is set to 5 W (./ = 6.45 456) for all decoupling schemes. The bandwidth of decoupling efficiency is indicated on top of each series of 1D-HSQC spectra.
19
10
30
(ppm)
40
13C
20
50 60 4
3
2 1H
1
(ppm)
20 40
13C (ppm)
0
60 80 3
4
2 1H
1
(ppm)
0
(ppm)
50
13C
-50
100 120 4
2
3 1H
1
(ppm)
Figure 5.UF-HSQC spectra of an ethanol sample, acquired with an optimized caWURST decoupling scheme, and increasing acquisition gradient durations and levels of interleaving. No interleaving artifacts are observable on projections of both signals.
20
Graphical abstract
21
S1090-7807(17)30223-9 http://dx.doi.org/10.1016/j.jmr.2017.08.015 YJMRE 6154
To appear in:
Journal of Magnetic Resonance
Received Date: Revised Date: Accepted Date:
18 May 2017 25 August 2017 29 August 2017
Please cite this article as: L. Rouger, S. Akoka, P. Giraudeau, Optimized decoupling schemes in ultrafast HSQC experiments, Journal of Magnetic Resonance (2017), doi: http://dx.doi.org/10.1016/j.jmr.2017.08.015
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 proof before it is published in its final 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.
Optimized decoupling schemes in ultrafast HSQC experiments
Laetitia Rouger,a Serge Akoka,a and Patrick Giraudeau*ab
a
CNRS UMR 6230 CEISAM, Université de Nantes, Nantes, France
e-mail: [email protected] b
Institut Universitaire de France, Paris, France
1
Abstract Ultrafast (UF) 2D NMR, which enables the acquisition of 2D spectra within a single scan, is nowadays used in a large array of applications. However, the use of UF-HSQC experiments is still limited by the need to compromise between spectral widths and resolution. Interleaved acquisitions can overcome this limitation, albeit at the cost of strong artifacts. We show that the occurrence of these artifacts is mainly related to the discontinuous decoupling scheme which is generally used in UF-HSQC. In this paper, four continuous decoupling schemes using adiabatic pulses are optimized for this kind of experiments, giving access to full-range UF-HSQC spectra.
Keywords: ultrafast NMR, adiabatic decoupling, HSQC, interleaving artifacts
2
Introduction Multidimensional NMR spectroscopy is a major tool for the elucidation of chemical or biochemical compounds, as well as for the analysis of complex mixtures. Although homonuclear experiments are characterized by a high sensitivity, such spectra are commonly crowded, thus complicating structural elucidation and/or quantification. 1 H-13C experiments, at the cost of a lower sensitivity, benefit from a larger spectral width leading to a lower spectral density and subsequently to a better peak separation. Inverse-detected experiments such as HSQC suffer from particularly long acquisition durations, due to the need to record numerous t1 increments to sample the indirect dimension. Several strategies have been proposed to overcome this speed limitation [1]. A first family of approaches consists in optimizing pulse angles to minimize the inter-scan delay, such as in the SOFAST or ASAP approaches [2,3]. Other strategies are based on the reduction of the number of time increments by spectral aliasing [4,5] or by a non-uniform sampling of the indirect dimension, in the latter case associated with nonlinear reconstruction methods [6–8]. Alternatives to Fourier transform (FT) have also been developed, such as Hadamard spectroscopy [9,10]. Fifteen years ago, a generic multidimensional approach was designed by Frydman and coworkers, making it possible to acquire 2D NMR spectra within a single scan, thus dramatically reducing the acquisition duration [11,12]. In this “ultrafast” (UF) method, a spatial encoding scheme replaces the conventional incremented evolution period t1. This encoding is based on a combination of bipolar gradients and linearly frequency-swept pulses [13]. The acquisition is performed thanks to an echo-planar spectroscopic imaging scheme [14]. The detailed principles of UF 2D NMR have been described in recent reviews [15–17]. Applications of UF NMR have been reported in a variety of fields such as reaction monitoring, metabolomics, or hyphenated techniques like chromatography or dissolution dynamic nuclear polarization [15,16]. UF-HSQC experiments were introduced from the very 3
beginning of this method [12]. However, their usage is still restricted by spectral width limitations intrinsic to UF experiments (see below). Indeed, although interleaving strategies are used to successfully overcome this limitation in homonuclear experiments [18], their use for heteronuclear UF experiments induces a significant signal deterioration. In this study, after describing the specificities of UF in terms of spectral width and resolution, we show that the spectrum quality losses in interleaved UF-HSQC experiments are significantly attributable to the discontinuous decoupling scheme used during detection. We suggest four continuous decoupling schemes optimized for such experiments, giving access to wide 1H and 13C spectral widths.
Spectral width issues in UF 2D NMR In UF 2D NMR experiments, the resolution is mainly limited in the spatially-encoded dimension (called “spen dimension” in the following, the FT EPSI dimension being called “conventional dimension”). For a given resolution in the spen dimension, the spectral widths in both dimensions are related to each other and limited by the maximal gradient amplitude available [19]: ∙ ∙ = 2 ∙
∙ ∆
Equation 1
In this equation, is the gyromagnetic ratio of the detected nucleus, is the amplitude of the gradients applied during acquisition, is the sample encoded length, and are the spectral widths in the spen and conventional dimensions respectively, and ∆ is the peak width in the spen dimension. For a typical commercial configuration (400 MHz spectrometer with maximum gradient power of 65 G/cm), the achievable spectral widths (13C x 1 H) in single-scan UF-HSQC are only 40 x 4 ppm, with a full-width at mid-height of 50 Hz in the spen dimension [15].
4
Several strategies have been proposed to circumvent this limitation. A first solution consists in classical peak aliasing along the conventional dimension to alleviate the compromise between the spectral widths in the two dimensions of UF spectra [20]. Although this strategy is not applicable in the spen dimension – since no Fourier Transform is performed – several efficient methods have been developed to observe signals lying out of the observed range by tuning the peak positions in the k-space. Indeed, a band-selective refocusing pulse flanked by a bipolar gradient pair can be added prior to the mixing step [20]. We also proposed a gradient-controlled aliasing [21], based on the use of suitably chosen gradients placed on each side of the mixing period – no selective pulse is required. Finally, a spatial/spectral encoding approach has been described by Shrot et al. [19,22], using selective pulses before the spatial encoding so that each peak undergoes a specific spectral encoding. All the previously described approaches give access to more observable peaks within a single scan, but require a priori knowledge of the spectral regions to be folded, moreover the folded peaks do not appear at their real chemical shifts. The latter aspects can be a limiting factor for the general use of UF NMR. If the experiment timescale allows acquiring a few scans, another approach can be used, consisting in interleaving several ultrafast acquisitions spanning different k-space trajectories. This interleaving procedure has been proposed since the early days of ultrafast NMR [12], and is directly inspired from interleaved EPSI [23,24]. Interleaved experiments rely on the repetition of the pulse sequence, while incrementing a pre-acquisition delay ( ) by
∙ ! , "
where # is the acquisition gradient duration (see UF-HSQC
pulse sequences, Fig. 1 a), and $ the number of interleaved scans. The $ acquired scans are then processed in an interleaved manner, reducing the effective dwell time in the conventional dimension by a factor $ . Thus, becomes = 5
" ∙ !
Equation 2
Using this interleaving procedure, can also be increased without reducing : since =
%! ∙&! ∙ ! ∙' ∙
Equation 3
when # is doubled to increase , $ needs to be doubled too in order to keep constant. Finally, this procedure can also be used to reduce the demand on the gradient coil during the acquisition. However, artifacts arise from this interleaving procedure, occurring at a distance
"
for each peak in the conventional dimension, where $ is the number of
interleaved scans [18]. The intensity of those artifacts is usually low compared to the peak they are arising from, although it strongly depends on the spectrometer. Nevertheless, the superposition of these artifacts with peaks of interest strongly degrades the spectrum readability, particularly in the cases of high dynamic ranges where artifacts arising from strong peaks can have a higher intensity than small peaks of interest. In UF-HSQC experiments, the relative intensity of such artifacts is magnified by the use of discontinuous decoupling, usually used in these experiments (pulse sequence shown in Fig. 1a). This limitation is illustrated in the two first columns of Figure 2. UF-HSQC spectra of an ethanol sample were recorded using discontinuous decoupling with different values of # . Even though long acquisition gradients lead to a very short spectral width resulting in aliasing in the conventional dimension, no artifacts are observed when no interleaving is used (first column). In the second column, for each value of # , the level of interleaving ($ ) was set to avoid aliasing and restore a full spectral width in the conventional dimension. In this case, strong artifacts are observed, occurring at
"
.
The relative intensity of the artifacts
increases with the level of interleaving. Projections of the peaks along the conventional dimension show that for 8 interleaved scans and # = 2 ms, the CH3 peak is indistinguishable from its related artifacts. However, this value of # is necessary to achieve a spectral width in 6
the spen dimension up to 250 ppm using near-maximum gradient strengths. No artifacts are observable in non-decoupled spectra acquired with similar acquisition gradient durations and levels of interleaving (see Figure S1 in Supplementary Information), so that the occurrence of such interleaving artifacts can be attributed to the decoupling scheme used. Post-processing methods have been developed to reduce the artifacts arising from interleaving.[18] The first method, based on the symmetrization of the spectrum, is unsuitable to heteronuclear spectra. The second one, consisting in the subtraction of the artifacts, requires careful peak picking and is restricted to spectra with artifacts of low relative intensity and small dynamic ranges. Moreover, this approach is difficult to implement automatically and requires time-consuming and manual analysis to identify the relevant peaks versus artifacts. This post-processing method cannot be applied in the case of the spectrum acquired with 8 interleaved scans and # = 2 ms, as the original CH3 peak is indistinguishable from its artifacts.
Results and discussion Decoupling during the incremented delay A first hypothesis to explain the high relative intensity of interleaving artifacts is the lack of 13
C decoupling during the delay incremented between interleaved scans ( ). Indeed, the
evolution of the system under JC-H couplings during this delay introduces inconsistencies between the different interleaved scans. One solution to alleviate these inconsistencies could be the use of a continuous decoupling during (Fig. 1 b). The GARP4 decoupling scheme was shown to be a large-bandwidth decoupling scheme and is typically used in conventional HSQC experiments [25]. Therefore, this scheme was chosen here, as no gradient-induced frequency dispersion occurs during . As shown in the third column of Figure 2, the addition
7
of this GARP4 decoupling during leads to a decrease of interleaving artifacts, particularly significant in the case of 8 interleaved scans and # = 2 ms. The evaluation of UF-HSQC using this decoupling scheme to record full-range spectra was performed on a sample offering a greater chemical shift range than ethanol, such as concentrated ibuprofen. To record the spectrum in Figure 3a, 32 increments were interleaved, and 2 scans accumulated for phase cycling, leading to an acquisition duration of 320 s. Acquisition gradients were set to 80% of the maximal amplitude available, in order to ease the hardware demand. Such acquisition conditions led to effective spectral widths of 250 ppm (13C) and 15 ppm (1H). Despite of the artifact decrease observed in ethanol spectra, the fullrange spectrum on ibuprofen reveals a disappointing sensitivity: only one signal is above the limit of detection. This poor performance may be attributed to a lack of homogeneity of the discontinuous decoupling used during the acquisition along the entire bandwidth. Continuous decoupling schemes Here, we propose the use of continuous decoupling schemes (as illustrated in Fig. 1c) to circumvent the homogeneity and sensitivity limitations of the above-mentioned decoupling scheme (Fig. 1b) and to record full-range UF-HSQC spectra with no artefacts and a reasonable sensitivity. However, conventional decoupling schemes of HSQC pulse sequences are not suitable since decoupling in the presence of strong magnetic field gradients requires a large decoupling bandwidth. The frequency dispersion induced by the acquisition gradients is: Δ) =
%*∙&! ∙' +
Equation 4
In this equation, - is the gyromagnetic ratio of the decoupled nuclei. For a conventional gradient system such as the one used in this study (up to 105.7 G/cm), the higher frequency dispersion expected is 144 kHz for 13C, corresponding to acquisition gradients limited to 80% of their maximal amplitude in order to limit the risk of hardware damage. This high frequency dispersion dictates the use of a broadband decoupling scheme. The average RF-power of the 8
different decoupling schemes is set to 5 W (./ = 6.45 456), in order to limit sample heating effects and the risk of hardware damage. In order to evaluate the bandwidth of decoupling efficiency for the different decoupling schemes, series of 1D-HSQC were recorded on a methanol sample, while varying the decoupling offset. The methanol molecule was chosen for its single resonance in 1D-HSQC, allowing to follow its intensity depending on the decoupling offset (Fig 4b-f). As mentioned above, the GARP4 decoupling scheme was shown to be a large-bandwidth decoupling scheme and is typically used in conventional HSQC experiments [25]. Nevertheless, even when calibrated with a relatively high RF-power (5 W, ./ = 6.45 456), its bandwidth efficiency is only 44 kHz: still more than 3 times smaller than the higher
13
C
frequency dispersion arising from our gradient system (144 kHz). Larger decoupling efficiency bandwidths can be obtained by the use of adiabatic pulses [26]. We chose to optimize a few of them to design optimized continuous decoupling schemes in UF-HSQC. Independently of the adiabatic pulse used, the phase cycling is M4P5: a nested combination of the MLEV phase cycle [27] and that proposed by Tycko et al. [28]. Indeed, this phase cycle was shown to be optimal to ensure an acceptable homogeneity of the decoupling efficiency through the entire bandwidth [29]. Four different types of adiabatic pulse shapes have been optimized: constant-adiabaticity WURST [30], hyperbolic secant [31], constant-adiabaticity cosine [32] and constantadiabaticity power hyperbolic secant [33]. The maximal amplitude of each kind of decoupling pulse is calculated to guaranty an average RF-power of 5 W (./ = 6.45 456). A preliminary optimization has been done by the use of simulations of the effective bandwidth via NMRsim, for each pulse. The evaluation of the experimental bandwidth of decoupling efficiency is performed by 1D-HSQC as above for the GARP4 decoupling scheme. The series of 1D-HSQC obtained (Fig. 4c-f) shows a similar effective bandwidth for every optimized
9
pulse: 116 kHz, close to the higher frequency dispersion expected for our gradient system (144 kHz).
Application to UF-HSQC experiments The use of one of the optimized continuous decoupling schemes (caWURST) in ultrafast HSQC experiments is compared to the discontinuous decoupling on an ethanol sample, in the same conditions as in Figure 2. The projections of the peaks along the conventional dimension clearly bring out the disappearance of the artifacts due to interleaving when optimized continuous decoupling schemes are used, as shown in Figure 5. This feature significantly improves the spectrum readability, and the sensitivity of UF-HSQC experiments in cases where long acquisition gradients have to be used in order to reach higher spectral widths. Similar results (in Supplementary Information) were obtained with the three other optimized decoupling schemes, highlighting the prevalence of the effective decoupling bandwidth criterion against the decoupling pulse shape used. To demonstrate the ability of UF-HSQC with optimized adiabatic decoupling to access large spectral widths, UF-HSQC spectra have been recorded on an ibuprofen sample (Figure 3b) in the same conditions as Figure 3a, except the decoupling which was replaced by an adiabatic scheme. Contrary to the spectrum recorded with GARP4 decoupling during and discontinuous decoupling during the acquisition, all the peaks are observable in this case. Indeed, projections of the only peak observable in Figure 3a show a clear increase of the signal-to-noise ratio when the optimized adiabatic decoupling is used. Similar results (in Supplementary Information) were obtained with the three other optimized decoupling schemes. In addition, a similar spectrum acquired using a continuous GARP4 decoupling is
10
presented in Figure 3c. Although the peaks of interest are present, the spectrum appears less clean, particularly due to an artifact line at 148 ppm, with an intensity similar to the one of the less intense peak of ibuprofen. This observation is coherent with the reduced decoupling bandwidth observed in Figure 4 a. Considering that the amount of energy deposited with the GARP4 decoupling is the same than with our optimized adiabatic decoupling schemes, the latter should be preferably used.
Conclusion The results described above demonstrate the efficiency of optimized continuous decoupling schemes for UF-HSQC interleaved experiments, thus pushing the boundaries of the latter in terms of spectral widths. Indeed, the use of continuous decoupling schemes makes it possible to acquire a full-range HSQC spectrum in a few minutes. While this approach suffers from the intrinsic sensitivity limitations of UF NMR, it could be used as an alternative to conventional HSQC for many structure elucidation applications where the amount of available material is not limited, or also for reaction monitoring purposes.
Experimental Sample preparation The ethanol and methanol sample was prepared by dissolving neat compounds in D2O (10% v/v). The ibuprofen sample was prepared by weighing 300 mg of ibuprofen and dissolving it in 600 µL of aceton-d6. NMR experiments All the NMR spectra are recorded at 298 K on a Bruker Avance III spectrometer equipped with a cryogenic probe 1H/13C and z-axis gradients, at a 1H frequency of 500.13 MHz. The /
INEPT delay was optimized for each sample, to be 7∙8 11
9:;
, using the average value of <=>? .
Decoupling schemes In the discontinuous decoupling scheme used, illustrated in Fig. 1a, 180°
13
C pulses are
applied between each acquisition gradients. In Figures 2 (third column) and 3a, the
13
C pulses used in the GARP4 decoupling during
were calibrated on a 1D 13C spectrum, with a duration of 40 µs. For all continuous adiabatic decoupling schemes, the phase cycling used is M4P5: @@@A@A where @ = 0° − 150° − 60° − 150° − 0° and @A is the same sequence with all radiofrequency phases reversed. All pulse shapes are calculated in Topspin, and defined by 250 points. The constant adiabaticity WURST pulse is defined by a total sweep-width of 123 kHz, a duration of 1024 µs, an amplitude power index of 2 and ./FG = 12.9 kHz. The hyperbolic secant pulse is defined by a spectral width of 130 Hz (based on a 1 s pulse), a truncation level of 32%, a duration of 1024 µs and ./FG = 9.36 kHz. The constant-adiabaticity cosine pulse is defined by a total sweep-width of 127 kHz, a duration of 1024 µs and ./FG = 12.9 kHz. Finally, the constant-adiabaticity power hyperbolic secant pulse is defined by a total sweepwidth of 123 kHz, a duration of 2000 µs, a β parameter of 1.5, an amplitude power index equal to 1 and ./FG = 8.55 kHz. 1D-HSQC experiments The pulse sequence used to record 1D-HSQC spectra shown in Fig. 3 corresponds to the first increment of the conventional 2D-HSQC pulse sequence [34]. The INEPT delay is set to 1.760 ms. UF experiments For all the ultrafast experiments, the spatial encoding is performed using a constant-time spatial encoding scheme [35] (Fig. 1) with two pairs of 5 ms smoothed chirp pulses on both sides of the central 1 H 180° pulse. The sweep range for this encoding pulses is 75 kHz, and 12
the amplitude of the encoding gradients is adapted to obtain a
13
C frequency dispersion
equivalent to the frequency range of the pulses (42.81 G/cm). The duration of the acquisition gradients is 2018 µs, and their amplitude 84.55 G/cm. The recovery delay is set to 5 s. For the ethanol sample, 8 scans are interleaved, leading to an experiment duration of 170 s. The INEPT delay is set to 1.866 ms. For the ibuprofen sample, 16 scans are interleaved, leading an experiment duration of 320 s. The INEPT delay is set to 1.724 ms. Processing The specific processing for the ultrafast spectra is performed using home-written routines in the Bruker program Topspin. It includes zero-filling in both dimensions, a Gaussian spatial apodization in the spen dimension [36], and a sinusoidal apodization in the conventional dimension.
Bibliography [1] L. Rouger, B. Gouilleux, P. Giraudeau, Fast n-Dimensional Data Acquisition Methods, in: J.C. Lindon, G.E. Tranter, D.W. Koppenaal (Eds.), Encyclopedia of Spectroscopy and Spectrometry (Third Edition), Academic Press, Oxford, 2017: pp. 588–596. doi:10.1016/B978-0-12-409547-2.12107-9. [2] D. Schulze-Sünninghausen, J. Becker, B. Luy, Rapid Heteronuclear Single Quantum Correlation NMR Spectra at Natural Abundance, J. Am. Chem. Soc. 136 (2014) 1242– 1245. doi:10.1021/ja411588d. [3] P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015. [4] D. Jeannerat, Computer optimized spectral aliasing in the indirect dimension of 1H-13C heteronuclear 2D NMR experiments. A new algorithm and examples of applications to small molecules., J. Magn. Reson. 186 (2007) 112–122. [5] M. Foroozandeh, D. Jeannerat, Deciphered Chemical Shifts in Aliased Spectra Recorded with two Slightly Different Narrow Windows or Differential Chemical Shift Evolution, ChemPhysChem. 11 (2010) 2503–2505. doi:10.1002/cphc.201000421. [6] A.S. Stern, K.-B. Li, J.C. Hoch, Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear-prediction extrapolation and maximum-entropy reconstruction, J. Am. Chem. Soc. 124 (2002) 1982–1993. [7] H. Barkhuijsen, R. De Beer, W.M.M.J. Bovée, D. Van Ormondt, Retrieval of frequencies, amplitudes, damping factors, and phases from time-domain signals using a linear least-squares procedure, J. Magn. Reson. 61 (1985) 465–481. 13
[8] J.C. Hoch, Maximum entropy signal processing of two-dimensional NMR data, J. Magn. Reson. 64 (1985) 436–440. [9] E. Kupce, R. Freeman, Frequency-domain Hadamard spectroscopy, J. Magn. Reson. 162 (2003) 158–165. [10] E. Kupce, R. Freeman, Two-dimensional Hadamard spectroscopy, J. Magn. Reson. 162 (2003) 300–310. [11] L. Frydman, T. Scherf, A. Lupulescu, The acquisition of multidimensional NMR spectra within a single scan, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15858–15862. [12] L. Frydman, A. Lupulescu, T. Scherf, Principles and features of single-scan twodimensional NMR spectroscopy, J. Am. Chem. Soc. 125 (2003) 9204–9217. [13] Y. Shrot, L. Frydman, Spatial encoding strategies for ultrafast multidimensional nuclear magnetic resonance, J. Chem. Phys. 128 (2008) 052209. [14] P. Mansfield, Multi-planar image formation using NMR spin echoes, J. Phys. C: Solid State Phys. 10 (1977) 55–58. [15] B. Gouilleux, L. Rouger, P. Giraudeau, Ultrafast Multi-dimensional NMR: Principles and Recent Applications, in: EMagRes, John Wiley & Sons, Ltd, 2007. http://dx.doi.org/10.1002/9780470034590.emrstm1494. [16] P. Giraudeau, L. Frydman, Ultrafast 2D NMR: An Emerging Tool in Analytical Spectroscopy, Annu. Rev. Anal. Chem. 7 (2014) 129–161. [17] A. Tal, L. Frydman, Single-scan multidimensional magnetic resonance, Prog. Nucl. Magn. Reson. Spectrosc. 57 (2010) 241–292. [18] L. Rouger, B. Charrier, M. Pathan, S. Akoka, P. Giraudeau, Processing strategies to obtain clean interleaved ultrafast 2D NMR spectra, J. Magn. Reson. 238 (2014) 87–93. doi:10.1016/j.jmr.2013.11.008. [19] Y. Shrot, L. Frydman, Spatial/spectral encoding of the spin interactions in ultrafast multidimensional NMR, J. Chem. Phys. 131 (2009) 224516. [20] P. Pelupessy, L. Duma, G. Bodenhausen, Improving resolution in single-scan 2D spectroscopy, J. Magn. Reson. 194 (2008) 169–174. [21] P. Giraudeau, S. Akoka, A new gradient-controlled method for improving the spectral width of ultrafast 2D NMR experiments, J. Magn. Reson. 205 (2010) 171–176. [22] Z.D. Pardo, G.L. Olsen, M.E. Fernández-Valle, L. Frydman, R. Martínez-Álvarez, A. Herrera, Monitoring Mechanistic Details in the Synthesis of Pyrimidines via Real-Time, Ultrafast Multidimensional NMR Spectroscopy, J. Am. Chem. Soc. 134 (2012) 2706– 2715. [23] F. Hennel, Image-Based Reduction of Artifacts in Multishot Echo-Planar Imaging, J. Magn. Reson. 134 (1998) 206–213. doi:10.1006/jmre.1998.1502. [24] S.B. Reeder, E. Atalar, A.Z. Faranesh, E.R. McVeigh, Multi-Echo Segmented k-space Imaging: An Optimized Hybrid Sequence for Ultrafast Cardiac Imaging, Magn. Reson. Med. 41 (1999) 375–385. [25] A.J. Shaka, P.B. Barker, R. Freeman, Computer-Optimized Decoupling Scheme for Wideband Applications and Low-Level Operation, J. Magn. Reson. 64 (1985) 547–552. [26] M. Garwood, L. DelaBarre, The Return of the Frequency Sweep: Designing Adiabatic Pulses for Contemporary NMR, J. Magn. Reson. 153 (2001) 155–177. doi:10.1006/jmre.2001.2340. [27] M.H. Levitt, R. Freeman, Composite pulse decoupling, J. Magn. Reson. (1969). 43 (1981) 502–507. doi:10.1016/0022-2364(81)90066-4. [28] R. Tycko, A. Pines, Iterative schemes for broad-band and narrow-band population inversion in NMR, Chem. Phys. Lett. 111 (1984) 462–467. doi:10.1016/00092614(84)85541-4.
14
[29] M.R. Bendall, Broadband and Narrowband Spin Decoupling Using Adiabatic Spin Flips, J. Magn. Reson. A. 112 (1995) 126–129. doi:10.1006/jmra.1995.1021. [30] E. Kupce, R. Freeman, Adiabatic pulses for wideband inversion and broadband decoupling, J. Magn. Reson. A. 115 (1995) 273–276. [31] M.S. Silver, R.I. Joseph, D.I. Hoult, Highly selective p/2 and p pulse generation, J. Magn. Reson. 1984 (1984) 347–351. [32] J.M. Böhlen, G. Bodenhausen, Experimental aspects of chirp NMR spectroscopy, J. Magn. Reson. A. 102 (1992) 293–301. [33] A. Tannús, M. Garwood, Improved Performance of Frequency-Swept Pulses Using Offset-Independent Adiabaticity, J. Magn. Reson. A. 120 (1996) 133–137. doi:10.1006/jmra.1996.0110. [34] N. Merchak, V. Silvestre, L. Rouger, P. Giraudeau, T. Rizk, J. Bejjani, S. Akoka, Precise and rapid isotopomic analysis by 1H–13C 2D NMR: Application to triacylglycerol matrices, Talanta. 156–157 (2016) 239–244. doi:10.1016/j.talanta.2016.05.031. [35] P. Pelupessy, Adiabatic single scan two-dimensional NMR spectroscopy, J. Am. Chem. Soc. 125 (2003) 12345–12350. [36] P. Giraudeau, S. Akoka, Sensitivity and lineshape improvement in ultrafast 2D NMR by optimized apodization in the spatially encoded dimension, Magn. Reson. Chem. 49 (2011) 307–313. doi:10.1002/mrc.2746.
15
Figures OP
I · KL
a 1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
ML - ML
L3
Incrementation of OP
b
I · KL
1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
GARP4
ML - ML
L3
Incrementation of OP
c
I · KL
1H
13C
G
H
H
H
H
MN
- MN
MN
- MN
adiabatic decoupling
ML - ML
L3
Incrementation of Figure 1. UF-HSQC pulse sequence in which decoupling is performed by (a) 180° 13C pulses between each acquisition gradient (discontinuous decoupling scheme), (b) a GARP4 decoupling during the interleaving delay additionally to discontinuous decoupling during acquisition, (c) adiabatic decoupling during both and acquisition. Narrow and wide black rectangles correspond to hard 90° and 180° pulses, respectively. Unnamed gradient shapes correspond to coherence selection gradients. Interleaving is performed by incrementing the delay as indicated.
16
Discontinuous decoupling
60
1.5
1H
(ppm)
2.0
2.4
2.2
13C
40
60
60
80
80 3
4
2 1H
(ppm)
20
(ppm)
80 4
0
(ppm)
50
50
120
120
(ppm)
1
(ppm)
0
100
2
1
-50
100
(ppm)
2 1H
0
1H
3
(ppm)
-50
3
40 60
1
-50
4
20
13C
(ppm)
-20 0
1.8
2.1
(ppm)
0
(ppm)
2.3 1H
1
-20
50
2.5
2 1H
(ppm)
13C
1H
2.2
3
0
40
2.4
60 4
1
-20
20
2.6
2
(ppm)
2.0
3
13C
2.5
4
40
(ppm)
60
30
50
50
50
1H
40
13C
13C
40
13C
20
30
(ppm)
10
20
13C
10
20
(ppm)
10
30
3.0
Discontinuous decoupling + GARP4
(ppm)
Discontinuous decoupling
Interleaved experiments
13C
Non-interleaved experiments
100 120 4
2
3 1H
1
(ppm)
Figure 2. UF-HSQC spectra of an ethanol sample, acquired with increasing acquisition gradient durations. The spectra in the left column were acquired with a discontinuous decoupling, without interleaving (which results in aliasing in the 1H dimension). The spectra in the central column were acquired with the same decoupling, but the level of interleaving was set to avoid aliasing in the conventional dimension. The spectra in the right column were also interleaved, but a GARP4 decoupling was used during additionally to the discontinuous decoupling used during the acquisition. Projections of both signals show strong interleaving artifacts, reduced by the use of GARP4 decoupling during .
17
c
b
100
150
8
6 1H
4
2
(ppm)
0
-2
100 150
200 10
8
6 1H
4
2
(ppm)
0
-2
(ppm)
(ppm)
50
150
200 10
0
13C
100
50
13C
50
0
(ppm)
0
13C
a
200 10
8
6 1H
4
2
0
-2
(ppm)
Figure 3. UF-HSQC spectrum of a concentrated ibubrofen sample acquired with 32 interleaved scans, and 2 scans accumulated, leading an acquisition duration of 5 min 30 s. (a) spectrum recorded using GARP4 decoupling during and discontinuous decoupling during acquisition. (b) spectrum recorded using an optimized caWURST decoupling scheme. (c) spectrum recorded using a continuous GARP4 decoupling.
18
44 kHz
a
-50
0
50
kHz
50
kHz
50
kHz
50
kHz
50
kHz
116 kHz
b
-50
0 116 kHz
c
-50
0
116 kHz
d
-50
0
116 kHz
e
-50
0
Figure 4. Series of 1D-HSQC spectra are recorded while varying the decoupling offset, for several decoupling schemes: GARP4 (a), optimized caWURST (b), optimized HSEC (c), optimized caCOS (d) and optimized caPowHSEC (e). The average RF-power is set to 5 W (./ = 6.45 456) for all decoupling schemes. The bandwidth of decoupling efficiency is indicated on top of each series of 1D-HSQC spectra.
19
10
30
(ppm)
40
13C
20
50 60 4
3
2 1H
1
(ppm)
20 40
13C (ppm)
0
60 80 3
4
2 1H
1
(ppm)
0
(ppm)
50
13C
-50
100 120 4
2
3 1H
1
(ppm)
Figure 5.UF-HSQC spectra of an ethanol sample, acquired with an optimized caWURST decoupling scheme, and increasing acquisition gradient durations and levels of interleaving. No interleaving artifacts are observable on projections of both signals.
20
Graphical abstract
21