DOI: 10.1002/chem.201501182
Communication
& Structure Elucidation
Fully Resolved NMR Correlation Spectroscopy Daisy Pitoux,[a] Bertrand Plainchont,[a] Denis Merlet,[a] Zhaoyu Hu,[b, c] David Bonnaff,[b] Jonathan Farjon,[a] and Nicolas Giraud*[a] dilution of heteronuclei (13C) to which protons are coupled to perform broadband homodecoupling.[3] Although being more sensitive than the ZS method, it can neither deal with protons from CH2 groups nor yield signals for protons that are not bound to the heteroatom under consideration. Third, the PSYCHE method involves implementing low flip angle sweptfrequency pulses in the presence of a weak magnetic field gradient.[2a, 4] This method is robust but requires finding a compromise between the sensitivity and the amount of spectral artifacts of the resulting spectra, which both depend on the flip angle of the swept-frequency pulses. Another major evolution is the implementation of pure-shift sequences in windowed acquisition schemes to perform the real-time acquisition of pureshift spectra.[1b, 2c] This approach has been enriched with real-time selective refocusing sequences derived from the G-SERF experiment,[5] which allow for reintroducing a limited number of couplings in the 1D pure-shift spectrum to probe a selected proton site.[6] However, a limitation of pure-shift methods is their ability to give rise to a (2D) correlation spectrum with optimized resolution in both dimensions, for two key reasons. On the one hand, all COSY-like pulse sequences that require the evolution of scalar couplings during an indirect evolution delay (t1) to correlate protons are incompatible with the implementation of pure-shift evolutions during t1. On the other hand, when a correlation is generated during a mixing period, very few pureshift methods allow for decoupling a given proton site during t1, and its correlated partner during t2, which is a prerequisite for obtaining a fully resolved cross peak. This issue could only be addressed for TOCSY spectra through a covariance processing of the data.[7] Here we present the push-G-SERF sequence (pure shift gradient-encoded selective refocusing spectroscopy; Figure 1) that combines the analytical potential of multi-dimensional correlation spectra with the resolution provided by pure shift and J-resolved spectroscopies. The evolution of the scalar couplings from a selected proton is recorded during t1 while every other spin interaction is refocused. The free induction decay (FID) is reconstructed from a series of chunks of data acquisition between which broadband homodecoupling is performed. Such an approach is fully compatible with the implementation of a z-filter to yield pure absorption, phased 2D spectra, which contributes to optimize the resolution. A push-G-SERF spectrum was recorded at 400 MHz on trans-2-hexen-1-al (1) (Figure 2 a) to probe the coupling network of H2 (Figure 2 b). The resulting correlations show a singlet and a doublet structure along the direct and indirect domains, respectively (Figure 2 c).
Abstract: A new correlation experiment cited as “push-GSERF” is reported. In the resulting phased 2D spectrum, the chemical shift information is selected along the direct dimension, whereas scalar couplings involving a selected proton nucleus are edited in the indirect domain. The robustness of this pulse sequence is demonstrated on compounds with increasing structural and spectral complexity, using state-of-the-art spectrometers. It allows for full resolution of both dimensions of the spectrum, yielding a straightforward assignment and measurement of the coupling network around a given proton in the molecule. This experiment is intended for chemists who want to address efficiently the structural analysis of molecules with an overcrowded spectrum.
The quest for high resolution in nuclear magnetic resonance has been enlightened by the breakthroughs accomplished in the field of “pure shift” NMR, which has allowed us to address the long-standing issue of the broadband homodecoupling of abundant spin nuclei such as protons. Three methods have emerged as the most efficient alternatives to remove scalar couplings from 1H spectra. First, the Zangger and Sterk method (ZS) allows for acting selectively on spin coherences in localized regions of the sample.[1] Despite being less sensitive, it has opened the way to several experiments within which each proton signal has a singlet structure.[1b, 2] Second, the bilinear rotation decoupling method (BIRD) relies on the isotopic [a] D. Pitoux, Dr. B. Plainchont, Prof. D. Merlet, Dr. J. Farjon, Dr. N. Giraud Equipe de RMN en milieu orient Universit Paris-Sud ICMMO, UMR 8182 (CNRS-UPS) 91405 Orsay cedex (France) E-mail: [email protected] Homepage: http://www.icmmo.u-psud.fr [b] Dr. Z. Hu, Prof. D. Bonnaff Equipe Mthodologies Synthses et Molcules Thrapeutiques Universit Paris-Sud ICMMO, UMR 8182 (CNRS-UPS), LabEx LERMIT 91405 Orsay Cedex (France) [c] Dr. Z. Hu Present address: Unit de Chimie des Biomolcules Institut Pasteur, UMR CNRS 3523 75724 Paris cedex (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501182. Chem. Eur. J. 2015, 21, 1 – 5
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Figure 1. The push-G-SERF pulse sequence. On the proton channels (1H), black, grey, and white ellipsoidal shapes correspond, respectively, to p/2 excitation, p/2 flip-back, and p refocusing semi-selective shaped pulses. The white bar corresponds to a p hard pulse. On the pulsed-field gradient channel (Gz), white rectangular bars refer to the application of a z field gradient (“rectangular” gradient), and white ellipsoidal shapes to a shaped z-field gradient. The phase cycle is f1 = (x, x); f2 = (x, x, x, x); f3 = (x, x, x, x); f4 = (x, x, x, x); f5 = (x, x, x, x); f6 = (x, y, x, y); f7 = (x, x); f8 = ( x, x); and frec = ( y, x).
The general structure of this spectrum corresponds to a J-resolved experiment: a suitable projection onto the direct dimension of the multiplets allows for generating a pure-shift spectrum, while the spectral width in the indirect domain is optimized for the measurement of multiplet splittings (Figure 2 d). In addition, it is combined with the simplified coupling edition of a G-SERF spectrum (Figure 2 e). The resulting spectrum leads to a straightforward and accurate assignment of the coupling partners around H2, namely H1 (9.50 ppm, J1– 3 4 2 = 7.9 Hz), H (6.85 ppm, J3–2 = 15.6 Hz), and H (2.32 ppm, J4– 2 = 1.4 Hz), which is a key feature of J-edited spectroscopy. Furthermore, linewidths of about 1 Hz and 5 Hz were measured in the direct domain for the G-SERF and the push-GSERF experiments, respectively. The line broadening in the push-G-SERF spectrum arises from transverse relaxation during the broadband homodecoupling block inserted between FID data chunks, as described elsewhere.[6b, 7] This linewidth is equivalent to those reported for 1D Quick G-SERF and realtime SERF spectra recorded on similar compounds.[6] It corresponds to an accuracy of 0.01 ppm at 400 MHz, which is suitable for an accurate measurement of chemical shifts. Moreover, Figure 3 shows the doublet splittings obtained through the projections calculated for protons H1, H3, and H4 on the G-SERF and push-G-SERF spectra (Figure 3 a, b, and c). These couplings allow for reconstructing the multiplet extracted for H2 from the J-resolved spectrum (Figure 3 d). For both pulse sequences, the efficiency of the gradient-encoded selective refocusing block led to linewidths of 1 Hz for these doublets. Such resolution allows for the measurement of the weak coupling J4–2 = 1.4 Hz (Figure 3 c), which would be out of reach if scalar couplings were edited through a real-time acquisition scheme. It is thus apparent that extracting scalar couplings can be performed with a better resolution along the indirect domain of a J-edited spectrum with optimized spectral width, than through a real-time windowed acquisition, regardless of the number of points that could be acquired in a direct time domain, because resolution is limited in the latter case by transverse relaxation effects and artifacts arising from direct chunked acquisition. &
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Figure 2. a) Molecular structure of trans-2-hexen-1-al 1. b) The 2D push-GSERF spectrum recorded on 1 to probe the coupling network around H2 (dH2 = 6.1 ppm). The standard proton spectrum is shown above the 2D map. Magnified regions of c) the push-G-SERF, d) the J-resolved and e) the G-SERF spectra recorded on 1 (see Supporting Information for experimental details).
As another illustration of its robustness, a push-G-SERF experiment was carried out at 1 GHz on the maltotrioside synthesis intermediate 2 to probe the coupling network of H2C (Figure 4 a). As is often the case with oligosaccharides,[8] 15 out of 2
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Figure 3. Projections of the columns calculated from the G-SERF (top) and push-G-SERF (bottom) spectra recorded on 1, for protons resonating at a) 9.50, b) 6.85, and c) 2.31 ppm. d) The projection calculated for H2 on the Jresolved spectrum.
the 27 protons from the three d-glucosyl units resonate around 3.5 ppm, in a range of only 0.35 ppm, which generally results in crowded 1H correlation spectra (the COSY spectrum recorded on 2 is shown in the Supporting Information). The push-G-SERF spectrum shows resolved signals for H1C (5.04 ppm), OH2C (5.92 ppm), and H3C (3.61 ppm), which are coupled to H2C (Figure 4 b and c). The usefulness of such analytical content is quite obvious here regarding the d-glycosyl unit C : the scalar couplings H2C–H1C (3.7 Hz) and H2C–H3C (9.1 Hz) allow for ascertaining the stereochemistry around H2C, and notably the configuration of the anomeric carbon. The observation of a coupling between H2C and OH2C (J = 6.1 Hz) is also a precious indirect clue of the regioselectivity of the addition of protecting groups on the hydroxyl functions. It should be highlighted that the coupling between H2C and H3C was extracted from a region of the spectrum where five protons resonate within 0.1 ppm (Figure 4 c). Such analysis would be more difficult using 1D real-time SERF or Quick G-SERF spectra, because the doublet would overlap with four other singlets (H5C, H6C, H6’C, and H3B) that, in addition, would be broader because of the direct chunked acquisition. We also emphasize the ability of J-edited spectroscopy to combine highly resolved chemical-shift assignments and coupling measurements within the same spectrum: by comparison, although DQF-COSY[9] or ECOSY[10] experiments in certain cases allow for assigning correlated protons with close chemical shifts, scalar couplings would be extracted from COSY-like spectra with a resolution Chem. Eur. J. 2015, 21, 1 – 5
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Figure 4. a) Molecular structure of 2. b) 2D push-G-SERF spectrum on 2 at 1 GHz to probe the couplings around H2C (dH2C = 3.43 ppm). In c) the standard 1H spectrum is shown above magnified regions of the push-G-SERF spectrum.
that would be poorer than in a J-edited spectrum, because it is limited by the spread of chemical shifts of the molecule. Progress on sensitivity aspects made by modern NMR spectrometers sheds new light on the relevance of multi-dimensional correlation spectroscopy based on the ZS method for the structure elucidation of complex molecules. The lower sensitivity of the ZS method, which arises from the fact that each correlation is generated by a spatially restricted cross section in the sample, has been thoroughly discussed elsewhere.[5a, 6b] 3
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Communication The model compound 1 was highly concentrated so as to confirm that very few artifacts were generated by this new sequence, whereas the 17 millimolar concentration of compound 2 (10 mg in 600 mL of solvent) is typical of those that are routinely prepared in research or industrial laboratories. For this latter sample, the decrease in sensitivity due to the spatial frequency encoding compared to a standard 1H spectrum was estimated at 98 %. This value is coherent with the sensitivity loss that can be predicted for a spectrum of width 3000 Hz encoded by a selective pulse of bandwidth 60 Hz, as described elsewhere.[11] We note that this roughly corresponds to the sensitivity penalty that has to be paid when dilute isotopes such as 13C nuclei are detected. It is interesting to observe that such a spectrum was however obtained in 30 min with a high signal-to-noise ratio, which seems reasonable regarding the analytical content made available. The gain in sensitivity resulting from the narrowing of broad multiplets for protons undergoing several couplings should also be highlighted. Finally, we note that this pulse sequence is compatible with recent methods that were introduced for improving the sensitivity of gradient-encoded experiments,[6b, 12] which makes J-edited spectroscopy suitable for implementation on spectrometers with lower magnetic fields or probes with lower sensitivity. In conclusion, we report a new push-G-SERF pulse sequence that achieves the combination within a single experiment of state-of-the-art resolution enhancement techniques with multidimensional correlation spectroscopy. It provides fully resolved, pure absorption 2D spectra that allow, at the same time, the assignment of proton resonances within a selected coupling network from resolved singlets, and measuring the corresponding scalar couplings with ultrahigh resolution. An improvement to this pulse sequence will of course be a generalized, fully resolved correlation experiment, in the spirit of the PCR-COSY.[13] Analytical strategies involving the systematic acquisition of push-G-SERF spectra to probe every detail from the structure of the molecule are already perfectly possible. This pulse sequence should be of interest for chemists looking for a simple and efficient tool that combines, in a single analysis, chemical-shift and scalar-coupling information, thus accelerating the conformational analysis.
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THC Fr3050 for conducting the research is also acknowledged, as well as the “Fondation Pierre Berg” & “SIDACTION” for the PhD grant of Z.H. The authors thank G. Tcherkez and C. Mauve for providing access to their NMR spectrometer at IBP laboratory, Universit Paris-Sud, Orsay. Keywords: NMR spectroscopy · oligosaccharides · pure shift · selective refocusing · structure elucidation
Acknowledgements
[1] a) K. Zangger, H. Sterk, J. Magn. Reson. 1997, 124, 486 – 489; b) N. H. Meyer, K. Zangger, ChemPhysChem 2014, 15, 49 – 55. [2] a) M. Nilsson, G. A. Morris, Chem. Commun. 2007, 933 – 935; b) N. Giraud, M. Joos, J. Courtieu, D. Merlet, Magn. Reson. Chem. 2009, 47, 300 – 306; c) N. H. Meyer, K. Zangger, Angew. Chem. Int. Ed. 2013, 52, 7143 – 7146; Angew. Chem. 2013, 125, 7283 – 7286; d) S. Glanzer, E. Schrank, K. Zangger, J. Magn. Reson. 2013, 232, 1 – 6; e) N. Helge Meyer, K. Zangger, Chem. Commun. 2014, 50, 1488 – 1490. [3] a) J. A. Aguilar, M. Nilsson, G. A. Morris, Angew. Chem. Int. Ed. 2011, 50, 9716 – 9717; Angew. Chem. 2011, 123, 9890 – 9891; b) A. Lupulescu, G. L. Olsen, L. Frydman, J. Magn. Reson. 2012, 218, 141 – 146; c) L. Paudel, R. W. Adams, P. Kirly, J. A. Aguilar, M. Foroozandeh, M. J. Cliff, M. Nilsson, P. Sndor, J. P. Waltho, G. A. Morris, Angew. Chem. Int. Ed. 2013, 52, 11616 – 11619; Angew. Chem. 2013, 125, 11830 – 11833. [4] a) G. Morris, J. Aguilar, R. Evans, S. Haiber, M. Nilsson, J. Am. Chem. Soc. 2010, 132, 12770 – 12772; b) M. Foroozandeh, R. W. Adams, N. J. Meharry, D. Jeannerat, M. Nilsson, G. A. Morris, Angew. Chem. Int. Ed. 2014, 53, 6990 – 6992; Angew. Chem. 2014, 126, 7110 – 7112. [5] a) N. Giraud, L. Beguin, J. Courtieu, D. Merlet, Angew. Chem. Int. Ed. 2010, 49, 3481 – 3484; Angew. Chem. 2010, 122, 3559 – 3562; b) D. Merlet, L. Beguin, J. Courtieu, N. Giraud, J. Magn. Reson. 2011, 209, 315 – 322. [6] a) N. Gubensk, W. M. F. Fabian, K. Zangger, Chem. Commun. 2014, 50, 12254 – 12257; b) N. Lokesh, S. R. Chaudhari, N. Suryaprakash, Chem. Commun. 2014, 50, 15597 – 15600. [7] M. Foroozandeh, R. W. Adams, M. Nilsson, G. A. Morris, J. Am. Chem. Soc. 2014, 136, 11867 – 11869. [8] a) W. A. Bubb, Conc. Magn. Reson. A 2003, 19, 1 – 19; b) M. D. Battistel, H. F. Azurmendi, B. Yu, D. I. Freedberg, Prog. Nucl. Magn. Reson. Spectrosc. 2014, 79, 48 – 68. [9] M. Rance, O. W. Sørensen, G. Bodenhausen, G. Wagner, R. R. Ernst, K. Wthrich, Biochem. Biophys. Res. Commun. 1983, 117, 479 – 485. [10] a) C. Griesinger, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 1985, 107, 6394 – 6396; b) C. Griesinger, O. W. Sorensen, R. R. Ernst, J. Magn. Reson. (1969 – 1992) 1987, 75, 474 – 492. [11] A. J. Pell, J. Keeler, J. Magn. Reson. 2007, 189, 293 – 299. [12] P. Sakhaii, B. Haase, W. Bermel, R. Kerssebaum, G. E. Wagner, K. Zangger, J. Magn. Reson. 2013, 233, 92 – 95. [13] N. Giraud, D. Pitoux, J. M. Ouvrard, D. Merlet, Chem. Eur. J. 2013, 19, 12221 – 12224.
This work was supported by the French Research Agency (ANR-2011-JS08-009-01). Financial support from the TGE RMN
Received: March 25, 2015 Published online on && &&, 0000
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COMMUNICATION & Structure Elucidation
Serfin’ NMR: We report a new correlation experiment (“push-G-Serf”) with full resolution along both spectral dimensions that allows for an easy and fast structural analysis in complex molecules through a straightforward assignment and measurement of the homonuclear coupling network around a selected proton in the molecule.
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D. Pitoux, B. Plainchont, D. Merlet, Z. Hu, D. Bonnaff, J. Farjon, N. Giraud* && – && Fully Resolved NMR Correlation Spectroscopy
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Fully Resolved NMR Correlation Spectroscopy Daisy Pitoux,[a] Bertrand Plainchont,[a] Denis Merlet,[a] Zhaoyu Hu,[b, c] David Bonnaff,[b] Jonathan Farjon,[a] and Nicolas Giraud*[a] dilution of heteronuclei (13C) to which protons are coupled to perform broadband homodecoupling.[3] Although being more sensitive than the ZS method, it can neither deal with protons from CH2 groups nor yield signals for protons that are not bound to the heteroatom under consideration. Third, the PSYCHE method involves implementing low flip angle sweptfrequency pulses in the presence of a weak magnetic field gradient.[2a, 4] This method is robust but requires finding a compromise between the sensitivity and the amount of spectral artifacts of the resulting spectra, which both depend on the flip angle of the swept-frequency pulses. Another major evolution is the implementation of pure-shift sequences in windowed acquisition schemes to perform the real-time acquisition of pureshift spectra.[1b, 2c] This approach has been enriched with real-time selective refocusing sequences derived from the G-SERF experiment,[5] which allow for reintroducing a limited number of couplings in the 1D pure-shift spectrum to probe a selected proton site.[6] However, a limitation of pure-shift methods is their ability to give rise to a (2D) correlation spectrum with optimized resolution in both dimensions, for two key reasons. On the one hand, all COSY-like pulse sequences that require the evolution of scalar couplings during an indirect evolution delay (t1) to correlate protons are incompatible with the implementation of pure-shift evolutions during t1. On the other hand, when a correlation is generated during a mixing period, very few pureshift methods allow for decoupling a given proton site during t1, and its correlated partner during t2, which is a prerequisite for obtaining a fully resolved cross peak. This issue could only be addressed for TOCSY spectra through a covariance processing of the data.[7] Here we present the push-G-SERF sequence (pure shift gradient-encoded selective refocusing spectroscopy; Figure 1) that combines the analytical potential of multi-dimensional correlation spectra with the resolution provided by pure shift and J-resolved spectroscopies. The evolution of the scalar couplings from a selected proton is recorded during t1 while every other spin interaction is refocused. The free induction decay (FID) is reconstructed from a series of chunks of data acquisition between which broadband homodecoupling is performed. Such an approach is fully compatible with the implementation of a z-filter to yield pure absorption, phased 2D spectra, which contributes to optimize the resolution. A push-G-SERF spectrum was recorded at 400 MHz on trans-2-hexen-1-al (1) (Figure 2 a) to probe the coupling network of H2 (Figure 2 b). The resulting correlations show a singlet and a doublet structure along the direct and indirect domains, respectively (Figure 2 c).
Abstract: A new correlation experiment cited as “push-GSERF” is reported. In the resulting phased 2D spectrum, the chemical shift information is selected along the direct dimension, whereas scalar couplings involving a selected proton nucleus are edited in the indirect domain. The robustness of this pulse sequence is demonstrated on compounds with increasing structural and spectral complexity, using state-of-the-art spectrometers. It allows for full resolution of both dimensions of the spectrum, yielding a straightforward assignment and measurement of the coupling network around a given proton in the molecule. This experiment is intended for chemists who want to address efficiently the structural analysis of molecules with an overcrowded spectrum.
The quest for high resolution in nuclear magnetic resonance has been enlightened by the breakthroughs accomplished in the field of “pure shift” NMR, which has allowed us to address the long-standing issue of the broadband homodecoupling of abundant spin nuclei such as protons. Three methods have emerged as the most efficient alternatives to remove scalar couplings from 1H spectra. First, the Zangger and Sterk method (ZS) allows for acting selectively on spin coherences in localized regions of the sample.[1] Despite being less sensitive, it has opened the way to several experiments within which each proton signal has a singlet structure.[1b, 2] Second, the bilinear rotation decoupling method (BIRD) relies on the isotopic [a] D. Pitoux, Dr. B. Plainchont, Prof. D. Merlet, Dr. J. Farjon, Dr. N. Giraud Equipe de RMN en milieu orient Universit Paris-Sud ICMMO, UMR 8182 (CNRS-UPS) 91405 Orsay cedex (France) E-mail: [email protected] Homepage: http://www.icmmo.u-psud.fr [b] Dr. Z. Hu, Prof. D. Bonnaff Equipe Mthodologies Synthses et Molcules Thrapeutiques Universit Paris-Sud ICMMO, UMR 8182 (CNRS-UPS), LabEx LERMIT 91405 Orsay Cedex (France) [c] Dr. Z. Hu Present address: Unit de Chimie des Biomolcules Institut Pasteur, UMR CNRS 3523 75724 Paris cedex (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501182. Chem. Eur. J. 2015, 21, 1 – 5
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Figure 1. The push-G-SERF pulse sequence. On the proton channels (1H), black, grey, and white ellipsoidal shapes correspond, respectively, to p/2 excitation, p/2 flip-back, and p refocusing semi-selective shaped pulses. The white bar corresponds to a p hard pulse. On the pulsed-field gradient channel (Gz), white rectangular bars refer to the application of a z field gradient (“rectangular” gradient), and white ellipsoidal shapes to a shaped z-field gradient. The phase cycle is f1 = (x, x); f2 = (x, x, x, x); f3 = (x, x, x, x); f4 = (x, x, x, x); f5 = (x, x, x, x); f6 = (x, y, x, y); f7 = (x, x); f8 = ( x, x); and frec = ( y, x).
The general structure of this spectrum corresponds to a J-resolved experiment: a suitable projection onto the direct dimension of the multiplets allows for generating a pure-shift spectrum, while the spectral width in the indirect domain is optimized for the measurement of multiplet splittings (Figure 2 d). In addition, it is combined with the simplified coupling edition of a G-SERF spectrum (Figure 2 e). The resulting spectrum leads to a straightforward and accurate assignment of the coupling partners around H2, namely H1 (9.50 ppm, J1– 3 4 2 = 7.9 Hz), H (6.85 ppm, J3–2 = 15.6 Hz), and H (2.32 ppm, J4– 2 = 1.4 Hz), which is a key feature of J-edited spectroscopy. Furthermore, linewidths of about 1 Hz and 5 Hz were measured in the direct domain for the G-SERF and the push-GSERF experiments, respectively. The line broadening in the push-G-SERF spectrum arises from transverse relaxation during the broadband homodecoupling block inserted between FID data chunks, as described elsewhere.[6b, 7] This linewidth is equivalent to those reported for 1D Quick G-SERF and realtime SERF spectra recorded on similar compounds.[6] It corresponds to an accuracy of 0.01 ppm at 400 MHz, which is suitable for an accurate measurement of chemical shifts. Moreover, Figure 3 shows the doublet splittings obtained through the projections calculated for protons H1, H3, and H4 on the G-SERF and push-G-SERF spectra (Figure 3 a, b, and c). These couplings allow for reconstructing the multiplet extracted for H2 from the J-resolved spectrum (Figure 3 d). For both pulse sequences, the efficiency of the gradient-encoded selective refocusing block led to linewidths of 1 Hz for these doublets. Such resolution allows for the measurement of the weak coupling J4–2 = 1.4 Hz (Figure 3 c), which would be out of reach if scalar couplings were edited through a real-time acquisition scheme. It is thus apparent that extracting scalar couplings can be performed with a better resolution along the indirect domain of a J-edited spectrum with optimized spectral width, than through a real-time windowed acquisition, regardless of the number of points that could be acquired in a direct time domain, because resolution is limited in the latter case by transverse relaxation effects and artifacts arising from direct chunked acquisition. &
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Figure 2. a) Molecular structure of trans-2-hexen-1-al 1. b) The 2D push-GSERF spectrum recorded on 1 to probe the coupling network around H2 (dH2 = 6.1 ppm). The standard proton spectrum is shown above the 2D map. Magnified regions of c) the push-G-SERF, d) the J-resolved and e) the G-SERF spectra recorded on 1 (see Supporting Information for experimental details).
As another illustration of its robustness, a push-G-SERF experiment was carried out at 1 GHz on the maltotrioside synthesis intermediate 2 to probe the coupling network of H2C (Figure 4 a). As is often the case with oligosaccharides,[8] 15 out of 2
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Figure 3. Projections of the columns calculated from the G-SERF (top) and push-G-SERF (bottom) spectra recorded on 1, for protons resonating at a) 9.50, b) 6.85, and c) 2.31 ppm. d) The projection calculated for H2 on the Jresolved spectrum.
the 27 protons from the three d-glucosyl units resonate around 3.5 ppm, in a range of only 0.35 ppm, which generally results in crowded 1H correlation spectra (the COSY spectrum recorded on 2 is shown in the Supporting Information). The push-G-SERF spectrum shows resolved signals for H1C (5.04 ppm), OH2C (5.92 ppm), and H3C (3.61 ppm), which are coupled to H2C (Figure 4 b and c). The usefulness of such analytical content is quite obvious here regarding the d-glycosyl unit C : the scalar couplings H2C–H1C (3.7 Hz) and H2C–H3C (9.1 Hz) allow for ascertaining the stereochemistry around H2C, and notably the configuration of the anomeric carbon. The observation of a coupling between H2C and OH2C (J = 6.1 Hz) is also a precious indirect clue of the regioselectivity of the addition of protecting groups on the hydroxyl functions. It should be highlighted that the coupling between H2C and H3C was extracted from a region of the spectrum where five protons resonate within 0.1 ppm (Figure 4 c). Such analysis would be more difficult using 1D real-time SERF or Quick G-SERF spectra, because the doublet would overlap with four other singlets (H5C, H6C, H6’C, and H3B) that, in addition, would be broader because of the direct chunked acquisition. We also emphasize the ability of J-edited spectroscopy to combine highly resolved chemical-shift assignments and coupling measurements within the same spectrum: by comparison, although DQF-COSY[9] or ECOSY[10] experiments in certain cases allow for assigning correlated protons with close chemical shifts, scalar couplings would be extracted from COSY-like spectra with a resolution Chem. Eur. J. 2015, 21, 1 – 5
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Figure 4. a) Molecular structure of 2. b) 2D push-G-SERF spectrum on 2 at 1 GHz to probe the couplings around H2C (dH2C = 3.43 ppm). In c) the standard 1H spectrum is shown above magnified regions of the push-G-SERF spectrum.
that would be poorer than in a J-edited spectrum, because it is limited by the spread of chemical shifts of the molecule. Progress on sensitivity aspects made by modern NMR spectrometers sheds new light on the relevance of multi-dimensional correlation spectroscopy based on the ZS method for the structure elucidation of complex molecules. The lower sensitivity of the ZS method, which arises from the fact that each correlation is generated by a spatially restricted cross section in the sample, has been thoroughly discussed elsewhere.[5a, 6b] 3
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Communication The model compound 1 was highly concentrated so as to confirm that very few artifacts were generated by this new sequence, whereas the 17 millimolar concentration of compound 2 (10 mg in 600 mL of solvent) is typical of those that are routinely prepared in research or industrial laboratories. For this latter sample, the decrease in sensitivity due to the spatial frequency encoding compared to a standard 1H spectrum was estimated at 98 %. This value is coherent with the sensitivity loss that can be predicted for a spectrum of width 3000 Hz encoded by a selective pulse of bandwidth 60 Hz, as described elsewhere.[11] We note that this roughly corresponds to the sensitivity penalty that has to be paid when dilute isotopes such as 13C nuclei are detected. It is interesting to observe that such a spectrum was however obtained in 30 min with a high signal-to-noise ratio, which seems reasonable regarding the analytical content made available. The gain in sensitivity resulting from the narrowing of broad multiplets for protons undergoing several couplings should also be highlighted. Finally, we note that this pulse sequence is compatible with recent methods that were introduced for improving the sensitivity of gradient-encoded experiments,[6b, 12] which makes J-edited spectroscopy suitable for implementation on spectrometers with lower magnetic fields or probes with lower sensitivity. In conclusion, we report a new push-G-SERF pulse sequence that achieves the combination within a single experiment of state-of-the-art resolution enhancement techniques with multidimensional correlation spectroscopy. It provides fully resolved, pure absorption 2D spectra that allow, at the same time, the assignment of proton resonances within a selected coupling network from resolved singlets, and measuring the corresponding scalar couplings with ultrahigh resolution. An improvement to this pulse sequence will of course be a generalized, fully resolved correlation experiment, in the spirit of the PCR-COSY.[13] Analytical strategies involving the systematic acquisition of push-G-SERF spectra to probe every detail from the structure of the molecule are already perfectly possible. This pulse sequence should be of interest for chemists looking for a simple and efficient tool that combines, in a single analysis, chemical-shift and scalar-coupling information, thus accelerating the conformational analysis.
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THC Fr3050 for conducting the research is also acknowledged, as well as the “Fondation Pierre Berg” & “SIDACTION” for the PhD grant of Z.H. The authors thank G. Tcherkez and C. Mauve for providing access to their NMR spectrometer at IBP laboratory, Universit Paris-Sud, Orsay. Keywords: NMR spectroscopy · oligosaccharides · pure shift · selective refocusing · structure elucidation
Acknowledgements
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This work was supported by the French Research Agency (ANR-2011-JS08-009-01). Financial support from the TGE RMN
Received: March 25, 2015 Published online on && &&, 0000
Chem. Eur. J. 2015, 21, 1 – 5
www.chemeurj.org
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2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Communication
COMMUNICATION & Structure Elucidation
Serfin’ NMR: We report a new correlation experiment (“push-G-Serf”) with full resolution along both spectral dimensions that allows for an easy and fast structural analysis in complex molecules through a straightforward assignment and measurement of the homonuclear coupling network around a selected proton in the molecule.
Chem. Eur. J. 2015, 21, 1 – 5
www.chemeurj.org
These are not the final page numbers! ÞÞ
D. Pitoux, B. Plainchont, D. Merlet, Z. Hu, D. Bonnaff, J. Farjon, N. Giraud* && – && Fully Resolved NMR Correlation Spectroscopy
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2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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