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Probing amide bond nitrogens in solids using 14N NMR spectroscopy
ARTICLE IN PRESS Solid State Nuclear Magnetic Resonance 33 (2008) 82– 87
Contents lists available at ScienceDirect
Solid State Nuclear Magnetic Resonance journal homepage: www.elsevier.com/locate/ssnmr
Probing amide bond nitrogens in solids using
14
N NMR spectroscopy
Sasa Antonijevic a,b,, Nicholas Halpern-Manners a a b
Department of Chemistry, University of California, Berkeley, 1 Cyclotron Road Building 11-D64, Berkeley, CA 94720, USA Materials Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road Building 11-D64, Berkeley, CA 94720, USA
a r t i c l e in f o
a b s t r a c t
Article history: Received 16 April 2008 Available online 20 April 2008
A novel two-dimensional nuclear magnetic resonance (NMR) experiment is proposed for indirect observation of 14N nuclei in various types of nitrogen-containing solids. In a method somewhat similar to the heteronuclear single-quantum correlation (HSQC) experiment widely used for protein structure determination in solutions, this technique correlates spin S ¼ 1/2 nuclei, e.g., 1H, 13C, with the 14N spin I ¼ 1 nucleus in solids. The present experiment, however, transfers coherence from neighboring 1H or 13 C nuclei to 14N via a combination of J-couplings and residual dipolar splittings (RDS). Projections of the two-dimensional NMR spectra onto the 14N dimension yield powder patterns that reflect the 14N quadrupolar interaction, which can be used to study molecular structure and dynamics. Indirect detection of amide nitrogen-14 via 1H and 13C is shown experimentally on a model compound of Nacetyl-glycine. & 2008 Elsevier Inc. All rights reserved.
Keywords: NMR spectroscopy Solid state Nitrogen-14 Peptides Proteins
Despite its presence in a wide range of materials and a natural abundance of 99.64%, nitrogen-14 remains relatively unexplored by nuclear magnetic resonance (NMR) techniques. The main reason for this is the large quadrupolar coupling which results in very broad 14N resonances in both liquids and solids. Broadening in liquids arises from very rapid quadrupolar relaxation that cannot be suppressed, while in solids the large anisotropic broadening can be reduced. There have been a number of techniques that utilize both direct and indirect detection to study 14 N nuclei in solids which include nuclear quadrupole resonance (NQR), overtone and NMR spectroscopy applied to study single crystals or powders combined with magic angle spinning (MAS), and dynamic angle spinning or double sample rotation [1–13]. The quadrupolar tensor parameters, which reflect the local electronic charge distribution, can be used to determine structural parameters of the studied material [7,14]. Indirect detection of 14N (spin I ¼ 1) by NMR spectroscopy of solids via spin S ¼ 1/2 nuclei has been demonstrated recently [15–24]. The experiment known as nitrogen excitation via residual dipolar splittings (NERDS) is analogous to the heteronuclear multiple-quantum correlation (HMQC) [25] experiment widely used to study molecules in solution. One of the principal differences is that the coherence transfer between the two different nuclei is conducted not only by J-couplings, JIS, but also by second-order quadrupole-dipole cross Corresponding author at: Department of Chemistry, University of California, Berkeley, 1 Cyclotron Road Building 11-D64, Berkeley, California 94720, USA. Fax: +1 510 486 5744. E-mail address: [email protected] (S. Antonijevic).
0926-2040/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ssnmr.2008.04.006
terms, also known as residual dipolar splittings (RDS), DRDS [26–31]. These cross-terms arise because the axis of the quantization of 14N nuclei is subject to the large quadrupolar interaction and is thus tilted from the direction of the static magnetic field, thereby preventing complete averaging of the dipolar interactions between 14N nuclei and neighboring S spins under MAS. As a result, the S resonances are split into 1:2 doublets where each component features a powder pattern, and the separation between the centers of mass of these two powder patterns is referred to here as the residual dipolar splitting, DRDS. The residual dipolar splitting is a purely through-space interaction with strength that depends upon the quadrupolar coupling constant (CQ), internuclear distance (rSI), relative orientation of the internuclear vector, the largest component of the electric field gradient tensor, and the static magnetic field strength, B0. In this work we present an experiment that provides a counterpart to the heteronuclear single-quantum correlation (HSQC) [32] experiment (where single-quantum (SQ) refers to the evolution of SQ coherences of one type of nucleus during the t1 interval). In our experiment it is only the 14N coherences which evolve, but one can choose to observe either the SQ or double-quantum (DQ) 14N coherences. The acronym sNERDS seems appropriate for coherences of a single (s) type of nucleus (14N) during t1 interval; likewise the acronym mNERDS seems appropriate for coherences of multiple (m) type nuclei. One application of this experiment probes amide bond nitrogen atoms in powdered solids, which is demonstrated on the model compound of N-acetyl-glycine (NAG). This work differs from and extends the work of Cavadini et al. [23] in the following ways: in this paper, the main focus was the
ARTICLE IN PRESS S. Antonijevic, N. Halpern-Manners / Solid State Nuclear Magnetic Resonance 33 (2008) 82–87
13C
(π/2)φ1 1H
sNERDS
Heteronuclear Decoupling
(CP)φ2 (π/2)φ4
13C
83
(π/2)φ7 τrec
(CP)φ3 τexc (τp)φ5
14N
t2
(τp)φ6 t1
(π/2)φ1 (π/2)φ2 1H
14N
1H
sNERDS
(π/2)φ5
τexc
τrec (τp)φ3
t2
(τp)φ4 t1
pC pH pN +1 +2 +1 +1 +1 0 +1 –1 +1 –2 0 +2 0 +1 0 0 0 –1 0 –2
14
N DQ
14
N SQ
–1 +2 –1 +1 –1 0 –1 –1 –1 –2 Fig. 1. Pulse sequences (A and B) and coherence transfer pathway diagrams (C) for the indirect detection of 14N single- (solid lines) and double-quantum (dashed lines) spectra by coherence transfer from (A) 13C or (B) 1H to 14N and back. Two separate experiments, utilizing the appropriate phase cycling, as indicated above with solid and dashed lines, can be used to observe 14N SQ and DQ spectra. Alternatively these spectra can be obtained in a single experiment using multiplex phase cycling, provided one settles on a compromise for the pulse width tp.
demonstration of a novel technique to study peptide bond nitrogen-14 nuclei, which are subjected to quadupolar coupling constants nearly three times larger than nitrogen-14 nuclei found in aminoacids studied by Cavadini et al. [23]. Peptide bond nitrogens have direct relevance to the major applications of these new techniques for studying biological solids. In addition, we propose to irradiate spy (spin S ¼ 1/2) nuclei in a timeresolved fashion (in comparison to nitrogen-14 nuclei); by doing so, it is possible to extend further our experiment to include sophisticated steps such as the introduction of pulsed fields gradients in order to improve performance. The final difference is in the approach taken to quantify the efficiency of the experiment. This work was originally submitted as a manuscript to another journal and a preprint was provided to Prof Geoffrey Bodenhausen who kindly acknowledges this in Cavadini et al. [23]. Pulse sequences and coherence transfer pathway diagrams for the indirect observation of 14N (spin I ¼ 1) via 13C or 1H (spin S ¼ 1/2) under MAS are shown in Fig. 1, while the phase cycles that can be used to obtain desired coherence selections are given in Table 1. Excitation of transverse magnetization of S spins, represented by a tensor operator T S1;1 [33], is achieved by means
of cross-polarization from 1H to 13C (when 13C is used as the ‘‘spy’’ nucleus) or by single radiofrequency pulse irradiation of 1H nuclei. After a delay texcE1/(2DRDS), the system reaches a state that can be loosely described by an antiphase operator T S1;1 T N 2;0. Since the coupling DRDS(14N-S) is anisotropic, and since the S nuclei magnetization often decays rapidly, the delay texc must be optimized empirically. A radiofrequency pulse with flip angle p/2 applied to the S spins followed by a pulse of duration tp applied near the center of the 14N spectrum leads to a partial S N S N 14 conversion into T S1;0 T N N 1;1 , T 1;0 T 2;1 and T 1;0 T 2;2 which contain N N N SQ (T 1;1 and T 2;1 ) and DQ (T 2;2 ) coherences only (unlike ordinary HSQC experiments with spin 1/2 nuclei where only SQ coherences are involved). These are allowed to evolve freely during an evolution interval t1 (where the increments Dt1 ¼ 1/nrot are synchronized with the spinning period), prior to reconversion into observable S coherence. A two-dimensional Fourier transformation yields a correlation spectrum with either 14N SQ or DQ signals in the o1 dimension and the conventional S nuclei spectrum in the o2 dimension. The SQ and DQ signal intensities can be optimized by choosing the intervals texc ¼ trec and the pulse width tp. These signals can be separated by phase cycling. It is also possible to obtain both SQ and DQ spectra in a single
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Table 1 Phase cycles suitable for use in the pulse sequences in Fig. 1 for the indirect observation of
14
N via (* indicates number of repetitions)
13
C nuclei selecting f1 f2 f3 f4 f5 f6 f7 Receiver
14
N SQ coherences during t1 interval ( C SQ sNERDS) (901,2701)*16 (01)*32 ((01)*2,(901)*2,(1801)*2,(2701)*2)*4 (01)*16,(1801)*16 ((01)*8,(1801)*8)*2 (01)*32 (01)*32 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701
13
14
N DQ coherences during t1 interval (13C DQ sNERDS) (901,2701)*32 (01)*64 ((01)*2,(901)*2,(1801)*2,(2701)*2)*8 (01)*32 (1801)*32 ((01)*8,(901)*8,(1801)*8,(901)*8)*2 (01)*64 (01)*64 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701
C nuclei selecting f1 f2 f3 f4 f5 f6 f7 Receiver
1 H nuclei selecting f1 f2 f3 f4 f5 Receiver
14
1
14
H nuclei selecting f1 f2 f3 f4 f5 Receiver
13
N SQ coherences during t1 interval (1H SQ sNERDS) (01,1801)*4 ((01)*2,(1801)*2)*2 (01)*4,(1801)*4 (01)*8 (01)*8 01,1801,1801,01,1801,01,01,1801 N DQ coherences during t1 interval (1H DQ sNERDS) (01,1801)*8 ((01)*2,(1801)*2)*4 (01)*4,(901)*4,(1801)*4,(2701)*4 (01)*16 (01)*16 01,1801,1801,01,1801,01,01,1801,01,1801,1801,01,1801,01,01,1801
experiment using multiplex phase cycling [34–36], provided one settles on a compromise for the pulse width tp. By selecting +1 S coherences (T S1;1 T N 2;0 ) in texc, which are then converted to 1 coherences that evolve in trec, there will be no net evolution under the chemical shifts (O), since exp(iOtexc)exp (iOtrec) ¼ 1 if texc ¼ trec. Thus, the chemical shift of the S spins and the inhomogeneous contributions to the S spin decay during texc and trec intervals can be eliminated. Despite these benefits, there is a loss of half of the signal in comparison to the analog HSQC experiment used for studying molecules in solutions. The second half of the signal can be recovered by selecting both T S1;1 T N 2;0 coherences during the texc interval, which will result in the observed signal being modulated by chemical shifts with a factor of cos(Otexc)exp(iOtrec). As a consequence of this modulation, resonances will be scaled in amplitude and will have different phases. Additionally, if both T S1;1 T N 2;0 are also selected during the trec interval, and the z-filter is implemented after the trec interval to observe the signal, then resonances will be scaled only in amplitude with a factor cos(Otexc)cos(Otrec) but would retain the same phase regardless of their chemical shifts. The conventional HSQC experiment uses non-selective p pulses simultaneously applied on both nuclei in the middle of the texc and trec periods to retain the signal intensity and avoid chemical shift phase modulation. However, due to the large 14N quadrupolar interaction, there is no instrumental possibility to apply radiofrequency pulses that would result in non-selective rotations of 14N magnetization.
Fig. 2 shows (A) 13C MAS NMR spectra and two-dimensional C–14N correlation (B) sNERDS and (C) mNERDS spectra of NAG. Projections onto the o1 dimension reveal a 14N powder pattern predominantly broadened by the second-order quadrupolar interaction. Both C0 and Ca projections reflect the same nitrogen while remaining different in appearance. One of the reasons for this lies in the unequal excitation of the powder pattern due to the difference in internuclear distances r C0 N ar Ca N , and the different relative orientations of the quadrupolar and dipolar tensors. Fig. 3 shows an analogous spectrum where 1H atoms are utilized as spy nuclei. It is vitally important to obtain narrow 1H resonances, which is accomplished through selective deuteration of the NAG molecule combined with rapid MAS. The experimental NERDS spectra offer information on the quadrupolar interaction of 14N nuclei, which can be related to the structure of a molecule [7,14]. The extraction of quadrupolar parameters from the experimental lineshapes can be a challenging process due to non-uniform excitation that leads to distortion of observed powder patterns as discussed previously [20]. The samples were packed in 4 mm Si3N4 rotors and spun at 15 kHz in a Doty triple resonance MAS probe at 16.4 T (1H, 13C and 14 N Larmor frequencies of 700.1, 176.1 and 50.6 MHz) and spectra were recorded using a Bruker Avance 700 spectrometer. CrossC polarization (CP) was used with nH RF ¼ 65 kHz, while nRF was ramped. Two pulse phase-modulation (TPPM) proton decoupling was used with nH RF ¼ 65 kHz, pulse widths of 4.5 ms, and a phase difference between two successive pulses of 301. With a 1000 W 13
ARTICLE IN PRESS S. Antonijevic, N. Halpern-Manners / Solid State Nuclear Magnetic Resonance 33 (2008) 82–87
13
176
13
C’
174
44
85
Cα
42
δ (ppm) 13
C’
13 α
C
250 300
δ1 (ppm)
350 400 450 500
250 300
δ1 (ppm)
350 400 450 500
176
44
174
42
δ2 (ppm) Fig. 2. (A) 13C MAS NMR and two-dimensional correlation 13C SQ NERDS NMR spectra of polycrystalline N-acetyl-glycine, 13C enriched in both Ca and acetyl carboxyl carbon C0 , recorded using a pulse sequence that selects (B) pure SQ 14N coherences (sNERDS) and (C) 13C and SQ 14N coherences (mNERDS) during the t1 evolution interval. The projections onto the o1 axis reveal quadrupolar powder patterns for the same 14N indirectly observed by the two different carbons. Spectra are the result of averaging 128 transients for each of 64 t1 increments with Dt1 ¼ 1/nrot ¼ 66.6 ms, with a relaxation interval of 10 s. The excitation and reconversion intervals texc ¼ trec were 1.2 ms, while the lengths of the two 14N pulses were tp ¼ 50 ms.
amplifier, the 14N pulses had a modest amplitude of nN RF ¼ 25 kHz, calibrated by direct detection of NH4Cl. The chemical shifts of 1H, 13 C and 14N are referenced relative to external standards of TMS and NH3 (liquid, 251). Typically 150 mg of material is used to obtain spectra shown in this work. In conclusion, we have demonstrated a NMR experiment for indirect detection of nitrogen-14 in solids, which is analogous to the HSQC experiment widely used for protein structure determination in solutions. The advantage of this sNERDS experiment over the mNERDS experiment introduced recently lies in the fact
that the indirect dimension is free from any broadening introduced from decoherence processes of the spy nuclei, as only 14N coherences are allowed to evolve during the t1 interval. This may be especially important when protons are used as spy nuclei due to their large refocused line widths. More generally, 14 N spectra offer a greater wealth of information in comparison to 15N spectra due to quadrupolar broadening that reflects the local charge distribution. It has been shown that these studies could provide more insight into the geometry of peptide bonds [7,14].
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16
14
12
10 8 δ (ppm)
6
4
2
16
14
12
10 8 δ2 (ppm)
6
4
2
250
300
δ1 (ppm)
350
400
450
500
250
300
δ1 (ppm)
350
400
450
500
Fig. 3. (A) 1H MAS NMR and two-dimensional correlation 1H SQ NERDS NMR spectra of polycrystalline N-acetyl-glycine (selectively deuterated so that the amide and carboxyl groups remain protonated), recorded using a pulse sequence that selects (B) pure SQ 14N coherences (sNERDS) and (C) 1H and SQ 14N coherences (mNERDS) during the t1 evolution interval. Spectra are the result of averaging 64 transients for each of 64 t1 increments with Dt1 ¼ 1/nrot ¼ 67.2 ms, with a relaxation interval of 10 s. The excitation and reconversion intervals texc ¼ trec were 0.8 ms, while the lengths of the two 14N pulses were tp ¼ 50 ms.
Acknowledgments This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division, of the US Department of Energy under Contract no. DE-AC03-76SF00098. S.A. acknowledges support from the
Lawrence Berkeley National Laboratory through a postdoctoral fellowship. N.H.-M. acknowledges support from the University of California Berkeley through a graduate fellowship. Chemicals used in this work were kindly donated by Dr Robert Lysek. This document was prepared as an account of work sponsored by the United States Government. While this document
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is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California. References [1] R. Blinc, et al., Pulsed nitrogen-proton double resonance study of ferroelectric transition in triglycine sulfate, J. Chem. Phys. 55 (1971) 4843. [2] M.J. Hunt, A.L. Mackay, Deuterium and nitrogen pure quadrupole-resonance in amino-acids.2, J. Magn. Reson. 22 (1976) 295–301. [3] R.E. Stark, R.A. Haberkorn, R.G. Griffin, N-14 NMR determination of Nh bond lengths in solids, J. Chem. Phys. 68 (1978) 1996–1997. [4] S.J. Opella, M.H. Frey, T.A. Cross, Detection of individual carbon resonances in solid proteins, J. Am. Chem. Soc. 101 (1979) 5856–5857. [5] P. Brunner, M. Reinhold, R.R. Ernst, Double quantum cross polarization—heteronuclear excitation and detection of Nmr double quantum transitions in solids, J. Chem. Phys. 73 (1980) 1086–1094. [6] R. Tycko, S.J. Opella, High-resolution N-14 overtone spectroscopy—an approach to natural abundance nitrogen Nmr of oriented and polycrystalline systems, J. Am. Chem. Soc. 108 (1986) 3531–3532. [7] R. Tycko, P.L. Stewart, S.J. Opella, Peptide plane orientations determined by fundamental and overtone N-14 Nmr, J. Am. Chem. Soc. 108 (1986) 5419–5425. [8] R. Tycko, S.J. Opella, Overtone Nmr-spectroscopy, J. Chem. Phys. 86 (1987) 1761–1774. [9] K.V. Ramanathan, S.J. Opella, Overtone decoupling—the effects of N-14 overtone irradiation on C-13 Nmr-spectra of a single-crystal, J. Magn. Reson. 78 (1988) 367–370. [10] C.P. Grey, W.S. Veeman, The detection of weak heteronuclear coupling between spin-1 and spin-1/2 nuclei in Mas Nmr-N-14/C-13/H-1 triple resonance experiments, Chem. Phys. Lett. 192 (1992) 379–385. [11] C.P. Grey, A.P.A.M. Eijkelenboom, W.S. Veeman, N-14 population transfers in 2-dimensional C-13-N-14-H-1 triple-resonance magic-angle-spinning nuclear-magnetic-resonance spectroscopy, Solid State Nucl. Magn. Reson. 4 (1995) 113–120. [12] S.S. Wi, L. Frydman, Heteronuclear recoupling in solid-state magic-anglespinning NMR via overtone irradiation, J. Am. Chem. Soc. 123 (2001) 10354–10361. [13] G. Jeschke, M. Jansen, High-resolution N-14 solid-state NMR spectroscopy, Angew. Chem. Int. Ed. 37 (1998) 1282–1283.
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[14] C.E. Hughes, R. Pratima, T. Karlsson, M.H. Levitt, Double-quantum solid-state NMR of C-13 spin pairs coupled to N-14, J. Magn. Reson. 159 (2002) 25–35. [15] A. Lupulescu, S. Antonijevic, G. Bodenhausen, Nitrogen-14 NMR using residual dipolar splitting in solids. In: Fourth European Conference on Solid-State NMR, Chamonix, France, September 2005. [16] Z.H. Gan, Measuring amide nitrogen quadrupolar coupling by high-resolution N-14/C-13 NMR correlation under magic-angle spinning, J. Am. Chem. Soc. 128 (2006) 6040–6041. [17] S. Cavadini, A. Lupulescu, S. Antonijevic, G. Bodenhausen, Nitrogen-14 NMR spectroscopy using residual dipolar splittings in solids, J. Am. Chem. Soc. 128 (2006) 7706–7707. [18] S. Cavadini, S. Antonijevic, A. Lupulescu, G. Bodenhausen, Indirect detection of nitrogen-14 in solids via protons by nuclear magnetic resonance spectroscopy, J. Magn. Reson. 182 (2006) 168–172. [19] S. Cavadini, A. Abraham, G. Bodenhausen, Proton-detected nitrogen-14 NMR by recoupling of heteronuclear dipolar interactions using symmetry-based sequences, Chem. Phys. Lett. 445 (2007) 1–5. [20] S. Cavadini, S. Antonijevic, A. Lupulescu, G. Bodenhausen, Indirect detection of nitrogen-14 in solid-state NMR spectroscopy, Chem. Phys. Chem. 8 (2007) 1363–1374. [21] Z.H. Gan, C-13/N-14 heteronuclear multiple-quantum correlation with rotary resonance and REDOR dipolar recoupling, J. Magn. Reson. 184 (2007) 39–43. [22] Z.H. Gan, J.P. Amoureux, J. Trebosc, Proton-detected N-14 MAS NMR using homonuclear decoupled rotary resonance, Chem. Phys. Lett. 435 (2007) 163–169. [23] S. Cavadini, A. Abraham, G. Bodenhausen, Coherence transfer between spy nuclei and nitrogen-14 in solids, J. Magn. Reson. 190 (2008) 160–164. [24] Z.H. Gan, Measuring nitrogen quadrupolar coupling with 13C detected wideline 14N NMR under magic-angle spinning, Chem. Commun. 7 (2008) 868–870. [25] L. Muller, Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence, J. Am. Chem. Soc. 101 (1979) 4481–4484. [26] S. Wi, L. Frydman, Residual dipolar couplings between quadrupolar nuclei in high resolution solid state NMR: description and observations in the highfield limit, J. Chem. Phys. 112 (2000) 3248–3261. [27] J. McManus, R. Kemp-Harper, S. Wimperis, Second-order quadrupolar-dipolar broadening in two-dimensional multiple-quantum MAS NMR, Chem. Phys. Lett. 311 (1999) 292–298. [28] J.G. Hexem, M.H. Frey, S.J. Opella, Influence of N-14 on C-13 Nmr-spectra of solids, J. Am. Chem. Soc. 103 (1981) 224–226. [29] A. Naito, S. Ganapathy, C.A. Mcdowell, High-resolution solid-state C-13-Nmr spectra of carbons bonded to nitrogen in a sample spinning at the magic angle, J. Chem. Phys. 74 (1981) 5393–5397. [30] R.K. Harris, A.C. Olivieri, Quadrupolar effects transferred to spin-1/2 magicangle spinning spectra of solids, Prog. NMR Spectrosc. 24 (1992) 435–456. [31] S. Antonijevic, G. Bodenhausen, High-resolution NMR spectroscopy in solids by truly magic-angle spinning, Angew. Chem. Int. Ed. 44 (2005) 2935–2938. [32] G. Bodenhausen, D.J. Ruben, Natural abundance N-15 Nmr by enhanced heteronuclear spectroscopy, Chem. Phys. Lett. 69 (1980) 185–189. [33] N. Muller, G. Bodenhausen, R.R. Ernst, Relaxation-induced violations of coherence transfer selection-rules in nuclear-magnetic-resonance, J. Magn. Reson. 75 (1987) 297–334. [34] N. Ivchenko, C.E. Hughes, M.H. Levitt, Multiplex phase cycling, J. Magn. Reson. 160 (2003) 52–58. [35] Z.H. Gan, H.T. Kwak, Enhancing MQMAS sensitivity using signals from multiple coherence transfer pathways, J. Magn. Reson. 168 (2004) 346–351. [36] N. Malicki, et al., Multiplex MQMAS NMR of quadrupolar nuclei, Solid State Nucl. Magn. Reson. 28 (2005) 13–21.
Contents lists available at ScienceDirect
Solid State Nuclear Magnetic Resonance journal homepage: www.elsevier.com/locate/ssnmr
Probing amide bond nitrogens in solids using
14
N NMR spectroscopy
Sasa Antonijevic a,b,, Nicholas Halpern-Manners a a b
Department of Chemistry, University of California, Berkeley, 1 Cyclotron Road Building 11-D64, Berkeley, CA 94720, USA Materials Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road Building 11-D64, Berkeley, CA 94720, USA
a r t i c l e in f o
a b s t r a c t
Article history: Received 16 April 2008 Available online 20 April 2008
A novel two-dimensional nuclear magnetic resonance (NMR) experiment is proposed for indirect observation of 14N nuclei in various types of nitrogen-containing solids. In a method somewhat similar to the heteronuclear single-quantum correlation (HSQC) experiment widely used for protein structure determination in solutions, this technique correlates spin S ¼ 1/2 nuclei, e.g., 1H, 13C, with the 14N spin I ¼ 1 nucleus in solids. The present experiment, however, transfers coherence from neighboring 1H or 13 C nuclei to 14N via a combination of J-couplings and residual dipolar splittings (RDS). Projections of the two-dimensional NMR spectra onto the 14N dimension yield powder patterns that reflect the 14N quadrupolar interaction, which can be used to study molecular structure and dynamics. Indirect detection of amide nitrogen-14 via 1H and 13C is shown experimentally on a model compound of Nacetyl-glycine. & 2008 Elsevier Inc. All rights reserved.
Keywords: NMR spectroscopy Solid state Nitrogen-14 Peptides Proteins
Despite its presence in a wide range of materials and a natural abundance of 99.64%, nitrogen-14 remains relatively unexplored by nuclear magnetic resonance (NMR) techniques. The main reason for this is the large quadrupolar coupling which results in very broad 14N resonances in both liquids and solids. Broadening in liquids arises from very rapid quadrupolar relaxation that cannot be suppressed, while in solids the large anisotropic broadening can be reduced. There have been a number of techniques that utilize both direct and indirect detection to study 14 N nuclei in solids which include nuclear quadrupole resonance (NQR), overtone and NMR spectroscopy applied to study single crystals or powders combined with magic angle spinning (MAS), and dynamic angle spinning or double sample rotation [1–13]. The quadrupolar tensor parameters, which reflect the local electronic charge distribution, can be used to determine structural parameters of the studied material [7,14]. Indirect detection of 14N (spin I ¼ 1) by NMR spectroscopy of solids via spin S ¼ 1/2 nuclei has been demonstrated recently [15–24]. The experiment known as nitrogen excitation via residual dipolar splittings (NERDS) is analogous to the heteronuclear multiple-quantum correlation (HMQC) [25] experiment widely used to study molecules in solution. One of the principal differences is that the coherence transfer between the two different nuclei is conducted not only by J-couplings, JIS, but also by second-order quadrupole-dipole cross Corresponding author at: Department of Chemistry, University of California, Berkeley, 1 Cyclotron Road Building 11-D64, Berkeley, California 94720, USA. Fax: +1 510 486 5744. E-mail address: [email protected] (S. Antonijevic).
0926-2040/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ssnmr.2008.04.006
terms, also known as residual dipolar splittings (RDS), DRDS [26–31]. These cross-terms arise because the axis of the quantization of 14N nuclei is subject to the large quadrupolar interaction and is thus tilted from the direction of the static magnetic field, thereby preventing complete averaging of the dipolar interactions between 14N nuclei and neighboring S spins under MAS. As a result, the S resonances are split into 1:2 doublets where each component features a powder pattern, and the separation between the centers of mass of these two powder patterns is referred to here as the residual dipolar splitting, DRDS. The residual dipolar splitting is a purely through-space interaction with strength that depends upon the quadrupolar coupling constant (CQ), internuclear distance (rSI), relative orientation of the internuclear vector, the largest component of the electric field gradient tensor, and the static magnetic field strength, B0. In this work we present an experiment that provides a counterpart to the heteronuclear single-quantum correlation (HSQC) [32] experiment (where single-quantum (SQ) refers to the evolution of SQ coherences of one type of nucleus during the t1 interval). In our experiment it is only the 14N coherences which evolve, but one can choose to observe either the SQ or double-quantum (DQ) 14N coherences. The acronym sNERDS seems appropriate for coherences of a single (s) type of nucleus (14N) during t1 interval; likewise the acronym mNERDS seems appropriate for coherences of multiple (m) type nuclei. One application of this experiment probes amide bond nitrogen atoms in powdered solids, which is demonstrated on the model compound of N-acetyl-glycine (NAG). This work differs from and extends the work of Cavadini et al. [23] in the following ways: in this paper, the main focus was the
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13C
(π/2)φ1 1H
sNERDS
Heteronuclear Decoupling
(CP)φ2 (π/2)φ4
13C
83
(π/2)φ7 τrec
(CP)φ3 τexc (τp)φ5
14N
t2
(τp)φ6 t1
(π/2)φ1 (π/2)φ2 1H
14N
1H
sNERDS
(π/2)φ5
τexc
τrec (τp)φ3
t2
(τp)φ4 t1
pC pH pN +1 +2 +1 +1 +1 0 +1 –1 +1 –2 0 +2 0 +1 0 0 0 –1 0 –2
14
N DQ
14
N SQ
–1 +2 –1 +1 –1 0 –1 –1 –1 –2 Fig. 1. Pulse sequences (A and B) and coherence transfer pathway diagrams (C) for the indirect detection of 14N single- (solid lines) and double-quantum (dashed lines) spectra by coherence transfer from (A) 13C or (B) 1H to 14N and back. Two separate experiments, utilizing the appropriate phase cycling, as indicated above with solid and dashed lines, can be used to observe 14N SQ and DQ spectra. Alternatively these spectra can be obtained in a single experiment using multiplex phase cycling, provided one settles on a compromise for the pulse width tp.
demonstration of a novel technique to study peptide bond nitrogen-14 nuclei, which are subjected to quadupolar coupling constants nearly three times larger than nitrogen-14 nuclei found in aminoacids studied by Cavadini et al. [23]. Peptide bond nitrogens have direct relevance to the major applications of these new techniques for studying biological solids. In addition, we propose to irradiate spy (spin S ¼ 1/2) nuclei in a timeresolved fashion (in comparison to nitrogen-14 nuclei); by doing so, it is possible to extend further our experiment to include sophisticated steps such as the introduction of pulsed fields gradients in order to improve performance. The final difference is in the approach taken to quantify the efficiency of the experiment. This work was originally submitted as a manuscript to another journal and a preprint was provided to Prof Geoffrey Bodenhausen who kindly acknowledges this in Cavadini et al. [23]. Pulse sequences and coherence transfer pathway diagrams for the indirect observation of 14N (spin I ¼ 1) via 13C or 1H (spin S ¼ 1/2) under MAS are shown in Fig. 1, while the phase cycles that can be used to obtain desired coherence selections are given in Table 1. Excitation of transverse magnetization of S spins, represented by a tensor operator T S1;1 [33], is achieved by means
of cross-polarization from 1H to 13C (when 13C is used as the ‘‘spy’’ nucleus) or by single radiofrequency pulse irradiation of 1H nuclei. After a delay texcE1/(2DRDS), the system reaches a state that can be loosely described by an antiphase operator T S1;1 T N 2;0. Since the coupling DRDS(14N-S) is anisotropic, and since the S nuclei magnetization often decays rapidly, the delay texc must be optimized empirically. A radiofrequency pulse with flip angle p/2 applied to the S spins followed by a pulse of duration tp applied near the center of the 14N spectrum leads to a partial S N S N 14 conversion into T S1;0 T N N 1;1 , T 1;0 T 2;1 and T 1;0 T 2;2 which contain N N N SQ (T 1;1 and T 2;1 ) and DQ (T 2;2 ) coherences only (unlike ordinary HSQC experiments with spin 1/2 nuclei where only SQ coherences are involved). These are allowed to evolve freely during an evolution interval t1 (where the increments Dt1 ¼ 1/nrot are synchronized with the spinning period), prior to reconversion into observable S coherence. A two-dimensional Fourier transformation yields a correlation spectrum with either 14N SQ or DQ signals in the o1 dimension and the conventional S nuclei spectrum in the o2 dimension. The SQ and DQ signal intensities can be optimized by choosing the intervals texc ¼ trec and the pulse width tp. These signals can be separated by phase cycling. It is also possible to obtain both SQ and DQ spectra in a single
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Table 1 Phase cycles suitable for use in the pulse sequences in Fig. 1 for the indirect observation of
14
N via (* indicates number of repetitions)
13
C nuclei selecting f1 f2 f3 f4 f5 f6 f7 Receiver
14
N SQ coherences during t1 interval ( C SQ sNERDS) (901,2701)*16 (01)*32 ((01)*2,(901)*2,(1801)*2,(2701)*2)*4 (01)*16,(1801)*16 ((01)*8,(1801)*8)*2 (01)*32 (01)*32 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701
13
14
N DQ coherences during t1 interval (13C DQ sNERDS) (901,2701)*32 (01)*64 ((01)*2,(901)*2,(1801)*2,(2701)*2)*8 (01)*32 (1801)*32 ((01)*8,(901)*8,(1801)*8,(901)*8)*2 (01)*64 (01)*64 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 01,1801,2701,901,1801,01,901,2701,1801,01,901,2701,01,1801,2701,901, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701, 1801,01,901,2701,01,1801,2701,901,01,1801,2701,901,1801,01,901,2701
C nuclei selecting f1 f2 f3 f4 f5 f6 f7 Receiver
1 H nuclei selecting f1 f2 f3 f4 f5 Receiver
14
1
14
H nuclei selecting f1 f2 f3 f4 f5 Receiver
13
N SQ coherences during t1 interval (1H SQ sNERDS) (01,1801)*4 ((01)*2,(1801)*2)*2 (01)*4,(1801)*4 (01)*8 (01)*8 01,1801,1801,01,1801,01,01,1801 N DQ coherences during t1 interval (1H DQ sNERDS) (01,1801)*8 ((01)*2,(1801)*2)*4 (01)*4,(901)*4,(1801)*4,(2701)*4 (01)*16 (01)*16 01,1801,1801,01,1801,01,01,1801,01,1801,1801,01,1801,01,01,1801
experiment using multiplex phase cycling [34–36], provided one settles on a compromise for the pulse width tp. By selecting +1 S coherences (T S1;1 T N 2;0 ) in texc, which are then converted to 1 coherences that evolve in trec, there will be no net evolution under the chemical shifts (O), since exp(iOtexc)exp (iOtrec) ¼ 1 if texc ¼ trec. Thus, the chemical shift of the S spins and the inhomogeneous contributions to the S spin decay during texc and trec intervals can be eliminated. Despite these benefits, there is a loss of half of the signal in comparison to the analog HSQC experiment used for studying molecules in solutions. The second half of the signal can be recovered by selecting both T S1;1 T N 2;0 coherences during the texc interval, which will result in the observed signal being modulated by chemical shifts with a factor of cos(Otexc)exp(iOtrec). As a consequence of this modulation, resonances will be scaled in amplitude and will have different phases. Additionally, if both T S1;1 T N 2;0 are also selected during the trec interval, and the z-filter is implemented after the trec interval to observe the signal, then resonances will be scaled only in amplitude with a factor cos(Otexc)cos(Otrec) but would retain the same phase regardless of their chemical shifts. The conventional HSQC experiment uses non-selective p pulses simultaneously applied on both nuclei in the middle of the texc and trec periods to retain the signal intensity and avoid chemical shift phase modulation. However, due to the large 14N quadrupolar interaction, there is no instrumental possibility to apply radiofrequency pulses that would result in non-selective rotations of 14N magnetization.
Fig. 2 shows (A) 13C MAS NMR spectra and two-dimensional C–14N correlation (B) sNERDS and (C) mNERDS spectra of NAG. Projections onto the o1 dimension reveal a 14N powder pattern predominantly broadened by the second-order quadrupolar interaction. Both C0 and Ca projections reflect the same nitrogen while remaining different in appearance. One of the reasons for this lies in the unequal excitation of the powder pattern due to the difference in internuclear distances r C0 N ar Ca N , and the different relative orientations of the quadrupolar and dipolar tensors. Fig. 3 shows an analogous spectrum where 1H atoms are utilized as spy nuclei. It is vitally important to obtain narrow 1H resonances, which is accomplished through selective deuteration of the NAG molecule combined with rapid MAS. The experimental NERDS spectra offer information on the quadrupolar interaction of 14N nuclei, which can be related to the structure of a molecule [7,14]. The extraction of quadrupolar parameters from the experimental lineshapes can be a challenging process due to non-uniform excitation that leads to distortion of observed powder patterns as discussed previously [20]. The samples were packed in 4 mm Si3N4 rotors and spun at 15 kHz in a Doty triple resonance MAS probe at 16.4 T (1H, 13C and 14 N Larmor frequencies of 700.1, 176.1 and 50.6 MHz) and spectra were recorded using a Bruker Avance 700 spectrometer. CrossC polarization (CP) was used with nH RF ¼ 65 kHz, while nRF was ramped. Two pulse phase-modulation (TPPM) proton decoupling was used with nH RF ¼ 65 kHz, pulse widths of 4.5 ms, and a phase difference between two successive pulses of 301. With a 1000 W 13
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13
176
13
C’
174
44
85
Cα
42
δ (ppm) 13
C’
13 α
C
250 300
δ1 (ppm)
350 400 450 500
250 300
δ1 (ppm)
350 400 450 500
176
44
174
42
δ2 (ppm) Fig. 2. (A) 13C MAS NMR and two-dimensional correlation 13C SQ NERDS NMR spectra of polycrystalline N-acetyl-glycine, 13C enriched in both Ca and acetyl carboxyl carbon C0 , recorded using a pulse sequence that selects (B) pure SQ 14N coherences (sNERDS) and (C) 13C and SQ 14N coherences (mNERDS) during the t1 evolution interval. The projections onto the o1 axis reveal quadrupolar powder patterns for the same 14N indirectly observed by the two different carbons. Spectra are the result of averaging 128 transients for each of 64 t1 increments with Dt1 ¼ 1/nrot ¼ 66.6 ms, with a relaxation interval of 10 s. The excitation and reconversion intervals texc ¼ trec were 1.2 ms, while the lengths of the two 14N pulses were tp ¼ 50 ms.
amplifier, the 14N pulses had a modest amplitude of nN RF ¼ 25 kHz, calibrated by direct detection of NH4Cl. The chemical shifts of 1H, 13 C and 14N are referenced relative to external standards of TMS and NH3 (liquid, 251). Typically 150 mg of material is used to obtain spectra shown in this work. In conclusion, we have demonstrated a NMR experiment for indirect detection of nitrogen-14 in solids, which is analogous to the HSQC experiment widely used for protein structure determination in solutions. The advantage of this sNERDS experiment over the mNERDS experiment introduced recently lies in the fact
that the indirect dimension is free from any broadening introduced from decoherence processes of the spy nuclei, as only 14N coherences are allowed to evolve during the t1 interval. This may be especially important when protons are used as spy nuclei due to their large refocused line widths. More generally, 14 N spectra offer a greater wealth of information in comparison to 15N spectra due to quadrupolar broadening that reflects the local charge distribution. It has been shown that these studies could provide more insight into the geometry of peptide bonds [7,14].
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16
14
12
10 8 δ (ppm)
6
4
2
16
14
12
10 8 δ2 (ppm)
6
4
2
250
300
δ1 (ppm)
350
400
450
500
250
300
δ1 (ppm)
350
400
450
500
Fig. 3. (A) 1H MAS NMR and two-dimensional correlation 1H SQ NERDS NMR spectra of polycrystalline N-acetyl-glycine (selectively deuterated so that the amide and carboxyl groups remain protonated), recorded using a pulse sequence that selects (B) pure SQ 14N coherences (sNERDS) and (C) 1H and SQ 14N coherences (mNERDS) during the t1 evolution interval. Spectra are the result of averaging 64 transients for each of 64 t1 increments with Dt1 ¼ 1/nrot ¼ 67.2 ms, with a relaxation interval of 10 s. The excitation and reconversion intervals texc ¼ trec were 0.8 ms, while the lengths of the two 14N pulses were tp ¼ 50 ms.
Acknowledgments This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division, of the US Department of Energy under Contract no. DE-AC03-76SF00098. S.A. acknowledges support from the
Lawrence Berkeley National Laboratory through a postdoctoral fellowship. N.H.-M. acknowledges support from the University of California Berkeley through a graduate fellowship. Chemicals used in this work were kindly donated by Dr Robert Lysek. This document was prepared as an account of work sponsored by the United States Government. While this document
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is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California. References [1] R. Blinc, et al., Pulsed nitrogen-proton double resonance study of ferroelectric transition in triglycine sulfate, J. Chem. Phys. 55 (1971) 4843. [2] M.J. Hunt, A.L. Mackay, Deuterium and nitrogen pure quadrupole-resonance in amino-acids.2, J. Magn. Reson. 22 (1976) 295–301. [3] R.E. Stark, R.A. Haberkorn, R.G. Griffin, N-14 NMR determination of Nh bond lengths in solids, J. Chem. Phys. 68 (1978) 1996–1997. [4] S.J. Opella, M.H. Frey, T.A. Cross, Detection of individual carbon resonances in solid proteins, J. Am. Chem. Soc. 101 (1979) 5856–5857. [5] P. Brunner, M. Reinhold, R.R. Ernst, Double quantum cross polarization—heteronuclear excitation and detection of Nmr double quantum transitions in solids, J. Chem. Phys. 73 (1980) 1086–1094. [6] R. Tycko, S.J. Opella, High-resolution N-14 overtone spectroscopy—an approach to natural abundance nitrogen Nmr of oriented and polycrystalline systems, J. Am. Chem. Soc. 108 (1986) 3531–3532. [7] R. Tycko, P.L. Stewart, S.J. Opella, Peptide plane orientations determined by fundamental and overtone N-14 Nmr, J. Am. Chem. Soc. 108 (1986) 5419–5425. [8] R. Tycko, S.J. Opella, Overtone Nmr-spectroscopy, J. Chem. Phys. 86 (1987) 1761–1774. [9] K.V. Ramanathan, S.J. Opella, Overtone decoupling—the effects of N-14 overtone irradiation on C-13 Nmr-spectra of a single-crystal, J. Magn. Reson. 78 (1988) 367–370. [10] C.P. Grey, W.S. Veeman, The detection of weak heteronuclear coupling between spin-1 and spin-1/2 nuclei in Mas Nmr-N-14/C-13/H-1 triple resonance experiments, Chem. Phys. Lett. 192 (1992) 379–385. [11] C.P. Grey, A.P.A.M. Eijkelenboom, W.S. Veeman, N-14 population transfers in 2-dimensional C-13-N-14-H-1 triple-resonance magic-angle-spinning nuclear-magnetic-resonance spectroscopy, Solid State Nucl. Magn. Reson. 4 (1995) 113–120. [12] S.S. Wi, L. Frydman, Heteronuclear recoupling in solid-state magic-anglespinning NMR via overtone irradiation, J. Am. Chem. Soc. 123 (2001) 10354–10361. [13] G. Jeschke, M. Jansen, High-resolution N-14 solid-state NMR spectroscopy, Angew. Chem. Int. Ed. 37 (1998) 1282–1283.
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