Chemical Physics Letters 488 (2010) 1016

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Chemical Physics Letters

journal homepage: www.elsevier.com/locate/cplett

Broadband magnetization transfer using moderate radio-frequency elds for NMR with very high static elds and spinning speeds
Markus Weingarth a, Geoffrey Bodenhausen a,b, Piotr Tekely a,*
a b

Dpartement de chimie, associ au CNRS, Ecole Normale Suprieure, 24 rue Lhomond, 75231 Paris, France Institut des Sciences et Ingnierie Chimiques, BCH, Ecole Polytechnique Fdrale de Lausanne, 1015 Lausanne, Switzerland

a r t i c l e

i n f o

a b s t r a c t
We introduce a new dipolar recoupling scheme, dubbed PARIS-xy (phase-alternated recoupling irradiation scheme using orthogonal radio-frequency phases), to promote efcient broadband magnetization exchange between 13C nuclei using moderate radio-frequency amplitudes at very high magnetic eld strengths and spinning speeds. Experimental observations for a wide range of spinning frequencies 30 6 mrot 6 60 kHz and magnetic eld strengths (B0 = 9.4, 17.6 and 21.2 T) are backed up by numerical simulations. 2010 Elsevier B.V. All rights reserved.

Article history: Received 4 January 2010 In nal form 29 January 2010 Available online 4 February 2010

1. Introduction The relentless expansion of solid-state NMR is largely driven by technical and methodological developments. Recent progress has paved the way, inter alia, to the determination of an increasing number of structures of microcrystalline proteins [13]. The efcient exchange of longitudinal magnetization between spins S and S0 such as pairs of non-equivalent carbon-13 nuclei is a prerequisite both for assignment and structure determination. The combination of high static elds and fast magic-angle spinning (MAS) is particularly attractive for structure determination of biomolecules [46]. However, fast spinning leads to the elimination of homoand heteronuclear dipolar interactions, and hence to a loss of efciency of SS0 magnetization transfer based on proton-driven spin diffusion (PDSD) [7,8]. It is therefore, necessary to recouple the dipolar interactions by suitable radio-frequency (rf) irradiation. Most established recoupling methods like rf-driven recoupling (RFDR) [9] and dipolar assisted rotational resonance (DARR) [10,11] have been developed for moderate static elds and spinning frequencies. DARR uses continuous-wave (CW) proton irradiation with an rf amplitude that matches the spinning speed (m1 = mrot). Recently, new approaches have been proposed for recoupling at high spinning frequencies [4,5,12]. In particular, we have introduced a phase-alternated recoupling irradiation scheme (PARIS), which proved to be effective over a broad bandwidth at moderate or fast spinning frequencies [6,13]. PARIS recoupling offers an attractive alternative to the popular DARR scheme [10,11] because of its inherent immunity to the inhomogeneity of the rf eld [13,14], its ability to achieve recoupling with rf amplitudes well below the rotary resonance condition [15] i.e., m1 < mrot, and
* Corresponding author. Fax: +33 1 44 32 33 97. E-mail address: Piotr.Tekely@ens.fr (P. Tekely). 0009-2614/$ - see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2010.01.072

its capacity to promote efcient magnetization transfer even when the spinning frequency mrot exceeds the difference of isotropic chemical shifts Dm(C0 , Cal) between spectrally distant carbons such as carboxyl (C0 ) and aliphatic (Cal) carbons, so that the rotational resonance (R2) condition mrot = Dm(C0 , Cal) [16] cannot be fullled. The original PARIS method (henceforth called basic PARIS in this Letter) merely requires toggling the phase of the proton rf eld between x and x at intervals sp = Nsrot, usually with N = or a multiple thereof [13]. This leads to efcient zeroth-order recoupling of heteronuclear 1H13C dipolar interactions [13]. The effect is most pronounced for N = (see Fig. 5 in [13]). Heteronuclear recoupling leads to a broadening of the 13C resonances, and hence to an overlap between them, so that the 13C spins can exchange magnetization by energy-conserving zero-quantum ip-op processes. The efciency of recoupling is largely independent of the proton rf amplitude [6,13,14]. We have shown that proton rf amplitudes as low as m1 = mrot/3 are sufcient to bring about efcient magnetization transfer if N = . At moderate spinning frequencies mrot = 23 kHz, the basic PARIS sequence can reveal a large number of contacts in carboncarbon correlation spectra of microcrystalline proteins recorded at 21.2 T (900 MHz for protons) [6]. Numerical simulations corroborate experimental observations and show that magnetization transfer is more efcient with PARIS than with DARR even in the absence of rf eld inhomogeneity [6,13]. Average Hamiltonian Theory (AHT) suggests that for N = the efciency of PARIS recoupling is not signicantly affected if one moves away from the rotary resonance condition m1 = mrot. This was conrmed experimentally and numerically by the existence of a broad plateau of proton rf amplitudes m1 where recoupling is efcient [6,13,14]. When the spinning frequency and the recoupling proton rf amplitude are matched, i.e., when m1 = mrot, the basic PARIS sequence corresponds to an R21 -type method in the termi1 nology of symmetric sequences [17] and leads to the recoupling

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of both heteronuclear IS and homonuclear II dipolar interactions. The basic PARIS method with N = can bring about efcient magnetization transfer between two aliphatic carbons such as Ca and Cb with a small isotropic chemical shift difference Dm(Ca, Cb). For spectrally distant carbons such as C0 and Cal and moderate spinning frequencies, basic PARIS with N = can benet from rotational resonance recoupling if mrot % Dm(C0 , Cal) . Like in DARR, the R2 condition mrot = Dm(C0 , Cal) is broadened by the recoupled heteronuclear protoncarbon dipolar interactions and, to a lesser extent, by protonproton dipolar couplings. Under such conditions, magnetization transfer is more efcient for basic PARIS than for DARR due to the compensation for the rf eld inhomogeneity and the presence of modulation sidebands (MS) which appear at mrot with respect to the parent signals. When the spinning speed exceeds the chemical shift difference, i.e., when mrot > Dm(C0 , Cal), it is obviously impossible to exploit rotational resonance R2 recoupling. Broadband magnetization transfer can then be restored by using longer pulse lengths sp = Nsrot, typically with N = 2 [13]. This causes the modulation sidebands to come closer in frequency space, since they now occur at mrot with respect to the parent signals. These sidebands can thus be matched to the difference in isotropic shifts Dm(C0 , Cal). However, the broadening of the resonance signals by the recoupled heteronuclear interactions, and thus the transfer between nearby carbons, is less effective for N = 2 than for N = and can only be restored by resorting to higher rf amplitudes (see below). In this work, we present an extension of the basic PARIS approach which provides efcient broadband magnetization transfer with moderate rf amplitudes even at spinning frequencies as high as mrot = 60 kHz and very high magnetic elds up to 21.2 T (900 MHz for protons). 2. Materials and experimental conditions Polycrystalline samples of uniformly 13C, 15N-labelled L-glycine (L-Gly) and 13C-labelled L-histidine (L-His) were used without further purication. The spectra of L-Gly were recorded on a Bruker spectrometer operating at 9.4 T (400 MHz 1H frequency) and mrot = 34 kHz. The spectra of L-His were obtained at 17.6 T (750 MHz) with mrot = 52 kHz, and at 21.2 T (900 MHz) with mrot = 39 and 60 kHz. Heteronuclear decoupling during the evolution and acquisition periods was achieved with the PISSARRO method [18,19]. In all experiments, cross-polarization (CP) was used with a ramped rf amplitude on the proton channel [20]. 3. PARIS-xy The new pulse sequence introduced in this work (see Fig. 1) will be referred to as PARIS-xy, which stands for phase-alternated recoupling irradiation scheme using orthogonal radio-frequency phases. The sequence consists of blocks of pulse pairs

Fig. 1. PARIS-xy sequence for transfer of longitudinal magnetization between like spins S and S0 by recoupling of heteronuclear dipoledipole interactions. The transfer is promoted by blocks of pulse pairs with orthogonal phases sx sx m sy sy m where m = 1, 2 . . . and the pulse width is sp = Nsrot with N = in this work.

e.g., switching to sx sx m sy sy m , does not have any signicant effect. It is possible to use different lengths, say m and m0 , of x and y blocks to broaden the spectral ranges that can be covered, but we shall only use symmetrical variants of PARIS-xy in this work. As mentioned above, the basic PARIS sequence causes modulation sidebands to appear at mrot with respect to the parent signals for N = , or at mrot for N = 2. These sidebands arise from the interference of spinning and phase-alternated irradiation [13,18]. Apart from modulation sidebands at mrot with respect to the parent signals, PARIS-xy creates further modulation sidebands:

mxy mrot Lmc

sx sx m sy sy m ;

where m = 1 or 2. All pulse lengths are equal, i.e., sx = sx = sy = sy = sp = Nsrot, and normally set to half the rotor period, i.e., sp = srot/2 (N = ) The duration tc of the cycle in the sense of average Hamiltonian theory is

t c 4msp
so that the cycle frequency is

mc 1=tc 4msp 1 :

This sequence ensures efcient zeroth-order recoupling of the heteronuclear interactions, leading to a pronounced broadening of the S spin resonances [13]. A sign reversal of the phases within a block,

with L = 1 and 2. The choice of the best variant of PARIS-xy will depend on the spinning frequency and on the differences Dm(S, S0 ) of the isotropic chemical shifts, which are proportional to the static eld. Nuclei other than 13C, which may have different distributions of chemical shifts, can be investigated by using different lengths m. The crucial advantage of PARIS-xy over basic PARIS is that broadband transfer at very fast spinning speeds can be achieved while maintaining N = , where the heteronuclear recoupling is most effective. For example, according to Eq. (4), PARIS-xy with m = 1 creates modulation sidebands at mrot while for PARIS-xy with m = 2 the modulation sidebands occur at mrot and 3=4 mrot. In general, we recommend the use of basic PARIS (N = ) for spinning frequencies equal or smaller than the chemical shift difference of spectrally distant resonances mrot 6 DmS; S0 and PARIS-xy if mrot > DmS; S0 . Thus efcient recoupling can be obtained over the whole range of spinning frequencies. Fig. 2 shows the 13C spectrum of L-Gly recorded at mrot = 34 kHz in the presence of proton irradiation with an rf amplitude m1 = 19 kHz. For PARIS-xy with m = 1, modulation sidebands occur at 17 kHz on either side of the Ca resonance. For PARIS-xy with m = 2, additional modulation sidebands appear at 25.5 kHz. These sidebands have a width of about 10 kHz.

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Fig. 2. Experimental carbon-13 spectra of L-glycine recorded at mrot = 34 kHz and B0 = 9.4 T (400 MHz for protons) using PARIS-xy with m = 1 and 2, both with a proton rf amplitude m1 = 19 kHz. The upper spectrum shows modulation sidebands associated with the Ca resonance with mmod = mrot = 17 kHz and mmod = mrot = 34 kHz. The latter are superimposed by spinning sidebands resulting from the modulation of internal interactions by macroscopic rotation. For PARIS-xy with m = 2 (lower spectrum) the modulation sidebands occur at mmod = mrot = 17 kHz, mmod = 3=4 mrot = 25.5 kHz, and mmod = mrot = 34 kHz. The arrows show the modulation sidebands and the asterisks indicate spinning sidebands associated with the chemical shift anisotropy (CSA) of the C0 carbon.

4. Results and discussion 4.1. Numerical simulations of magnetization transfer The exchange of longitudinal magnetization between Sz and S0z results from multi-spin processes involving higher order terms in the effective Hamiltonian. An analytical treatment of such processes is very demanding. We have therefore carried out numerical simulations to obtain insight into the mechanism of magnetization exchange by PARIS-xy irradiation. All simulations were carried out with SPINEVOLUTION [21]. Zero-quantum ip-op processes are energy conserving when corresponding resonance lines at least partially overlap. To promote efcient magnetization transfer through zero-quantum exchange, the matching condition for a suitable modulation sideband is

mmod Dm(C0 , Ca) by changing Dm(C0 , Ca). The resulting prole is

symmetric with respect to the exact matching condition of Eq. (5). Expanding the simulations from a simple C2H2 to a C2H5 system leads to a broadening of the transfer prole to a width of about 15 kHz with a broad maximum in the central region. This leads to a more uniform transfer for longer mixing times, bearing in mind that the plateau is reached later. Fig. 3b shows that the width and transfer efciency are largely independent of the rf amplitude. Indeed, the magnetization transfer proles are almost the same for recoupling rf amplitudes m1 = 60 and 40 kHz. Below a threshold m1 = mrot/3, both the transfer efciency and bandwidth slowly decrease. Low rf amplitudes can nevertheless be used to promote magnetization transfer, albeit with longer mixing times. Finally, we examined the inuence of homonuclear dipolar proton-proton couplings on the transfer prole (Fig. 3c). These interactions were found to broaden the width of the matching condition without improving the efciency of the transfer. The simulated rf dependence of the transfer efciency is in good agreement with the experimental observations (see below). 4.2. Simultaneous magnetization exchange among aliphatic carbons and between carbonyl and aliphatic carbons Experiments were carried out to probe the efciency of PARISxy with m = 1 at two spinning frequencies (mrot = 52 and 60 kHz) and two static elds (B0 = 17.6 and 21.2 T). Fig. 4 shows a 13C13C

mmod DmS; S0 :

Because the proles have a width of about 15 kHz (see below), this matching condition need not be fullled precisely. Consider the magnetization transfer between C0 and Ca in L-serine using PARISxy (m = 1) with mrot = 60 kHz and B0 = 21.2 T, where Dm(S, S0 ) = 30 kHz. Under such experimental conditions, the modulation sidebands appear at 30 kHz from the parent Ca resonance. In Fig. 3a and b, the efciency of the magnetization transfer, dened as the cross-peak amplitude aSS0 (sm > 0) divided by diagonal peak intensity aSS (sm = 0), was calculated as a function of the deviation

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correlation spectrum of a powder sample of L-His containing a mixture of two polymorphic forms with different levels of protonation [22,23] one of which making up about 15% of the sample. The spectrum of Fig. 4 was recorded at B0 = 17.6 T and mrot = 52 kHz using PARIS-xy with m = 1 and a proton rf amplitude m1 = 18 kHz applied during sm = 300 ms. Thus, mmod = mrot = 26 kHz, so that the matching condition of Eq. (5) is fullled for Dm(C0 , Cb) = 25.5 kHz, but that the mismatch is more than 3 kHz for Dm(C0 , Ca) = 22.33 kHz. Since modulation sidebands can promote efcient magnetization transfer over a range of at least 15 kHz, exact matching is not necessary. Indeed, strong C0 Cb and C0 Ca cross-peaks can be seen in Fig 4, with relative intensities in the range of 3565% and 2535%, respectively, compared to the relevant diagonal peaks in the row. The presence of cross-peaks reecting all possible one- and twobond correlations in both polymorphs proves the efciency of PARIS-xy with low proton rf amplitudes. Fig. 5a shows a PARIS-xy spectrum obtained with m = 1 at B0 = 21.2 T, mrot = 60 kHz, sm = 175 ms, and a proton rf amplitude m1 = 25 kHz. Under these conditions, Dm(C0 , Ca) = 28.8 kHz and Dm(C0 , Cb) = 33.3 kHz. The modulation sidebands at mmod = mrot = 30 kHz are placed roughly half-way between the two matching conditions. Again, intense C0 Cb and C0 Ca cross-peaks appear (in addition to CaCb cross-peaks). Since the mixing time sm was much shorter, all cross-peaks are weaker in Fig. 5a than in Fig. 4. Instead of using PARIS-xy with m = 1, one can also use basic PARIS with N = 2, which also generates modulation sideband at mmod = mrot. However, the data shown in Fig. 6 prove that at high spinning speeds, PARIS-xy promotes more efcient transfer between the aliphatic carbons and allows lower proton rf amplitudes. Finally, we compared PARIS-xy with the phase-alternated version of mixed rotational and rotary resonance (MIRROR) [24], choosing a modulation frequency matched to the chemical shift difference Dm(C0 , Ca). MIRROR performed much better than PARIS-xy for the selected C0 Ca correlation (Fig. 6c), but the Ca Cb correlation with MIRROR is no better than can be achieved by proton-driven spin diffusion (PDSD), and the CcCd correlation is worse than can be achieved with PDSD. These observations show that MIRROR is well suited for a band-selective transfer but less appropriate to cover large spectral regions. 4.3. Broadband magnetization exchange between aliphatic, aromatic and carboxyl carbons Figs. 4 and 5a do not reveal all cross-peaks between the aromatic Car and aliphatic Cal carbons, nor all cross-peaks between Car and carboxylic C0 carbons, when using PARIS-xy with m = 1, because of the violation of the matching condition of Eq. (5). All contacts that are missing in Fig. 4 can be recovered, and the intensity of the C0 Cal cross-peaks can be increased, provided the modulation sidebands are simultaneously matched, at least approximately, with the corresponding spectral regions. The broadband features of PARIS-xy recoupling at high spinning speeds (mrot = 39 kHz) and high elds (B0 = 21.2 T) are demonstrated in Fig. 5c and d. Broadband magnetization transfer between three spectrally distant regions can be promoted simultaneously by using PARIS-xy with m = 2, which leads to mmod = mrot = 19.5 kHz, appropriate to promote magnetization transfer between aromatic Car and aliphatic Cal carbons, as well as between aromatic Car and carboxyl C0 regions, and to mmod = 3=4 mrot = 29.25 kHz, suitable for matching between C0 and Cal carbons. Correlation spectra using DARR (Fig. 5b) and PARIS-xy with m = 2 (Fig. 5c) were recorded with mrot = 39 kHz, a mixing time sm = 100 ms and a proton recoupling rf amplitude m1 = 39 kHz (i.e., m1 = mrot to full the n = 1 rotary resonance condition, which is required for DARR.) We found the transfer efciency to be signicantly enhanced over the whole spectrum obtained with PARIS-xy. A dramatic enhance-

Fig. 3. (a) Numerical simulations of the C0 Ca magnetization transfer in L-serine as a function of mismatch, i.e. of the violation of Eq. (5), for PARIS-xy with m = 1 at mrot = 60 kHz and B0 = 21.2 T and a recoupling proton rf amplitude m1 = 60 kHz. Simulations were carried out for increasing numbers of protons and different mixing times sm. The short-dashed line corresponds to a CaC0 HaHb sub-system and 0 sm = 20 ms, the continuous line to CaC0 HaHbHb and sm = 30 ms, the long-dashed 0 line to CaC0 HaHbHb HN1HN2 and sm = 60 ms. (b) Similar simulations for a 0 CaC0 HaHbHb HN1HN2 spin system with recoupling proton rf amplitudes m1 = 60 kHz (long-dashed lines), 40 kHz (continuous lines) and 20 kHz (short-dashed lines). (c) 0 The same as (a) for a CaC0 HaHbHb HN1HN2 spin system with (long-dashed lines) and without (short-dashed lines) proton-proton dipolar couplings.

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Fig. 4. Carboncarbon correlation spectrum of fully 13C labelled L-histidine, containing two differently protonated polymorph forms, measured at B0 = 17.6 T with mrot = 52 kHz, using PARIS-xy with m = 1 with a proton recoupling rf amplitude m1 = 18 kHz during sm = 300 ms and low-power PISSARRO decoupling during the evolution and detection intervals (m1 = 14 kHz, sp = 140 ls). Some cross-sections of Cb (I) and Ca (II) are shown.

ment of about 230% can be observed for the CaCb cross peak. Moreover, the C0 Cal cross-peaks, which are very weak with DARR, show relative intensities of 34% for C0 Ca and 63% for C0 Cb, compared to the relevant diagonal peaks. This results from the approximate matching of the modulation sidebands (mmod = 3=4 mrot = 29.25 kHz) with the average of the shift differences Dm(C0 , Ca) = 28.8 kHz and Dm(C0 , Cb) = 33.3 kHz. All CarCal cross-peaks between aromatic and aliphatic carbons, with the exception of the CeCa correlation (4.7 ), are observed because of the approximate matching between mmod = mrot = 19.5 kHz and Dm(Car, Cal). This is in good agreement with the simulated proles of Fig. 3, which predict efcient magnetization transfer as far as 7.5 kHz from the exact matching condition. Further experiments (not shown) demonstrated that shorter mixing times sm % 50 ms were sufcient to obtain intense correlations. This could be relevant to attenuate misleading relayed transfer effects. When using Paris-xy with m = 2, the efciency of the transfer induced by matching of isotropic shift differences Dm(S,S0 ) with mmod = 3=4 mrot is in general better than for mmod = mrot. We also recorded PARIS-xy spectra with m = 2 and mrot = 39 kHz with proton rf recoupling amplitudes m1 = 20 kHz (Fig. 5d) and m1 = 15 kHz (not shown), well below the rotary resonance condition m1 = mrot required for DARR, combined with longer mixing times (sm = 150 and 200 ms). Both spectra bear witness to the efciency of broadband magnetization transfer. With m1 = 20 kHz (Fig. 5d), between 24% and 71% of the magnetization is transferred, while with m1 = 15 kHz, the efciency lies in the range 2063%. In both cases, the most efcient transfer was observed from Cb and the weakest from Ce. At B0 = 21.2 T (900 MHz), PARIS-xy with m = 2 can be used up to mrot = 55 kHz. At higher spinning speeds, simultaneous matching of sideband frequencies mmod = mrot and 3=4 mrot with Dm(C0 , Car) is no longer possible, although at higher static elds (e.g., at B0 = 23.5 T) one could still use spinning speeds mrot > 60 kHz. If one wishes to increase the spinning speed at a given static eld, it may be benecial to increase the pulse length sp = Nsrot from N = to 3=4 . Indeed, zeroth-order recoupling of heteronuclear interactions is also reasonably efcient at sp = 3=4 srot, although recoupling is more efcient for N = (sp = srot), in particular when lower proton rf amplitudes m1 are used [13]. The broadband character and the efciency of magnetization exchange under PARIS-xy with m = 2 at very high spinning speeds and very high elds with low rf amplitudes result from the fact that the whole spectrum is covered with modulation sidebands associ-

ated with signals that are broadened because of the recoupling of the heteronuclear interactions. For protonated carbons (and for all associated sidebands), this broadening is on the order of 15 kHz. The sidebands associated with each parent signal cover a spectral width of at least 50 kHz. Actually, the bandwidth is even larger since rst order modulation sidebands appear at mmod = mrot in all variants of PARIS-xy, like for basic PARIS with N = . The bandwidth of PARIS-xy is much broader than for the MIRROR technique, which requires variable rf amplitudes to enhance the transfer bandwidth [24]. PARIS-xy provides not only broadband transfer at very high spinning frequencies, but also in the intermediate regime 10 < mrot < 50 kHz. The efciency of PARIS-xy improves with increasing static elds. Indeed, for very high static elds, very high spinning frequencies and low rf amplitudes, PARIS-xy unfolds its full potential since several modulation sidebands can be exploited simultaneously (Fig. 5d). In some cases, for instance in NCACB transfer schemes, it is desirable to avoid promoting correlations between carboxylic and aliphatic carbons, to favour transfer processes between Ca and Cb carbons. Indeed, for the CaCb transfer, basic PARIS with N = , mrot = 39 kHz, m1 = 39 kHz, and sm = 100 ms performs 25% better than PARIS-xy with m = 2. In comparison to DARR, the enhancement for the CaCb transfer achieved with basic PARIS is 280%. This advantage of basic PARIS with respect to PARIS-xy results from the narrow-band character of basic PARIS with N = at very high spinning speeds. Both PARIS methods are efcient with proton rf amplitudes as low as m1 % mrot/3. At medium elds (B0 < 11.7 T) and high spinning frequencies (mrot > 40 kHz), the performance of PARIS-xy with N = is not optimum, since the modulation sidebands often lie outside the relevant spectral window. In such cases, it may be better to resort to PARIS-xy with N = 3=4 , which generates more closely spaced modulation sidebands. PARIS-xy with N = 3=4 also yields effective zerothorder recoupling of the heteronuclear dipolar interactions (see Fig. 5 in [13]).

5. Conclusions The novel PARIS-xy recoupling scheme can promote efcient broadband magnetization transfer at very high spinning frequencies. The new pulse sequence demonstrates high recoupling ef-

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Fig. 5. Carboncarbon correlation spectra of L-histidine measured at 21.2 T and (a) mrot = 60 kHz or (bd) mrot = 39 kHz. The magnetization exchange was promoted with (a) PARIS-xy with m = 1 (recoupling rf amplitude m1 = 25 kHz, mixing time sm = 175 ms), (b) DARR (m1 = 39 kHz, sm = 100 ms), (c) PARIS-xy with m = 2 (m1 = 39 kHz, sm = 100 ms) and (d), PARIS-xy with m = 2 (m1 = 20 kHz, sm = 200 ms). Cross-sections of Cb (I) and Ca (II) are shown for each spectrum. The cross-sections of (d) were normalised to the heights of the diagonal peaks in (c). The intensities of (a) are not directly comparable with (bd) as the cross-polarization efciency depends on the spinning speed. Heteronuclear decoupling with a proton rf eld amplitude m1 = 114 kHz was applied during both evolution and acquisition periods. Although this rf amplitude is close to 2mrot = 120 kHz (i.e., close to the n = 2 rotary resonance condition), excellent decoupling efciency has been obtained with PISSARRO decoupling.

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bands. The rf amplitude can be freely chosen since it does not need to match any resonance condition, and the method is very robust and exible over a wide range of spinning frequencies and static magnetic elds. The efciency, robustness and ease of implementation of PARIS-xy bode well for structural and dynamic studies of biomolecules while its low-power features appear particularly attractive for heat-sensitive samples such as hydrated proteins. Acknowledgments Financial support from the Agence Nationale de la Recherche (ANR) (Programme Blanc 2009) and from the Fdration de recherche (FR3050) Trs Grands Equipements de Rsonance Magntique Nuclaire Trs Hauts Champs (TGE RMN THC) of the CNRS is gratefully acknowledged. References
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Fig. 6. The fraction of exchanged magnetization for some representative crosspeaks in L-histidine as a function of the recoupling rf amplitude for PARIS-xy with m = 1 (blue circles, continuous line), basic PARIS using N = 2 (orange squares, dashed line), PDSD (black lozenges) and the phase-alternated version of MIRROR (green triangles). The latter was optimised for an exchange between Ca and C0 with Dm(C0 , Ca) = 22.33 kHz, by periodically reversing the phase after sp = 44.79 ls. All measurements were carried out at B0 = 17.6 T, mrot = 52 kHz and sm = 80 ms. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

ciency with low rf amplitudes over a broad range of isotropic chemical shift differences Dm(S, S0 ). The efciency of PARIS-xy can be attributed to the presence of a manifold of modulation side-