Minimizing the t1-noise when using an indirect 1H high-resolution detection of unlabeled samples.

The most utilized through-space correlation 1H-{X} methods with proton indirect detection use two consecutive transfers, 1H â†’ X and then X â†’ 1H, with the evolution time t1 in the middle. When the X isotope is not 100% naturally abundant (NA), only the signal of the protons close to these isotopes is modulated by the 1H-X dipolar interactions. This signal is theoretically disentangled with phase-cycling from the un-modulated one. However, this separation is never perfect and it may lead to t1-noise in case of isotopes with very small NA, such as 13C or even worse 15N. One way to reduce this t1-noise is to minimize, 'purge', during t1 the un-modulated 1H magnetization before trying to suppress it with phase-cycling. We analyze experimentally several sequences following the HORROR condition, which allow purging the 1H transverse magnetization. The comparison is made at three spinning speeds, including very fast ones for 1H resolution: 27.75, 55.5 and 111 kHz. We show (i) that the efficiency of this purging process increases with the spinning speed, and (ii) that the best recoupling sequences are the two simplest ones: XY and S1 = SR212. We then compare the S/N that can be achieved with the two most used 1H-{X} 2D methods, called D-HMQC and CP-CP. The only difference in between these two methods is that the transfers are done with either two π/2-pulses on X channel (D-HMQC), or two Cross-Polarization (CP) transfers (CP-CP). The first method, D-HMQC, is very robust and should be preferred when indirectly detecting nuclei with high NA. The second method, CP-CP, (i) requires experimental precautions to limit the t1-noise, and (ii) is difficult to use with quadrupolar nuclei because the two CP transfers are then not efficient nor robust. However, CP-CP is presently the best method to indirectly detect isotopes with small NA, such as 13C and 15N.

Accurate NMR determination of C-H or N-H distances for unlabeled molecules.

Cross-Polarization with Variable Contact-time (CP-VC) is very efficient at ultra-fast MAS (νR ≥ 60 kHz) to measure accurately the dipolar interactions corresponding to C-H or N-H short distances, which are very useful for resonance assignment and for analysis of dynamics. Here, we demonstrate the CP-VC experiment with (1)H detection. In the case of C-H distances, we compare the CP-VC signals with direct ((13)C) and indirect ((1)H) detection and find that the latter allows a S/N gain of ca. 2.5, which means a gain of ca. 6 in experimental time. The main powerful characteristics of CP-VC methods are related to the ultra-fast spinning speed and to the fact that most of the time only the value of the dipolar peak separation has to be used to obtain the information. As a result, CP-VC methods are: (i) easy to set up and to use, and robust with respect to (ii) rf-inhomogeneity thus allowing the use of full rotor samples, (iii) rf mismatch, and (iv) offsets and chemical shift anisotropies. It must be noted that the CP-VC 2D method with indirect (1)H detection requires the proton resolution and is thus mainly applicable to small or perdeuterated molecules. We also show that an analysis of the dynamics can even be performed, with a reasonable experimental time, on unlabeled samples with (13)C or even (15)N natural abundance.

Observation of proximities between spin-1/2 and quadrupolar nuclei: which heteronuclear dipolar recoupling method is preferable?

We have recently shown that the dipolar-mediated heteronuclear multiple-quantum coherence (D-HMQC) method allows observing through-space proximities between spin-1/2 ((1)H, (13)C, (31)P...) and quadrupolar ((23)Na, (27)Al...) nuclei. However, the D-HMQC effectiveness depends on the choice of the heteronuclear dipolar recoupling sequence. Here, we compare the efficiency and the robustness of four rotor-synchronized sequences: the symmetry-based ones, R4(1)(2)R4(1)(-2) and its super-cycled version, SR4(1)(2), and two schemes based on simultaneous amplitude and frequency modulations, denoted SFAM-1 and SFAM-2. For the SFAM methods, we point out efficient recoupling conditions that facilitate their experimental optimization and we introduce analytical expressions for the buildup of D-HMQC signal in the case of an isolated spin pair. We show that the main differences between these four sequences lie in the number of adjustable parameters and in their robustness with respect to chemical shift and homonuclear dipolar interactions. The relative performances of these four recoupling sequences are analyzed using average Hamiltonian theory, numerical simulations, and (27)Al-{(31)P} D-HMQC experiments on crystalline aluminophosphate.

Structural characterisation of phosphate materials: new insights into the spatial proximities between phosphorus and quadrupolar nuclei using the D-HMQC MAS NMR technique.

We show in this article how the spatial proximity between phosphorus and quadrupolar nuclei can be efficiently and easily investigated with the D-HMQC (Dipolar Hetero-nuclear Multiple-Quantum Coherences) NMR technique. Compared to the commonly used CP-HETCOR (Cross-Polarisation HETero-nuclear CORrelation) sequence, the D-HMQC pulse scheme exhibits a higher sensitivity and a better robustness with respect to spinning frequency, electronic shielding and quadrupole interaction, and thus does not require time-consuming and complicated optimisation procedures. The advantages of the D-HMQC are demonstrated in this article through the acquisition of (31)P/S through-space two-dimensional correlation NMR spectra providing unreported structural information on (i) a sodium alumino-silicate glass doped with only 3% of P(2)O(5), (ii) a potassium boro-phosphate glass containing BO(3) and BO(4) groups and (iii) a crystalline zirconium vanado-phosphate. All these systems, representative of the most important mixed phosphate network materials, cannot be correctly investigated with the conventional CP-HETCOR NMR technique.

Comparison of several hetero-nuclear dipolar recoupling NMR methods to be used in MAS HMQC/HSQC.

We compare several hetero-nuclear dipolar recoupling sequences available for HMQC or HSQC experiments applied to spin-1/2 and quadrupolar nuclei. These sequences, which are applied to a single channel, are based either on the rotary resonance recoupling (R3) irradiation, or on two continuous rotor-synchronized modulations (SFAM1 and SFAM2), or on four symmetry-based sequences (R2(1)1,SR4(1)2,R12(3)5,R20(5)9), or on the REDOR scheme. We analyze systems exhibiting purely hetero-nuclear dipolar interactions as well as systems where homo-nuclear dipolar interactions need to be canceled. A special attention is given to the behavior of these sequences at very fast MAS. It is shown that R3 methods behave poorly due to the narrowness of their rf-matching curves, and that the best methods are SR4(1)2 and SFAM (SFAM1 or SFAM2 if homo-nuclear interactions are not negligible). REDOR can also recouple efficiently hetero-nuclear dipolar interactions, provided the sequence is sent on the non-observed channel and homo-nuclear dipolar interactions are negligible. We anticipate that at ultra-fast spinning speed, SFAM1 and SFAM2 will be the most efficient methods.

Through-space MP-CPMAS experiments between spin-1/2 and half-integer quadrupolar nuclei in solid-state NMR.

We present a new CPMAS method that allows the acquisition of through-space 2D HETCOR spectra between spin-1/2 nuclei and half-integer quadrupolar nuclei in the solid state. It uses rotor-synchronized selective pulses on the quadrupolar nucleus and continuous-wave RF irradiation on the spin-1/2 nucleus to create hetero-nuclear dipolar coherences. The method is more robust, more efficient, and easier to set up than the standard CPMAS transfer.

Heteronuclear dipolar decoupling effects on multiple-quantum and satellite-transition magic-angle spinning NMR spectra.

We here report on the influence of heteronuclear dipolar decoupling on the (27)Al 3QMAS, 5QMAS, and the double-quantum filter-satellite-transition magic-angle spinning (DQF-STMAS) spectra of a strongly dipolar-coupled system, gibbsite. The requirements for heteronuclear dipolar decoupling increase with the order of coherence evolving in the indirect dimension of a two-dimensional (2D) experiment. The isotropic line width of the high-resolution 2D spectra, in samples like gibbsite, is composed of four parts: the distribution of isotropic shifts (delta(ISO), delta(QIS)), the homogeneous broadening related to the proton-proton flip-flop terms, the (27)Al-(27)Al homonulcear dipolar couplings, and the (1)H-(27)Al heteronuclear dipolar couplings. It is shown that, even in the case of gibbsite, where a strong proton-proton bath exists, the main resolution limiting factor in these experiments resides in the (1)H-(27)Al dipolar interaction.

HMQC and refocused-INEPT experiments involving half-integer quadrupolar nuclei in solids.

Hetero-nuclear coherence transfers in HMQC and refocused-INEPT experiments involving half-integer quadrupolar nuclei in solids are analyzed. 1D and 2D schemes are considered under MAS for the general case of multi-spin systems SI(n) (n4), where S is an observed nucleus. These results are also discussed in the context of high-resolution schemes featuring MQMAS or STMAS. The theoretical predictions are verified experimentally in a series of 1D and 2D experiments performed at 9.4 and 18.8T.

Through-space R3-HETCOR experiments between spin-1/2 and half-integer quadrupolar nuclei in solid-state NMR.

We present several new methods that allow to obtain through-space 2D HETCOR spectra between spin-1/2 and half-integer quadrupolar nuclei in the solid state. These methods use the rotary-resonance concept to create hetero-nuclear coherences through the dipolar interaction instead of scalar coupling into the HMQC and refocused INEPT experiments for spin n/2 (n>1). In opposite to those based on the cross-polarization transfer to quadrupolar nuclei, the methods are very robust and easy to set-up.

Resolution enhancement using a new multiple-pulse decoupling sequence for quadrupolar nuclei.

A new decoupling composite pulse sequence is proposed to remove the broadening on spin S=1/2 magic-angle spinning (MAS) spectra arising from the scalar coupling with a quadrupolar nucleus I. It is illustrated on the (31)P spectrum of an aluminophosphate, AlPO(4)-14, which is broadened by the presence of (27)Al/(31)P scalar couplings. The multiple-pulse (MP) sequence has the advantage over the continuous wave (CW) irradiation to efficiently annul the scalar dephasing without reintroducing the dipolar interaction. The MP decoupling sequence is first described in a rotor-synchronised version (RS-MP) where one parameter only needs to be adjusted. It clearly avoids the dipolar recoupling in order to achieve a better resolution than using the CW sequence. In a second improved version, the MP sequence is experimentally studied in the vicinity of the perfect rotor-synchronised conditions. The linewidth at half maximum (FWHM) of 65 Hz using (27)Al CW decoupling decreases to 48 Hz with RS-MP decoupling and to 30 Hz with rotor-asynchronised MP (RA-MP) decoupling. The main phenomena are explained using both experimental results and numerical simulations.

Measurement of through-space connectivities between spin-1/2 and quadrupolar nuclei in solid-state NMR: the TEDOR-MQMAS method.

We present the transferred echo double-resonance multiple-quantum MAS (TEDOR-MQMAS) method that allows to analyze under high resolution the through-bond connectivities between spin-1/2 and quadrupolar nuclei. This method avoids some of the limitations related to the spin-lock of half-integer quadrupolar nuclei under MAS. However, the losses observed during the TEDOR transfer are related to the T'(2) constants, and they may thus be more important than those observed during the CP-MAS transfer, which are related to T(1rho) > T'(2).

Homogeneous broadenings in 2D solid-state NMR of half-integer quadrupolar nuclei.

The question of the homogeneous broadening that occurs in 2D solid-state NMR experiments is examined. This homogeneous broadening is mathematically introduced in a simple way, versus the irreversible decay rates related to the coherences that are involved during t1 and t2. We give the pulse sequences and coherence transfer pathways that are used to measure these decay rates. On AlPO4 berlinite, we have measured the 27Al echo-type relaxation times of the central and satellite transitions on 1Q levels, so that of coherences that are situated on 2Q, 3Q, and 5Q levels. We compare the broadenings that can be deduced from these relaxation times to those directly observed on the isotropic projection of berlinite with multiple-quantum magic-angle spinning (MAS), or satellite-transition MAS. We show that the choice of the high-resolution method, should be done according to the spin value and the corresponding homogeneous broadening.

Implementing SPAM into STMAS: a net sensitivity improvement in high-resolution NMR of quadrupolar nuclei.

Gan and Kwak recently introduced two new tools for high-resolution 2D NMR methods applied to quadrupolar nuclei: double-quantum filtering in STMAS (DQF-STMAS) and the soft-pulse added mixing (SPAM) idea. Double-quantum filtering suppresses all undesired signals in the STMAS method with limited loss in sensitivity. With SPAM, all pathways are added constructively after the second hard-pulse instead of using a single pathway as previously. Here, the sensitivity, advantages and drawbacks of DQF-STMAS are compared to 3QMAS. Additionally, SPAM can be included into DQF-STMAS method, resulting in a net sensitivity gain with respect to 3QMAS of ca. 10-15.

Increasing the sensitivity of 2D high-resolution NMR methods applied to quadrupolar nuclei.

Gan and Kwak recently proposed a soft-pulse added mixing (SPAM) idea in the classical two-pulse multiple-quantum magic-angle spinning scheme. In the SPAM method, a soft pi/2 pulse is added after the second hard-pulse (conversion pulse) and all coherence orders in between them are constructively used to obtain the signal. We, here, further extend this idea to distributed samples where the signal mainly results from echo pathways and that from anti-echo pathways dies out after a few t1 increments. We show that, with a combination of SPAM and collection of fewer anti-echoes, an enhancement of the signal to noise ratio by a factor of ca. 3 may be obtained over the z-filtered version. This may prove to be useful even for samples with long T2' relaxation times.

Theoretical study of CP-VC: a simple, robust and accurate MAS NMR method for analysis of dipolar C-H interactions under rotation speeds faster than ca. 60 kHz.

We show that Cross-Polarization with Variable Contact-time (CP-VC) allows an accurate determination of C-H dipolar interactions, which permits an easy detailed analysis of bond lengths and local dynamics, e.g. in biomolecules. The method presents a large dipolar scaling factor of 1/√2, leading to a better determination of dipolar interactions, especially for long C-H distances, and it allows the observation of very small local details such as those related either to CH(2) three spin systems, or even to hydrogen bonds. CP-VC is very simple to set up and very robust with respect to most experimental parameters, such as: rf-offsets, chemical-shift anisotropies, imperfect Hartmann-Hahn setting, and rf-inhomogeneity. The only required condition is the use of a sufficiently fast MAS spinning speed of at least ca. 60 kHz.

Evaluation of human recombinant bone morphogenetic protein-2-loaded tricalcium phosphate implants in rabbits' bone defects.

Porous beta-tricalcium phosphate (betaTCP) has osteoconductive properties. The adsorption of human recombinant bone morphogenetic protein-2 (rhBMP-2) onto TCP could realize an osteoinductive bone substitute. We evaluated it on an animal model using dual-energy X-ray absorptiometry (DEXA) and solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. BetaTCP cylinders loaded with rhBMP-2 were implanted into rabbits' femoral condyle bone defects, and betaTCP alone as control into the contralateral femur. We studied two different doses of rhBMP-2 (10 and 40 microg) on two groups of four animals. Evaluation consisted in radiography, histology, and histomorphometry, DEXA, and NMR spectroscopy using an original method of quantification. With both doses of rhBMP-2, we observed on radiographs an increase of trabecular bone around implants. Histology showed resorption of the ceramic, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous betaTCP by new bone formed. Histomorphometry showed that the osteoid surface (OS/BS) was greatest with the high dose of rhBMP-2. The difference was slight between the low dose of rhBMP-2 and control. DEXA showed a dose-dependent increase of bone mineral density of rhBMP-2-loaded betaTCP vs. control. NMR spectroscopy confirmed that the amount of new bone formed in betaTCP was greater when betaTCP carried rhBMP-2, and increased with the dose of rhBMP-2 used. We showed that betaTCP was a good matrix for rhBMP-2, which gave it osteoinductive properties in an orthotopic site, in a dose-dependent manner. Thus, such composite biomaterial seems to be of great interest in reconstructive bone surgery. Further studies are needed in clinical practice to determine optimal doses.

On the conversion of triple- to single-quantum coherences in MQMAS NMR.

A systematic experimental and numerical evaluation of several basic approaches to multiple-quantum magic angle spinning (MQMAS) NMR is presented for spin-32 nuclei. The approaches use identical MQ excitation, via a single RF pulse of high power, and three types of methods for conversion to observable coherence: (a) nutation by strong continuous wave pulse; (b) rotation-induced adiabatic coherence transfer (RIACT), and (c) fast amplitude modulation (FAM-1). The optimization strategies and maximum achievable MQMAS efficiencies of (87)Rb in RbNO(3) and LiRbSO(4) are investigated using several coherence transfer schemes under a wide range of experimental parameters. These parameters include the strength of the RF magnetic field nu(RF), the sample rotation speed nu(R), the length of the conversion period, and the modulation frequency in FAM-1. The data provide new insights into the spin dynamics involved in these techniques and the experimental guidelines for achieving the best sensitivity. The RF requirements for maximum efficiency of conversion depend on the method to be used. In general, FAM-1 performs better than the nutation and RIACT methods in terms of efficiency and off-resonance behavior, especially when nu(RF) is small compared to the quadrupole frequency nu(Q). The experiments performed using nutation, RIACT, and FAM-1 methods yield similar resolution in the isotropic dimension, regardless of nu(RF).

Refinement of solid-state MAS NMR spectra of quadrupolar nuclei: application to the analysis of some 51V compounds.

Magic angle solid-state 51V NMR spectra of several vanadium powder compounds have been recorded. A refinement of these spectra using theoretical simulations of the powder spectra have been performed assuming the simultaneous anisotropic effects of electric field gradients (EFG) and of electronic shielding. It is shown that the relative orientation of the principal axes of the two tensors have to be considered.

Improving the resolution in proton-detected through-space heteronuclear multiple quantum correlation NMR spectroscopy.

Connectivities and proximities between protons and low-gamma nuclei can be probed in solid-state NMR spectroscopy using two-dimensional (2D) proton-detected heteronuclear correlation, through Heteronuclear Multiple Quantum Correlation (HMQC) pulse sequence. The indirect detection via protons dramatically enhances the sensitivity. However, the spectra are often broadened along the indirect F1 dimension by the decay of heteronuclear multiple-quantum coherences under the strong (1)H-(1)H dipolar couplings. This work presents a systematic comparison of the performances of various decoupling schemes during the indirect t1 evolution period of dipolar-mediated HMQC (D-HMQC) experiment. We demonstrate that (1)H-(1)H dipolar decoupling sequences during t1, such as symmetry-based schemes, phase-modulated Lee-Goldburg (PMLG) and Decoupling Using Mind-Boggling Optimization (DUMBO), provide better resolution than continuous wave (1)H irradiation. We also report that high resolution requires the preservation of (1)H isotropic chemical shifts during the decoupling sequences. When observing indirectly broad spectra presenting numerous spinning sidebands, the D-HMQC sequence must be fully rotor-synchronized owing to the rotor-synchronized indirect sampling and dipolar recoupling sequence employed. In this case, we propose a solution to reduce artefact sidebands caused by the modulation of window delays before and after the decoupling application during the t1 period. Moreover, we show that (1)H-(1)H dipolar decoupling sequence using Smooth Amplitude Modulation (SAM) minimizes the t1-noise. The performances of the various decoupling schemes are assessed via numerical simulations and compared to 2D (1)H-{(13)C} D-HMQC experiments on [U-(13)C]-L-histidine⋅HCl⋅H2O at various magnetic fields and Magic Angle spinning (MAS) frequencies. Great resolution and sensitivity enhancements resulting from decoupling during t1 period enable the detection of heteronuclear correlation between aliphatic protons and ammonium (14)N sites in L-histidine⋅HCl⋅H2O.