Correlation 2D-NMR experiments involving both 13C and 2H isotopes in oriented media: methodological developments and analytical applications.

Special issue review Received: 7 May 2014

Revised: 6 July 2014

Accepted: 7 July 2014

Published online in Wiley Online Library: 10 September 2014

(wileyonlinelibrary.com) DOI 10.1002/mrc.4118

Correlation 2D-NMR experiments involving both 13C and 2H isotopes in oriented media: methodological developments and analytical applications† Philippe Lesot,a* Olivier Lafonb and Philippe Berdaguéa Correlation 2D-NMR experiments for 13C and 2H isotopes turn out to be powerful methods for the assignment of the quadrupolar doublets in the 2H NMR spectra of isotopically modified (polydeuterated or perdeuterated) or unmodified solutes in homogeneously oriented solvents, such as thermotropic systems or lyotropic liquid crystals. We review here the different pulse sequences, which have been employed, their properties, and their most salient applications. These 2D-NMR sequences have been used for (i) 13C–2H correlation with and without 1H relay and (ii) 2H–2H correlation with 13C relay. The 13C–2H correlation experiments without 1H relay have been achieved for specifically deuterated or non-selectively deuterated analytes, but also more recently for isotopically unmodified ones thanks to the high sensitivity of very high-field NMR spectrometers (21.1 T) equipped with cryogenic probes. The 13C–2H correlation 2D-NMR experiments are especially useful for the assignment of overcrowded deuterium spectra because the 2H signals are correlated to 13C signals, which benefit from a much larger dispersion of chemical shifts. In this contribution, particular attention will be paid to the use of correlation 2D-NMR experiments for 2H and 13C nuclei in weakly aligning, polypeptide oriented chiral solvents, because these methods are useful and original tools for enantiomeric and enantiotopic analyses. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: heteronuclear 2D-NMR; quadrupolar interactions; deuterated molecules; (13C–2H)-isotopomers; chiral alignment media; thermotropics

General Introduction

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* Correspondence to: Philippe Lesot, RMN en Milieu Orienté, ICMMO, UMR-CNRS 8182, Université de Paris-Sud, Orsay, F-91405 Orsay CEDEX, France. E-mail: [email protected] †

This article is published in Magnetic Resonance in Chemistry as a special issue on the NMR of Liquid Crystals by Ronald Y. Dong (Department of Physics and Astronomy, UBC, Vancouver, Canada).

a RMN en Milieu Orienté, ICMMO, UMR-CNRS 8182, Université de Paris-Sud, Orsay, F-91405 Orsay CEDEX, France b Unité de Catalyse et de Chimie du Solide, UMR-CNRS 8181, Univ Lille 1, F-59652 Lille CEDEX, France Abbreviations: CDCOM, Carbon and Deuterium Correlation in Oriented Medium; CLC, Chiral Liquid Crystal; COMARO, COmposite Magic-Angle ROtation; DAPT, Dipolar Assisted Polarization Transfer; DECOR, DEuterium-Carbon CORrelation; NASDAC, Natural Abundance Spectroscopy for Deuterium and Carbon; PBLG, Poly-γ-BenzylL-glutamate; PBDG, Poly-γ-Benzyl-D-Glutamate; PCBLL, Poly-ε-Carbobenzyloxy-LLysine; QD, Quadrupolar Doublet; QUOSY, QUadrupole Ordered SpectroscopY; RQC, Residual Quadrupolar Coupling; SPINAL, Small Phase INcremental ALternation; TPPI, Time-Proportional Phase Incrementation; 5CB, 4-n-pentyl-4′-cyanobiphenyl.

Copyright © 2014 John Wiley & Sons, Ltd.

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Deuterium NMR spectroscopy on deuterated molecules or at natural abundance level (1.55 × 102% compared to 1H) noted thereafter natural abundance deuterium (NAD) is a powerful tool for a broad range of applications in (bio)chemistry. This includes the following: (i) the elucidation of chemical reaction mechanisms,[1,2] (ii) the determination of metabolic pathways,[3–6] (iii) the identification of geographical or botanical origin and the fight against counterfeiting,[7,8] (iv) the study of orientational order in anisotropic media (liquid crystals, membranes, and confined or stretched polymer chains),[9–16] and (v) the analysis of atom-scale dynamics in various systems, including biomolecules and advanced materials.[17–19] Recently, 2H NMR using chiral anisotropic solvents has been proposed as an efficient and versatile technique for stereochemical analysis.[20,21] Thus, this technique has been successfully applied: (i) discriminate enantiomers, including isotopic ones, and enantiotopic elements in prochiral molecules,[22] (ii) to measure enantiomeric excesses (ee) associated with asymmetric synthesis or biosynthetic pathways,[23] (iii) to determine the relative[24] and absolute[25] configuration of small molecules, (iv) to study conformational exchange,[26,27] and (v) to measure the isotopic profile of natural products.[28,29] Interestingly, 2H nuclei have a spin I = 1 and, hence, a quadrupolar electric moment, eQD, with e the elementary charge and QD = 0.286 fm2. This quadrupolar moment is small, and hence, 2H NMR spectra recorded in solutions or in mesophases benefit from high resolution. Furthermore, the low gyromagnetic ratio of 2H isotope

(γ(2H) = 0.153γ(1H)) leads to small 2H–2H dipolar coupling constants, even in perdeuterated solids.[30] Consequently, 2H NMR spectra of mesophases or solids often exhibit higher resolution than the 1H spectra.[31] In anisotropic fluids and solids, the 2H spectra are dominated by the quadrupolar interaction originating from the quadrupolar moment and the electric field gradient at the position of the 2 H nucleus. This interaction provides valuable information on molecular orientational ordering and dynamics. The flip side of

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the low value of γ(2H) is the weaker sensitivity of 2H NMR compared to 1H NMR. Nevertheless, the continuous improvements in NMR instrumentation and methodology (high magnetic field, cryogenic probe, advanced electronic, fast processing, …) have enabled the acquisition of NAD spectra in steadily lower concentration, in both isotropic and anisotropic fluids as well as in solids, even if the natural abundance of 2H is only 1.55 × 102%.[20,21,32] The assignment of one-dimensional (1D) 2H NMR spectra of polydeuterated or isotopically unmodified solutes (NAD NMR) is often difficult, owing to the limited range of 2H chemical shifts (20 ppm). Furthermore, this range in Hertz is only 15% of 1H one because γ(2H) = 0.153γ(1H). The assignment is even more challenging in chiral oriented media because the spectral enantiodiscrimination increases significantly the number of 2H signals. Various multidimensional (nD) NMR experiments have been proposed to facilitate the assignment of anisotropic 2H signals. These NMR experiments include the following: (i) 2H autocorrelation two-dimensional (2D) and three-dimensional (3D) methods, which correlate the two components of the quadrupolar doublets and permit to assign them on the basis of the 2H chemical shifts,[33–36] (ii) 2H homonuclear correlation 2D experiments via 2H–2H scalar (nJDD) and dipolar (nDDD) couplings[37–39] or with 13C relay,[40] and (iii) 13C–2H correlation 2D experiments, which correlate the 2H signal with that of the covalently bonded 13C nucleus and thus allow the assignment of 2H signals on the basis of 13C chemical shifts.[1–5,17,18,31,38,41–56] The assignment in 2H autocorrelation and homonuclear correlation methods is based on the 2H chemical shifts, which correspond to a narrow frequency range, as explained earlier. Furthermore, 2H–2H couplings are often too weak to produce visible correlation peaks, especially for weakly aligned solutes or for NAD experiments. 2H–13C correlation 2D experiments benefit from (i) the larger dispersion of 13 C chemical shifts (200 ppm), which is about tenfold larger in parts per million than that of 2H chemical shifts and (ii) the non-negligible 2 H–13C heteronuclear (1JCD and 1DCD) couplings between covalently bonded nuclei. Given the low natural abundance of isotopomers containing both 2H and 13C nuclei (1.55 102% × 1.1%), 2H–13C correlation 2D spectra have been mainly acquired for deuterated compounds. However, the first acquisitions of 2H–13C correlation 2D spectra of isotopically unmodified solutes dissolved in solutions or in weakly ordering liquid crystals have been recently achieved using high-field NMR spectrometer equipped with a cryogenic probe.[48] 2 H–13C correlation 2D-NMR experiments have been employed for solutes dissolved in isotropic liquids[1–5] or weakly aligned in chiral liquid crystals[38,45,46,48] as well as for mesogenic molecules of thermotropic liquid crystals[41–44,49] and for the solid state of aminoacids, peptides, and proteins.[17,18,31,50–56] For isotropic liquids, 2H–13C 2D correlation has been achieved using 2H → 13C INEPT transfer[1–5,57] because it is usually more sensitive than other methods, such as HMQC or HSQC.[4] Note also that 2H → 13C INEPT and DEPT 1D experiments as well as 2H → 13C and 13C → 2H cross-polarization (CP) ones using isotropic solvents have been reported in literature.[57–59] For liquid crystals and solids, 2H–13C correlation experiments have employed either 2H → 13C INEPT transfer,[38,45–48] 13C → 2H → 13C HMQC scheme,[43,44,50,52] 2H → 13C DAPT,[49] or 2H → 13C CP transfer.[17,18,31,41,42,51,53,56] For strongly aligned compounds or solids, the CP transfer is rendered difficult by the intricate spin dynamics of 2 H coherences in the presence of first-order quadrupolar interaction, which is about 170–210 kHz, and generally exceeds typical rf field strengths.[41,42,55,60] For solids under MAS conditions, it has been shown that CP transfer employing a train of rotor-synchronized pulses on the 2H channel are more efficient, more robust, and requires lower rf field than those employing continuous rf irradiation.[55,61]

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In this review, we present and discuss the correlation 2D experiments involving 2H and 13C isotopes in oriented media. We especially focus on the use of these NMR methods in weakly orienting chiral media. 2

H and 13C 1D NMR Spectra in Weakly Ordering Chiral Media

In the last two decades, various chiral oriented media, such as liquid-crystalline systems (polypeptide, DNA …)[62–66] or stretched polymer gels (gelatin, collagen, polysaccharrides…),[67,68] have been used as chiral orienting solvents for stereochemical NMR analysis. In these chiral orienting environments, two enantiomers (R/S) or enantiotopic directions (pro-R/pro-S) in prochiral molecules are oriented differently on average with respect to the magnetic field (Bo) of the NMR spectrometer. These different orientations result in distinct anisotropic NMR observables, such as RQC for 2H nuclei or chemical shift anisotropies (CSA) for 13C nuclei. Therefore, in chiral oriented solvents, enantiomers as well as enantiotopic directions exhibit in principle distinct proton-decoupled 2H (2H–{1H}) and proton-decoupled 13C (13C–{1H}) NMR signals (Fig. 1).[20,21,62] From an analytical viewpoint, 2H NMR in a CLC is the most NMR sensitive method for spectral enantiodiscrimination purposes,[69] mainly due to the large magnitude of 2H quadrupolar coupling constant, C DQ , which ranges from 170 to 210 kHz depending on the hybridization state of the covalently bonded carbon atom.[62] The average orientation of a given bond C–D is described quantitatively by the order parameter, SC D. In unwound uniaxial chiral nematic liquid crystals, such as those formed by PBLG in organic solvents, the molecules are homogeneously aligned, and the first-order quadrupolar interaction splits the 2H signal into one QD. The splitting of the doublet is called the RQC, and assuming no asymmetry parameter of the 2 H electric field gradient, the expression of RQC associated to an enantio-related pair of C–D vectors can be rewritten as 3 or B Δv AQi or B ¼ C DQi SACD i 2 A or B * + B0 2 2 1 3 e QD qCDi 3 cos θCDi ¼ 2 h 2

(1)

0 In Eqn (1), θBCD is the angle between the C–D direction and the i

static magnetic field B0, eqC–Di is the component of the electric field gradient along the C–D direction at the position of 2H nucleus, h is the Planck constant while the superscripts ‘A’ and ‘B’ stand for the stereodescriptors R and S (enantiomers) and pro-R and pro-S (enantiotopic directions). In chiral orienting solvents, the C–D bonds of distinct enantiomers or enantiotopic C–D bonds exhibit a priori different order parameter, and hence different RQCs according to Eqn (1) (Fig. 1a). This difference in RQCs corresponds to the spectral enantiodiscriminations in 2H–{1H} NMR spectra. Enantiodiscrimination in chiral ordering solvents has been first detected using 2H–{1H} NMR of selectively deuterated solutes.[20,21] However, this method requires the isotopic enrichment of studied analytes. Although numerous strategies exist, this preliminary step is often considered by chemists as either time-consuming or arduous. Alternatively, NMR techniques avoiding any chemical modifications of molecules were explored to overcome this limitation. These methods include proton-decoupled 19F (19F–{1H}) NMR for fluorinated compounds[70,71] or the 13C–{1H} NMR,[62,72–75] which is applicable for all isotopically unmodified organic and organometallic compounds. In this latter approach, the 13Ci sites of enantiomers or


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Anisotropic deuterium-carbon 2D-NMR

13

1

2

1

Figure 1. Schematic principle of the spectral enantiomeric discrimination in CLC using (a) C–{ H} and (b) H–{ H} 1D-NMR spectroscopy. The R/S assign2 13 ment of resonances is arbitrarily defined. Similar spectral patterns are obtained for a pair of enantiotopic H or C nuclei (or group of nuclei).

the enantiotopic 13Ci sites can be spectrally differentiated on the basis of 13C CSA’s according to Eqn (2).[62] νAi or B ¼

γ A or B 1 σ iso B0 i Δσ i 2π

(2)

In the aforementioned equation, σ iso and Δσi are the isotropic i and anisotropic contributions to the electronic shielding around the 13C nucleus, respectively. Δσi depends on the average orientation of the principal axis system of the CSA tensor for 13Ci site. When the difference, ΔσAi ΔσBi, is larger than the linewidths, two spectrally distinct 13C resonances are observed on 13C–{1H} 1D spectra as shown in Fig. 1b. The efficiency of the method depends both on the strength of the magnetic field B0 (Eqn 2) but also the hybridization state of the 13C atoms; the best separations being generally observed for sp2 and sp carbons.[62] The main advantage of the proton-decoupled natural abundance 13 C NMR stems from the detection of all 13C isotopomers associated to (chiral) molecule in a single experiment. Hence, each 13C site becomes a potential spy to visualize the spectral enantiodifferentiation in a CLC. From 1998, this principle was successfully transposed to the case of 2H–{1H} 1D-NMR at natural abundance level by Lesot et al.[76] We discuss in the succeeding text correlation 2D NMR experiments involving 2H and 13C nuclei in chiral ordering solvents.

HETCOR Scheme Versus HSQC/HMQC Schemes 2

H–13C correlation for ordered solutes can be achieved using various schemes, such as 2H → 13C HETCOR sequences based on INEPT

transfer[38,45–48] (Fig. 2a) as well as 13C → 2H → 13C HSQC and HMQC schemes.[44,50,52] In the succeeding text, 2H–13C HETCOR, HMQC, and HSQC denote sequences for which the indirectly detected F1 dimension corresponds to 2H dimension and the acquisition F2 dimension corresponds to 13C dimension. 13C–2H HETCOR, HMQC, or HSQC notations refer to the reverse situation. We show in the succeeding text that 2H–13C correlation based on HETCOR scheme is usually more sensitive than those based on HMQC or HSQC. Under ideal conditions, the sensitivity of HETCOR, HMQC, and HSQC 2D experiments is proportional to[48,77]

S 1 TR pffiffiffiffiffiffiffiffi ∝f ϕγðexcÞγ3=2 ðdetÞ pffiffiffiffiffi 1 exp exc T1 N T tot TR (3)

where S/N is the signal-to-noise ratio, Ttot is the total experiment time, γ(exc) and γ(det) are the gyromagnetic ratios of the excited and detected isotopes, respectively, TR is the recycling delay, that is, the average delay between two consecutive experiments, and T1exc is the relaxation time of z-magnetization for the excited isotope. ϕ = 1 for 2D spectra displayed in phase mode and ϕ = 1/√2 for 2D spectra displayed in magnitude mode and f is the efficiency of the magnetization transfer between the excitation and the detection nuclei. Compared to a 13C/1H system, the case of 13C and 2H isotopes is different for two reasons: (i) the absence of NOE from 1H to 2H nuclei because the relaxation of 2H nuclei is mainly governed by 2 H quadrupolar interaction and not by the 1H–2H dipolar

1

1

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Figure 2. (a) Basic H-decoupled Y-X HETCOR 2D-NMR scheme with ϕ 1 = {x,x}, ϕ 2 = {x,x}, ϕ 3 = {x,x}, and ϕ 4 = {x,x}. H decoupling during acquisition can be performed with WALTZ, GARP, COMARO, COmposite Magic-Angle Rotation or SPINAL, Small Phase Incremental Alternation composite pulse sequences. To 1 limit the heating of the samples, the H decoupling can only start just before the first 90° pulse. (b) Dependence of the sensitivity as function of the TR delay for 2 13 2 13 13 2 13 2 H– C HETCOR (continuous line), H– C HMQC/HSQC (dot-dash line), C– H HETCOR (dashed line), and C– H HMQC/HSQC (dotted line). The sensitivity is 2H 13C calculated from Eqn (3), assuming T1 = 0.25 s and T1 = 1.5 s. Figure is adapted from Refs. (38) and (48).

P. Lesot, O. Lafon and P. Berdagué interaction[78] and (ii) the spin value I = 1 of 2H nuclei. Two important consequences derive from the integer spin of 2H. First, the difference in transfer efficiencies between the various 2H–13C correlation methods stems from the higher efficiency of 2H → 13C transfer compared to 13C → 2H transfer because the central component of 13C triplet coupled to 2H nuclei does not evolve under the onebond 13C–2H total coupling, 1TCD, sum of one-bond 13C–2H J and dipolar couplings, and hence cannot be transferred to 2H. Thus, the f prefactor is equal to 4/3 for 2H–13C HETCOR; 1 for 13 C–2H HETCOR, HMQC, and HSQC; and 2/3 for 2H–13C HMQC and HSQC schemes.[48] Second, owing to the evolution of 2H singlequantum coherences under the effect of quadrupolar interaction (I = 1) during the defocusing and refocusing delays, the contour plot of 2H–13C and 13C–2H HETCOR as well as 13C–2H HMQC and HSQC experiments cannot be phased in pure absorption. Hence, these 2D spectra must be displayed in magnitude mode resulting in a loss by a factor of √2 in sensitivity (ϕ = 1/√2 in Eqn 3). Actually, only the 2H–13C HMQC and HSQC schemes can be phased in pure absorption and then benefits from ϕ = 1; however, these schemes remain unfavorable in terms of overall sensitivity. Figure 2b indicates clearly that 2H–13C HETCOR sequence with 13 C detection displays the highest sensitivity, especially for short TR delays. Assuming a T1(2H) value equal to 0.25 s, the optimal sensitivity for 2H–13C HETCOR scheme is obtained for TR ≈ 0.3 s. However, as simultaneous 1H and 2H decoupling is applied during the acquisition period of these experiments, too short recycle delays (TR ≈ 0.3 s) result in a high duty cycle for the rf field, which is incompatible with the high temperature stability required for anisotropic samples or even with the rf specifications of a cryogenic probe. Even using suboptimal conditions (TR ≈ 0.6–1 s), the sensitivity of 2H–13C HETCOR is still 2.3–1.7 times larger than that of 2H–13C HMQC and HSQC with 13C detection. Furthermore, for weakly aligned solutes (PBLG system), the width of 2H–{1H} spectra is usually narrower than that of 13C–{1H} spectra. Hence, the sequence for which F1 dimension corresponds to the 2H dimension, such as 2H–13C HETCOR scheme, requires a smaller number of increments for the indirect evolution period, t1, and hence results in shorter acquisition times. Finally, HETCOR-type and HSQC-type experiments benefit from narrower linewidths in the F1 dimension than those for the HMQC ones. Indeed, in the latter scheme, the linewidths are mainly governed by the relaxation rate of multiple quantum coherences, which often lead to broadening because multiple quanta usually relax faster than single-quantum coherences.[79]

2

H–13C Correlation 2D Experiments on Isotopically Enriched Solutes

The CDCOM 2D method

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For weakly aligned (polydeuterated or perdeuterated) solutes, 2 H–13C correlation has been achieved using the proton-decoupled 2 H–13C HETCOR 2D sequence with 13C detection. This method has been named CDCOM.[38] The basic scheme is shown in Fig. 2a. The 180° pulse on 13C channel in the middle of the indirect evolution period, t1, refocuses the total 13C–2H couplings, 1TCD, which correspond to the sum of scalar coupling, 1JCD, and dipolar ones, 1DCD (1TCD = 1JCD + 21DCD). The 1TCD couplings are also eliminated during the acquisition by applying a composite pulse decoupling scheme on the 2H channel. To remove artifacts and reduce offset effects, the first 180° pulse can be replaced by a 180° composite, adiabatic, or universal rotation pulse.[80–83]


After a two-step phase cycle and disregarding all relaxation terms and phase factors, the simplest expression of the NMR signal, S(t1,t2), for CDCOM signal associated to coupled 2H–13C pair is Sðt1 ; t2 Þ ∝f cos½ð2πνD πΔνQ Þðt1 þ τ Þ þ cos½ð2πνD þ πΔνQ Þðt1 þ τ Þg sin π 1 T CD τÞ sin 2π 1 T CD τ’Þ exp½i2πνC ðτ’ þ t 2 Þg (4) In this equation, νD and νC denote the 2H and 13C frequencies while ΔνQ and 1TCD denote the 2H RQCs and the 13C–2H total spin-spin couplings, respectively, each of them being expressed in Hz. The double FT of this signal allows us to correlate 13C chemical shifts in the F2 dimension to the 2H QDs centered on their respective chemical shifts in the F1 dimension. Using standard spectrometers (7–14 T) and conventional probes, CDCOM 2D experiments can be acquired with acceptable S/N and suitable F1 resolution within 10 h with a number of scans varying between 64 and 128, depending on the solute concentration. Analysis of CDCOM maps of perdeuterated chiral molecules Carbon and deuterium correlation in oriented medium 2D experiments are particularly useful to simplify the analysis of (chiral or achiral) molecules having long alkyl chains, because for these compounds, the dispersion of 2H chemical shifts is generally small. To illustrate this point, Fig. 3 compares the dispersion of QDs on the 2D map along the F2 dimension on the CDCOM and tilted Q-COSY Fz 2D map of (±)-[2H15]-2-ethylhexanoic acid dissolved in a polypeptide chiral mesophase. As seen, the dispersion of 13C signals is about 30-fold larger than that of 2H signals along the F2 dimension of the tilted Q-COSY Fz 2D map. This effect is particularly visible for the methyl groups 6 and 8 (Zoom II of Fig. 3a). Thus, the QDs associated to D6 and D8 methyl sites are perfectly separated in the CDCOM 2D spectrum but not in the tilted Q-COSY one. Another advantage of the CDCOM experiment compared to tilted Q-COSYtype (or tilted Q-resolved-type) experiments lies in the possibility to correlate the pair of QDs corresponding to the diasterotopic 2H sites of a given methylene group for chiral compounds. For instance, the four QDs of diasterotopic 2H sites are correlated to a single 13C resonance.[38] Besides, the univocal assignment of 13C signals in this example permits a direct assignment of QDs, while the assignment on the basis of 2H chemical shift remains ambiguous. As a second illustrative example, Fig. 4 shows an example of F1symmetrical CDCOM 2D map recorded for another perdeuterated chiral molecule, the (±)-[2H17]-2-ethylhexanol oriented in the chiral mesophase PBLG/CHCl3. In this refocused variant of the CDCOM experiment (denoted R-CDCOM), the 2H chemical shifts are refocused in the indirect dimension by shifting the 180° pulse from the 13C channel to the 2H channel. This modification leads to symmetrical 2 H spectrum in F1 dimension. The reasons and the advantages in refocusing δ(2H) in t1 dimension of the CDCOM will be examined in details in the section entitled “2H–13C Correlation for Isotopically Unmodified Solutes: a New Frontier”. This R-CDCOM spectrum has been recorded at 21.1 T using the cryoprobe technology. Interestingly, the high sensitivity of such an instrument has permitted to reduce significantly the number of scans (here, four scans are added per t1 increment), leading to the total acquisition time of 30 min. Similarly to the first example reported in the preceding text, the CDCOM map of (±)-[2H17]-2-ethylhexanol oriented in a chiral mesophase allows to spread the QDs on a large spectral range


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2

Figure 3. Comparison between the (a) 9.4 T CDCOM and (b) 9.4 T tilted Q-COSY map of (±)-[ H15]-2-ethylhexanoic acid oriented in the PBLG/CHCl3 system 2 at 300 K acquired with a triple-resonance inverse probe (TXI Bruker model) and selective excitation H probe, respectively. In F1, the projection of both maps 2 1 displayed the H–{ H}1D-NMR spectrum. In subfigure 3a, the zoom I shows the region corresponding to inner quadrupolar doublets associated with C-3 (for which the intensity values of the contour levels are four times lower than in the whole spectrum) and the zoom II shows the region corresponding to the signal of methyl groups (for which the intensity values of the contour levels are eight times higher than in the whole spectrum). In subfigure 3b, the zoom I displays the region corresponding to the signals of the methyl groups. Full experimental details can be found in Ref. 38. Figure is adapted from Ref. (38).

2

Figure 4. The 21.1 T R-CDCOM 2D spectra of (±)-[ H17]-2-ethylhexanol oriented in PBLG/CHCl3 at 305 K acquired with triple-resonance inverse cryoprobe 2 (TCI Bruker model). Here, the H chemical shifts have been refocused during the t1 dimension. The 2D spectrum has been recorded as a 6400 (t2) × 512 (t1) data matrix with four scans per t1 increment, TR = 0.6 s and τ = 19 ms. The zoom (b) displays an expansion of the region of methyl groups where the presence of cross peaks associated to CD2H and CD3 groups is resolved (see text). Unpublished results.

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effects on the 13C chemical shifts will be discussed in the section “2H–13C Correlation for Isotopically Unmodified Solutes: a New Frontier”. Revealing the binuclear spectral enantiodiscriminations As explained in 2H and 13C 1D NMR Spectra in Weakly Ordering Chiral Media section (Eqns 1 and 2), the spectral discriminations of enantiomers or enantiotopic directions in prochiral molecules can be detected on the basis of the difference of 2H RQC and/or 13 C CSA.[62] Thus, the 2H and 13C signals associated with a coupled



599

(70 ppm) in the F2 dimension, thus facilitating their assignment on the basis of 13C chemical shifts. As mentioned previously, the assignment of various QDs associated to diastereotopic 2H sites is clearly facilitated on the map (see signals of methylene group 1). More interestingly, we can see in this second example how the CDCOM experiments can separate the 2H–13C correlation peaks associated to fully deuterated and partly deuterated groups. This effect is particularly evident for the methyl groups (see the zoom in Fig. 4). Such a distinction is basically impossible with 2H homonuclear QUOSY experiments. Another example illustrating the 2H isotope

P. Lesot, O. Lafon and P. Berdagué 2

H–13C pair can be enantiodifferentiatied for both nuclei, simultaneously. Such spectral occurrences are named ‘binuclear spectral enantiodiscriminations’.[45] For a scalemic mixture of enantiomers (namely, an enantiomeric mixture with 0 < ee < 100%), the coupled R/S assignments of 2H and 13C resonances are trivial due to the difference of peak intensity originating from the ee. For a racemic mixture (ee = 0%), the intensities (or integrals) of 2H and 13C signals corresponding to the enantiomers are identical, and hence, only 2D correlation experiments allow then to pair up the 2H and 13C signals belonging to the same enantiomer. Nevertheless, the absolute configuration of signals remains unknown. Similarly, in prochiral compounds, the NMR signals of enantiotopic nuclei have identical intensities, and 2H–13C correlation is one of the only method that is able to pair up the 2H and 13C signals for enantiotopic pairs in prochiral compounds. Binuclear spectral enantiodiscriminations revealed on a CDCOM 2D map have been observed so far in the case of a monodeuterated chiral molecule, the (±)-[1-2H]-1-octyn-3-ol, and a perdeuterated prochiral aromatic molecule, the [2H11]dibenzylmethanol, both dissolved in a polypeptide CLC (Fig. 5). In the first example, the analysis of CDCOM map shows that (i) only the acetylenic carbon atom bearing the 2H nucleus exhibits heteronuclear cross-correlation on the 2D map (Fig. 5a), indicating that the adequate choice of τ and τ′ values achieves a selective one-bond 2H–13C magnetization transfer and (ii) the existence of two pairs of 2H–13C correlations located on two distinct 13C schemical shifts indicates clearly that the chiral discrimination occurs on both nuclei, (iii) the outer and inner QD (ΔνAQ and ΔνBQ ) correlate with the shielded carbon signal (δΑ) and the deshielded carbon atom (δΒ), respectively. From an analytical viewpoint, it exists a

mathematical relationship between the relative anisotropic chemical shift in ppm of the acetylenic 13C resonances (δΑ and δΒ) and the relative magnitude of 2H quadrupolar doublets (ΔνAQ and ΔνBQ ), assuming that the quadrupolar and nuclear shielding tensors are axial and collinear for the alkyne fragment (Fig. 5a): ΔνAQ ΔνBQ ¼

9 C DQ A e2 QD qCD δ δB with C DQ ¼ 4 ζ h

(5)

where ζ is the main component of the electronic screening (expressed in ppm) along the C–D bond.[45] Interestingly, from Eqn (5), it is possible to determine the absolute sign of the RQCs in alkyne fragment when spectral enantiodiscrimination is observed on both nuclei and the 2H and 13C signals are correlated. The analysis of CDCOM map of the [2H11]-dibenzylmethanol has evidenced that the most shielded 13C resonances correlate with the largest deuterium quadrupolar doublet for both the orthocarbon and meta-carbon atoms, but not for the para-carbon atom (Fig. 5b). This opposite behavior proves that there is no simple relationship between the magnitude of the 2H quadrupolar doublets and chemical shifts of covalently bonded 13C nucleus, when the 13 C shielding tensor is not of axial symmetry. Conclusively, these binuclear spectral enantiodiscriminations are useful to investigate orientational behavior. Conformational analysis of perdeuterated cis-decaline The combined analysis of the conformational dynamics and the orientational behavior of flexible cyclic chiral solutes using NMR spectroscopy in CLC is a noteworthy challenge. Recently, such a work has been performed in the case of the perdeuterated cis-decalin, a

2

2

600

Figure 5. A 9.4 T proton-decoupled CDCOM 2D map of (a) (±)-[1- H]-1-octyn-3-ol and (b) [ H11]-dibenzylmethanol, showing the simultaneous 13 2 2 enantiodiscrimination for both C and H NMR signals. Note that no C–D correlations were observed for non- H labeled carbon atoms. Experimental details can be found in Ref. (45). Figure is adapted from Ref. (45).



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Anisotropic deuterium-carbon 2D-NMR molecular structure of biochemical interest, because its derivatives are commonly encountered in several natural products.[46,84] The cis-decalin is an apolar bicycloalkane, which interconverts between two enantiomeric conformers of C2 symmetry (that we note thereafter CDC-a and CDC-b).[84] As the activation enthalpy of this process is around 120 kJ mol1 (about 50-fold larger than the thermal energy at 300 K), the exchange between the two stereoisomers can be almost frozen, on the NMR time scale, at low temperatures (T < 250 K).[85,86] Consequently, far below the coalescence point, Tc, the 2H signals of two enantiomers of cisdecalin can be spectrally discriminated when a CLC is used as NMR solvent. In contrast at high temperature, far above Tc, the racemization process between CDC-a and CDC-b is fast enough on the NMR timescale, and yields a single high-resolution 2H spectrum for both isomers. Interestingly, the 2H spectrum far

2

13

Figure 6. 3D structures of cis-[ H18]-decaline along with the C atomic numbering and symmetry elements. Top: enantiomeric conformers (CDCa and CDC-b) of C2 symmetry that can be spectrally differentiated using NMR in CLC at low temperature. Bottom: average structure (CDC-c) of C2v symmetry in which enantiotopic elements can be spectrally differentiated using NMR in CLC at high temperature. Figure is adapted from Ref. (84).

above the coalescence temperature can be analyzed by considering an ‘average 3D structure’ of C2v symmetry denoted here as CDC-c.[46] The three 3D structures of cis-decalin and associated elements of symmetry at high and low temperatures are depicted in Fig. 6.[46] The CDC-c structure predicts the existence of enantiotopic 2H–13C directions that are susceptible to be spectrally discriminated in a CLC. However, it cannot be applied to determine consistent orientational order parameters because the quantitative analysis of enantiodiscrimination for flexible molecules in the fast exchange regime requires the determination of the orientational order parameters of each conformer.[46] Figure 7 shows that 2H–{1H} and 13C–{2H} signals of cis-decaline dissolved in the chiral PBLG mesophase strongly differ between 243 and 356 K. In this example, the CDCOM 2D experiments were efficiently used to help the assignment of the 2H–{1H} spectra. Here again, the CDCOM technique offers a much greater resolving power compared to QUOSY experiments. This situation avoids any mis-assignment of QDs in F1 dimension that would be unsuitable to correctly determine the order parameters of the Saupe matrix and then analyze the orientational behavior of cis-decaline enantiomers at 243 K.[46] Besides, the CDCOM experiments allow pairing up the QDs associated to axial and equatorial geminal deuterons of each methylene group, even if the absolute assignment remains impossible. The analysis of CDCOM maps at 243 K shows that CDC-a and CDC-b stereoisomers are spectrally discriminated along the 2H dimension because a doubling of all QDs is observed in chiral mesophase compared to achiral mesophase, containing an equimassic mixture of PBLG and PBDG (PBLG enantiomer). Although the spectral differentiation of CDC-a and CDC-b based on differences of 13C CSA is theoretically possible (see the section “2H and 13C 1D NMR Spectra in Weakly Ordering Chiral Media”), two main reasons explain the absence of enantiodiscrimination in 13 C dimension: (i) the 13C CSAs are generally very small for sp3 carbons in apolar compounds, and (ii) the low temperature of the sample increases significantly the linewidths, masking the smallest enantiodiscriminations. At 356 K, the number of QDs and 13C resonances is reduced in agreement with the proposed average structure, which possesses higher symmetry (C2v) than the individual enantiomer (C2). Thus, only three 13C resonances (junction site and α and β sites) are observed (instead of five ones at 243 K). Except for the 2H–13C pairs associated to the junction site that is

2

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601

Figure 7. The 9.4 T CDCOM 2D spectra of cis-[ H18]-decaline in PBLG/CHCl3 at (a) low temperature (243 K) and (b) high temperature (356 K) recorded in around 2 1 15 h. For both maps, the true 2D projection is displayed on the F2 dimension while for a better resolution of peaks, the H–{ H} 1D spectrum is shown in the F1 dimension. Note that in Figure 7a, the largest doublet of site β2 is not shown. Experimental details can be found in Ref. (46). Figure is adapted from Ref. (46).

P. Lesot, O. Lafon and P. Berdagué homotopic and insensitive to the conformational exchange, the 13C resonances of α and β sites are expected to be correlated with two pairs of 2H QDs in chiral mesophase, each pair corresponding to the geminal deuterons of a methylene group (see Fig. 6, bottom). Experimentally, only the 13C signal associated with the α sites (1,5/4,8) presents a correlation with four doublets. For the β sites, the discrimination of 2H–13C enantiotopic directions is very weak (e,e′) or null (b,b′), and only two doublets are observed (Fig. 7). Thus, the postulated average structure of C2v symmetry allows an easy qualitative interpretation of the CDCOM 2D spectrum in the fast exchange regime. 1

H Relayed 13C–2H Heteronuclear Correlation 2D Experiments

Problem statement The evaluation of enantiomeric and diastereomeric excesses (ee and de) in mixtures of dideuterated unlike/like (u,l) stereoisomers with two remote stereogenic centers of different stereochemistry for each asymmetric carbon is an interesting and challenging analytical problem from the NMR viewpoint. Examples of typical molecules of interest are shown in Fig. 8. In these examples, the unlike and like isomers correspond to the achiral compound (RS) (denoted also meso form) and the enantiomers (RR/SS), respectively. For such mixtures, the discrimination of enantiomers (RR/SS) is basically impossible using isotropic NMR, because generally, achiral solvents are used. Additionally, the spectral discrimination of diastereoisomeric forms (u/l) in isotropic solutions is not always guaranteed, in particular, when the stereogenic centers are far from each other as in the case of mixtures 1 and 2 (Fig. 8a).[47] One possible analytical solution for deuterated solutes consists in using 2H–{1H} or 13C–{1H} 1D NMR (deuterated solutes) in CLC that can afford efficient way to separate the signals of three stereoisomers of mixture (the meso compound and the two enantiomers) on the basis of 2 H RQCs or 13C CSA differences.[62] Examples of schematic spectral situations showing the discrimination of all species using 2H–{1H} or 13C–{1H} are displayed in Fig. 8b. Assuming that diastereoisomers and enantiomers for mixtures 1 or 2 are discriminated in a CLC, four distinct quadrupolar doublets are expected to be observed on 2H–{1H} spectra, two of them

originate from the discrimination of enantiomers, two others originating from the discrimination of enantiotopic deuterons of the meso isomers. From the 13C–{1H} NMR point of view, the mixtures 1 and 2 must be considered separately. For mixture 1, four 13C resonances are expected to be observed for each nonequivalent carbon site if the three stereoisomers are spectrally discriminated on the basis of 13C CSA (Fig. 8b). For mixture 2, four 13C resonances are expected for sites 1–5/2–4, but only three resonances for sites 3 and 6 as these latter ones belong to the symmetry plane in the meso molecule (Fig. 8a), and hence do not exhibit enantiotopic discriminations. The most difficult situation for the analysis of (u,l)-mixtures occurs when the ee and de are both null. In that particular case, four QDs of equal intensity are expected, which makes difficult their assignment. Various strategies have been developed to solve this problem. These assignment strategies are all based on the fact that only the meso compound possesses two deuterons that can be potentially correlated. Three possibilities can be proposed: (i) the use of 2H–2H correlations through direct dipolar couplings, (ii) the 2 H–2H correlations via multiple polarization transfers involving heteronuclear atoms (1H or 13C) as relay, using ‘DHD’ and ‘DCD’ experiments (see the section “13C Relayed 2H–2H Homonuclear 2D Experiments for Weakly Aligned Solutes”), and (iii) the use of 1H relayed 13 2 C– H correlation if a specific spectral property exists, which can be exploited in 13C NMR (Fig. 9). The 1H relayed 13C–2H correlation 2D experiments denoted ‘D(H)nC’ (with n = 1, 2) involve two heteronuclear polarization transfers with one or two proton relays (2Hi → 1Hj → 13Ck or 2Hi → 1Hj → 1Hk → 13Cl).[47] Schematic pulse sequences of these 13 2 C– H correlation experiments with a single or multiple 1H relay are depicted in Fig. 9 and commented in the succeeding text.

Description of the pulse schemes The basic principle of these sequences is to transfer the 2H polarization to 13C nuclear via one or two 1H relays using INEPT-type transfers. Another method would consist in the creation of effective trilinear coupling terms, but to the best of our knowledge, this method has not been reported yet for 1H-relayed 2H–13C correlation experiments.[87] Figure 9a and 9b shows the pulse schemes of ‘DHC’ 2D experiments relying on INEPT transfer. The first INEPT block transfers 2H magnetization into 1H single-quantum coherence (SQC’s),

602

Figure 8. (a) Two examples of structures of (u,l)-type isomers containing remote asymmetric centers around an aromatic core: the case of 1,41 4 2 1 3 2 benzenedimethan-α -α -[ H2]-ol (mixture 1) and the 1,3-benzenedimethan-α -α -[ H2]-ol (mixture 2). Because of the C2 axis in the enantiomers (RR and 2 1 13 1 SS), the two deuterons are homotopic, and so magnetically equivalent. (b) Schematic H–{ H} and C–{ H} 1D spectral patterns expected to be observed for various stereoisomers in mixtures 1 and 2 when dissolved in chiral mesophases. All compounds are assumed to be spectrally discriminated, but the as13 signment shown is arbitrary. The magnitude of ΔνQ’s as well as the position of C peaks is arbitrarily chosen. Figure is adapted from Ref. (47).



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Figure 9. Pulse schemes of (a and b) the ‘DHC’ and (c) ‘DHHC’ 2D experiments using two INEPT-type transfers. The ‘DHC’ sequence (a) is optimal when ∣2TDH∣ > ∣TCH∣, whereas the sequence (b) must be used when ∣2TDH∣ < ∣TCH∣. For the ‘DHHC’ sequence, we assume that |THH| < |TCH|. Phase cycles for a, b, and c are given in Ref. (47). (d) ‘D(H)nC’ sequence including an MLEV spin-lock sequence (flanked by two trim pulses). Figure is adapted from Ref. (47).

when setting τ 1 = τ 2 + τ 3. As seen in the aforementioned equation (lines 2 and 3), the effective evolution periods due to couplings TDH and TCH are equal to (τ 1 τ 2 + τ 3) and (τ 1 + τ 2 + τ 3) for the ‘DHC’ sequence of Fig. 9a, and (τ 1 + τ 2 + τ 3) and (τ 1 τ 2 + τ 3) for the ‘DHC’ sequence of Fig. 9b.

assuming that |TH k H l| < |TC j H l| and τ 2 = τ 3 + τ 4 in order to eliminate the evolution under 1H chemical shifts. In the simple case where m = n = 1, the ideal values for the refocusing periods τ 2, τ 3, and τ 4 are as follows: τ 2 = 1/∣4THH∣, τ 3 = 1/∣4THH∣ 1/∣4TCH∣ and τ 4 = 1/ ∣4TCH∣. The ideal value for delay τ 1 results in a subtle compromise

f corr ðτ DH ; τ 1 ; τ 2 ; τ 3 ; τ CH Þe i½2πνC ð τDH þ

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t 2 Þ



603

In this equation, the correlation function, fcorr(τ DH, τ 1, τ 2, τ 3, τ CH), is denoted hereafter fcorr({τ p}). For a ‘Di–Hkn–Hlm–Cj’ spin system where Hk and Hl protons are weakly coupled, its analytical expression is 8 9 sin πT Di Hk τ DH cosn1 πT Di Hk τ DH > > > > > > > > > > sin 2πT i k ðτ 1 ±τ 2 þ τ 3 Þ > > DH > > > > > > > > < = sin πT Cj Hk ðτ 1 ∓τ 2 þ τ 3 Þ 2 f corr τ p ¼ n cosn1 3πD k k ðτ þ τ þ τ Þ (7) 1 2 3 > > H H > > > > > > > > ∏ cos πT Hk Hl ðτ 1 þ τ 2 þ τ 3 Þ > > > > > > > l > > > : ; n1 sin πT Cj Hk τ Cj Hk cos πT Cj Hk τ Cj Hk

When n = 1 and m = 0, fcorr({τ p}) of the ‘DHC’ sequence of Fig. 9b is maximal when τ DH = 1/∣2TDH∣, τ 1 = 1/∣8TDH∣, τ 2 = 1/∣8TDH∣ 1/ ∣4TCH∣ with ∣2TDH∣ < ∣TCH∣, τ 3 = 1/∣4TCH∣ and τ CH = 1/∣2TCH∣. For the ‘DHC’ sequence of Fig. 9a, optimal conditions are obtained when using τ 1 = 1/∣4TCH∣, τ 2 = 1/∣4TCH∣ 1/∣8TDH∣ and τ 3 = 1/∣8TDH∣. To transfer 2H magnetization to a remote carbon atom through a pair of coupled protons, the solution consists of implementing a 90° pulse at the end of the refocusing delay of 1H–2H coupling leading to the ‘DHHC’ sequence (Fig. 9c). The ideal values for τ DH and τ CH are equal to 1/∣2TDH∣ and 1/∣2TCH∣, as in the case of the ‘DHC’ sequence. During the τ 1 delay, the 1H SQC’s evolve under the effect of couplings, TDH and THH, then the evolution of these coherences under the effect of total couplings, TCH and THH, proceeds for the effective periods τ 2 τ 3 + τ 4 and τ 2 + τ 3 + τ 4, respectively. If the expression of the NMR signals for the ‘DHHC’ correlation experiment is identical to that of Eqn (6), the transfer function differs from that associated with the ‘DHC’ sequence. Thus, for a ‘Di–Hkn–HIm–Cj’ spin system, where m and n equivalent protons are weakly coupled (first order), the analytical expression of fcorr({τ p}) is now 9 8 sin πT Di Hk τ DH cosm1 πT Di Hk τ DH > > > > > > sin 2πT i k τ cosm1 3πD k k τ > > > > > > 1 1 D H H H > > > > > > n1 > > sin πT πT k l τ 1 cos k l τ1 > > H H H H > > > > > > < = sin πT Cj Hl ðτ 2 τ 3 þ τ 4 Þ 2 f corr τ p ¼ ðnmÞ (8) n1 > > 3πDHl Hl ðτ 2 þ τ 3 þ τ 4 Þ > cos > > > > > > > > > sin πT Hk Hl ðτ 2 þ τ 3 þ τ 4 Þ > > > > > > > > n1 > cos > > > πT k l ðτ 2 þ τ 3 þ τ 4 Þ > > H H > > : ; n1 sin πT Cj Hl τ CH cos πT Cj Hl τ CH

while the second heteronuclear INEPT transfer converts the 1H SQC’s into an observable 13C magnetization during acquisition. The combination of three 180° pulses allows the evolution of 1H SQC’s under the effect of 2H–1H and 1H–13C total couplings (J + 2D) with the elimination of the 1H chemical shift evolution when τ 1 = τ 2 + τ 3.[88] In practice, the position of 2H and 13C 180° pulses depends on the magnitude of ∣2TDH∣ and ∣TCH∣ (Fig. 9a and 9b). Note here that if the proton (or group of equivalent protons) used as relay is also coupled with other protons, then the 1H magnetization evolves also under the effect of coupling, ∣THH∣ during τ 1 + τ 2 + τ 3. After a two-step phase cycle and disregarding the relaxation terms, the general form of expression of the NMR signal for a ‘DHC’ 2D experiment is given by ( ) cos½ð2πνD þ πΔνQ Þðt1 þ τ DH Þ Sðt 1 ; t2 Þ∝ (6) þ cos½ð2πνD πΔνQ Þðt1 þ τ DH Þ

P. Lesot, O. Lafon and P. Berdagué between the refocusing of 1H magnetization with the TDH coupling and its defocusing with THH coupling. Mathematically, this value corresponds to the maximum of the function constituted by the four terms depending of τ 1 in Eqn (8). From the relaxation viewpoint, fcorr({τ p}) of the both ‘DHC’ sequences depends on the following term, i j k expð−τ DH =T D2 Þexp −ðτ 1 þ τ 2 þ τ 3 Þ=T H2 expð−τ CH =T C2 Þ (8a) while for the ‘DHHC’ sequence, this dependence is equal to. i k expð−τ DH =T D2 Þexpð−τ 1 =T H2 Þexp −ðτ 2 þ τ 3 j l þ τ 4 Þ=T H2 expð−τ CH =T C2 Þ (8b) In both experiments, the comparison of the values of total heteronuclear coupling constants to the relaxation rates 1/T2(1H, 2H and 13C) indicates that the T2 relaxation time mainly affects the optimal value of τ DH delay. Experimental model examples The efficiency and robustness of the ‘DHC’ and ‘DHHC’ sequences were experimentally tested on two model (achiral) molecules, the (S)-[1-2H1]-2,3,4-trihydro-1-naphtalenol (denoted (S)-THN) and [2,4,6-2H4]-3-methylphenol (denoted MP) dissolved in PBLG/CHCl3 and PCBLL/CHCl3 mesophases, respectively.[47] Figure 10a shows the ‘DHC’ 2D map obtained for (S)-THN when all refocusing delays of the ‘DHC’ sequence are optimized for correlating deuteron D1 to carbon atom C-8. As seen, no further correlation is observed on the 2D map. Therefore, correlations obtained on the 2D map allow to unambiguously the carbon signal C-8 on the aromatic ring to be assigned. The assignment has been also reconfirmed by recording the isotropic 13C–13C INADEQUATE 2D-NMR spectrum. This example illustrates perfectly the interest of ‘DCH’ experiments in structural analysis. Indeed, the assignment of the 13C peaks around 128.5 ppm is not trivial using the additivity rules of substituents for instance, and contradictory assignments for carbon atoms, C-8 and C-5, can be found using NMR prediction softwares or databases. The ‘DHHC’ 2D correlation experiments on (S)-THN permits us to correlate the signal associated with the D1 and C-7 nuclei through two 1H relays, thus allowing an easy assignment of 13C signal for carbon 7. This assignment agrees with the INADEQUATE 2D spectrum. Considering the interest of ‘DHHC’ 2D experiments in structural analysis, the extension of these experiments to the case of ‘D(H)nD’ 2D experiments with n > 2 is noteworthy. Such type of experiment can be achieved by replacing the 90° pulse used for 1H relay by a multi-pulse MLEV-17 or DIPSI-2 mixing sequence flanked with two trim pulses.[89,90]

Finally, as seen in Fig. 10c, ‘DHC’ experiments can also be carried out via a group of equivalent protons in freely rotating methyl groups as in case of MP. The defocusing and refocusing delays of ‘DHC’ sequence are optimized for correlating deuteron D2 to carbon C-7. In this example, as ∣2TCH∣ > ∣TDH∣, the ‘DHC’ pulse sequence of Fig. 9b must be applied. If the equation of the NMR signals of the 2D map is identical to Eqn (6), the transfer function differs because of the further modulation of signal by the evolution under the homonuclear coupling between H7 nuclei and the heteronuclear coupling between D4 and H7 nuclei during the effective period τ 1 τ 2 + τ 3. Thus, the expression of fcorr({τ p}) becomes 8 9 > > sin½π 4 T D2 H7 τ DH cos2 ½π 4 T D2 H7 τ DH > > > > > > > > 4 > > 2 7 ½ T ð τ τ þ τ Þ sin 2π 1 2 3 > > D H > > > > > > 4 > 4 7 ðτ 1 τ 2 þ τ 3 ÞÞ=3 > 1 þ 2 cos 2π ð ½ T > > D H < = 2 2 f corr τ p ¼ 9 cos ½3π DH7 H7 ðτ 1 þ τ 2 þ τ 3 Þ (9) > > > > 5 > > > cos½π T H5 H7 ðτ 1 þ τ 2 þ τ 3 Þ > > > > > > > > > 1 7 7 > > ½ ð Þ sin π T τ þ τ þ τ > > 1 2 3 C H > > > > : ; 1 7 7 2 1 7 7 sin½π T C H τ CH cos ½π T C H τ CH Applications to a mixture of (u/l)-stereoisomers ‘DHC’ or ‘DHHC’ 2D experiments in the case of mixtures 1 and 2 of (u/l)-stereoisomers have been simulated spectra using NMRSIM 4.3 software. Figure 11 reports the ‘DHC’ map for mixture 2 when all heteronuclear couplings are nonzero. The simulated spectrum proves the usefulness of ‘DHC’ sequence to distinguish the NMR signals of (u/l)-stereoisomers. However, none of the experimental ‘DHC’ spectra acquired for both mixtures dissolved in a chiral polypeptide (PBLG or PCBLL) mesophase did exhibit the desired cross peaks between 2H and 13C6 signals. Various sample compositions of the CLC and different temperatures were tested, but the magnitude 2 H–1H total couplings for the meso isomer and enantiomers remains too weak in mixtures 1 and 2 to detect any 2H–13C correlation in the ‘DHC’ 2D spectra. These weak 2H–1H total couplings stemmed from (i) the long D–H distance in these molecules and (ii) the fast rotation of the lateral groups around the sp2–sp3 single bonds. 13

C Relayed 2H–2H Homonuclear 2D Experiments for Weakly Aligned Solutes

Principle of 13C relayed 2H–2H homonuclear experiments 13

C relayed 2H–2H correlation NMR experiments have been also proposed by Lafon et al. to distinguish meso from d,l stereoisomers

2

1

604

Figure 10. (a) ‘DHC’ and (b) ‘DHHC’ anisotropic 2D-NMR spectra of (S)-[1- H1]-2,3,4-trihydro-1-naphtalenol showing the correlation between D and C-8 8 1 8 7 2 nuclei through proton H and between D and C-7 through protons H and H , respectively. (c) ‘DHC’ anisotropic 2D-NMR spectra of [2,4,6- H4]-32 13 methylphenol showing the H– C correlation through the methyl group. All experiments have been recorded at 9.4 T and 300 K. Experimental details can be found in Ref. (47). Figure is adapted from Ref. (47).



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Anisotropic deuterium-carbon 2D-NMR transfers involved one-bond 2H–13C total couplings (1TCD) that are generally much larger than geminal 2H–2H total couplings (2TDD) in a CD2 group, except if the coupling is fortuitously null (1JCD = 21DCD).

The INEPT-DECADENCY and DEPT-DECADENCY 2D sequences

Figure 11. Simulated 9.4 T ‘DHC’ 2D spectrum of mixture 2 dissolved in a chi2 13 ral mesophase when H and C signals (aromatic carbon 6) for each stereoiso2 13 mer are spectrally discriminated. H RQCs and C chemical shifts used for the 2 1 simulations are similar to the values measured on experimental H–{ H} and 13 1 C–{ H} 1D spectra of mixture 2 dissolved in the PCBLL/CHCl3 phase at 317 K. The sign of RQC’s for the meso compound and enantiomers is assumed to be the same (positive or negative). Other details can be found in Ref. (47). Figure is adapted from Ref. (47).

in a mixture and then to determine their diastereoisomeric and enantiomeric purity using polypeptide mesophases.[40] The proposed sequences are applicable for particular compounds containing a ‘CD2’ group as those reported in Fig. 12, but the concept can be extended to other molecules. In this example, the proton-decoupled 2 H–2H COSY 2D experiments in chiral and achiral anisotropic phases have allowed to pair up and assign QDs belonging to meso and d,l stereoisomers on the basis of 2H–2H correlation peaks on the 2D map.[40] However, this simple strategy is only suitable when the 2 H–2H total couplings between geminal 2H nuclei are sufficiently large in magnitude to produce cross-correlation peaks (CP) visible on the 2D spectrum. We have proposed an alternative approach based on 2H–2H correlation experiments with a 13C relay and denoted for DEuterium CArbon DEuterium Nuclear Correlation spectroscopy (DECADENCY).[40] Advantageously, the magnetization

Deuterium-carbon deuterium nuclear correlation spectroscopy 2D experiments are homonuclear experiments based on a relayed heteronuclear transfer mechanism. They belong to the class of Xrelayed Y,Y-COSY 2D experiments that was pioneered independently by Lallemand and Wüthrich in the case of an 1H-X-1H fragment.[91,92] After a double FT, the 2D map of DECADENCY experiments is formally equivalent to the 2H–2H COSY 2D map. It contains the same kind of spectral information, namely, autocorrelation peaks (AP), cross peaks (CP) and diagonal peaks (DP) for a 13CD2 spin system. CP’s correspond to the case where polarization is transferred from deuterium to carbon-13 nucleus, and then transferred back to the second deuterium (Dk → 13C → Dl) in a CD2 group. On the other hand, AP’s and DP’s correspond to the case where polarization is transferred from deuterium to carbon-13 nucleus and then returns to the same deuterium (Dk → 13C → Dk). In DECADENCY experiments, two mechanisms of C–D polarization transfers involving either a double INEPT-type or double DEPT-type process can be considered.[40] The corresponding pulse schemes are presented in Fig. 13a and 13b along with the associated coherence transfer pathways, respectively. The INEPT-DECADENCY sequence based on two consecutive INEPT-type polarization transfers, again derives from the HETCOR scheme as in the case of the CDCOM,[38] but a second INEPT transfer of polarization returns the magnetization from the 13C to 2H nuclei, just before the acquisition period of signal. The AP, CP, and DP expressions for both sequences are listed in Table 1. It could be noted that possible alternative to the ‘DCD’ (DECADENCY) experiment consists in using 2D experiments with a ‘DHD’-type transfer where the carbon relay is replaced by a proton relay. In that case, the 2H magnetization is transferred via a highly abundant nucleus (99.985%), thus increasing the intrinsic sensitivity of the experiment. However, the weak magnitude of 1H–2H total couplings may lead to weak transfer efficiency between 1H and 2 H isotopes, and hence limits the use of this method. Owing to the modulation of signal by RQCs during the defocusing and refocusing τ delays (Table 1), it is not possible to phase all resonances in pure absorption mode in F1 and F2 dimensions after the double Fourier transform. Consequently, the 2D maps of DECADENCY maps are displayed in magnitude mode.

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605

Figure 12. Two-step reaction pathway leading to a mixture of meso and d,l stereoisomers containing a ‘CD2’probe from a racemic mixture of the ‘anti’-(E)benzyl 5-iodo-4-methylhex-2-enoate. Figure is adapted from Ref. (39)


Figure 13. Pulse schemes and associated coherence transfer pathway diagrams for DECADENCY experiments based on either two consecutive (a) INEPT1 type and (b) or DEPT-type transfers. Similar to CDCOM experiments, the heating can be reduced by starting the H decoupling just before the first 90° pulse. Phase cycling can be found in Ref. (40). Quadrature detection in F1 dimension uses TPPI, Time-Proportional Phase Incrementation method. Figure is adapted from Ref. (40).

The implementation of 90° pulses to refocus 2H quadrupolar interaction during the fixed delays could be considered, but this solution generates several unwanted terms in the density operators and hence decreases the sensitivity.[40] In the case of INEPT-DECADENCY 2D experiment, a possible alternative consists however of simply removing the τ delays from the initial sequence and allowing the 13C–2H couplings to evolve during t1 and t2 periods. In practice, the 13C π pulse in the midpoint of t1 and the two τ intervals can be removed while the 2H decoupling is turned off during t2. After a double FT, a phased 2D map is obtained with the same spectral information (δD and ΔνQ), but the 2H signals are splitted by 1TCD coupling. Note here that this solution is not applicable for the DEPT-DECADENCY 2D experiment because the signal evolves under the effect of RQC during the τ′/2 intervals (Table 1).

INEPT-DECADENCY map obtained when the delays τ and τ′ are set to 1/(2TCD) and 1/(4ΤCD). The difference in the intensity of pairs of QDs for analytes 1 and 2 (that can be seen in the F1/F2 projection of the map) is a direct consequence of a larger 2H–2H total coupling for solute 1 that increases the linewidth, and hence reduces the intensity of corresponding QDs. As seen, the CP’s are the dominant peaks on the 2D maps, and allow the correlation between the two pairs of QDs associated with geminal 2H nuclei in solutes 1 and 2. Note here that the position of the most intense CPs on the DEPT-DECADENCY 2D map (map not shown) indicates that the QDs have the same sign, namely, positive or negative.[40] This conclusion has been confirmed by recording the 2H–{1H} 1D spectrum of the equimolar mixture of 1 and 2 dissolved in the achiral PBG/CHCl3 mesophase.

Experimental examples

2

To experimentally explore and illustrate the analytical potentialities of the DECADENCY 2D experiments, the phenyl[2H2]methanol (PM) dissolved in the PBLG/CHCl3 phase at 300 K has been used as model molecule. In a chiral oriented medium, the C–D enantiotopic directions of this prochiral molecule of Cs symmetry in average are nonequivalent and exhibit two QDs (and two total couplings, 1TC–D) corresponding to the pro-R and pro-S 2H atoms and denoted A and B. Figure 14a presents the INEPT-DECADENCY map of PM displayed at low contour plot level when τ = 1/(2ΤCD) and τ′ = 1/(4TCD) with TCD set to the average value of ∣1TCD∣’s, i.e. 29 Hz. As expected, 16 peaks (4 × 4) corresponding to the various types of peaks generated by the sequence are observed. The presence of CPs allows the correlation between geminal deuterium nuclei DA and DB bonded to the prostereogenic carbon of the molecule. The equations of Table 1 indicate that the intensities of AP, CP, and DP depend on the length of τ and τ′ delays. The intensity of all peaks is maximal for τ = 1/(2ΤCD). The intensity of CP is maximal for τ′ = 1/(4TCD), whereas the intensities of AP and DP cancels for τ′ = 1/(3TCD) (Fig. 14b). Application to mixture of two prochiral molecules

606

The interest of DECADENCY correlation experiments was first illustrated for separating the signals of two dideuterated prochiral solutes in a mixture. For this purpose, an equimolar mixture of PM (1) and 1–chloro[1–2H2]nonane (2) dissolved in the PBLG/CHCl3 phase (Fig. 15a) has been prepared. Figure 15b presents the


H–13C Correlation for Isotopically Unmodified Solutes: a New Frontier

Introduction In 1964, Diehl and Leipert reported the first experimental 2H signal at natural abundance level recorded using a 1.17 T (50 MHz for 1H) continuous-wave NMR spectrometer.[93] Since this pioneer 1D-NMR experiment, numerous important instrumental and methodological developments (advanced electronic, high magnetic field, deuterium cryoprobes, and data processing) have dramatically improved the sensitivity of NMR spectroscopy and hence have facilitated the acquisition of NAD NMR spectra of solids, liquid crystals, and liquids within reasonable acquisition times.[20,21] For instance, NAD 2D spectra of weakly aligned solutes can be acquired in a few hours using 600 MHz spectrometer equipped with a cryoprobe. Considering the natural isotopic abundance of deuterium nuclei (0.0155%) and carbon-13 nuclei (1.1%) in any molecule, the natural abundance of isotopomers containing both 2H and 13C nuclei is about 1.7 × 104%. In other words, the acquisition of 2H–13C NMR correlation 2D spectra at natural abundance level entails the detection and the selection of one molecule over 580 000 ones. The first successful experimental detection of natural abundance 2H–13C isotopomers have been reported in 2012 by Lesot and Lafon using PM (MW = 108 g mol1) dissolved in chloroform and in PBLG/ chloroform chiral mesophase.[48] The pulse sequences used for these NASDAC 2D-NMR experiments are described in the succeeding text.


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wileyonlinelibrary.com/journal/mrc 1

1

13

1 SDP ðt 1 ; t 2 Þ∝ i sin ωDk;l ðt 1 þ τ Þ cos ωQk;l ðt1 þ τ Þ 3 ) ( 2 sin ωCDk;l τ cos 2ωCDk;l τ’ 1 þ 2cos 2ωCDl;k τ’ n o expi ½ωDk;l ðτþt2 ÞωQk;l ðτþt2 Þ þ expi½ωDk;l ðτþt2 ÞþωQk;l ðτþt2 Þ

2 SCP ðt1 ; t2 Þ∝ i sin ωDk;l ðt1 þ τ Þ cos ωQk;l ðt1 þ τ Þ 3 ( ) sin½ωCDk;l τ sin½2ωCDk;l τ’ sin½2ωCDl;k τ’sin½ωCDl;k τ 9 8 < expi½ωDl;k ðτ þ t2 Þ ωQl;k ðτ þ t2 Þ = ; : þexpi½ωDl;k ðτ þ t2 Þþ ωQl;k ðτ þ t2 Þ

1 SAP ðt1 ; t2 Þ∝ i sin ωDk;l ðt1 þ τ Þ cos ωQk;l ðt1 þ τ Þ 3 ) ( 2 sin ωCDk;l τ cos 2ωCDk;l τ’ 1 þ 2cos 2ωCDl;k τ’ 9 8 < expi½ωDk;l ðτ þ t2 Þ ωQk;l ðτ þ t2 Þ = ; : þexpi½ωDk;l ðτ þ t2 Þþ ωQk;l ðτ þ t2 Þ

INEPT-DECADENCY

2

DEPT-DECANDENCY

1

1

1 SDP ðt 1 ; t 2 Þ∝ cos ωDk;l ðt1 þ τ þ τ’=2Þ 6 ( ) sin2 ωCDk;l τ cos2 ωCDk;l τ’=2 1 þ 4 cos ωCDl;k τ’ þ cos 2ωCDl;k τ’ 9 8 < expi½ωQk;l ðt1 þ τ þ τ’=2Þ þ ðωDk;l ωQk;l Þðτ=2 þ τ þ t2 Þ = ; : þexpi½ωQk;l ðt1 þ τ þ τ’=2Þ þ ðωDk;l þ ωQk;l Þðτ’=2 þ τ þ t2 Þ

( ) sin½ωCDk;l τ sin½ωCDk;l τ’ 1 SCP ðt1 ; t2 Þ∝ 3 sin½ωCDl;k τ’ sin½ωCDl;k τ 8 9 expi½ðωDl;k þωQl;k Þðτ’=2 þ τ þ t2 Þ > > > > > > > > > !> > > > > k;l l;k k;l k;l cos ω ½ ð þ ω Þτ’=2 sin ½ ð ω ω Þ ð t þ τ þ τ’=2 Þ CD CD D Q 1 > > > > > > > > > > > > < þ cos½ðωCDk;l ωCDl;k Þτ’=2 sin½ðωDk;l þ ωQk;l Þðt1 þ τ þ τ’=2Þ = > > > > þexpi½ðωDl;k ωQl;k Þðτ’=2 þ τ þ t2 Þ > > > > > > > > ! > > > > cos½ðωCDk;l þ ωCDl;k Þτ’=2 sin½ðωDk;l þ ωQk;l Þðt1 þ τ þ τ’=2Þ > > > > > > > > > > : ; þ cos½ðωCDk;l ωCDl;k Þτ’=2 sin½ðωDk;l ωQk;l Þðt 1 þ τ þ τ’=2Þ

1 SAP ðt 1 ; t 2 Þ∝ i sin ωDk;l ðt1 þ τ þ τ’=2Þ 6 ) ( 1 þ 4 cos ωCDl;k τ’ þ cos 2ωCDl;k τ’ sin2 ½ωCDk;l τ sin2 ωCDk;l τ’=2 9 8 < expi½ωQk;l ðt1 þ τ þ τ’=2ÞþðωDk;l ωQk;l Þðτ’=2 þ τ þ t2 Þ= ; : þexpi½ωQk;l ðt1 þ τ þ τ’=2ÞþðωDk;l þ ωQk;l Þðτ’=2 þ τ þ t2 Þ

In these equations, ωD = 2πνD, ωQ = πΔνQ, and ωCD = π TCD, where TCD is the one-bond C– H total coupling ( JCD + 2 DCD).

a

DP

607

Magn. Reson. Chem. 2014, 52, 595–613

CP

AP

Peaks

Table 1. Signal equations of autocorrelation (AP), cross-correlation (CP), and diagonal peaks (DP) for two deuterons, k and l, after a four-step phase cycle and disregarding all relaxation terms and phase



2

Figure 14. INEPT-DECADENCY 2D spectra of phenyl[ H2]methanol obtained when (a) τ = 1/(2TCD) and τ′ = 1/(4TCD) and (b) τ = 1/(2TCD) and τ′ = 1/(3TCD) with 2 1 TCD = 29 Hz. Other experimental parameters are identical for both spectra. For both 2D spectra, F1 dimension displays the H–{ H} 1D spectrum , while F2 dimension displays the projection of the 2D map. The assignment of QDs (A and B) relative to the molecular numbering is arbitrary. Other details can be found in Ref. (40). Figure is adapted from Ref. (40).

Figure 15. (a) Structure of the prochiral solutes, compounds 1 and 2, of model mixture. (b) INEPT-DECADENCY 2D spectrum of the equimolar mixture of 1 and 2 1 2 obtained when τ = 1/(2TCD) and τ′ = 1/(4TCD). QDs marked by a star are associated with deuterated impurities. Projections correspond to the 1D H–{ H} spectrum. QDs of solutes 1 and 2 are labeled with open and solid circles, respectively. Other details can be found in Ref. (40). Figure is adapted from Ref. (40).

The NASDAC and R-NASDAC 2D sequences As the CDCOM 2D experiments applied for deuterated molecules (see the section “2H–13C Correlation 2D Experiments on Isotopically Enriched Solutes”), the NASDAC 2D experiment uses a sequence derived from the 2H–13C HETCOR sequence with 13C detection. Here again, this scheme exhibits a higher sensitivity than HMQC and HSQC ones, in particular for short recycling delays.[48] Contrary to CDCOM, the coherence pathway selection is achieved by combining both pulsed B0 field gradients and a phase cycling of 128 steps. The pulse scheme and the associated coherence pathway is shown in Fig. 16a. Disregarding all relaxation terms and phase factors, the general expression of 2D-NMR signal for a coupled 2H–13C pair recorded with NASDAC sequence is given by 2 Sðt 1 ; t 2 Þ ∝ f exp½ið2πv D πΔv Q Þðt1 þ τ Þ þ exp½ið2πv D þ πΔv Q Þðt 1 þ τ Þg 3 f sinðπT CD τ Þ sinð2πT CD τ’Þ exp½i2πv C ðτ’ þ t 2 Þg

(10)

608

where ωD = 2πν2Haniso, ωC = 2πν13Caniso, ωQ = πΔνQ, and ωCD = π 1TCD. For isotropic systems, all anisotropic NMR interactions are averaged out to zero. In other words, the 2H and 13C resonance frequencies, vD and vC, correspond to isotropic chemical shifts and 1TCD = 1JCD. A variant of the NASDAC entitled R-NASDAC has been also proposed (Fig. 16b). In this new sequence, the composite 180° pulse


during t1 is applied on the 2H channel in order to refocus the 2H chemical shifts in F1. The expression of R-NASDAC signal simply derives from Eqn (10), but the term vD is removed. For this experiment, the coherence pathway selection is achieved by combining both pulsed field gradients and an extended phase cycling of 256 steps. For both 2D sequences, the intensity of the signal depends on the lengths of defocusing and refocusing delays, τ and τ′. At natural abundance level, only isolated 2H–13C pairs are detected, and hence the optimal defocusing and refocusing delays are τ = 1/ (2∣1TCD∣) and τ′ = 1/(4∣1TCD∣). While in isotropic media, the RNASDAC is obviously useless, this experiment is more advantageous in anisotropic media for two reasons: (i) the reduction of the F1 spectral width, which limits the possible truncation effect of signals, and (ii) the 2D map can be symmetrized in F1 dimension. Filtering one molecule over 580 000 ones The success of NASDAC experiments requires the efficient elimination of all signals of unwanted molecules, namely, the (1H–13C)isotopomers. The (1H–13C)-isotopomers signals in NASDAC and R-NASDAC result from the coherence pathway with zero 2H coherence order (p(2H) = 0) and 13C coherence orders, p(13C) = 0 before the first 90° pulse on 13C channel and p(13C) = 1 after. Assuming a gradient pulse based-selection, the intensity ratio of the (1H–13C)isotopomer and (2H–13C)-isotopomer signal can be evaluated as


Magn. Reson. Chem. 2014, 52, 595–613


2

13

Figure 16. (a) Variant of H– C HETCOR pulse sequence used in NASDAC experiments along with the diagram of coherence transfer pathways. The 1 13 refocusing of TCD coupling is achieved by applying a 180° composite inversion pulse on the C channel, but other solutions can be applied. (b) R-NASDAC scheme along with the diagram of coherence transfer pathways. Phase cycling of both sequences can be found in Ref. (48). Figure is adapted from Ref. (48).

I1 H13 C ¼ I2 H13 C

NAð HÞ 43 12 γð2 HÞγ3=2 ð13 CÞ fz 2 γ5=2 ð13 CÞ 1

NAð HÞ

n o R 1 exp TT13C n 1 o 1 exp TT2HR 1

(11) where fz is the attenuation of the 1H–13C signal by the gradient,[48] NA (1H) = 99.9845% and NA(2H) = 0.0155% are the natural abundances of 1 H and 2H isotopes, while 4/3 is the transfer efficiency of 2H–13C HETCOR and 1/2 stems from the twofold decrease in signal intensity owing to the selection of 2H (+1)-coherence using gradient. Assuming T12H = 0.25 s, T13C 1 = 1.5 s, and TR = 0.6 s, we found a ratio I1H13C/I2H13C ≈ 15. Consequently, the pulsed field gradients are not sufficient to filter out more than 99.9845% of (1H–13C)-isotopomers signal that is essential to properly detected (2H–13C)-isotopomers. Hence, an additional phase cycling must be employed in combination with pulse gradients. The application of randomization pulses on the 13C channel before the first 2H pulse might be also proposed to facilitate the suppression of (1H–13C)-isotopomers signal.[94]

First experimental examples The NASDAC 2D experiments were successfully tested with the unmodified PM dissolved in isotropic solvents, and then in the case of chiral oriented solvents as seen in Figs 17a and 18a. The concentration of a given isomer containing both 2H and 13C nuclei ([2H–13C]) was estimated at about 9 μmol L1 in both solvents. In both media, 2D spectra were recorded in 18 h with a 2D matrix of 6400 (t2) × 64 (t1) data points and 1080 scans added for each t1 increment and TR equal to 0.6 s. Even using a 21.1 T magnetic field and a tripleresonance inverse (TCI) cryoprobe, a large number of scans are required to detect the 2H–13C isotopomers with a sufficient S/N ratio. To suppress the truncation effects in F1 due to the small numbers of point in the indirect dimension, reconstruction methods must be applied. Here, linear prediction approach has been applied. The detection of the 2H–13C isotopomers of PM in liquids (and then anisotropic systems) was evidenced by two facts: (i) the observation of CP on 2D maps and (ii) the small but systematic upfield shifting of 13C resonances observed on the F2 projection of the map compared to their positions on the 1D 13C–{1H} 1D-NMR spectrum (Fig. 17b). These

Magn. Reson. Chem. 2014, 52, 595–613



609

Figure 17. (a) Two expanded regions (aromatic and methylenic) of 21.1 T NASDAC 2D spectrum (along with full F1 and F2 projections) of isotopically unmod13 1 ified PM dissolved in chloroform at 305 K. (b) Comparison between the isotropic 226.4 MHz natural abundance C–{ H} 1D spectrum (bottom), the F2 1D pro13 1 jection of the isotropic NASDAC 2D experiment of isotopically unmodified PM (middle), and the linear combination of C–{ H} 1D spectra and F2 projection (top), in which the intensity of the F2 projection is multiplied by 15. Other details can be found in Ref. (48). Figure is adapted from Ref. (48).


Figure 18. Two expanded regions (aromatic and methylenic) of (a) NASDAC and (b) symmetrized R-NASDAC 2D map (with associated F1 and F2 projections) 2 of PM in PBLG/CHCl3 at 305 K. The assignment of H quadrupolar doublets for the H2a/H2b positions is arbitrary. Other details can be found in Ref. (48). Figure is adapted from Ref. (48).

spectral shifts originate from the influence of 1H/2H substitution on 13 C chemical shift, an isotopic effect varying from 273 to 327 ppb in the case of PM.[95] This remarkable result has emphasized for the first time that the observation and the determination 2H isotopic effects on 13C chemical shifts was possible without any isotopic labeling. As expected, in the anisotropic chiral media (Fig. 18a), 2H signals consist of sum of quadrupolar doublets. The R-NASDAC experiment has been also successfully acquired for PM dissolved in the chiral mesophase using the same experimental conditions chosen for NASDAC but with a reduction of the F1 spectral width of 40%. The corresponding symmetrized map is reported in Fig. 18b. The enhanced spectral quality in particular in F1 dimension shows clearly the advantage of the R-NASDAC experiment compared to NASDAC one. Finally, the analysis of both 2D maps demonstrates that (2H–13C)(enantio)isotopomers with a natural abundance concentration of ≈9 μmol L1 can be experimentally detected in a CLC. The constant performance improvements of NMR hardwares should permit to record soon isotropic and anisotropic NASDAC experiments with much lower (2H–13C) isotopomers concentrations.

Correlation Experiments Applied to Thermotropic Mesophases The DECOR 2D sequence All previous examples of correlation NMR experiments involving 2H and 13C isotopes were acquired for weakly aligned solutes in

polypeptide, chiral orienting media. However, the first 2H–13C correlation 2D-NMR experiment in oriented media, called DECOR, was described in 1997 by Auger et al. for analyzing perdeuterated mesogenic molecules such as the 4-n-pentyl-4′-cyanobiphenyl (5CB) forming a thermotropic nematic liquid crystal between 291 and 308 K.[41,42] The 5CB molecules (Fig. 20a) in nematic phase are strongly aligned with the magnetic field with order parameters in the order of 101,[96] and hence the RQCs range from 9 to 52 kHz (in absolute value) in the case of 5CB.[42] For their purpose, they utilized 2H → 13C CP transfer to correlate 2 H and 13C signals of 5CB under static conditions (Figs 19a and 20). However, the maximal rf field strength on 2H channel delivered by common solid-state NMR probe is often comparable to the RQCs, and the first-order quadrupolar interaction interferes with the rf field applied to 2H nuclei during the CP step.[41,42,55,60] This issue has been circumvented by transferring only one component of the quadrupolar doublets. This frequency-selective transfer was achieved by (i) placing the transmitter frequency of the 2H pulse during the CP step at the frequency of one component of the quadrupolar doublet and (ii) using an rf field on 2H channel much lower than the RQCs. However, when using constant rf field amplitude on both 13C and 2H channels during the CP transfer, the efficiency of the CP transfer strongly depends on the RQC value.[42,60] The robustness to RQC values was improved by sweeping linearly the amplitude of 2H rf field during CP step. Nevertheless, even for ramped CP step, the efficiency of 2H → 13C transfer still strongly depends on the magnitude of RQCs,[41,60] and only a single component of the

(b)

(a)

2

13

2

2

610

Figure 19. (a) DECOR pulse sequence based on H → C CP transfer. A π pulse can be applied to the H nuclei at the middle of the t1 period to refocus H 13 2 2 2 13 chemical shifts and C– H total couplings. ΩCP denotes the offset of the transmitter frequency on the H channel during the CP transfer. (b) H– C HMQC pulse sequence. The defocusing and refocusing delays are denoted τ. Figure is adapted from Refs. (41) and (43). (Courtesy of L. Emsley et al. and D. Sanström et al., reprinted with permission from the American Chemical Society).



Magn. Reson. Chem. 2014, 52, 595–613


(a)

(b)

2

Figure 20. (a) The 61.4 MHz deuterium 1D-NMR spectra of a static sample of fully deuterated [ H19]-5CB acquired in its nematic phase (298 K) with a one2 pulse sequence. The chemical structure of 5CB is shown along with the atom labeling. (b) A 9.4 T DECOR 2D map of [ H19]-5CB. 1D and 2D spectra were acquired under static conditions using a 7 mm triple-resonance CP-MAS probe without sample rotation. A total of 128 t1 increments with 96 scans each were collected. A 4 s recycle delay was used between scans (to avoid sample heating problems). The contact time for CP was set to 5 ms using an rf field strength of 2 13 2 13.8 kHz at the center of the ramp for H and 19.5 kHz for C. The rf field on H channel was linearly ramped from 9.2 to 18.4 kHz. During the CP step, the deuterium frequency was moved to 17.8 kHz off resonance to allow single-quantum CP. Other details can be found in Ref. (41). Figure is adapted from Ref. (41). (Courtesy of L. Emsley et al., reprinted with permission from the American Chemical Society).

2

13

2

Magn. Reson. Chem. 2014, 52, 595–613



611

Figure 21. A 9.4 T H– C HMQC 2D spectrum of [ H19]-5CB acquired under static conditions with a τ delay equal to 200 μs. The total acquisition time was 24 h. Other experimental details can be found in Ref. (43). Figure is adapted from Ref. (43). (Courtesy of D. Sandström et al., reprinted with permission from the American Chemical Society).

P. Lesot, O. Lafon and P. Berdagué quadrupolar doublets is transferred to the 13C nuclei. Furthermore, the CP transfer of DECOR sequence requires the optimization of several parameters, including the 2H transmitter frequency, the mean 2H rf field, and the slope of linear ramp. HMQC 2D sequence applied to the analysis of 5CB As shown by Sandström and Zimmermann, a simpler approach to correlate 2H and 13C signals of 5CB consists in the use of the standard 2H–13C HMQC sequence (Fig. 19b).[43,44] As this method only uses hard pulses on 2H channel, except for the 2H decoupling, the sequence is more broadband and more robust to the RQC values. Furthermore, the number of experimental parameters to optimize is lower. The transmitter frequency is placed at the center of the spectrum and the maximal 2H rf field compatible with probe specifications is used. The 2H–13C HMQC method has been employed to correlate 2H and 13C signals of perdeuterated 5CB under static conditions (Fig. 21) and under off MAS.[43,44] Off MAS reduces the magnitude of anisotropic interactions, including RQCs and 1H–2H dipolar couplings.[97] Hence, this method allows the use of lower decoupling rf field on 2H channel. However, the spectral resolution of the 2H dimension of 2H–13C HMQC 2D spectrum was lower under off MAS than under static conditions. To the best of our knowledge, neither 13C–2H HMQC-type 2D sequence with 2H detection nor HETCOR-type 2D sequences have been employed to correlate 2H and 13C signals of strongly aligned molecules.

Conclusion NMR in anisotropic media has proven to be a powerful tool for the structural analysis of complex small organic molecules.[98–100] Among magnetically active nuclei, deuterons provide remarkable nuclear spies that can be used for numerous and original analytical applications ranging from the structural or stereochemical analysis to natural isotope fractionation. This quadrupolar nucleus of weak quadrupolar moment and ubiquitous in all organic molecules provides useful insights into the molecular orientational ordering and dynamics in anisotropic media and solids. Furthermore, it can now be detected in natural abundance by NMR experiments in a few hours. Various specifically designed NMR tools have been introduced in the last decade to facilitate the analysis of complex/overcrowded anisotropic 2H 1D-NMR spectra, using homonuclear or heteronuclear correlation experiments. In this contribution, we have presented a compendium of the correlation 2D-NMR experiments involving 2H and 13C nuclei in oriented media. In particular, we have described the experiments, which have been used in chiral oriented systems. The advent of very high-magnetic field spectrometers equipped with 2 H cryogenic probes is expected to generalize the use of these analytical tools, which are now applicable for isotopically unmodified solutes dissolved in liquids or weakly ordering media. These correlation 2D experiments involving 2H and 13C nuclei will have implication for various fields, including organic chemistry, biochemistry, food sciences, pharmacology, catalysis, and material sciences. Acknowledgements

612

P. L. and O. L. thank the CNRS for its financial support. Financial support from the TGIR-RMN-THC Fr3050 CNRS for conducting some NASDAC 2D-NMR experiments at 21.1 T is also gratefully acknowledged. P. L. and O. L. also thank the restaurant ‘La Chicorée’ in Lille for serving food and Belgian beers at midnight. We appreciated it after optimizing NASDAC 2D experiments late in the evening.


References [1] J. R. Wesener, P. Schmitt, H. Günther. Org. Mag. Reson. 1984, 22, 468–470. [2] J. R. Wesener, H. Günther. J. Am. Chem. Soc. 1985, 107, 1537–1540. [3] G. D. Brown. Phytochem. Rev. 2003, 2, 45–49. [4] T. A. Bartholomeusz, F. Mesnard, F.-X. Felpin, J. Lebreton, R. J. Robins, A. Roscher. C. R. Chimie 2006, 9, 514–519. [5] G. D. Brown, L.-K. Sy. Tetrahedron 2007, 63, 9548–9566. [6] B. Schneider. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 155–198. [7] G. J. Martin, M. L. Martin, G. S. Remaud, in Modern Magnetic Resonance (Ed: G. A. Webb), Springer, Berlin, 2006, pp. 1647–1657. [8] C. Aghemo, A. Albertino, R. Gobettoa, F. Spanna. J. Sci. Food Agric. 2011, 91, 2088–2094. [9] R. Y. Dong, Nuclear Magnetic Resonance of Liquid Crystals, 2nd edn, Springer-Verlag, New York, 1997. [10] R. Y. Dong, in Encyclopedia of Magnetic Resonance (eMagRes) (Eds: R. K. Harris, R. Wasylishen), John Wiley & sons, Chichester, Posted on-line th 15 March 2007. DOI: 10.1002/9780470034590.emrstm0265 [11] S. Rivillon, P. Auroy, B. Deloche. Phys. Rev. Lett. 2000, 84, 499–502. [12] S. Valic, P. Judeinstein, B. Deloche. Polymer 2003, 44, 5263–5267. [13] V. Domenici, M. Geppi, C. A. Veracini. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 50, 1–50. [14] S. D. Cady, K. Schmidt-Rohr, J. Wang, C. S. Soto, W. F. DeGrado, M. Hong. Nature 2010, 463, 689–693. [15] V. Domenici. Prog. Nucl. Magn. Reson. Spectrosc. 2012, 63, 1–32. [16] B. Bechinger, E. S. Salnikov. Chem. Phys. Lipids 2012, 165, 282–301. [17] M. Hologne, K. Faelber, A. Diehl, B. Reif. J. Am. Chem. Soc. 2005, 127, 11208–11209. [18] M. Hologne, Z. Chen, B. Reif. J. Magn. Reson. 2006, 179, 20–28. [19] T. Gutmann, I. del Rosal, B. Chaudret, R. Poteau, H.-H. Limbach, G. Buntkowsky. ChemPhysChem 2013, 14, 3026–3033. [20] P. Lesot, in Encyclopedia of Magnetic Resonance (eMagRes), vol. 2(3) (Eds: R. K. Harris, R. E. Wasylishen), John Wiley & Sons, Chichester, th Posted online 15 October 2013, pp. 315–334, DOI: 10.1002/ 9780470034590.emrstm1318. [21] P. Lesot, J. Courtieu. Prog. Nucl. Magn. Reson. Spectrosc. 2009, 55, 128–155, and references therein. [22] P. Lesot, M. Sarfati, J. Courtieu. Chem. Eur. J. 2003, 9, 1724–1745. [23] A. Parenty, J.-M. Campagne, C. Aroulanda, P. Lesot. Org. Lett. 2002, 2, 1663–1666. [24] C. M. Thiele, A. Marx, R. Berger, J. Fischer, M. Biel, A. Giannis. Angew. Chem. Int. Ed. 2006, 45, 4455–4460. [25] L. Ziani, P. Lesot, A. Meddour, J. Courtieu. Chem. Commun. 2007, 4737–4739. [26] P. Lesot, O. Lafon, H. B. Kagan, C.-A. Fan. Chem. Commun. 2006, 389–391. [27] O. Lafon, P. Lesot, C.-A. Fan, H. B. Kagan. Chem. Eur. J. 2007, 13, 3772–3786. [28] P. Lesot, C. Aroulanda, I. Billault. Anal. Chem. 2004, 76, 2827–2835. [29] P. Lesot, V. Baillif, I. Billault. Anal. Chem. 2008, 80, 2963–2972. [30] C. Brevard, P. Granger, Handbook of High Resolution Multinuclear NMR, J. Wiley & Sons, New York, 1981. [31] V. Agarwal, K. Faelber, P. Schmieder, B. Reif. J. Am. Chem. Soc. 2009, 131, 2–3. [32] K. Kazimierczuk, O. Lafon, P. Lesot. Analyst 2014, 139, 2702–2713. [33] D. Merlet, B. Ancian, J. Courtieu, P. Lesot. J. Am. Chem. Soc. 1999, 121, 5249–5258. [34] O. Lafon, P. Lesot, D. Merlet, J. Courtieu. J. Magn. Reson. 2004, 171, 135–142. [35] O. Lafon, P. Lesot. Chem. Phys. Lett. 2005, 404, 90–94. [36] P. Lesot, O. Lafon. Chem. Phys. Lett. 2008, 458, 219–222. [37] J. W. Emsley, D. L. Turner. Chem. Phys. Lett. 1981, 82, 447–450. [38] P. Lesot, M. Sarfati, D. Merlet, B. Ancian, J. W. Emsley, B. A. Timini. J. Am. Chem. Soc. 2003, 125, 7689–7695. [39] H. Villar, F. Guibé, C. Aroulanda, P. Lesot. Tetrahedron: Asymmetry 2002, 13, 1465–1475. [40] O. Lafon, P. Lesot. J. Magn. Reson. 2005, 174, 254–264. [41] C. Auger, A. Lesage, S. Caldarelli, P. Hodgkinson, L. Emsley. J. Am. Chem. Soc. 1997, 119, 12000–12001. [42] C. Auger, A. Lesage, S. Caldarelli, P. Hodgkinson, L. Emsley. J. Phys. Chem. B 1998, 120, 3718–3723. [43] D. Sandström, H. Zimmermann. J. Phys. Chem. B 2000, 104, 1490–1493. [44] D. Sandström, H. Zimmermann. Mol. Phys. 2002, 100, 1935–1940.


Magn. Reson. Chem. 2014, 52, 595–613

Anisotropic deuterium-carbon 2D-NMR [45] O. Lafon, P. Lesot, P. Berdagué. Phys. Chem. Chem. Phys. 2004, 6, 1080–1084. [46] C. Aroulanda, O. Lafon, P. Lesot. J. Phys. Chem. B, 2009, 113, 10628–10640. [47] K. Ben Ali, O. Lafon, H. Zimmermann, E. Guittet, P. Lesot. J. Magn. Reson. 2007, 187, 205–215. [48] P. Lesot, O. Lafon. Anal. Chem. 2012, 84, 4569–4573. [49] S. Jayanthi, K. V. Ramanathan. Chem. Phys. Lett. 2010, 487, 122–128. [50] D. Sandström, M. Hong, K. Schmidt-Rohr. Chem. Phys. Lett. 1999, 300, 213–220. [51] P. Robyr, B. H. Meier. Chem. Phys. Lett. 2000, 327, 319–324. [52] H. Mengezaz, S. Hediger, L. Emsley, D. Sandström. Polym. Bull. 2001, 46, 183–190. [53] Ü. Akbey, F. Camponeschi, B.-J. van Rossum, H. Oschkinat. ChemPhysChem 2011, 12, 2092–2096. [54] D. Lalli, P. Schanda, A. Chowdhury, J. Retel, M. Hiller, V. A. Higman, L. Handel, V. Agarwal, B. Reif, B. van Rossum, U. Akbey, H. Oschkinat. J. Biomol. NMR 2011, 51, 477–485. [55] D. Wei, U. Akbey, B. Paaske, H. Oschkinat, B. Reif, M. Bjerring, N. C. Nielsen. J. Phys. Chem. Lett. 2011, 2, 1289–1294. [56] X. Shi, J. L. Yarger, G. P. Holland. J. Magn. Reson. 2013, 226, 1–12. [57] P. L. Rinaldi, N. J. Baldwin. J. Am. Chem. Soc. 1982, 104, 5791–5793. [58] T. T. Nakashima, R. E. D. McClung, B. K. John. J. Magn. Reson. 1984, 58, 27–36. [59] B. Luy, S. J. Glaser. Chem. Phys. Lett. 2000, 323, 377–381. [60] P. Hodgkinson, C. Auger, L. Emsley. J. Chem. Phys. 1998, 109, 1873–1884. [61] S. K. Jain, A. B. Nielsen, M. Hiller, L. Handel, M. Ernst, H. Oschkinat, U. Akbey, N. C. Nielsen. Phys. Chem. Chem. Phys. 2014, 16, 2827–2830. [62] M. Sarfati, P. Lesot, D. Merlet, J. Courtieu. Chem. Commun. 2000, 2069–2081 and references therein. [63] P. Lesot, O. Lafon, C. Aroulanda, R. Y. Dong. Chem. Eur. J. 2008, 14, 4082–4092. [64] S. Sau, K. V. Ramanathan. J. Phys. Chem. B 2009, 113, 1530–1532. [65] M. Chan-Huot, P. Lesot, P. Pelupessy, L. Duma, G. Bodenhausen, P. Duchambon, M. D. Toney, U. V. Reddy, N. Suryaprakash. Anal. Chem. 2013, 85, 4694–4697. [66] Lokesh, N. Suryaprakash. Chem. Eur. J. 2012, 18, 11560–11563. [67] K. Kobzar, H. Kessler, B. Luy. Angew. Chem. Int. Ed. 2005, 44, 3145–3147. [68] C. Naumann, P. W. Kuchel. Chem. Eur. J. 2009, 15, 12189–12191. [69] The term “enantiodiscrimination” is used to refer to both enantiomeric and enantiotopic discriminations observed in CLCs. [70] M. Jakubcova, A. Meddour, J.-M. Péchiné, A. Baklouti, J. Courtieu. J. Fluorine Chem. 1997, 86, 149–153. [71] V. Madiot, P. Lesot, D. Grée, J. Courtieu, R. Grée. Chem. Commun. 2000, 169–170. [72] A. Meddour, P. Berdagué, A. Hedli, J. Courtieu, P. Lesot. J. Am. Chem. Soc. 1997, 119, 4502–4508. [73] P. Tzvetkova, B. Luy, S. Simova, Topics in Chemistry and Material Science 5 (2011) pp. 70–77 of Current Issues in Organic Chemistry, (Eds: R. D.

[74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100]

Nikolova, S. Simova, P. Denkova, G. N. Vayssilov), Heron Press Ltd, Birmingham, 2011. M. Rivard, F. Guillen, J.-C. Fiaud, C. Aroulanda, P. Lesot. Tetrahedron: Asymmetry 2003, 14, 1141–1152. O. Lafon, P. Lesot, M. Rivard, M. Chavarot, F. Rose-Munch, E. Rose. Organometallics 2005, 24, 4021–4028. P. Lesot, D. Merlet, A. Meddour, A. Loewenstein, J. Courtieu. Tetrahedron: Asymmetry 1998, 9, 1871–1881. R. R. Ernst, G. Bodenhausen, A. Wokaun, Principle of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press, Oxford, 1987. D. Neuhaus, in Encyclopedia of Magnetic Resonance (Eds: R. K. Harris, R. E. Wasylichen), John Wiley & sons, Chichester, posted online on th 15 June 2011. J. Norwood, J. Boyd, J. E. Heritage, N. Soffe, I. D. Campbell. J. Magn. Reson. 1990, 87, 488–501. M. H. Levitt, in Encyclopedia of Magnetic Resonance, (Eds: R. K. Harris, R. E. Wasylichen), John Wiley & sons, Chichester, posted online on th 15 March 2007. M. Garwood, L. Delabarre. J. Magn. Reson. 2001, 153, 155–177. C. Thibaudeau, G. Rémaud, V. Silvestre, S. Akoka. Anal. Chem. 2010, 82, 5582–5590. K. Kobzar, S. Ehni, T. E. Skinner, S. J. Glaser, B. Luy. J. Magn. Reson. 2012, 225, 142–160. M. Sarfati, C. Aroulanda, J. Courtieu, P. Lesot. Tetrahedron: Asymmetry 2001, 12, 737–744. D. K. Dalling, D. M. Grant, L. F. Johnson. J. Am. Chem. Soc. 1971, 93, 3678–3682. D. K. Dalling, D. M. Grant, E. G. Paul. J. Am. Chem. Soc. 1973, 95, 3718–3724. T. O. Reiss, N. Khaneja, S. J. Glaser. J. Magn. Reson. 2003, 165, 95–101. L. Kay, E. Ikura, A. Bax. J. Magn. Reson. 1991, 91, 84–92. A. Bax, D. G. Davis. J. Magn. Reson. 1985, 65, 355–360. P. Lesot, J. Courtieu, D. Merlet, J. W. Emsley. Liq. Cryst. 1996, 21, 427–435. M. A. Delsuc, E. Guittet, N. Trotin, J.-Y. Lallemand. J. Magn. Reson. 1984, 56, 163–166. D. Neuhaus, G. Wider, K. Wagner, K. Wüthrich. J. Magn. Reson. 1984, 57, 164–168. P. Diehl, T. Leipert. Helv. Chim. Acta 1964, 47, 545–557. M. R. Bendall, D. T. Pegg, D. M. Doddrell. J. Magn. Reson. 1981, 45, 8–29. P. E. Hansen. Ann. Report NMR Spectrosc. 1983, 15, 105–231. J. W. Emsley, P. Lesot, G. De Luca, A. Lesage, D. Merlet, G. Pileio. Liq. Cryst. 2008, 35, 443–464. J. Courtieu, J.-P. Bayle, B. M. Fung. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 141–169. G. Kummerlöwe, B. Luy. Trends Anal. Chem. 2009, 28, 483–493. R. R. Gil. Angew. Chem. Int. Ed. 2011, 50, 7222–7224. C. M. Thiele. Concept Magn. Reson. A 2007, 30, 65–80.

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Magn. Reson. Chem. 2014, 52, 595–613



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