Chiral recognition by dissolution DNP NMR spectroscopy of 13

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Article

Chiral recognition by dissolution DNP NMR spectroscopy of C-labeled DL-methionine 13

Eva Monteagudo, Albert Virgili, Teodor Parella, and Míriam Pérez-Trujillo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00156 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 17, 2017

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Analytical Chemistry

Chiral recognition by dissolution DNP NMR spectroscopy of 13C-labeled DL-methionine

Eva Monteagudo,† Albert Virgili,‡ Teodor Parella† and Míriam Pérez-Trujillo*† 5

†Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Barcelona, Spain ‡Departament de Química, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del 10

Vallès, Barcelona, Spain

* Corresponding Author *E-mail: [email protected]. Phone: +34 935813785

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ACS Paragon Plus Environment

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ABSTRACT A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a 20

millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized 13

C-labeled DL-methionine enantiomers were differently observed with a single-scan

13

C

NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is 25

limited; e.g. when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like

13

C, is required. The advantages and current limitations of the

developed method, as well as, future perspectives are discussed.

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Keywords: Chiral recognition, DNP, NMR,

13

C NMR, dissolution DNP, DL-methionine,

chiral solvating agent, CSA, hyperpolarization


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The recognition of enantiomeric molecules by chemical analytical techniques is still a 35

challenge.1,2 Among the different techniques, NMR spectroscopy together with the use of chiral auxiliaries (CA) has given excellent results being nowadays an active field of research.3-5 Compared to other CA, chiral solvating agents (CSA) present interesting advantages, such as no derivatization of the analyte or chromatographic steps, no problems of kinetic resolution and a quick way to perform the experiment.5-7 Enantiomeric

40

differentiation is especially challenging when the chiral recognition is meant to be done under biological sample conditions – i.e. in aqueous media, at millimolar concentration and within complex matrices. This is the case of chiral metabolomics,8 a concept we recently introduced to integrate chiral recognition into metabolomic studies and which comprehends all the aforementioned complexity. As evidence of its potential, a racemic analyte (RS-

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ibuprofen) was 1H NMR enantiodifferentiated in a body fluid (urine) using a CSA (βcyclodextrin); the enantiodiscrimination experiment was conducted directly in the intact biological sample without prior simplification of the mixture. Though 1H has been the preferred nucleus in NMR-aided chiral recognition (mainly for reasons of sensitivity and high presence in organic molecules), 1H observation entails the intrinsic problem of poor

50

spectral dispersion and signal overlapping produced by the small chemical shift range of 1H and the broad width of some individual signals due to J multiplicity splitting. This problem becomes aggravated in chiral recognition studies by the presence of the CSA (typically present in a higher amount than the analyte and therefore showing intense signals). The problem is still more significant when dealing at the same time with complex mixtures.

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Signal multiplicity in 1H spectra can be reduced using J-resolved8 or pure-shift9,10 NMR


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experiments, although they are still not completely effective with strong coupled signals. Another drawback owing to signal multiplicity in 1H spectra is related to the resolution of the enantiodifferentiated (split) peak. To be completely resolved into two observable signals (one corresponding to each enantiomer), a multiplet needs a higher 60

enantiodifferentiation value (∆∆δ) than a singlet. To consider this issue, we defined recently the enantioresolution quotient, E,11 of an enantiodiscriminated signal; a parameter that shows the goodness of the enantioresolution of a split signal, which is proportional to ∆∆δ and decreases with signal width. Multiplicity, signal overlapping and spectral dispersion problems can be overcome by monitoring the chiral recognition experiment

65

through 13C NMR spectroscopy.12 13C is a nucleus with high presence in organic molecules, with an intrinsic broader chemical shift range and higher spectral dispersion than 1H, and with an all-singlet spectrum easily achieved by broadband 1H decoupling. In turn, 13C has been chosen to observe enantiodifferentiation by NMR in the solid-state,13 as well as to monitoring metabolomic studies.14,15

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As well known, the main drawback of observing

13

C is its intrinsic low sensitivity

(due to a low natural abundance and gyro-magnetic ratio), which results in long experimental times and sometimes even in the impracticality of the experiment. Different hyperpolarization methods have been developed to enhance significantly the NMR signal.16 Among them, dynamic nuclear polarization (DNP)17,18 allows to increase the NMR signal 75

intensity by several orders of magnitude by the transference of electron spin polarization to atomic nuclei via microwave irradiation at (or near) the electron Larmor frequency, enhancing nuclear spin polarization. Dissolution DNP (d-DNP)19 is based on the


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combination of solid-state DNP with subsequent rapid dissolution to generate a solution of hyperpolarized nuclei, making DNP accessible for solution-state NMR and MRI, and 80

encouraging the development of multiple in-vitro and in-vivo applications.20-23 Recent studies by d-DNP 13C NMR include the analysis of complex biological mixtures.21-23 In the present work, a method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in aqueous solution, at a millimolar concentration and using a CSA was

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performed. The selected compound was the essential amino acid methionine, which is the primary source of sulphur in the body and plays an important role in the biosynthesis of proteins, in the growth of new blood vessels and in the normal body metabolism, especially in the metabolism of lipids.24,25 As a proof of concept, enantiodiscriminated

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using

CSA

13

C-labeled DL-methionine was

(-)-(18-crown-6)-2,3,11,12-tetracarboxylic

acid

(18C6H4)12 and the process was conducted by d-DNP 13C NMR spectroscopy.

Experimental Section Reagents and chemicals 95

DL-Methionine, DL-[1-13C]-methionine, (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4), [1-13C] pyruvic acid, trityl radical OX63, glycerol, H2O (LC-MS grade) and 3-(trimethylsilyl)-[2,2,3,3-2H4]-propionic acid sodium salt (TSP) were purchased from Sigma-Aldrich S.A. (Tres Cantos, Madrid, Spain). Deuterium oxide (99.96 % D) was obtained from Cortecnet (Voisins-le-Bretonneux, France).


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100 d-DNP NMR spectroscopy The study was carried out with a HyperSense® (Oxford Instruments Molecular Biotools, Oxford, UK) commercial polarizer working at 3.35 T and 1.4 K. It was combined with a Bruker AVANCE-III 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, 105

Germany), equipped with a 5 mm broadband TXI inverse probehead incorporating a zgradient coil and working at field strength of 14.1 T (600.13 MHz and 150.92 MHz, 1H and 13

C frequencies respectively). The selection of the optimal

13

C microwave irradiation frequency26 was done by

irradiating a sample of [1-13C] pyruvic acid (50 µl, 305 mM) doped with trityl radical 110

OX63 (15 mM) into H2O:glycerol (1:1) at several monitoring the

13

C microwave frequencies, while

13

C NMR signal. A range of frequencies between 94.000 GHz and 94.200

GHz was swept by irradiating the sample during 3 min at several frequencies (see Supporting Information, Figure S1). For the enantiodifferentiation experiment performed through d-DNP 115

13

C NMR, a

sample consisting on DL-[1-13C]-methionine (224 mM) plus trityl radical OX63 (15 mM) in H2O:glycerol (1:1) was prepared. Then, an aliquot of 50 µl was polarized at 94.078 GHz (100 mW) for 9000 s at 1.4 K of temperature. After that, the DL-[1-13C]-methionine frozen sample was rapidly dissolved into a 5 ml room temperature dissolution of D2O containing ()-18C6H4 (33.6 mM). The resulting dissolution was automatically transferred to a 5 mm

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NMR tube, which was adequately set in the NMR magnet (ca. 5 s). Then, the

13

C NMR

experiment was automatically started. The SSFT/FLASH method27-29 applied to d-DNP


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NMR was used to acquire hyperpolarized decoupled

13

13

C NMR decay data. Power gated proton

C NMR experiments were recorded using a 10° flip-angle RF pulse, an

acquisition time of 0.53 s and a relaxation delay of 1 s. Data were collected into 32 K data 125

points and with a spectral width of 30864 Hz. Resulting FIDs were Fourier transformed without any apodization function. Spectra were manually phased and baseline corrected.13C NMR signal intensity decay curves of hyperpolarized C1 of DL-[1-13C]-methionine (with and without CSA) were obtained by fitting signal intensity values to a monoexponential decay curve using MestreNova10 software.

130 NMR spectroscopy NMR experiments were performed on the AVANCE III 600 MHz NMR spectrometer previously described. 1D

13

C NMR standard experiments were performed using the

parameters described before. A capillary with TSP (3% in D2O) was introduced in the 135

NMR tube for external referencing. The integration of split signals was carried out by peak deconvolution using the GSD application of MestreNova10.

Results and discussion 140

Initially, racemic DL-methionine, at natural abundance and in aqueous solution (2.4 mM), was enantiodifferentiated through conventional

13

C NMR spectroscopy using (-)-

18C6H4 as chiral solvating agent. The racemate solution was titrated directly in the NMR tube with the CSA until a total amount of 19 equivalents of (-)-18C6H4 was added.


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Standard 1D 13C NMR experiments were performed requiring long acquisition times (expt 145

24h 9 min). 13C NMR spectra corresponding to initial and last titration points (0 and 19 eq of (-)-18C6H4 respectively) are depicted in Figure 1. After the addition of 19 eq of CSA, all

five

13

C

signals

of

DL-methionine

were

enantiodiscriminated,

with

enantiodifferentiation values (∆∆δ) ranging from 43 to 261 ppb and enantioresolution quotients (E)11 from 1.1 to 4.7. As shown, a clean spectrum with no signal overlapping was 150

obtained. Intense signals of CSA are indicated with asterisks in the spectrum.


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Figure 1. 150.92 MHz 13C NMR spectra of a) DL-methionine (2.4 mM) in D2O (expt 24 h 155

9 min) and b) DL-methionine (2.4 mM) in D2O after the addition of 19 eq of (-)-18C6H4 (expt 24 h 9 min). Asterisks denote signals corresponding to the chiral auxiliary.

After that, a method integrating d-DNP in the

13

C NMR-aided study of chiral

recognition was designed. The method was based on the initial polarization of solely the 160

analyte followed by a quick mix with the transfer solvent containing the CSA. In this way, the hyperpolarized chiral analyte could be fast detected and, moreover, the signals of the CSA would be minimized or not detected in the resulting spectrum. Specifically, the procedure consisted of the following sequential steps: i) preparation of the sample containing the analyte into the sample cup, ii) insertion of the sample into the dynamic

165

nuclear polarizer, iii) hyperpolarization of the sample at low temperature, iv) insertion of the transfer solvent with the CSA, v) sample dissolution, vi) fast transfer of the resulting dissolution into the NMR magnet and vii) rapid acquisition of the

13

C NMR experiment

(Figure 2).


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Figure 2. Schematic protocol for the study of chiral recognition by d-DNP NMR spectroscopy using a chiral solvating agent (CSA).

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Like most hyperpolarization techniques, the d-DNP method is limited by the fact that the high polarization is available only for a short time window, of the order of the longitudinal relaxation time (T1) of the nuclei. C1 isotopically labelled DL-[1-13C]methionine was used as analyte in order to build-up the procedure and to conduct a preliminary experiment. The choice was made taking into account the characteristic loss of

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hyperpolarization during the transfer of the dissolution to the NMR magnet and considering the expected longer T1 of the carboxyl carbon compared to the others. Initially, the optimal experimental conditions for sample preparation and polarization of DL-[1-13C]-methionine were determined. Trityl radical OX63 and H2O:glycerol (1:1) were chosen as polarizing


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and glassing agents respectively.30 The optimal 185

13

C microwave irradiation frequency was

determined considering the selected free radical and glassing agents.26 For that, a

13

C

microwave DNP microsweep was acquired with [1-13C] pyruvic acid (50 µl, 305 mM) doped with trityl radical OX63 (15 mM) into H2O:glycerol (1:1). A range of 13C microwave frequencies between 94.000 GHz and 94.200 GHz was swept by irradiating the sample at several frequencies while monitoring the 190

13

C NMR signal intensity. The curve showed a

positive maximum polarization peak at 94.078 GHz, with a separation between P(+) and P(-) of 85 MHz (Experimental Section and Figure S1 of the Supporting Information). Next, the optimal irradiation time for the polarization of DL-[1-13C]-methionine was determined. The solid-state 13C polarization build-up curve of the analyte (50 µl, 224 mM) (Figure S2 of the Supporting Information) showed 9000 s as an optimal irradiation time. To our

195

knowledge, these are the first described conditions for the polarization of methionine as a free amino acid and in a low concentration (i.e. under molar range in the glassing matrix); analyte concentrations in the gel matrix are typically on the molar range.30 The d-DNP 13C NMR enantiodifferentiation experiment consisted on the following. A sample consisting on DL-[1-13C]-methionine (224 mM) plus trityl radical OX63 (15 mM)

200

in H2O:glycerol (1:1) was prepared. Then, a 50 µl sample aliquot was polarized at 94.078 GHz (100 mW) for 9000 s at 1.4 K. Once polarized, frozen DL-[1-13C]-methionine sample was rapidly dissolved into a 5 ml solution of D2O containing CSA (-)-18C6H4 (33.6 mM). The resulting dissolution was automatically transferred to a 5 mm NMR tube adequately set into the magnet. The overall time of both processes - mixing frozen polarized analyte

205

mixture with CSA solution plus transferring the resulting dissolution to the NMR tube -


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was of ca. 5 s. After this time, the Hyperpolarized

13

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C NMR experiment started automatically.

C NMR decay data was acquired using the SSFT/FLASH method27-29

applied to d-DNP NMR, which yielded 1D 13C NMR spectra at every scan and allowed the measurement of heteronuclear T1(13C) (Figure 3). The experiment was identically repeated 210

without CSA in the transfer solvent. T1 values of C1 of dissolved DL-[1-13C]-methionine with and without (-)-18C6H4 in the dissolution were 8.3 and 12.5 s respectively (see Experimental Section). As expected, T1(13C) decreased by the formation of the diastereoisomeric complexes between the enantiomeric analytes and the CSA that takes place in the enantiodifferentiation experiment.4-7 This is an important aspect to consider

215

when designing new experiments for new samples.


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Figure 3.

13

C NMR signal intensity decay curves of hyperpolarized C1 of DL-[1-13C]-

methionine without CSA (white circles, T1(13C) = 12.5 s) and with CSA (black squares, 220

T1(13C) = 8.5 s). T1(13C) obtained by fitting signal intensity values to a monoexponential decay curve.

Figure 4 shows the d-DNP 13C NMR spectrum of the resulting dissolution containing 2.2 mM DL-[1-13C]-methionine and 15 eq of (-)-18C6H4 in D2O (plus trityl radical, 225

glycerol and H2O).. The spectrum was recorded with just a single scan, meaning an NMR acquisition time of 1 s. The DNP-enhanced enantiodifferentiation experiment yielded a clean and simple 1D spectrum, which showed just the signal of the hyperpolarized metabolite (labelled carbon C1) and two residual signals of glycerol, being free from CSA peaks. Results clearly showed the differentiation of the carboxylic carbon signals of the two

230

enantiomers; a split of the peak with enantiodifferentiation value, ∆∆δ, of 40 ppb and enantioresolution quotient, E,11 of 0.4. Integration values were close to the real enantiomeric ratio (er 1.0).


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235 Figure 4. 150.92 MHz d-DNP 13C NMR spectrum (1 scan, expt 1 s) of hyperpolarized DL[1-13C]-methionine (2.2 mM) during the enantiodifferentiation experiment with CSA (-)18C6H4 (15 eq). Asterisks denote peaks corresponding to glycerol.

240

Once the sample analyte was at thermal equilibrium (after 1 h), an analogous singlescan

13

C spectrum with same acquisition parameters was recorded. The spectrum showed

no signals, neither of analyte C1 nor of other compounds in the solution (Figure S3 of the Supporting Information). After that, the same experiment at thermal equilibrium was repeated with a longer acquisition time (ns 1024, expt 43 min), observing the C1 peak of 245

DL-[1-13C]-methionine, the signals of the CSA and the peaks of glycerol (Figure 5). At


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these conditions, C1 signal was enantiodifferentiated with ∆∆δ of 50.1 ppb and enantioresolution quotient E of 0.9; the er corresponded to 1 as expected for a racemate.

250 Figure 5. 150.92 MHz

13

C NMR spectrum (1024 scans, expt 43 min) of DL-[1-13C]-

methionine (2.2 mM) at thermal equilibrium with CSA (-)-18C6H4 (15 eq). The sample contains also trityl radical OX63, glycerol and H2O. Asterisks and circles denote peaks corresponding to glycerol and CSA, respectively. 255 In NMR-aided chiral recognition using a CSA, due to the nature and origin of ∆∆δ,5 magnetically different diastereoisomeric complexes or complexes with different association constants lead to a differentiation in δ (∆∆δ). ∆∆δ depends on the nature of the analyte and the CSA and it is affected (increasing) by the number of equivalents of CSA added and by


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the concentration of the analyte, at a given temperature and solvent.11 Obtained results showed that, when the enantiomeric mixture was hyperpolarized, the ∆∆δ was very similar to that at the thermal equilibrium (∆∆δ 40.4 vs 50.1 ppb, respectively); results suggested that the complexation between the analyte and the CSA is not affected by the prior hyperpolarization of the analyte. Also, the measurement of split peaks integrals under

265

hyperpolarization conditions was analogous to that at thermal equilibrium and close to the real er of 1. Though caution should be used when DNP-based techniques are utilized for quantification, our results regarding the relative integrals of split peaks suggested that both enantiomers were equally hyperpolarized and that a reliable determination of er could be successfully performed. Future studies applying the developed method to different chiral

270

analytes browsing an er range (from 0 to 1) must be done to confirm it. Finally, though the integral ratio was consistent with the real er, a decrease in the enantioresolution of the split peak compared to that at thermal equilibrium was observed (E 0.4 vs 0.9, respectively). The loss of enantioresolution was attributed to the hindrance for a fine shims optimization prior to the required fast data acquisition, meaning a broadening of the signals and a consequent

275

decrease of E. As discussed in a previous work,11 a decrease in signal enantioresolution (E) can affect the quality of the er measurement; this is of special importance when measuring enantiomeric mixtures with an er far from a racemic situation.

This work presents an NMR approach for the enantiorecognition of small molecules 280

when sensitivity is limited; e.g. when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like

13

C, is required. This last case is typical


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when the observation of 1H is ruled out due to: i) 1H poor analytes with none or low enantiodiscrimination observed in the proton spectrum; ii) analyte peaks of interest severely overlapped by CSA signals (a common situation when a high amount of CSA is required); 285

iii) severe signal overlapping in 1H spectrum before the addition of the CSA. The described method enhances the sensitivity of the conventional NMR-based method by the previous hyperpolarization of the analyte and it is applicable to the observation of other magnetically active nuclei, such as 15N. Besides, the way the addition of the CSA in the experiment was integrated into the d-DNP NMR method allowed the selective hyperpolarization of the

290

chiral analyte, which lightens the common problem of signal overlapping between analyte and CSA. The conventional CSA addition method for the chiral recognition of small molecules by NMR spectroscopy is a very reproducible robust methodology based on the rapid formation of diastereoisomeric complexes between the analyte (enantiomeric mixture) and the chiral auxiliary (commonly an enantiopure molecule). CSAs generally

295

undergo fast exchange with substrates; the NMR spectrum is a weighted average of the proportion of bound and unbound substrate.5-7 Our results showed that the formation of diastereoisomeric complexes took place when the analyte was hyperpolarized and that they remained when the system recovered thermal equilibrium. In the example utilized hyperpolarized 13C labeled racemic methionine was enantiodiscriminated by crown ether (-

300

)-18C6H4. At the same time as developing the method, the enantiorecognition of a naturally occurring metabolite in aqueous solution wanted to be studied under hyperpolarization conditions. The sufficient hyperpolarization level of DL-[1-13C]methionine under optimized conditions, allowed us to demonstrate the feasibility of the developed methodology. However, based on the results obtained, which suggests the


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formation of hyperpolarized analyte-CSA diastereoisomeric complexes (analogous to those without analyte hyperpolarization), the described d-DNP NMR-based method for chiral recognition should not be limited to labeled molecules. The hyperpolarization of natural abundance molecules by dissolution DNP, due to the transference of electron spin polarization to 13C nuclei via microwave irradiation (as here described), followed by single-

310

scan

13

C NMR spectroscopy has been widely reported.31,32 A natural abundance chiral

analyte reaching a hyperpolarization level enough to be observed by a single-scan

13

C

NMR experiment, should be enantiodifferentiated by d-DNP NMR spectroscopy given an adequate CSA – i.e. a CSA that forms magnetically different diastereoisomeric complexes with the enantiomeric analytes. In the case of an analyte with poor hyperpolarization level, 315

the application of 1H-13C cross polarization to d-DNP would help to increase it; the implementation of CP would require hardware and software adaptation.33 The analyte concentration detection limit of the d-DNP NMR-based method is given by the instrument used (magnetic field, kind of probe), the nucleus observed, the experiment performed and the hyperpolarization level reached by the analyte (nature of analyte and conditions of

320

hyperpolarization). The integration of d-DNP in the conventional NMR-based method for chiral recognition brings along some still not resolved issues related to the nature and current state of the d-DNP NMR technique; mainly, to the short time available before starting the NMR experiment, which is a consequence of the short life of hyperpolarized states. This

325

condition difficult i) the shimming process previous the acquisition and ii) a robust reproducible transfer of the dissolution to the NMR tube, which affects, respectively, the enantioresolution E of the split peaks and the global reproducibility and robustness of the


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method. d-DNP NMR technology is advancing very fast and last progresses, especially those focused on preserve enhance magnetization (e.g. long-lived states)34-36 and the use of 330

specially designed sample injectors,37 will help overcome the aforementioned limitations, allowing a broader development of the described methodology for chiral recognition studies. The present work paves the way to future studies, such as the application of the method to chiral analytes at natural abundance, pure enantiomeric mixtures and enantiomers within complex mixtures and the extension to other heteronuclei, like 15N.

335

Conclusions To conclude, the feasibility of chiral recognition by dissolution DNP

13

C NMR

spectroscopy was demonstrated for the first time. The present work presents an NMR approach for the enantiorecognition of small molecules when sensitivity is limited; e.g. 340

when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like

13

C, is required. A method integrating d-DNP and

13

C NMR-aided

enantiodifferentiation using chiral solvating agents was developed, in which only the chiral analyte was hyperpolarized and selectively observed by NMR spectroscopy. The described method enhances the sensitivity of the conventional NMR-based method and lightens the 345

common problem of signal overlapping between analyte and CSA. The differentiation of the hyperpolarized DL-[1-13C]-methionine enantiomers using CSA (-)-18C6H4 was efficiently and quickly observed from a single-scan

13

C NMR experiment. The outcome

was a clean spectrum with a clear enantiodifferentiated C1 signal of the racemic amino acid and no signals of the chiral auxiliary. Under hyperpolarization of the analyte,


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enantiodifferentiation ∆∆δ and relative integration values of split peaks were similar to those obtained at thermal equilibrium, whereas the enantioresolution quotient E decreased. The integration of d-DNP in the conventional NMR-based method for chiral recognition brings along some still not resolved issues related to the nature and current state of the dDNP NMR technique. Last advances in d-DNP NMR, especially in preserve enhance

355

magnetization (e.g. long-lived states), will help to improve the robustness and reproducibility of the method as well as maintain the split peak enantioresolution. The present work paves the way to future studies, such as its application to chiral analytes at natural abundance, to pure enantiomeric mixtures and enantiomers within complex mixtures and the extension to other heteronuclei, like 15N.

360

Acknowledgments Financial

support

from

the

MINECO

(CTQ2015-64436-P)

is

gratefully

acknowledged. NMR studies were carried out at the joint NMR facility of the Universitat 365

Autònoma de Barcelona and CIBER-BBN (Cerdanyola del Vallès).

Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org. 370

Figure S1, curve for the determination of the optimal frequency; Figure S2, solid-state

13

13

C microwave irradiation

C polarization build-up curve of DL-[1-13C]-


20

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methionine and Figure S3,

13

C NMR spectra of d-DNP NMR enantiodifferentiated

sample at thermal equilibrium acquired with 1 scan (PDF).

375

Author Information Corresponding Author *E-mail: [email protected]. Phone: +34 935813785 ORCID 380

Míriam Pérez-Trujillo: 0000-0002-6919-7417

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For TOC only

455


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150.92 MHz 13C NMR spectra of a) DL-methionine (2.4 mM) in D2O (expt 24 h 9 min) and b) DL-methionine (2.4 mM) in D2O after the addition of 19 eq of (-)-18C6H4 (expt 24 h 9 min). Asterisks denote signals corresponding to the chiral auxiliary. 115x91mm (600 x 600 DPI)


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Schematic protocol for the study of chiral recognition by d-DNP NMR spectroscopy using a chiral solvating agent (CSA). 88x50mm (600 x 600 DPI)



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13

C NMR signal intensity decay curves of hyperpolarized C1 of DL-[1-13C]-methionine without CSA (white circles, T1(13C) = 12.5 s) and with CSA (black squares, T1(13C) = 8.5 s). T1(13C) obtained by fitting signal intensity values to a monoexponential decay curve. 95x88mm (600 x 600 DPI)


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150.92 MHz d-DNP 13C NMR spectrum (1 scan, expt 1 s) of hyperpolarized DL-[1-13C]-methionine (2.2 mM) during the enantiodifferentiation experiment with CSA (-)-18C6H4 (15 eq). Asterisks denote peaks corresponding to glycerol. 101x75mm (600 x 600 DPI)



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150.92 MHz 13C NMR spectrum (1024 scans, expt 43 min) of DL-[1-13C]-methionine (2.2 mM) at thermal equilibrium with CSA (-)-18C6H4 (15 eq). The sample contains also trityl radical OX63, glycerol and H2O. Asterisks and circles denote peaks corresponding to glycerol and CSA, respectively. 90x59mm (600 x 600 DPI)


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55x20mm (600 x 600 DPI)


Chiral recognition by dissolution DNP NMR spectroscopy of 13

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