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“Precise determination of enantiomeric excess by a sensitivity enhanced two-dimensional band-selective pure-shift NMR” Kavitha Rachineni, Veera Mohana Rao Kakita, Satya Narayana Dayaka, Sahithya Phani Babu Vemulapalli, and Jagadeesh Bharatam Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01288 • Publication Date (Web): 19 Jun 2015 Downloaded from http://pubs.acs.org on June 23, 2015

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Analytical Chemistry

1

Precise determination of enantiomeric excess by a

2

sensitivity

3

selective pure-shift NMR

enhanced

two-dimensional

band-

4 5

Kavitha Rachineni,a,‡ Veera Mohana Rao Kakita,a,‡ Satyanarayana Dayaka,a,‡ Sahithya

6

Phani Babu Vemulapalli,a and Jagadeesh Bharatam*a

[a]

Centre for NMR and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad-500 007, India.

7 8 9 10

Keywords

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Enantiomeric excess, zCOSY, band-selective homodecoupling, enhanced resolution,

12

enhanced sensitivity

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Abstract

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Unambiguous identification and precise quantification of enantiomers in chiral

3

mixtures is crucial for enantiomer specific synthesis as well as chemical analysis.

4

The task is often challenging for mixtures with high enantiomeric excess and for

5

complex molecules with strong 1H-1H scalar (J) coupling network.

6

advancements in 1H-1H decoupling strategies to suppress the J-interactions, offered

7

new possibilities for NMR based unambiguous discrimination and quantification

8

enantiomers.

9

zCOSY NMR method with homonuclear band-selective decoupling in both the F1

The recent

Herein, we discuss a high resolution two-dimensional pure-shift

10

and F2 dimensions (F1F2-HOBS-zCOSY).

This advanced method shows a sharp

11

improvement in resolution over the other COSY methods, and also eliminates the

12

problems associated with the overlapping decoupling sidebands. The efficacy of this

13

method has been exploited for precise quantification of enantiomeric excess (ee) ratio

14

(R/S) up to 99:1 in the presence of very low concentrations of chiral lanthanide shift

15

reagents (CLSR) or chiral solvating agents (CSA).

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simple and can be easily implemented on any modern NMR spectrometers, as a

17

routine analytical tool.

The F1F2-HOBS-zCOSY is

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1

Introduction

2

Enantiomer specific biological and pharmacological activities of natural products

3

have stimulated enormous interest in chemists to synthesize enantiomerically pure molecules.

4

This in turn has highlighted the necessity of developing various analytical methods such as

5

HPLC,1 CD,2 X-Ray3 and NMR4 for unambiguous discrimination and precise quantification

6

of stereo-isomers in enantiomeric mixtures. In this regard, NMR spectroscopy has been

7

proved to be highly useful and different methods are developed over the years for quantifying

8

the enantiomeric excess (ee) of a chiral mixture.5,

9

protons of R and S enantiomers of a chiral compound exhibit isochronous NMR signals,

10

which limit their discrimination by using NMR spectroscopy. However, by incorporating

11

chiral derivatizing agents (CDA),7, 8 or by simply dissolving chiral solvating agents (CSA),9,

12

10

13

(enantiomers) can be converted into NMR-observable diastereomers (magnetically non-

14

equivalent).

15

resolving agents17 are also employed to discriminate and determine the ee. In this regard, the

16

chiral auxiliaries, CLSR and CSA, which yield diastereomeric complexes via non-covalent

17

bond interactions, are attractive, as they are less expensive and can be easily employed.

18

Nevertheless, irrespective of the choice of chiral auxiliaries, the accurate quantification of

19

diastereomeric ratio or ee warrants experimental approaches that yield a good spectral

20

resolution and signal sensitivity, particularly for quantifying diastereomers of very low

21

population, i.e., high ee.

6

In fact, the magnetically equivalent

and chiral lanthanide shift reagents (CLSR)11 in the analyte solution, the substrates

Chiral alignment media12-16 and more recently specially synthesized chiral

22

Chemical shift separation between the two enantiomers depends on the concentration

23

of chiral auxiliaries dissolved, and increased concentrations of CLSR or CSA are often

24

required to resolve the enantiomers. This situation leads to spectral overlap or/and line

25

broadening. For example, in the case of CLSR, due to the paramagnetic interaction, spectral 3 ACS Paragon Plus Environment


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lines are severely broadened resulting in poor resolution and sensitivity. On the other hand,

2

low dissolutions of CLSR or CSA do not yield the desired resolution of enantiomers (Figure

3

1). Furthermore, as the inherent 1H-1H scalar couplings (J) split the individual spectral lines

4

into multiplets, the poorly resolved enantiomer signals manifest into broader complex

5

multiplets, thereby hamper the analysis.

6

sensitivity are of a serious concern in the conventional NMR spectroscopy. Therefore, a

7

high-resolution and high-sensitive experimental approach that works well at low

8

concentrations of CLSR or CSA and effectively suppresses the undesired 1H-1H scalar

9

coupling information to yield precise quantifications even for higher ee, is essential. The

10

present work addresses this issue by proposing an advanced 2D NMR scheme that involves

11

suppressing of J-coupling multiplets into individual singlets in both the dimensions, which

12

yields high resolution and sensitivity even at low dissolutions of CLSR or CSA.

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These two sources for poor resolution and

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H-1H homodecoupling as a means of suppressing the scalar coupling multiplets has

14

been realized long back. 18-23 For example, the potential of Zangger-Sterk (ZS) decoupling22

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and its variants are widely explored, which formed basis for the subsequent advancements in

16

the recent years, such as pure-shift NMR24-41 that yield singlets for each 1H chemical site,

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thereby provide high resolution. Despite the earlier methods are impressive, they suffer from

18

poor sensitivity and prolonged experimental times, particularly for broadband decoupling.

19

Alternately, the observation of other nuclei, such as

20

discriminate the enantiomers, but the low sensitivity of

21

utility for a routine screening.42-44

13

C, while decoupling the 1H allows to 13

C (~1%) restricts their practical

22

Very recently, Morris et al., have introduced a superior PSYCHE (Pure Shift Yielded

23

by CHirp Excitation) broadband homodecoupling scheme that circumvents the sensitivity

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issues to a larger extent.45, 46 Nevertheless, in terms of the sensitivity, real-time homonuclear

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band-selective decoupling (HOBS) is superior to that of broadband decoupling.47-52 The 4 ACS Paragon Plus Environment

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advanced HOBS scheme relies on periodic interruption of the acquisition by real-time

2

decoupling blocks in one and multi dimensional NMR that eliminates the time consuming

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pseudo-dimensional acquisitions and special spectral reconstruction processing.31-38,

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Accordingly, the one-dimensional (1D) HOBS scheme has been demonstrated for the

5

quantification of diastereomers (11.6:88.4 R/S) 53 and enantiomers of only relatively low ee %

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samples (35:65 R/S, 30% ee).54, 55 On the other hand, unambiguous identification and precise

7

quantification of minor enantiomers in high ee samples is a challenging task. For example,

8

the 1D HOBS approach suffers due to the sideband artefacts that appear at 2n/AQ Hz (n and

9

AQ are the number of interruptions and acquisition time, respectively) positions,54, 55 which

10

may overlap with the required pure-shift resonances and hamper the analysis. As shown

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below, the problem is more acute, especially for high ee samples. Suryaprakash and co-

12

workers have shown homodecoupling along the F1-dimension in conventional 2D-COSY56

13

and 2D-TOCSY57 schemes that involves selective excitation followed by band-selective

14

decoupling (soft and hard 180o pulses), and its applications for ee determinations ~33% and

15

80%, respectively. The later scheme, termed as RES-TOCSY,57 is conceptually related to the

16

1D-TOCSY.58 Despite these schemes are impressive and easy to implement, they suffer from

17

poor resolution along the direct dimension (F2).

47-55

18

In the present work, we discuss the high resolution 2D- homonuclear band-selective

19

pure-shift z-COSY (real-time) with 1H homodecoupling along (i) the F1-dimension (F1-

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HOBS-zCOSY), (ii) the F2-dimension (F2-HOBS-zCOSY) and (iii) both the F1 and F2-

21

dimensions (F1F2-HOBS-zCOSY).

22

improvement in the sensitivity over the present F1-HOBS-zCOSY or the earlier 2D-COSY

23

method.56 Whereas, as shown below, the F1F2-HOBS-zCOSY yields significant

24

enhancement in the resolution and sensitivity, over the other two techniques. This superior

25

method can be applied for routine analysis of low as well as high ee mixtures and other

The F2-HOBS-zCOSY has shown a marginal



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complex molecules. We exemplify this general approach for menthol, methyl benzyl amine

2

(MBA) and 1-aminoindan of different enantiomeric ratios, in the presence of low

3

concentrations

4

(trifluoromethylhydroxymethylene)-(+)-camphorate] (ETHM, CLSR) or triphenoxyborane +

5

R-binol (CSA), and show a precise quantification of minor enantiomers down to ~1%.

of

different

chiral

auxiliaries,

europium

tris[3-

6 7

Experimental Section

8

The proposed two-dimensional HOBS-zCOSY schemes, particularly the superiority

9

of the F1F2-HOBS-zCOSY, are demonstrated for three different samples: racemic menthol

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(R/S 50:50, 0% ee), enantiomeric methyl benzyl amine (MBA) at different ratios (R/S 50:50,

11

70:30, 90:10, 95:5 and 99:1, correspond to 0%, 40%, 80%, 90% and 98% ee, respectively)

12

and enantiomeric 1-aminoindan at different ratios (R/S 50:50 and 95:5, correspond to 0% and

13

90% ee, respectively).

14

(trifluoromethylhydroxymethylene)-(+)-camphorate] (ETHM), R-binol and triphenoxyborane

15

are purchased from M/s Sigma-Aldrich, USA. By dissolving 25 mg of the readily available

16

racemic menthol in 0.5 ml of CDCl3 (M/s CIL, USA), the resultant 0% ee sample is directly

17

used for NMR studies, whereas, different ee % samples of MBA are prepared from the R and

18

S enantiomer stock solutions in CDCl3. The amount of R and S enantiomers present in the

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MBA samples are as follows: 12.5 mg of R and 12.5 mg of S (0% ee), 17.5 mg of R and 7.5

20

mg of S (40% ee), 22.5 mg of R and 2.5 mg of S (80% ee), 23.75 mg of R and 1.25 mg of S

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(90% ee) and 25.0 mg of R and 0.25 mg of S (98% ee). For menthol and MBA, ETHM,

22

which is a paramagnetic salt, is used as a CLSR to convert the enantiomers into the NMR

23

observable diastereomers.

The racemic menthol, MBA, 1-aminoindan, europium tris[3-


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The utility of F1F2-HOBS-zCOSY has also been explored for the CSA induced

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enantiomeric discrimination of chiral amines as well. Herein, the derivatization protocol

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involves the formation of chiral boronate esters from the 1-aminoindan (4 equivalents, 4.58

4

mg of R/S), R-binol (4 equivalents, 9.8 mg) and triphenoxyborane (1 equivalent, 2.5 mg) in

5

0.5 ml of CDCl3, where the derivatization can directly be acheived in NMR tubes.59 The

6

resultant chiral boronate esters of 1-aminoindan facilitate their ee quantifications.

7

NMR experiments were carried out on Bruker Avance-III 500 (5mm, BBFO probe) or 700

8

MHz (5mm, cryo cooled probe) spectrometers, at 25 oC. Prior to the Fourier transformation,

9

the resulted 2D data sets are zero filled to 1024 points in both the dimensions. Line

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broadening of 0.3 Hz and a 90o phase-shifted sine-bell apodization are applied in F2 and F1

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dimensions, respectively. Base line correction is applied, wherever necessary, which did not

12

result in any adverse effect on the R/S quantifications.

All the

13 14

Results and Discussion

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Due to the R and S enantiomer specific interaction of the paramagnetic ETHM

16

(CLSR) with Hα of MBA (quartet pattern) and with H7 of menthol (multiplet pattern),

17

separate resonance line for each enantiomer is expected. Initially, the effect of ETHM

18

concentration on the resolution of enantiomeric mixtures of different ratios is tested for

19

MBA (Figure 1). As expected, the normal 1H NMR spectrum of MBA (without ETHM,

20

Figure 1a.i) shows a single J-coupled quartet pattern for Hα (~ 4.15 ppm). Upon the addition

21

of 3 mM ETHM, a sextet pattern for Hα is realized, which is due to an overlap of two

22

independent quartet patterns corresponding to the R and S enantiomers, respectively. This

23

observation signifies an onset of the separation of the two enantiomers and is well evident

24

for racemic (R/S 50:50) MBA (Figure 1a) due to their equal intensities. Further, it also

25

suggests that, at 3 mM concentration, the line width is predominantly governed by the intra7 ACS Paragon Plus Environment


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molecular J-coupling interactions of Hα and the intermolecular ETHM-MBA interactions

2

play rather a minor or comparable role. A further increase in ETHM concentration beyond

3

3mM has shown notable features for different R/S ratios that suggest some limitations of this

4

conventional 1D NMR approach for ee determination. In the case of racemic mixture (R/S

5

50:50) of MBA, increase in the chemical shift separation between the two signals (Figure

6

1a) is clearly observed with the increase in ETHM. But the individual lines are found to be

7

more broadened (due to the increased contribution of paramagnetic relaxation) and the J-

8

coupling patterns are gradually buried. Nevertheless, as the intensities of the lines are equal

9

in the case of racemic mixture, the R/S quantification is straight forward. Similarly, the ee

10

quantifications are not hampered for samples up to 70% (R/S 85:15). On the other hand, for

11

samples of 80% ee (R/S 90:10) in the presence of ETHM concentration >3 mM (Figure 1b),

12

the relatively low intense peak corresponding to the S isomer gets further broadened thereby

13

making the accurate discrimination/ quantification more difficult. This is also evident for

14

95% ee sample (Figure 1c). Further, for 98% ee sample, the very low populated S isomer

15

could not be identified (Figure 1d). These observations suggest that at higher concentrations

16

(~10 mM or above) of CLSR (ETHM), which typically is the case with the earlier NMR

17

studies, the identification and quantification of R/S enantiomers may have to be restricted

18

only for racemic or low ee samples.

19

It is important to note that the homodecoupling mediated pure-shift NMR involves,

20

collapse of the multiplet structure of the individual lines into singlets, wherein, the area

21

under each multiplet is re-distributed as singlet, thereby high sensitivity is achieved along

22

with the high resolution.

23

concentrations of the paramagnetic CLSR is more appropriate, so that the line-width is still

24

predominantly governed by the scalar coupling interactions. As discussed above, in the

25

present study, CLSR (ETHM) ~3 mM has been found to be optimum. On the other hand, if

In order to exploit this powerful feature, usage of low


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high concentrations of CLSR are used (Figure 1), the paramagnetic relaxation broadens the

2

lines much beyond the widths of scalar coupling multiplets, and the suppression of scalar

3

coupling will not yield any appreciable high resolution and sensitivity. This would be a

4

serious setback, particularly for the high ee mixtures > 80% (R/S 90:10). In order to

5

overcome the difficulty of estimating high ee, the recently reported 1D-HOBS scheme47 is

6

explored.

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Figure 1. Comparison of expanded 1D-1H NMR regions of Hα of MBA, by changing the concentration of CLSR (ETHM) for different enantiomeric excess ratios: a) 0% ee, b) 80% 9 ACS Paragon Plus Environment


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ee, c) 90% ee and d) 98% ee. The Figure 1(c) shows very low intense and broad signal for Senantiomer of 90% ee sample and the Figure 1(d) suggests that the identification and quantification of the low populated enantiomer (98% ee) is practically difficult from the conventional one-dimensional spectrum. The broad line seen in some spectra, which shifts down-field with the increase in ETHM concentration, belongs to the NH2 protons.

6 7

1D-HOBS decoupled spectra of enantiomeric mixtures

8

Figure 2 compares the expanded regions of conventional and 1D-HOBS spectra of 0

9

% ee (50:50), 80% ee (90:10) and 98% ee (99:1) samples of MBA in the presence of 3 mM

10

ETHM. As can be seen, the usual multiplet structures are nicely collapsed into pure-shift

11

resonances in all the cases. For the racemic sample, the individual singlets corresponding to

12

R and S can easily be seen in Figure 2a. Herein the broad satellites represented by asterisk

13

symbols indicate the side-bands that arise in HOBS spectra.54, 55 For 80% ee sample (Figure

14

2b), the pure-shift signal intensities are found to be higher than those of the conventional 1D-

15

1

16

low, their poor signal intensities are dominated by the side-bands corresponding to the major

17

enantiomer (R), which hampered the accurate quantification of the minor (S) enantiomer.

18

This is clearly evident for 98% ee sample (Figure 2c). Further, the problem is more serious

19

for the cases where the side-bands of the major enantiomer and the pure-shift resonance of

20

the minor enantiomer overlap.

H NMR spectra. However, as the relative population of the minor enantiomer (S) is very

21

The limitations of conventional 1D 1H and 1D HOBS schemes can be circumvented

22

by the 2D F1F2-HOBS-zCOSY method reported here. Herein the sidebands and the required

23

pure-shift resonances are well-resolved, thereby provide accurate means of identification and

24

quantification of enantiomers of high ee.


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Figure 2. Comparison of expanded regions of the conventional 1D 1H and 1D HOBSdecoupled spectra of Hα proton of MBA, for three different R/S ratios: (a) racemic (50:50), (b) 80% ee (90:10) and (c) 98% ee (99:1) samples. The spectra are recorded at 3.0 mM of ETHM concentration and the asterisk symbols represent the decoupling sidebands. As discussed in the main text, the unambiguous identification and quantification of the minor S enantiomer of MBA is not possible even from the 1D HOBS-decoupled spectrum. All the experiments are recorded under the identical experimental conditions.

9 10

Two-dimensional HOBS-zCOSY pulse schemes

11

Figure 3 illustrates the pulse schemes for two-dimensional a) F2-HOBS-zCOSY and

12

b) F1F2-HOBS-zCOSY, which are the hybrids of z-COSY60 and HOBS decoupling47 blocks.

13

The F2-HOBS-zCOSY is designed by interrupting the acquisition in the direct dimension

14

(F2) by a real-time HOBS decoupling element comprised of soft band-selective and hard

15

180o pulses,20 which suppresses the homonuclear 1H-1H scalar coupling along F2. Whereas,

16

in the case of F1F2-HOBS-zCOSY, both the indirect evolution (t1) and acquisition (t2) time

17

periods are interrupted once and ~20 times (n), respectively, by the HOBS decoupling

18

elements, which resulted in complete suppression of scalar coupling multiplets in both the



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1

dimensions. The present F2-HOBS-zCOSY and F1F2-HOBS-zCOSY are versatile, and by

2

simply setting n=1, these schemes reduce to the conventional selective refocused COSY (SR-

3

zCOSY) and F1-HOBS-zCOSY, respectively, which are also recorded for comparison.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3. Schematic representation of real-time homonuclear F2-decoupled HOBS zCOSY (F2-HOBS-zCOSY) (a) and F1F2-decoupled HOBS zCOSY (F1F2-HOBS-zCOSY) (b), pulse sequences. By setting n=1, the F1F2-HOBS-zCOSY is reduced to F1-HOBS-zCOSY. All the three sequences are tested independently for the samples mentioned in the text. The filled and open rectangles represent the 90° and 180° pulses, respectively. The small flip angle ‘β’ equals to 20o. The chirp61 shaped inversion pulse of 20 ms length is applied during the spatial encoding gradients, which suppresses the zero quantum magnetization and yields only the pure in-phase signals. The shaped pulses, which are marked with 'r' represent the Reburp refocusing pulses.62 Specific parameters for the present pulse sequences: phase cycling: φ1 = x, -x; φ2 = x; φ3 = -x; φrec = x, -x; pulsed field gradients: G1,2,3,4,5 (1 ms)= 5%, 15%, 11%, 31% and 2%, respectively of 53.5G/cm gradient strength. The Reburp shaped pulses of ~ 20 ms are used to refocus the interested spectral region. For quadrature, STATESTPPI63 technique is used. Two signal accumulations are made for each experiment. In all the cases, 400 Hz of spectral width is used for both the F1 and F2 dimensions. The corresponding acquisition times and dwelling increments are 0.64s/0.16s and 512/128 along the F2/F1 dimensions, respectively. Total time required for each experiment is ~10 min.

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1


Pure-shift 2D-F1F2-HOBS-zCOSY of racemic menthol

2

The performance of the proposed F2-HOBS-zCOSY and F1F2-HOBS-zCOSY

3

schemes with respect to the earlier reported methods is demonstrated initially on racemic

4

menthol. As discussed above, in order to minimize the CLSR induced line broadening and to

5

gain in sensitivity via homodecoupling, an optimized concentration of ETHM (3 mM) is used

6

for all the samples.

7

Initially, the choice of spectral bands for HOBS decoupling /ee quantification are

8

examined. The enantiomer specific interaction of the paramagnetic ETHM (CLSR) imparts

9

discrete R/S-specific chemical shifts for protons around the binding site. This translation aids

10

the R/S enantiomers distinguishable, particularly with the suppression of J-multiplets to

11

singlets by applying the homodecoupling, as described above. The onset of resolution in the

12

multiplicity pattern of the normal 1H NMR spectrum in the presence of low concentration of

13

CLSR, guides the selection of the chemical shift region for the homodecoupling (Figure S.1

14

of supporting information).

15

signals/regions to choose could be one or even more.

16

quantifications it is desirable to choose the R/S signal pairs with relatively higher chemical

17

shift separation. For example, in the presence of 3mM CLSR, the 1D-HOBS spectra of

18

methanol have shown three pairs of signals for H7 (2.39 ppm), H8 (0.99 ppm) and H10 (0.94

19

ppm) resonances, with each pair exhibiting separation of 9.0, 2.2 and 1.1 Hz, respectively,

20

corresponding to the R/S enantiomers (Figure 4.I). Resonances of the remaining 7 protons

21

did not show any observable R/S separations. Therefore, herein, only two independent F1F2-

22

HOBS-zCOSY experiments covering ~2.6 to ~2.1 ppm for H7 proton and ~1.1 to ~0.8 ppm

23

for H8 and H10 protons, are adequate. Nevertheless, due to the higher R/S chemical shift

24

separation, the obvious choice for R/S quantification would be H7 proton. However, as

25

shown in the supporting information (Figure S.2), the F1F2-HOBS-zCOSY of H8 proton with

Specific to the sample under investigation, the number of However, for unambiguous ee



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1

2.2 Hz R/S separation has also allowed the differentiation of the enantiomers as well as the

2

cross-verification of the data obtained from the H7 resonance, whereas the resolution of H10

3

signal with 1.1 Hz R/S separation is found not sufficient for the analysis. These findings have

4

also suggested a possible limit of R/S separation >2.0 Hz for this method.

5

Figure 4.II compares SR-zCOSY, F1-HOBS-zCOSY, F2-HOBS-zCOSY and F1F2-

6

HOBS-zCOSY of H7. As can be seen, the overlapped complex multiplet (60 Hz) of H7 could

7

not be resolved by the conventional SR-zCOSY. Further, despite the sensitivity exhibited by

8

the F2-HOBS-zCOSY is better compared to F1-HOBS-zCOSY, the spectral resolution

9

achieved by these two methods is inadequate, particularly for high ee samples. In a clear

10

contrast to these observations, the pure-shift resonances recorded by using the F1F2-HOBS-

11

zCOSY, have shown profound improvement in the resolution of R and S enantiomers. These

12

are clearly resolved as diagonal peaks, while the corresponding sidebands are separated out as

13

off-diagonal peaks.

14

As the spin-density of each enantiomer is directly proportional to the volume

15

integration of the respective diagonal peak with the corresponding, an unambiguous

16

identification and estimation of ee is much simple with this method. Therefore, unlike the

17

1D-HOBS decoupling schemes, this method does not suffer from the overlapped sideband

18

artefacts.

19

revealed a sensitivity gain of ~ 2 for the F1F2-HOBS-zCOSY with respect to the

20

conventional SR-zCOSY and F1-HOBS-zCOSY.

Further, the comparison of the internal projections /volume integrations has


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1 2 3 4 5 6 7 8 9 10 11 12 13


Figure 4. I. Comparison of different regions of the conventional and HOBS decoupled 1H NMR spectra of menthol, in the presence of 3mM ETHM (CLSR), which shows resolved resonances specific to R/S enantiomers. These spectra facilitate the selection of appropriate resonances based on the separation of R/S chemical shifts (Hz). The resonances with higher R/S separation are of usual choice for the unambiguous quantifications as explained in the text. Herein, the R/S separations corresponding to H7 (9 Hz) (a) and H8 (2.2 Hz) (b), protons are used for the analysis and the results are found to be consistent. II. Comparison of the expanded regions of zCOSY spectra for H7 of menthol recorded by using the conventional SR-zCOSY (a), F1-HOBS-zCOSY (b), F2-HOBS-zCOSY (c) and F1F2-HOBS-zCOSY (d). The internal spectral projections are the sum of the individual contributions of R and S enantiomers. As can be seen, a significant improvement in the signal to noise ratio and resolution are realized for the F1F2-HOBS-zCOSY spectrum compared to the other three methods. The asterisk symbols in the internal projections represent the decoupling sidebands.

14 15 16



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1

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Pure-shift 2D-F1F2-HOBS-zCOSY of methyl benzyl amine (MBA)

2

The utility of the F1F2-HOBS-zCOSY method is explored for MBA samples

3

with different R/S ratios (0 to 98% ee). Figure 5 compares the performance of F1F2-

4

HOBS-zCOSY with that of F1-HOBS-zCOSY, F2-HOBS-zCOSY and conventional

5

SR-zCOSY. As evident from these studies, the SR-zCOSY could not resolve the

6

enantiomers,

7

satisfactory identification and quantification of the enantiomers, only up to ~80% ee.

8

Impressively, the F1F2-HOBS-zCOSY could discriminate and precisely quantify the

9

minor enantiomer, down to 1%, with high resolution. In this case, the identification

10

and quantification of the minor S-enantiomer by using F1-HOBS-zCOSY (Figure 5j)

11

and F2-HOBS-zCOSY (Figure 5o) has become difficult, particularly, in the F2-

12

HOBS-zCOSY the sideband intensities correspond to the major R-enantiomer,

13

overlapped with the minor S-enantiomer, making the analysis not possible (Figure

14

5o). On the other hand, in the F1F2-HOBS-zCOSY, the relatively intense sidebands

15

of R-enantiomer are separated out as off-diagonal peaks and the minor S-enantiomer

16

is nicely resolved with an enhanced sensitivity (Figure 5t).

whereas

the

F1-HOBS-zCOSY

and

F2-HOBS-zCOSY

enabled

17

The ee values are estimated from the volume integrations of the sum of

18

diagonal peaks (with and without considering the decoupling sidebands), as discussed

19

below, which are found to be consistent with those known (w/w %) from the

20

gravimetric preparation (Table 1).

21

estimation of ee was straightforward for samples exhibiting well-resolved diagonal

22

peaks and for those of high concentrations or low ee. For these samples, the ee

23

quantification is made by taking initially the diagonal peaks alone and later it has

24

been repeated by considering the side-bands as well. In both the cases, the relative

This is clearly evident from Figure 6. The


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1

intensities are found to be consistent with the expected ee values (Figure S.3 of

2

supporting information).

3

Furthermore, in order to verify the influence of the side-bands on the ee

4

estimations, particularly for high ee samples, the volume integrations are performed

5

over a rectangle region covering the decoupling sidebands centered around the

6

diagonal peak of the major enantiomer, say R (Figure S.4 of supporting information).

7

The similar area has also been used for the volume integration of the corresponding

8

minor enantiomer, S, despite its side-bands are not visible / buried in the noise, and

9

the estimated ee values are found to be almost similar. Further, the noise levels are

10

also estimated over the same area in peak-free regions, which are about ~ 1000 times

11

lower compared to the volume integration of R (please see supporting information,

12

Figure S.4). Therefore, the error/ confident limits are ignored in the correlation plot.

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 5. Comparison of the expanded diagonal Hα regions of enantiomeric MBA of different R/S ratios recorded by using the conventional SR-zCOSY (a to e, upper row), F1HOBS-zCOSY (f to j, second row), F2-HOBS-zCOSY (k to o, third row) and their corresponding F1F2-HOBS-zCOSY spectra (p to t, bottom row). The spectra are recorded for different ee of MBA, as mentioned in the figure. A systematic decrease (0 to 98% ee) in the population of the S-enantiomer is reflected in the intensity of the signal corresponding to the S-enantiomer, which is shown by a blue dotted line in F1F2-HOBS-COSY. The internal spectral projections are the sum of the individual contributions of R and S enantiomers. However, the accurate enantiomeric discrimination and quantification of ee are achieved from the well-resolved 2D pure-shift F1F2-HOBS-zCOSY contours, as explained in the text. The decoupling sidebands for the internal projections are shown by asterisk symbols.

15


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18


Figure 6. Correlation between the ee ratios known from the w/w% and those derived from the volume integrals of the corresponding 2D F1F2-HOBS-zCOSY cross-peaks.

19 20 21 22 23

Table 1. Enantiomeric excess (ee) is determined for different R/S ratios of MBA. The magnitudes of the integral volumes estimated from the diagonal cross-peaks (pure-shift resonances) corresponding to the each enantiomer and the w/w % (R/S) used for preparing the samples, are given below. w/w composition of R and S-enantiomers is used for preparing the samples Weight of Renantiomer (mg)

Weight of Senantiomer (mg)

12.5

12.5

17.5

ee calculated from the w/w %

Normalized volume integrals derived from F1F2-HOBSzCOSY, including the decoupling side-bands

ee calculated from the volume integrations ee =

R-enantiomer

S-enantiomer

( ܴ െ ܵൗܴ ൅ ܵ ) × 100

0%

100

99.2

0.4%

7.5

40%

100

42.7

40.2%

22.5

2.5

80%

100

12.1

78.4%

23.75

1.25

90%

100

5.4

89.8%

25.0

0.25

98%

100

1.1

97.8%

24 25 19 ACS Paragon Plus Environment


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1

High ee determination in the presence of CSA

2 3

The versatility of the F1F2-HOBS-zCOSY is extended to CSA based chiral

4

discrimination also. Accordingly, the racemic as well as 90% ee R/S (5:95) mixture of 1-

5

aminoindan, where CSA (triphenoxyborane + R-binol) is used as a chiral auxiliary, instead of

6

CLSR.

7

The Hd and Hc protons in the conventional 1D 1HNMR spectrum of 1-aminoindan

8

show scalar coupling patterns: triplet of doublets (with the adjacent He, Hc and Hb protons)

9

and a triplet of doublet of doublets (with the adjacent Hd, He, Hb and Ha protons), at 1.47 ppm

10

and 2.23 ppm, respectively. In the presence of CSA, the enantiomeric mixtures with different

11

R/S ratios show complex multiplet pattern for both Hd (8 lines) and Hc (16 lines), which is

12

due to the overlapped Hd and Hc J-multiplets of the corresponding enantiomers.

13

Similar to the results of the CLSR added MBA samples discussed above, herein too,

14

the conventional SR-zCOSY is not found suitable for the discrimination/quantification of

15

high ee samples (Figure 7). On the other hand, the F1F2-HOBS-zCOSY method has shown

16

dramatic improvement in the resolution and the dominant sidebands are separated out as off-

17

diagonal peaks and enabled the observation of low concentrated enantiomers with enhanced

18

sensitivity. Further, the average ee ratios are calculated from the repeated F1F2-HOBS-

19

zCOSY experiments, which are found to be accurate to within ± 0.5% and ± 1.5%, with

20

reference to the w/w % racemic and high ee samples, respectively.


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1 2 3 4 5 6 7 8

Figure 7. Comparison of the expanded regions of Hd and Hc protons of enantiomeric 0% (I) and 90% (II) ee of 1-aminoindan recorded by using the conventional SR-zCOSY (Ia and Ib ; IIa and IIb, respectively) and F1F2-HOBS-zCOSY (Ic and Id ; IIc and IId, respectively) in the presence of low concentrations of CSA. The captions and the legends on the figures are self explanatory. An excellent resolution in the F1F2-HOBS-zCOSY allows the clear identification of the enantiomers and their precise quantification.

9

Conclusions

10

In conclusion, the present manuscript reports a high-sensitive two-dimensional (2D)

11

NMR method, F1F2-HOBS-zCOSY and its application to the precise determination of high



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

enantiomeric excess. In contrast to the conventional methods, the F1F2-HOBS-zCOSY

2

results in excellent resolution for low concentrations of chiral auxiliaries such as chiral

3

lanthanide shift reagents (CLSR) or chiral solvating agents (CSA). The enhanced sensitivity

4

and resolution are achieved by effectively suppressing the 1H-1H scalar couplings in both the

5

F1 and F2 dimensions. The resultant pure-shift resonances allow unambiguous identification

6

and quantification of the minor enantiomers, down to 1%, (which amounts to ~98% ee). The

7

results obtained over a wide range of ee (0% to ~98%) samples are found to be in excellent

8

agreement with the known w/w % of the enantiomers. The studies highlight the superiority

9

of the F1F2-HOBS-zCOSY scheme over the conventional methods, which can easily be

10

implemented on any high-field NMR spectrometer for routine use.

11 12

AUTHOR INFORMATION

13

Corresponding Author Email: [email protected] Tel: +91 40 2719 3976

14

Author Contributions

15

The manuscript was written through contributions of all authors. All authors have given

16

approval to the final version of the manuscript.

17

‡These authors contributed equally.

18

Funding Sources

19

CSIR-IICT, India

20


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1

Notes

2

The authors declare no competing financial interest.

3 4 5 6

ACKNOWLEDGEMENTS Authors thank CMET and AARF projects of CSIR-IICT, India for funding. KR, VMRK, SD and VSPB thank CSIR, India for fellowships.

7 8

SUPPORTING INFORMATION AVAILABLE

9

This information is available free of charge via the Internet at http://pubs.acs.org/.

10 11

REFERENCES

12

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Table of Contents Graphic

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Comparison of expanded 1D-1H NMR regions of Hα of MBA, by changing the concentration of CLSR (ETHM) for different enantiomeric excess ratios: a) 0% ee, b) 80% ee, c) 90% ee and d) 98% ee. The Figure 1(c) shows very low intense and broad signal for S-enantiomer of 90% ee sample and the Figure 1(d) suggests that the identification and quantification of the low populated enantiomer (98% ee) is practically difficult from the conventional one-dimensional spectrum. The broad line seen in some spectra, which shifts downfield with the increase in ETHM concentration, belongs to the NH2 protons. 1393x1393mm (56 x 56 DPI)

ACS Paragon Plus Environment


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Comparison of expanded regions of the conventional 1D 1H and 1D HOBS-decoupled spectra of Hα proton of MBA, for three different R/S ratios: (a) racemic (50:50), (b) 80% ee (90:10) and (c) 98% ee (99:1) samples. The spectra are recorded at 3.0 mM of ETHM concentration and the asterisk symbols represent the decoupling sidebands. As discussed in the main text, the unambiguous identification and quantification of the minor S enantiomer of MBA is not possible even from the 1D HOBS-decoupled spectrum. All the experiments are recorded under the identical experimental conditions. 1393x1393mm (56 x 56 DPI)


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Schematic representation of real-time homonuclear F2-decoupled HOBS zCOSY (F2-HOBS-zCOSY) (a) and F1F2-decoupled HOBS zCOSY (F1F2-HOBS-zCOSY) (b), pulse sequences. By setting n=1, the F1F2-HOBSzCOSY is reduced to F1-HOBS-zCOSY. All the three sequences are tested independently for the samples mentioned in the text. The filled and open rectangles represent the 90° and 180° pulses, respectively. The small flip angle ‘β’ equals to 20o. The chirp61 shaped inversion pulse of 20 ms length is applied during the spatial encoding gradients, which suppresses the zero quantum magnetization and yields only the pure inphase signals. The shaped pulses, which are marked with 'r' represent the Reburp refocusing pulses.62 Specific parameters for the present pulse sequences: phase cycling: φ1 = x, -x; φ2 = x; φ3 = -x; φrec = x, -x; pulsed field gradients: G1,2,3,4,5 (1 ms)= 5%, 15%, 11%, 31% and 2%, respectively of 53.5G/cm gradient strength. The Reburp shaped pulses of ~ 20 ms are used to refocus the interested spectral region. For quadrature, STATES-TPPI63 technique is used. Two signal accumulations are made for each experiment. In all the cases, 400 Hz of spectral width is used for both the F1 and F2 dimensions. The corresponding acquisition times and dwelling increments are 0.64s/0.16s and 512/128 along the F2/F1 dimensions, respectively. Total time required for each experiment is ~10 min. 1393x1393mm (56 x 56 DPI)



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I. Comparison of different regions of the conventional and HOBS decoupled 1H NMR spectra of menthol, in the presence of 3mM ETHM (CLSR), which shows resolved resonances specific to R/S enantiomers. These spectra facilitate the selection of appropriate resonances based on the separation of R/S chemical shifts (Hz). The resonances with higher R/S separation are of usual choice for the unambiguous quantifications as explained in the text. Herein, the R/S separations corresponding to H7 (9 Hz) (a) and H8 (2.2 Hz) (b), protons are used for the analysis and the results are found to be consistent. II. Comparison of the expanded regions of zCOSY spectra for H7 of menthol recorded by using the conventional SR-zCOSY (a), F1HOBS-zCOSY (b), F2-HOBS-zCOSY (c) and F1F2-HOBS-zCOSY (d). The internal spectral projections are the sum of the individual contributions of R and S enantiomers. As can be seen, a significant improvement in the signal to noise ratio and resolution are realized for the F1F2-HOBS-zCOSY spectrum compared to the other three methods. The asterisk symbols in the internal projections represent the decoupling sidebands. 1393x1393mm (56 x 56 DPI)


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Comparison of the expanded diagonal Hα regions of enantiomeric MBA of different R/S ratios recorded by using the conventional SR-zCOSY (a to e, upper row), F1-HOBS-zCOSY (f to j, second row), F2-HOBS-zCOSY (k to o, third row) and their corresponding F1F2-HOBS-zCOSY spectra (p to t, bottom row). The spectra are recorded for different ee of MBA, as mentioned in the figure. A systematic decrease (0 to 98% ee) in the population of the S-enantiomer is reflected in the intensity of the signal corresponding to the S-enantiomer, which is shown by a blue dotted line in F1F2-HOBS-COSY. The internal spectral projections are the sum of the individual contributions of R and S enantiomers. However, the accurate enantiomeric discrimination and quantification of ee are achieved from the well-resolved 2D pure-shift F1F2-HOBS-zCOSY contours, as explained in the text. The decoupling sidebands for the internal projections are shown by asterisk symbols.

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Correlation between the ee ratios known from the w/w% and those derived from the volume integrals of the corresponding 2D F1F2-HOBS-zCOSY cross-peaks. 1393x1393mm (56 x 56 DPI)


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Comparison of the expanded regions of Hd and Hc protons of enantiomeric 0% (I) and 90% (II) ee of 1aminoindan recorded by using the conventional SR-zCOSY (Ia and Ib ; IIa and IIb, respectively) and F1F2HOBS-zCOSY (Ic and Id ; IIc and IId, respectively) in the presence of low concentrations of CSA. The captions and the legends on the figures are self explanatory. An excellent resolution in the F1F2-HOBSzCOSY allows the clear identification of the enantiomers and their precise quantification. 1393x1393mm (56 x 56 DPI)



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Precise Determination of Enantiomeric Excess by a ... - ACS Publications

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