- Email: [email protected]
CH-RES-TOCSY: Enantiomers spectral resolution and measurement of heteronuclear residual dipolar couplings
Accepted Manuscript Title: CH-RES-TOCSY: Enantiomers Spectral Resolution and Measurement of Heteronuclear Residual Dipolar Couplings Author: N. Lokesh N. Suryaprakash PII: DOI: Reference:
S0009-2614(15)00098-6 http://dx.doi.org/doi:10.1016/j.cplett.2015.02.016 CPLETT 32809
To appear in: Received date: Revised date: Accepted date:
7-1-2015 3-2-2015 9-2-2015
Please cite this article as: N. Lokesh, N. Suryaprakash, CH-RES-TOCSY: Enantiomers Spectral Resolution and Measurement of Heteronuclear Residual Dipolar Couplings, Chem. Phys. Lett. (2015), http://dx.doi.org/10.1016/j.cplett.2015.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Unraveling of complete spectra of both enantiomers Determination of C-H RDCS of selected carbon Identification of symmetric isomers
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New NMR experimental technique
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*Graphical Abstract (pictogram) (for review)
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CH-RES-TOCSY: Enantiomers Spectral Resolution and Measurement of Heteronuclear Residual Dipolar Couplings N. Lokesh and N. Suryaprakash
Indian Institute of Science, Bangalore-560012
cr
Email id- [email protected]
ip t
NMR Research Centre and Solid State and Structural Chemistry Unit
Unraveling of complete spectra of both enantiomers
an
Determination of C-H RDCS of selected carbon
us
Highlights
Identification of symmetric isomers
d
M
New NMR experimental technique
te
Abstract: A new 2D NMR technique cited as CH-RES-TOCSY, for complete unraveling the spectra of enantiomers and for the measurement of structurally important C-H RDCs is reported. The spectral overlap and complexity of peaks were reduced by the blend of selective excitation
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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 61 62 63 64 65
and homo-decoupling. Differential values of C-H RDCs of enantiomers (R and S) are exploited to separate the enantiomeric peaks. The complete unraveling of the spectra of both the enantiomers is achieved by incorporating a TOCSY mixing block prior to signal acquisition. The additional application of the method is demonstrated for the assignment of symmetric isomers. 1. Introduction
In many fields, particularly in pharmaceutical research [1,2] and asymmetric synthesis [3,4], the differentiation of enantiomers and their analysis are significantly important. It is due to the differential physico-chemical properties of enantiomers in a chiral environment. Enantiomers are commonly analyzed by NMR [5-8], X-ray [9,10], ORD, CD[11] and chromatographic techniques [12,13]. The easy accessibility and the availability of experimental techniques renders NMR spectroscopy as an indispensable tool to analyze enantiomers. It differentiates enantiomers
Page 3 of 14
by the use of chiral auxiliaries, which introduces diastereomeric interactions with the enantiomers [5-8]. Chiral auxiliaries, such as, chiral derivatizing agents, chiral solvating agents and chiral lanthanide complexes are specific to functional groups. Thus different types of chiral auxiliaries are essential to differentiate different class of enantiomers (amines, alcohols, acids,
ip t
etc). Furthermore, the chemical shift difference between the discriminated peaks is the only parameter used in isotropic medium.
cr
On the other hand a chiral weak aligning medium differentiates different class of enantiomers [14,15] including the non-polar chiral molecules [16]. Such a discrimination arises due to [17]. The differentiation is
us
differential ordering and orientation of enantiomers (R & S)
visualized through multiple order dependent NMR parameters, such as, chemical shift anisotropy
an
(CSA), residual dipolar couplings (RDCs) and quadrupole couplings (QCs), which give additional structural information [18-21]. However, it is difficult to extract these structural
M
parameters from the conventional 1D 1H spectrum, due to severe overlap and complexity arising from multiple scalar and dipolar interactions among coupled spins.
d
Over the years many NMR experiments have been developed to differentiate enantiomers and to access the structural parameters. The simple experiment is 1D 2H NMR, which differentiates
te
enantiomers due to their differential quadrupolar couplings [20], but it demands partial or completely deuterated samples. In circumventing this, Natural Abundance 2H NMR cited as NAD NMR has been extensively utilized [20,22-25]. However, 1H NMR method is an ideal
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choice due to high natural abundance of protons in organic molecules and its high gyro-magnetic ratio. Number of 1H NMR techniques have been reported for differentiation of enantiomeric peaks, which are based on selective excitation and/or partial homo-decoupling [26]. But most of these methods resolves only certain enantiomeric peaks and fails to separate complete spectra of enantiomers. Recently we have successfully achieved complete separation of enantiomeric spectra using the technique of selective double quantum excitation [27]. The method differentiates enantiomers and provides multiple 1H-1H RDCs. Nevertheless it does not provide hetero-nuclear couplings (C-H RDCs), which are significantly important in the structure calculations of both small organic molecules and large biomolecules [19,21,28]. In the present study we are reporting a new 2D NMR method cited as CH-RES-TOCSY, which gives complete resolved spectra of both the enantiomers in addition to providing both long and
Page 4 of 14
short range sign sensitive C-H RDCs of a selected carbon. The developed method is based on the initial selective excitation of
13
C attached protons with homo-decoupling in indirect-time
dimension, which allows evolution of only selected C-H RDCs of enantiomers and hence the enantiomeric peaks separation due to differential RDCs. The complete spectra of enantiomers
ip t
could be achieved by a TOCSY mixing prior to signal acquisition. The present method can be utilized in conjunction with the reported double quantum experiment [23] to determine both HH
cr
and CH RDCs. The applicability of this novel technique is demonstrated for the determination of scalar couplings in isotropic medium, assignment of symmetric isomers, in addition to the
us
differentiation of enantiomers and the measurement of RDCs in the aligning medium on chosen examples.
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2. Experimental Section
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2.1 Sample Preparation and Experimental details
All the commercially available chemicals were purchased and used without any further purification. Chemicals used are poly-γ-benzyl-L-glutamate (PBLG), propylene carbonate, cis-
d
and trans- stilbene. Three different isotropic solutions were prepared using 8 mg of propylene
te
carbonate in 500 μl of CDCl3, 8 mg of trans-stilbene in 500 μl of CDCl3 and 8 mg of cis-stilbene in 500 μl CDCl3. For the aligned sample, 50 mg of racemic mixture of propylene carbonate and 85 mg of PBLG were taken in 450 mg of CDCl3 and the medium was prepared according to the
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reported procedure [27]. 2.2 NMR Experiment
Experiments were carried out using 400 MHz and 800 MHz spectrometers. The maximum gradient strength of the coil is 53.5 Gcm-1. For selective excitation and refocousing EBurp1 and ReBurp pulses of pulse lengths 20 ms and 28 ms were used ( band width ≈ 220 Hz) [29]. For TOCSY mixing, isotropic mixing time of 60 ms was used. The matrix of 8192 X 256 time domain data points were collected and are zero filled with 16384 X 512 points. 2.3 XH-RES-TOCSY Pulse Sequence The CH-RES-TOCSY pulse sequence is reported in Fig. 1. Intial 1JCH INEPT block transfers magnetization from 1H to 13C, followed by purge gradient pulse G1, which dephases 12C attached
Page 5 of 14
proton magnetization. The subsequent x-filter (two 900 pulses on carbon with phases x, x and x, x respectvely) suppresses any residual bulk
13
12
C attached proton magnetization. From the resulted
C attached proton magnetization, desired protons were selectively excited. During t1
evolution, combined selective and non-selective 1800 pulses were used, to achieve homo-
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decoupling. Selective 1800 pulse rotates selected proton magnetization by 1800 and non-selective 1800 pulse rotates complete proton magnetization by 1800 resulting in effective 3600 (net 00)
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rotation on selected proton and 1800 on rest of proton magnetization, which makes selective and non-selective protons out of phase by 1800, causing selective proton-proton decoupling during
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the indirect dimension. This ensures the evolution of only chemical shifts of selected protons and one-bond C-H coupling in the indirect dimension. Prior to the signal acqusition, an isotropic TOCSY (MLEV-17) mixing block was used to transfer magnetization to other
12
C attached
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protons of the molecule to get completely unraveled spectra and nJCH.
13
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Thus the experiment gives selective 1JCH in the indirect dimension and nJCH (n>1) from the C α/β cross peaks in the direct dimension. The required
displacement of spin-state selective
coherence order was selected by the use of z-gradients G2, -G2, G3 and –G3 (Fig. 1). To
3. Results and Discussion
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applied before or after the gradients.
d
compensate the evolved magnetization during gradient pulse, a 1800 pulse and equal delays were
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3.1 Measurement of scalar couplings in propylene carbonate: Intially the CH-RES-TOCSY was demonstrated for the measurement of nJCH in propylene carbonate in CDCl3 solvent.
Initially
12
C attached protons were filtered, dephased and
suppressed by INEPT block, purge gradient and X-filter. From the resultant 13C attached proton magnetization, the proton 3 (Fig. 2) was selectively excited and was decoupled from the remaining protons during indirect dimension. Therefore only chemical shift and 1JCH of proton 3 was evolved in the indirect dimension. Before signal acquisition, the magnetization of proton 3 was transfered to other coupled protons through TOCSY mixing. The obtained CH-RES-TOCSY spectrum of propylene carbonate is reported in Fig. 2, from which 1JCH and nJCH of carbon 3 can be measured. The nJCH were measured in direct dimension (F2) and are reported in the spectrum.
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The relative signs were assigned based on the sense of displacement of cross sections with respect to that of proton 3.
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3.2 Unraveling of enantiomers spectra in PBLG aligning medium
After demonstrating the CH-RES-TOCSY for the measurement of scalar couplings in propylene
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carbonate, the potential utlity of the experiment was demonstrated for differentiating enantiomers and measurement of C-H RDCs. The experiment was carried out on a racemic mixture of
experimental section). As discussed previously the
12
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propylene carbonate aligned in PBLG medium (sample preparation is discussed in the C attached proton magnetization was
filtered, dephased and suppressed before selectively exciting the proton 3. The excited proton 3
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was decoupled in the indirect dimensioin which permitted the evolution of only 1TCH (=JCH+2DCH) and CSA pertaining to proton 3. Due to differential 1TCH and CSA of proton 3 in R-
M
and S-propylene carbonate, both enantiomeric peaks got separated out along F1 dimension. The separated peaks of R and S proton magnetization was transferred to all other coupled protons
d
through TOCSY mixing. The resulted spectrum exhibits four-arrays of peaks along F1-dimension and is given Fig. 3. Two-arrays, each for both R and S due to evolution of 1TCH in the indirect
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dimension are different for both R and S enantiomers (assignment of peaks is based on ref [30]). The nTCH were measured along F2-dimension as discussed in the previous section and are
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reported in Fig. 3. The sense of displacement of peaks provided relative signs. The advantage of the CH-RES-TOCSY experiment is clearly evident that it not only achieved complete separation of enantiomers spectra but also yielded selective C-H RDCs.
3.3 Assignment of cis- and trans- isomers of stilbene. Finally the application of CH-RES-TOCSY is demonstrated for the determination of 2JCH and 3
JHH cis- and trans- isomers, where magnetic equivalence of protons prevents the measurement
of 3JHH and hence their recognition [31,32]. The CH-RES-TOCSY spectra were recorded on trans- and cis- stilbene by selectively exciting one of the
13
C attached aliphatic proton, marked
with red color in the chemical structure (Fig. 4). The obtained CH-RES-TOCSY spectra provided 1JCH in the F1/F2 dimension and 2JCH in the direct dimension, which are reported in Fig.
Page 7 of 14
4. Furthermore, each of these peaks exhibited a doublet pattern in both the dimensions due to 3
JHH, which aided the distinction of trans- and cis- stilbene. The couplings measured are
reported in the Fig. 4. This is another significant utility of CH-RES-TOCSY experiment where JCH and 3JHH can be measured in symmetric molecules, which aided in assigning them. Conclusions
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4
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n
A simple and an elegant 2D NMR technique has been developed for the complete unravelling of
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the overlapped spectra of enantiomers in addition to providing both long and short range sign sensitive C-H RDCs for a selected carbon. The application of the designed sequence has also been demonstrated for the measurement of scalar couplings and also differential RDCs of
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enantiomers for a chosen molecule in isotropic and anisotropic phases respectively. Another significant utility of the developed methodology is it permits the measurement of nJCH and 3JHH
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in cis- and trans- symmetric isomers, enabling their assignment. Thus the developed technique finds enormous utility in number of applications.
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Acknowledgements
NL thanks IISc for SRF. NS gratefully acknowledges the generous financial supported by Board of Research in Nuclear Sciences, Mumbai (Grant No. 2013/37C/4/BRNS).
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
P.M. Dewick, Medicinal Natural Products, John Wiley & Sons, Ltd, 2009, p. 1. J. Gal, Chirality in Drug Research, Wiley-VCH Verlag GmbH & Co. KGaA, 2006, p. 1. R.E. Gawley, J. Aubé, in: R.E.G. Aubé (Ed.), Principles of Asymmetric Synthesis (Second Edition), Elsevier, Oxford, 2012, p. 1. C.M. Reeves, B.M. Stoltz, Asymmetric Synthesis II, Wiley-VCH Verlag GmbH & Co. KGaA, 2012, p. 1. J.M. Seco, E. Quiñoá, R. Riguera, Chem. Rev. 104 (2004) 17. J.M. Seco, E. Quiñoá, R. Riguera, Chem. Rev. 112 (2012) 4603. T.J. Wenzel, C.D. Chisholm, Prog. Nucl. Magn. Reson. Spectrosc. 59 (2011) 1. T.J. Wenzel, C.D. Chisholm, Chirality 23 (2011) 190. N. Harada, Chirality 20 (2008) 691. J. Trotter, Acta Crystallogr., Sect. B: Struct. Sci. 37 (1981) 493.
Page 8 of 14
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cr
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[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
d
[12] [13] [14] [15] [16] [17] [18]
N. Harada, K. Nakanishi, N. Berova, Comprehensive Chiroptical Spectroscopy, John Wiley & Sons, Inc., 2012, p. 115. T.J. Ward, D.-M. Hamburg, Anal. Chem. 76 (2004) 4635. T.J. Ward, K.D. Ward, Anal. Chem. 82 (2010) 4712. Lokesh, N. Suryaprakash, Chem. Eur. J. 18 (2012) 11560. U.V. Reddy, N. Suryaprakash, Chem. Commun. 47 (2011) 8364. M. Sarfati, J. Courtieu, P. Lesot, Chem. Commun. (2000) 1113. M. Sarfati, P. Lesot, D. Merlet, J. Courtieu, Chem. Commun. (2000) 2069. F. Hallwass, M. Schmidt, H. Sun, A. Mazur, G. Kummerlöwe, B. Luy, A. NavarroVázquez, C. Griesinger, U.M. Reinscheid, Angew. Chem. Int. Ed. 50 (2011) 9487. G. Kummerlöwe, B. Luy, TrAC, Trends Anal. Chem. 28 (2009) 483. P. Lesot, J. Courtieu, Prog. Nucl. Magn. Reson. Spectrosc. 55 (2009) 128. C.M. Thiele, Eur. J. Org. Chem. 2008 (2008) 5673. D. Merlet, B. Ancian, J. Courtieu, P. Lesot, J. Am. Chem. Soc. 121 (1999) 5249. P. Lesot, M. Sarfati, J. Courtieu, Chem. Eur. J. 9 (2003) 1724. K. Kazimierczuk, O. Lafon, P. Lesot, Analyst 139 (2014) 2702. Z. Serhan, L. Martel, I. Billault, P. Lesot, Chem. Commun. 46 (2010) 6599. N. Nath, S. Hebbar, U. Ramesh Prabhu, N. Suryaprakash, J. Indian. Inst. Sci. 90 (2010) 1. S. Hebbar, U.R. Prabhu, N. Suryaprakash, J. Magn. Reson. 215 (2012) 23. B.R. Donald, J. Martin, Prog. Nucl. Magn. Reson. Spectrosc. 55 (2009) 101. H. Geen, R. Freeman, J. Magn. Reson. (1991) 93. N. Nath, N. Suryaprakash, J. Phys. Chem. B 115 (2011) 6868. S.R. Chaudhari, N. Nath, N. Suryaprakash, RSC Adv. 2 (2012) 12915. L. Lunazzi, A. Mazzanti, J. Am. Chem. Soc. 126 (2004) 12155.
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[11]
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Page 9 of 14
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Graphical Abstract
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Page 10 of 14
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Fig 1. The CH-RES-TOCSY pulse sequence (A) and the coherence pathway (B). In Fig. A, filled and unfilled rectangular bars pertain to 1800 and 900 hard pulses. Filled and unfilled shaped bars
d
on 1H channel are 1800 and 900 selective pulses. Rectangular grey filled bar is isotropic mixing pulse (MLEV-17). Δ/2=(1/4*1JCH) used for INEPT. G1 is gradient purge pulse. The coherence
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path way (Fig. B, P1H for proton and P13C for carbon) was selected by G2, -G2, G3 and –G3 gradient pulses. All pulses have phase x unless otherwise mentioned. The four-step phase cycle
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is used; ϕ1= y, ϕ2=x, –x, –x, x and ϕrec= x, –x, –x, x. The spectra were recorded in magnitude mode.
Page 11 of 14
ip t cr us an M d
Fig 2. The CH-RES-TOCSY spectrum and the chemical structure of propylene carbonate. 1
13
C
n
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proton (3, marked as red) is selectively excited. The JCH and JCH of carbon 3 were obtained along F1/F2 and F2 dimensions respectively. The frequency separations giving 1JCH and nJCH are marked with arrows and the values are given in Hz. The relative signs of couplings are assigned
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based on direction of displacement of cross sections in the direct dimension (assuming 1JCH is positive sign)
Page 12 of 14
ip t cr us an M
Fig 3. The CH-RES-TOCSY spectrum and the chemical structure of propylene carbonate.
13
C
attached proton (3, marked as red) was selectively excited in the experiment. The resolved R and
d
S isomer spectra are marked (assignment is based on ref [30]), the JCH (taken from Fig. 2) and
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measured TCH values of carbon 3 are tabulated.
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Page 13 of 14
ip t cr us an M d
Fig 4. The CH-RES-TOCSY spectra and the chemical structure of trans- (A, C) and cis- (B,D)
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stilbene. One of the aliphatic 13C attached protons was selectively excited (marked as red). The measured 1JCH, 2JCH of marked carbon and 3JHH of both isomers are reported in the respective spectrum. The relative signs of couplings are assigned based on the direction of displacement of
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peaks in the direct dimension (assuming the sign 1JCH is positive).
Page 14 of 14
S0009-2614(15)00098-6 http://dx.doi.org/doi:10.1016/j.cplett.2015.02.016 CPLETT 32809
To appear in: Received date: Revised date: Accepted date:
7-1-2015 3-2-2015 9-2-2015
Please cite this article as: N. Lokesh, N. Suryaprakash, CH-RES-TOCSY: Enantiomers Spectral Resolution and Measurement of Heteronuclear Residual Dipolar Couplings, Chem. Phys. Lett. (2015), http://dx.doi.org/10.1016/j.cplett.2015.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Unraveling of complete spectra of both enantiomers Determination of C-H RDCS of selected carbon Identification of symmetric isomers
Ac
ce pt
ed
M
an
us
cr
ip t
New NMR experimental technique
Page 1 of 14
Ac
ce
pt
ed
M
an
us
cr
i
*Graphical Abstract (pictogram) (for review)
Page 2 of 14
CH-RES-TOCSY: Enantiomers Spectral Resolution and Measurement of Heteronuclear Residual Dipolar Couplings N. Lokesh and N. Suryaprakash
Indian Institute of Science, Bangalore-560012
cr
Email id- [email protected]
ip t
NMR Research Centre and Solid State and Structural Chemistry Unit
Unraveling of complete spectra of both enantiomers
an
Determination of C-H RDCS of selected carbon
us
Highlights
Identification of symmetric isomers
d
M
New NMR experimental technique
te
Abstract: A new 2D NMR technique cited as CH-RES-TOCSY, for complete unraveling the spectra of enantiomers and for the measurement of structurally important C-H RDCs is reported. The spectral overlap and complexity of peaks were reduced by the blend of selective excitation
Ac ce p
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 61 62 63 64 65
and homo-decoupling. Differential values of C-H RDCs of enantiomers (R and S) are exploited to separate the enantiomeric peaks. The complete unraveling of the spectra of both the enantiomers is achieved by incorporating a TOCSY mixing block prior to signal acquisition. The additional application of the method is demonstrated for the assignment of symmetric isomers. 1. Introduction
In many fields, particularly in pharmaceutical research [1,2] and asymmetric synthesis [3,4], the differentiation of enantiomers and their analysis are significantly important. It is due to the differential physico-chemical properties of enantiomers in a chiral environment. Enantiomers are commonly analyzed by NMR [5-8], X-ray [9,10], ORD, CD[11] and chromatographic techniques [12,13]. The easy accessibility and the availability of experimental techniques renders NMR spectroscopy as an indispensable tool to analyze enantiomers. It differentiates enantiomers
Page 3 of 14
by the use of chiral auxiliaries, which introduces diastereomeric interactions with the enantiomers [5-8]. Chiral auxiliaries, such as, chiral derivatizing agents, chiral solvating agents and chiral lanthanide complexes are specific to functional groups. Thus different types of chiral auxiliaries are essential to differentiate different class of enantiomers (amines, alcohols, acids,
ip t
etc). Furthermore, the chemical shift difference between the discriminated peaks is the only parameter used in isotropic medium.
cr
On the other hand a chiral weak aligning medium differentiates different class of enantiomers [14,15] including the non-polar chiral molecules [16]. Such a discrimination arises due to [17]. The differentiation is
us
differential ordering and orientation of enantiomers (R & S)
visualized through multiple order dependent NMR parameters, such as, chemical shift anisotropy
an
(CSA), residual dipolar couplings (RDCs) and quadrupole couplings (QCs), which give additional structural information [18-21]. However, it is difficult to extract these structural
M
parameters from the conventional 1D 1H spectrum, due to severe overlap and complexity arising from multiple scalar and dipolar interactions among coupled spins.
d
Over the years many NMR experiments have been developed to differentiate enantiomers and to access the structural parameters. The simple experiment is 1D 2H NMR, which differentiates
te
enantiomers due to their differential quadrupolar couplings [20], but it demands partial or completely deuterated samples. In circumventing this, Natural Abundance 2H NMR cited as NAD NMR has been extensively utilized [20,22-25]. However, 1H NMR method is an ideal
Ac ce p
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 61 62 63 64 65
choice due to high natural abundance of protons in organic molecules and its high gyro-magnetic ratio. Number of 1H NMR techniques have been reported for differentiation of enantiomeric peaks, which are based on selective excitation and/or partial homo-decoupling [26]. But most of these methods resolves only certain enantiomeric peaks and fails to separate complete spectra of enantiomers. Recently we have successfully achieved complete separation of enantiomeric spectra using the technique of selective double quantum excitation [27]. The method differentiates enantiomers and provides multiple 1H-1H RDCs. Nevertheless it does not provide hetero-nuclear couplings (C-H RDCs), which are significantly important in the structure calculations of both small organic molecules and large biomolecules [19,21,28]. In the present study we are reporting a new 2D NMR method cited as CH-RES-TOCSY, which gives complete resolved spectra of both the enantiomers in addition to providing both long and
Page 4 of 14
short range sign sensitive C-H RDCs of a selected carbon. The developed method is based on the initial selective excitation of
13
C attached protons with homo-decoupling in indirect-time
dimension, which allows evolution of only selected C-H RDCs of enantiomers and hence the enantiomeric peaks separation due to differential RDCs. The complete spectra of enantiomers
ip t
could be achieved by a TOCSY mixing prior to signal acquisition. The present method can be utilized in conjunction with the reported double quantum experiment [23] to determine both HH
cr
and CH RDCs. The applicability of this novel technique is demonstrated for the determination of scalar couplings in isotropic medium, assignment of symmetric isomers, in addition to the
us
differentiation of enantiomers and the measurement of RDCs in the aligning medium on chosen examples.
an
2. Experimental Section
M
2.1 Sample Preparation and Experimental details
All the commercially available chemicals were purchased and used without any further purification. Chemicals used are poly-γ-benzyl-L-glutamate (PBLG), propylene carbonate, cis-
d
and trans- stilbene. Three different isotropic solutions were prepared using 8 mg of propylene
te
carbonate in 500 μl of CDCl3, 8 mg of trans-stilbene in 500 μl of CDCl3 and 8 mg of cis-stilbene in 500 μl CDCl3. For the aligned sample, 50 mg of racemic mixture of propylene carbonate and 85 mg of PBLG were taken in 450 mg of CDCl3 and the medium was prepared according to the
Ac ce p
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 61 62 63 64 65
reported procedure [27]. 2.2 NMR Experiment
Experiments were carried out using 400 MHz and 800 MHz spectrometers. The maximum gradient strength of the coil is 53.5 Gcm-1. For selective excitation and refocousing EBurp1 and ReBurp pulses of pulse lengths 20 ms and 28 ms were used ( band width ≈ 220 Hz) [29]. For TOCSY mixing, isotropic mixing time of 60 ms was used. The matrix of 8192 X 256 time domain data points were collected and are zero filled with 16384 X 512 points. 2.3 XH-RES-TOCSY Pulse Sequence The CH-RES-TOCSY pulse sequence is reported in Fig. 1. Intial 1JCH INEPT block transfers magnetization from 1H to 13C, followed by purge gradient pulse G1, which dephases 12C attached
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proton magnetization. The subsequent x-filter (two 900 pulses on carbon with phases x, x and x, x respectvely) suppresses any residual bulk
13
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C attached proton magnetization. From the resulted
C attached proton magnetization, desired protons were selectively excited. During t1
evolution, combined selective and non-selective 1800 pulses were used, to achieve homo-
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decoupling. Selective 1800 pulse rotates selected proton magnetization by 1800 and non-selective 1800 pulse rotates complete proton magnetization by 1800 resulting in effective 3600 (net 00)
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rotation on selected proton and 1800 on rest of proton magnetization, which makes selective and non-selective protons out of phase by 1800, causing selective proton-proton decoupling during
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the indirect dimension. This ensures the evolution of only chemical shifts of selected protons and one-bond C-H coupling in the indirect dimension. Prior to the signal acqusition, an isotropic TOCSY (MLEV-17) mixing block was used to transfer magnetization to other
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C attached
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protons of the molecule to get completely unraveled spectra and nJCH.
13
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Thus the experiment gives selective 1JCH in the indirect dimension and nJCH (n>1) from the C α/β cross peaks in the direct dimension. The required
displacement of spin-state selective
coherence order was selected by the use of z-gradients G2, -G2, G3 and –G3 (Fig. 1). To
3. Results and Discussion
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applied before or after the gradients.
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compensate the evolved magnetization during gradient pulse, a 1800 pulse and equal delays were
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3.1 Measurement of scalar couplings in propylene carbonate: Intially the CH-RES-TOCSY was demonstrated for the measurement of nJCH in propylene carbonate in CDCl3 solvent.
Initially
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C attached protons were filtered, dephased and
suppressed by INEPT block, purge gradient and X-filter. From the resultant 13C attached proton magnetization, the proton 3 (Fig. 2) was selectively excited and was decoupled from the remaining protons during indirect dimension. Therefore only chemical shift and 1JCH of proton 3 was evolved in the indirect dimension. Before signal acquisition, the magnetization of proton 3 was transfered to other coupled protons through TOCSY mixing. The obtained CH-RES-TOCSY spectrum of propylene carbonate is reported in Fig. 2, from which 1JCH and nJCH of carbon 3 can be measured. The nJCH were measured in direct dimension (F2) and are reported in the spectrum.
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The relative signs were assigned based on the sense of displacement of cross sections with respect to that of proton 3.
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3.2 Unraveling of enantiomers spectra in PBLG aligning medium
After demonstrating the CH-RES-TOCSY for the measurement of scalar couplings in propylene
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carbonate, the potential utlity of the experiment was demonstrated for differentiating enantiomers and measurement of C-H RDCs. The experiment was carried out on a racemic mixture of
experimental section). As discussed previously the
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propylene carbonate aligned in PBLG medium (sample preparation is discussed in the C attached proton magnetization was
filtered, dephased and suppressed before selectively exciting the proton 3. The excited proton 3
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was decoupled in the indirect dimensioin which permitted the evolution of only 1TCH (=JCH+2DCH) and CSA pertaining to proton 3. Due to differential 1TCH and CSA of proton 3 in R-
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and S-propylene carbonate, both enantiomeric peaks got separated out along F1 dimension. The separated peaks of R and S proton magnetization was transferred to all other coupled protons
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through TOCSY mixing. The resulted spectrum exhibits four-arrays of peaks along F1-dimension and is given Fig. 3. Two-arrays, each for both R and S due to evolution of 1TCH in the indirect
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dimension are different for both R and S enantiomers (assignment of peaks is based on ref [30]). The nTCH were measured along F2-dimension as discussed in the previous section and are
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reported in Fig. 3. The sense of displacement of peaks provided relative signs. The advantage of the CH-RES-TOCSY experiment is clearly evident that it not only achieved complete separation of enantiomers spectra but also yielded selective C-H RDCs.
3.3 Assignment of cis- and trans- isomers of stilbene. Finally the application of CH-RES-TOCSY is demonstrated for the determination of 2JCH and 3
JHH cis- and trans- isomers, where magnetic equivalence of protons prevents the measurement
of 3JHH and hence their recognition [31,32]. The CH-RES-TOCSY spectra were recorded on trans- and cis- stilbene by selectively exciting one of the
13
C attached aliphatic proton, marked
with red color in the chemical structure (Fig. 4). The obtained CH-RES-TOCSY spectra provided 1JCH in the F1/F2 dimension and 2JCH in the direct dimension, which are reported in Fig.
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4. Furthermore, each of these peaks exhibited a doublet pattern in both the dimensions due to 3
JHH, which aided the distinction of trans- and cis- stilbene. The couplings measured are
reported in the Fig. 4. This is another significant utility of CH-RES-TOCSY experiment where JCH and 3JHH can be measured in symmetric molecules, which aided in assigning them. Conclusions
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n
A simple and an elegant 2D NMR technique has been developed for the complete unravelling of
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the overlapped spectra of enantiomers in addition to providing both long and short range sign sensitive C-H RDCs for a selected carbon. The application of the designed sequence has also been demonstrated for the measurement of scalar couplings and also differential RDCs of
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enantiomers for a chosen molecule in isotropic and anisotropic phases respectively. Another significant utility of the developed methodology is it permits the measurement of nJCH and 3JHH
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in cis- and trans- symmetric isomers, enabling their assignment. Thus the developed technique finds enormous utility in number of applications.
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Acknowledgements
NL thanks IISc for SRF. NS gratefully acknowledges the generous financial supported by Board of Research in Nuclear Sciences, Mumbai (Grant No. 2013/37C/4/BRNS).
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
P.M. Dewick, Medicinal Natural Products, John Wiley & Sons, Ltd, 2009, p. 1. J. Gal, Chirality in Drug Research, Wiley-VCH Verlag GmbH & Co. KGaA, 2006, p. 1. R.E. Gawley, J. Aubé, in: R.E.G. Aubé (Ed.), Principles of Asymmetric Synthesis (Second Edition), Elsevier, Oxford, 2012, p. 1. C.M. Reeves, B.M. Stoltz, Asymmetric Synthesis II, Wiley-VCH Verlag GmbH & Co. KGaA, 2012, p. 1. J.M. Seco, E. Quiñoá, R. Riguera, Chem. Rev. 104 (2004) 17. J.M. Seco, E. Quiñoá, R. Riguera, Chem. Rev. 112 (2012) 4603. T.J. Wenzel, C.D. Chisholm, Prog. Nucl. Magn. Reson. Spectrosc. 59 (2011) 1. T.J. Wenzel, C.D. Chisholm, Chirality 23 (2011) 190. N. Harada, Chirality 20 (2008) 691. J. Trotter, Acta Crystallogr., Sect. B: Struct. Sci. 37 (1981) 493.
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[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
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[12] [13] [14] [15] [16] [17] [18]
N. Harada, K. Nakanishi, N. Berova, Comprehensive Chiroptical Spectroscopy, John Wiley & Sons, Inc., 2012, p. 115. T.J. Ward, D.-M. Hamburg, Anal. Chem. 76 (2004) 4635. T.J. Ward, K.D. Ward, Anal. Chem. 82 (2010) 4712. Lokesh, N. Suryaprakash, Chem. Eur. J. 18 (2012) 11560. U.V. Reddy, N. Suryaprakash, Chem. Commun. 47 (2011) 8364. M. Sarfati, J. Courtieu, P. Lesot, Chem. Commun. (2000) 1113. M. Sarfati, P. Lesot, D. Merlet, J. Courtieu, Chem. Commun. (2000) 2069. F. Hallwass, M. Schmidt, H. Sun, A. Mazur, G. Kummerlöwe, B. Luy, A. NavarroVázquez, C. Griesinger, U.M. Reinscheid, Angew. Chem. Int. Ed. 50 (2011) 9487. G. Kummerlöwe, B. Luy, TrAC, Trends Anal. Chem. 28 (2009) 483. P. Lesot, J. Courtieu, Prog. Nucl. Magn. Reson. Spectrosc. 55 (2009) 128. C.M. Thiele, Eur. J. Org. Chem. 2008 (2008) 5673. D. Merlet, B. Ancian, J. Courtieu, P. Lesot, J. Am. Chem. Soc. 121 (1999) 5249. P. Lesot, M. Sarfati, J. Courtieu, Chem. Eur. J. 9 (2003) 1724. K. Kazimierczuk, O. Lafon, P. Lesot, Analyst 139 (2014) 2702. Z. Serhan, L. Martel, I. Billault, P. Lesot, Chem. Commun. 46 (2010) 6599. N. Nath, S. Hebbar, U. Ramesh Prabhu, N. Suryaprakash, J. Indian. Inst. Sci. 90 (2010) 1. S. Hebbar, U.R. Prabhu, N. Suryaprakash, J. Magn. Reson. 215 (2012) 23. B.R. Donald, J. Martin, Prog. Nucl. Magn. Reson. Spectrosc. 55 (2009) 101. H. Geen, R. Freeman, J. Magn. Reson. (1991) 93. N. Nath, N. Suryaprakash, J. Phys. Chem. B 115 (2011) 6868. S.R. Chaudhari, N. Nath, N. Suryaprakash, RSC Adv. 2 (2012) 12915. L. Lunazzi, A. Mazzanti, J. Am. Chem. Soc. 126 (2004) 12155.
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[11]
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Graphical Abstract
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Fig 1. The CH-RES-TOCSY pulse sequence (A) and the coherence pathway (B). In Fig. A, filled and unfilled rectangular bars pertain to 1800 and 900 hard pulses. Filled and unfilled shaped bars
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on 1H channel are 1800 and 900 selective pulses. Rectangular grey filled bar is isotropic mixing pulse (MLEV-17). Δ/2=(1/4*1JCH) used for INEPT. G1 is gradient purge pulse. The coherence
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path way (Fig. B, P1H for proton and P13C for carbon) was selected by G2, -G2, G3 and –G3 gradient pulses. All pulses have phase x unless otherwise mentioned. The four-step phase cycle
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is used; ϕ1= y, ϕ2=x, –x, –x, x and ϕrec= x, –x, –x, x. The spectra were recorded in magnitude mode.
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Fig 2. The CH-RES-TOCSY spectrum and the chemical structure of propylene carbonate. 1
13
C
n
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proton (3, marked as red) is selectively excited. The JCH and JCH of carbon 3 were obtained along F1/F2 and F2 dimensions respectively. The frequency separations giving 1JCH and nJCH are marked with arrows and the values are given in Hz. The relative signs of couplings are assigned
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based on direction of displacement of cross sections in the direct dimension (assuming 1JCH is positive sign)
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Fig 3. The CH-RES-TOCSY spectrum and the chemical structure of propylene carbonate.
13
C
attached proton (3, marked as red) was selectively excited in the experiment. The resolved R and
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S isomer spectra are marked (assignment is based on ref [30]), the JCH (taken from Fig. 2) and
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measured TCH values of carbon 3 are tabulated.
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Page 13 of 14
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Fig 4. The CH-RES-TOCSY spectra and the chemical structure of trans- (A, C) and cis- (B,D)
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stilbene. One of the aliphatic 13C attached protons was selectively excited (marked as red). The measured 1JCH, 2JCH of marked carbon and 3JHH of both isomers are reported in the respective spectrum. The relative signs of couplings are assigned based on the direction of displacement of
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peaks in the direct dimension (assuming the sign 1JCH is positive).
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