1530
J. Phys. Chem. B 2009, 113, 1530–1532
Visualization of Enantiomers in the Liquid-Crystalline Phase of a Fragmented DNA Solution Sujay P. Sau and K. V. Ramanathan* NMR Research Centre, Indian Institute of Science, Bangalore 560012, India ReceiVed: July 24, 2008; ReVised Manuscript ReceiVed: October 17, 2008
The use of the liquid-crystalline phase of fragmented DNA solution for enantiomeric differentiation by NMR is reported. The lyotropic cholesteric liquid crystal system formed, orients in a magnetic field and is able to discriminate water soluble enantiomeric mixtures in a simple 2D J-resolved NMR experiment. The use of NMR in the analysis of enantiomers has been well-known for a very long time. In recent years, there has been a renewed interest in this area in view of the requirement for pharmaceutical industries to produce single enantiomeric drug molecules. As a result, methods that can distinguish enantiomers and estimate their relative ratios have gained importance. In this context, the use of chiral liquid crystals is found to be very promising. In the past decade, Courtieu and co-workers establishedtheuseofasolutionofPBLG(poly-γ-benzyl-L-glutamate),1-3 a synthetic polymer, in various organic solvents as a powerful tool for enantiomeric analysis by NMR. This technique makes use of the differential ordering effect (DOE)4 of the chiral liquidcrystalline phase on the left- and right- handed species for the enantiomeric differentiation. This method is found to be useful not only for systems that possess a chiral center but also for those that exhibit enantiomerism due to the presence of either a chiral axis or a chiral plane5 and also for atropisomers.6,7 Though the use of the PBLG liquid crystal has proven to be a powerful tool, its application is limited to molecules soluble in organic solvents. Over the last few years, there has been an extensive search for a suitable water-soluble lyotropic cholesteric liquid-crystal (LCLC) system to extend the potentials of the technique for the analysis of water-soluble enantiomers, and there are a few reports of such systems in the literature.8,9 However, unlike the PBLG system, these are surfactant-based lyotropic liquid crystals that utilize the lamellar-liquid crystalline phase for enantiomeric differentiation. In the search for a system similar to the PBLG liquid-crystalline system, we have investigated the possibility of the use of DNA as a water-soluble LCLC medium. Though the liquid-crystalline (LC) property of DNA10-17 has been studied since 1961 for the purpose of understanding the packing and organization of DNA inside of the cell nucleus, there is no report of using this LC phase of DNA as an orienting medium. In this article, we report the first use of the LC phase of DNA solution for orienting small molecules, particularly for the purpose of differentiation of water-soluble enantiomeric mixtures. High-molecular-weight DNA from salmon sperm testes was purchased from Sigma-Aldrich. The DNA was checked for protein contamination and found to be free of protein content. The DNA was dissolved in autoclaved water and sonicated18,19 (with Vibracell sonicator) in an ice bath for 8-10 min with 20 s pulses and 10 s of gap between pulses. The size distribution of sonicated DNA samples was determined by gel electrophore* To whom correspondence should be addressed.
sis on a 1.3% agarose TAE gel (tris-acetate EDTA buffer) with a PCR (polymerase chain reaction) marker. After determination of the size distribution, the DNA was precipitated by isopropanol, washed twice with 70% ethanol, and finally lyophilized overnight. From the agarose gel, the size of fragmented DNA was found to be within 100-500 base pairs. For NMR studies, the lyophilized DNA fragments were directly weighed into a NMR tube and dissolved in saline/ buffer solution (D2O). Orientation of DNA in the magnetic field is slow and takes about 3-4 h for the molecules to orient, which was monitored by following the deuterium quadrupolar doublet and the 23Na quadrupole split triplet of the sodium in the buffer and their line widths. Once a steady state was reached, the deuterium spectrum showed typically a doublet of 48 Hz separation with a line width of 15 Hz for each line of the doublet. The 23Na spectrum showed a separation of 500 Hz between a satellite transition and the central transition, with a line width of 75 Hz for the central transition. Typical spectra are displayed in the Supporting Information. For monitoring the alignment of molecules in the magnetic field, the 1D proton spectra are not useful, as shown below, compared to 2H and 23Na spectra. This is because 2H and 23Na have smaller gyromagnetic ratios, which make them less sensitive to field inhomogeneity and magnetic susceptibility effects. They also have quadrupolar couplings very much larger in magnitude compared to the dipolar and J couplings experienced by protons. To check the possibility of the use of the DNA solution for enantiomeric differentiation, 6 mg of DL-alanine was dissolved in a liquid-crystalline DNA solution. Subsequently J-resolved20 and selective (methyl proton) J-resolved (SERF)21 experiments were performed. Figure 1 shows both the 1D and 2D J-resolved spectra of DL-alanine dissolved in the liquid-crystalline phase of the DNA solution. In this cholesteric liquid-crystalline phase, the 1D 1H NMR spectrum is featureless, and the multiplet structure is not visible due to sample and magnetic field inhomogeneities and is not useful for analysis. However, in the 2D proton J-resolved experiment, which removes the problem arising due to inhomogeneous line broadening along the F1 dimension,22,23 one should observe a set of quartets for the CH proton from each enantiomer due to total coupling (Tij ) (J + 2D)ij) between CH and CH3 protons, whereas for the methyl protons of alanine, one should observe a set of a doublet (1:1) of a triplet (1:2:1) from each enantiomer due to intramethyl dipolar coupling (Dij) (the triplet) and total
10.1021/jp806534v CCC: $40.75 2009 American Chemical Society Published on Web 01/09/2009
Liquid-Crystalline Phase of Fragmented DNA Solution
J. Phys. Chem. B, Vol. 113, No. 5, 2009 1531
Figure 2. 500 MHz selective (methyl proton) J-resolved (SERF) spectrum of alanine oriented in DNA solution. The spectrum was recorded at 300 K by adding 32 scans per t1 with a total of 64 increments, zero-filled to 512 points in the F1 dimension. The selective excitation of the methyl peaks was achieved with the band selective excitation pulses24 with a typical bandwidth of 300 Hz.
expectation. The chemical shifts of CH3 and CH protons are 1.51 and 3.82 ppm. The magnitudes of the coupling constants obtained from the spectra are as shown in the table below.
Figure 1. (a) 500 MHz 1H NMR spectrum obtained for a racemic mixture of alanine dissolved in a DNA-D2O buffer liquid-crystalline system. The composition of the DNA solution is (Ala/DNA/D2O buffer) ) 6 mg:33.6 mg:190 µL. (b) 1H tilted J-resolved 2D NMR spectrum of the same system. The spectra were recorded at 300 K on a Bruker DRX-500 NMR spectrometer by adding 8 scans per t1 increment with a total of 256 increments and zero-filled to 1024 points in the F1 dimension. A relaxation delay of 5 s was used between scans. The spectrum was processed with a sine-bell window function in both the dimensions and plotted in the magnitude mode in the F1 dimension. The cross sections corresponding to the R and β protons are shown along the left and right margins of the 2D spectrum, respectively, and the peaks corresponding to the two enantiomers are identified by filled and open circles. The contours of the 2D spectrum as well as the F2 projection show an additional peak of lesser intensity accompanying all of the peaks caused by temperature fluctuation during the experiment possibly due to local rf heating. This has, however, no effect on the coupling pattern as seen in the F1 cross sections and the visualization of the enantiomers. At the center of the cross sections, some additional peaks are seen. We believe that they are zero-frequency artifacts, however other possibilities exist. Nevertheless we ignore the same.
coupling (Tij) between CH3 and CH protons (the doublet). In the case of the SERF experiment (Figure 2), made up of methyl selective π/2 and π pulses, the splitting of methyl protons reduces to a set of triplets from each enantiomer with merged central peaks. The observed splitting pattern along the F1 dimension for R and β protons in 2D J-resolved experiment is as expected for the enantiomeric differentiation of DL-alanine in a cholesteric liquid crystal. A pair of quartets (8 lines) for the R proton and a pair of doublets of triplets (12 lines) for the β protons are observed as shown in Figure 1. The observed splitting pattern for the methyl protons in the SERF experiment (Figure 2) is also according to the
In conclusion, we have shown here that the LCLC phase of a concentrated aqueous solution of DNA of considerably smaller size (in our case, 100-500 base pairs) can orient enantiomers differently that can be visualized by 2D J-resolved 1H NMR spectroscopy. Acknowledgment. The authors gratefully acknowledge Prof. D. N. Rao (Dept. of Biochemistry) and his group members, especially Ms. Swayamprabha, for their great help in DNA sample preparation. The use of the DRX-500 NMR spectrometer funded by the Department of Science and Technology (DST), New Delhi at the NMR Research Centre, Indian Institute of Science, Bangalore is gratefully acknowledged. Supporting Information Available: 2H and 23Na spectra. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Sarfati, M.; Lesot, P.; Merlet, D.; Courtieu, J. Chem. Commun. 2000, 1113, and references therein. (2) Aroulanda, C.; Merlet, D.; Courtieu, J.; Lesot, P. J. Am. Chem. Soc. 2001, 123, 12059. (3) Farjon, J.; Merlet, D.; Lesot, P.; Courtieu, J. J. Magn. Reson. 2002, 158, 169. (4) Lesot, P.; Merlet, D.; Courtieu, J.; Emsley, J. W.; Rantala, T. P.; Jokisaari, J. J. Phys. Chem. A 1997, 101, 5719. (5) Smadja, W.; Auffret, S.; Berdague, P.; Merlet, D.; Canlet, C.; Courtieu, J.; Legros, J.; Boutros, A.; Fiaud, J. Chem. Commun. 1997, 2031. (6) Meddour, A.; Berdague, P.; Hedil, A.; Courtieu, J.; Lesot, P. J. Am. Chem. Soc. 1997, 119, 4502. (7) Lesot, P.; Lafon, O.; Kagan, H. B.; Fan, C. Chem. Commun. 2006, 389. (8) Baczko, K.; Larpent, C.; Lesot, P. Tetrahedron: Asymmetry 2004, 15, 971.
1532 J. Phys. Chem. B, Vol. 113, No. 5, 2009 (9) Solgadi, A.; Meddour, A.; Courtieu, J. Tetrahedron: Asymmetry 2004, 15, 1315. (10) Robinson, C. Tetrahedron 1961, 13, 219. (11) Rill, R. L.; Hilliard, P. R., Jr.; Levy, G. C. J. Biol. Chem. 1983, 258, 250. (12) Merchant, K.; Rill, R. L. Macromolecule 1994, 27, 2365. (13) Zakharova, S. S.; Jesse, W.; Backendorf, C.; van der Maarelohan, J. R. C. Biophys. J. 2002, 83, 1119. (14) Rill, R. L.; Strzelecka, T. E.; Davidson, M. W.; Van Winkle, D. H. Physica A 1991, 176, 87. (15) Strzelecka, T. E.; Rill, R. L. J. Am. Chem. Soc. 1987, 109, 4513. (16) Rolf, Brandes.; David R., Kearns. Biochemistry 1986, 25, 5890, and references therein.
Sau and Ramanthan (17) Van Winkle, D. H.; Chatterjee, A.; Link, R.; Rill, R. L. Phys. ReV. E 1997, 55, 4354. (18) Merchant, K.; Rill, R. L. Biophys. J. 1997, 73, 3154. (19) Strzelecka, T. E.; Rill, R. L. Biopolymer 1990, 30, 57. (20) Aue, W. P.; Karhan, J.; Ernst, R. R. J. Chem. Phys. 1976, 64, 4226. (21) Facke, T.; Berger, S. J. Magn. Reson., Ser. A 1995, 113, 114. (22) Bodenhausen, G.; Freeman, R.; Niedermeyer, R.; Turner, D. E. J. Magn. Reson. 1997, 26, 133. (23) Levitt, M. H. Spin Dynamics, 2nd Ed.; Wiley, Chichester, U.K., 2007. (24) Freeman, R. Prog. Nucl. Magn. Reson. 1998, 32, 59.
JP806534V
