Journal of Chromatography A

Journal of Chromatography A, 1274 (2013) 1 5 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma Review Development and application of chiral crown ethers as selectors for chiral separation in high-performance liquid chromatography and nuclear magnetic resonance spectroscopy Man-Jeong Paik b, Jong Seong Kang c, Bin-Syuan uang d, James R. Carey d,, Wonjae Lee a, a College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea b College of Pharmacy, Sunchon National University, Sunchon, Jeonnam, 540-950, Republic of Korea c College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea d Department of Applied Chemistry, National University of Kaohsiung, 700, Kaohsiung University Rd., Nanzih District, Kaohsiung 811, Taiwan a r t i c l e i n f o Article history: Received 17 ctober 2012 Received in revised form 30 November 2012 Accepted 30 November 2012 Available online 10 December 2012 Keywords: Chiral crown ether 18-Crown-6-2,3,11,12-tetracarboxylic acid Enantiomer separation Chiral stationary phase Chiral selector Chiral solvating agent a b s t r a c t Chiral crown ethers have been widely used in the resolution of various chiral compounds containing a primary amino group. Covalently bonded chiral stationary phases derived from (18-crown-6)-2,3,11,12- tetracarboxylic acid (18-C-6-TA) were developed in our groups and utilized for the resolution for several types of analytes. By use of NMR spectroscopy, chiral discrimination studies were performed for -amino acids and their esters using 18-C-6-TA. ere, advances in the development and application of chiral stationary phases and chiral solvating agents using 18-C-6-TA for enantiomer resolution are described in relationship to recent chiral recognition mechanism studies. 2012 Elsevier B.V. All rights reserved. Contents 1. Introduction.......................................................................................................................................... 1 2. Chiral separation using 18-C-6-TA chiral crown ether in high-performance liquid chromatography............................................. 2 3. Chiral separation using 18-C-6-TA chiral crown ether in NMR..................................................................................... 3 4. Chiral recognition mechanisms using 18-C-6-TA as a chiral selector............................................................................... 4 5. Conclusion............................................................................................................................................ 4 References........................................................................................................................................... 4 1. Introduction A number of chiral selectors for chiral stationary phases (CSPs) have been developed and applied for enantiomer separation of a Part of the Chiral Separations and Enantioselectivity special issue, Vol. 1269, 21 December 2012. Corresponding author. Tel.: +886 7 591 9778; fax: +886 7 591 9348. Corresponding author at: College of Pharmacy, Chosun University, 375 Seoseok- Dong, Dong-Ku, Gwangju 501-759, Republic of Korea. Tel.: +82 62 230 6376; fax: +82 62 222 5414. E-mail addresses: jcarey@nuk.edu.tw (J.R. Carey), wlee@chosun.ac.kr (W. Lee). variety of chiral compounds [1]. Among these selectors are crown ethers, a class of synthetic host polyether molecules that bind certain cations such as alkali ions or protonated primary amines with high selectivity and affinity. Chiral crown ethers derived from CSPs have aroused considerable interest since Cram, a pioneer in host guest chemistry, immobilized chiral crown ethers onto solid supports such as silica gel [2,3]. nce the work by Cram, a number of chiral crown ether selectors, using techniques such as liquid liquid extraction, have been applied to the resolution of racemic -amino acids and primary chiral amines [2 4]. A distinctive feature of crown ether CSPs is the ability to resolve enantiomers of chiral compounds containing a primary amine. Resolution of the enantiomers in the absence of a primary amino group is seldom effective by competing columns. 0021-9673/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.11.086

2 M.-J. Paik et al. / J. Chromatogr. A 1274 (2013) 1 5 C C C C Fig. 1. The structures of chiral crown ethers derived from bis-(1,1 -binaphthyl)-22- crown-6 for Crownpak CR (left) and 18-C-6-TA derived from l-tartaric acid (right), respectively. Among several chiral crown ethers, (3,3 -diphenyl-1,1 - binaphthyl)-20-crown-6 was chosen to be dynamically coated on an octadecylsilica (DS) gel. The resulting column, shown in Fig. 1, led to the very successful CSP which was commercialized as Crownpak CR (Daicel Chemical Industries, Japan) (Fig. 1) [5,6]. This chiral crown ether type CSP is useful for the resolution of various chiral primary amines including amino acids. owever, due to its dynamically coated nature, the Crownpak CR has severe limitations regarding the use of mobile phases [6]. In light of these limitations, we sought to develop a new chiral crown ether type CSP that can beimmobilized directly to a solid support. ur studies focused on the use of chiral crown ether of (+)-(18-crown-6)- 2,3,11,12-tetracarboxylic acid (18-C-6-TA) used by Kuhn s group as a chiral selector in capillary electrophoresis for resolution of primary amino compounds including amino acids (Fig. 1) [7 10]. ence, we developed CSP 1 by covalently bonding (+)-18-C-6-TA to aminopropyl silica gel and applied it to the chromatographic resolution of several racemic -amino acids and primary amino compounds [11 18]. Using NMR, we studied the chiral selector (+)-18-C-6-TA for chiral discrimination of these analytes [19 21]. erein, we describe the development and application of the chiral crown ether of 18-C-6-TA as a selector for chiral discrimination using PLC and NMR experiments. The results are discussed in relationship to chiral recognition mechanism studies. 2. Chiral separation using 18-C-6-TA chiral crown ether in high-performance liquid chromatography As described previously, the Crownpak CR is a unique chiral crown ether type CSP derived from bis-(1,1 -binaphthyl)-22- crown-6 coated with DS silica gel [5]. Therefore, use of a mobile phases containing more than 15% methanol in water is not permitted for column safety [5,6]. If a mobile phase containing more than 15% methanol in water is used, the chiral selector material of the CSP leaches from the column. Due to the limited selection of mobile phases, hydrophobic analytes such as quinolone compounds have significantly long retention times [16,22]. In addition, when preparative separations are performed, there is a high possibility that the coated chiral crown ether can be co-eluted with the analytes. To overcome these drawbacks, we developed covalently bonded type CSP 1 derived from (+)-18-C-6-TA, which can be used with a wide variety of solvents. This advantage also extends the applicability of CSP 1 towards hydrophobic analytes [16]. Independently, the Machida s group developed a similar covalently bound column CSP 2 derived from (+)-18-C-6-TA using an alternative synthetic method [23,24]. As shown in Fig. 2, the chemical structure of CSP 1 is significantly different from that of CSP 2 [12,17,23]. The authors reported that the bonded form of (+)-18- C-6-TA to the silica gel appears to be through both monoamide and diamide linkages. When the same analytes were resolved on CSP 1 and CSP 2 by PLC, CSP 1 provided much better chiral separation than CSP 2 [17,23]. Specifically, all of the examined amino acids were well resolved with base-line separation on CSP 1. n the other hand, the examined amino acids were poorly resolved on CSP 2. Additionally, four amino acid analytes could not be resolved on CSP 2. [17]. In addition to the amino acids, various types of chiral primary amines such as amino alcohols, aryl -amino ketones and fluoroquinolone including gemifloxacin were well-resolved on CSP 1 [11 18]. Currently, the chiral crown ether covalent type CSP 1 is commercially available as Chirol RCA [(+)-CSP 1] derived from (+)-18-C-6-TA and Chirol SCA [( )-CSP 1] derived from ( )-18- C-6-TA (RS Tech Corp., Daejeon, Korea). Chirol RCA and Chirol SCA can be used to invert the elution order as desired [18]. Fig. 3 shows typical chromatographic resolution chromatograms of an enantiomerically enriched DPA (l:d = 100:1) and racemic DPA on (+)-CSP 1 and/or ( )-CSP 1, respectively. nce the second minor peak of (d)-dpa is embedded in the first major peak of (l)-dpa on (+)-CSP 1, the exact determination of the enantiomeric purity might be precluded. For a practical chiral analysis process, the inversion of the elution order offers a strong advantage. Because the minor enantiomer of (d)-dpa is eluted in the front of the major enantiomer of (l)-dpa on ( )-CSP 1, for ease of analysis, determination of enantiomeric purity should be performed using ( )-CSP 1. Like the chiral crown ether coated type Crownpak CR, a dynamic CSP was prepared by hydrophobically bonding the N-dodecyl diamide of 18-C-6-TA to octadecyl silica gel and used to resolve various chiral compounds containing a primary amino moiety such N-R R-N a,b (+)-18-C-6-TA c C 3 C 3 C 3 C 3 C 3 C 3 C 3 N N N Fig. 2. The structures of CSP 1 (R ) and CSP 1a (R C 3) (left) developed by our group, and CSP 2 (right) by Machida s group, which are derived from the same chiral selector (+)-18-C-6-TA. (a) Acetyl chloride (b) aminopropyl silica gel, Et 3N (c) EEDQ, aminopropyl silica gel.

M.-J. Paik et al. / J. Chromatogr. A 1274 (2013) 1 5 3 Fig. 3. Chromatograms of the resolution of an enantiomerically enriched DPA (l:d = 100:1) on (+)-CSP 1 (left) and ( )-CSP 1(middle) as well as racemic DPA on ( )-CSP 1(right). as amino acids [25]. In general, this coated type CSP showed poorer resolution than the corresponding covalently bonded CSP 1. Furthermore, much effort has been made to develop chiral stationary phases based on (+)-18-C-6-TA with a view of enhancing chiral recognition efficiency and stability of CSP 1 [26]. For example, the N in the silica tether from both amides of CSP 1 was replaced by an amine (N C 3 ) in CSP 1a (Fig. 1) [27]. CSP 1 and CSP 1a were comparable to each other for the resolution of amino acids but CSP 1a was superior to CSP 1 in the resolution of amino acid esters. In addition, CSP 1a gave improved enantiomeric separation of primary aromatic amines as compared to CSP 1. This study demonstrated that changing the linkages of these two CSPs can influence their ability to resolve analytes, and may alter the chiral recognition mechanism related to separation [27]. In addition, we have attempted to extend the application of crown ether derived CSP 1 to chiral acids such as 2- aryloxypropionic acids [28]. nce the direct enantiomer resolution of 2-aryloxypropionic acids lacking a primary amino moiety was not accomplished on CSP 1, the corresponding N-hydrazide derivatives were synthesized for complexation between the N-hydrazide and the crown ether moiety. Indeed, several 2-aryloxypropionic acids as N-hydrazide derivatives were well resolved on CSP 1 using this indirect method. 3. Chiral separation using 18-C-6-TA chiral crown ether in NMR nce successful chiral separation of amino acids and their derivatives was obtained on CSP 1 derived from (+)-18-C-6-TA using PLC, it was expected that (+)-18-C-6-TA could be useful as a chiral selector in the enantiodiscrimination of these analytes using solution NMR [19 21]. When (+)-18-C-6-TA was used in an enantiodiscrimination experiment using NMR spectroscopy, several amino acids and amino acid esters showed high chemical shift nonequivalences ( ı) of their -protons [29]. In general, upon chiral complexation in presence of the (+)-18-C-6-TA, the chemical shift-changes of the -protons of the analytes are greater than those of other peaks [29,30]. As the ratio of 18-C-6-TA to analytes was increased, the chemical shift changes of the -protons of the analytes gradually increased [20]. In addition, all -proton chemical shift changes from the d-enantiomers were consistently greater than those of l-enantiomers upon complexation, indicating that the d-enantiomers interact more strongly with (+)-18-C-6-TA than the l-enantiomers. These NMR results are consistent with the PLC data obtained on (+)-CSP 1 derived from (+)-18-C-6-TA [17,19,29]. Largely, the amino acid methyl esters investigated in this study showed larger chemical shift nonequivalences than the corresponding amino acids using the 18-C-6-TA chiral selector [29]. n the contrary, all of the amino acids gave higher PLC separation factors than the corresponding amino acid methyl esters on CSP 1 [12]. Importantly, it should be noted that the magnitude of chemical shift nonequivalence in NMR has no direct relationship to the separation factors observed from PLC, because the chemical shift nonequivalence in NMR depends directly on the structural changes resulting from complexation between the chiral selector and each enantiomer, and not on a solid support. Particularly, the leucine methyl ester and the phenylalanine methyl ester did not show chiral separation on CSP 1 derived from 18-C-6-TA, whereas they showed considerably high chemical shift differences of their protons in the presence of 18-C-6-TA as determine by NMR [12,29]. This implies that the NMR analytical method using 18-C-6-TA as a chiral solvating agent may be complementary to the PLC analytical method using 18-C-6-TA derived CSP 1. Fig. 4 shows typical 1 NMR spectra of three racemic amino acids in the presence of equimolar (+)-18-C-6-TA. milarly, Wenzel and Machida reported their chiral discrimination results using 18-C-6-TA for various primary amines as well as amino acids and their analogs using NMR spectroscopy [30 33]. Consequently, it is expected that the chiral selector of 18-C-6-TA can be used to determine enantiomeric purity by measuring chemical shift nonequivalences in NMR spectroscopy [20,30]. For the determination of enantiomeric purity of these analytes, in general, the NMR analytical method using 18-C-6-TA as a chiral solvating agent shows lower precision and accuracy than the PLC analytical method on 18-C-6-TA derived CSP [18,34]. owever, because Fig. 4. 1 NMR spectra of racemic amino acid in the presence of equimolar (+)-18-C-6-TA; (A) phenylglycine (PG), (B) phenylalanine (Phe), (C) isoleucine (Ile).

4 M.-J. Paik et al. / J. Chromatogr. A 1274 (2013) 1 5 Fig. 5. Schematic representation of proposed chiral recognition mechanism (left) between the chiral selector of the CSP 1 derived from (+)-18-C-6-TA and the second eluted enantiomer of the d-amino acids or esters, based on complex structure (right) of (+)-18-C-6-TA/D-PG generated from NMR NE data and molecular dynamics calculations. It shows intermolecular hydrogen bonding (dotted line) between the chiral selector of the (+)-CSP 1 and the carbonyl oxygen of the d-amino acid or its ester. The tripodal hydrogen bonds inside the 18-crown-6 ring of the CSP are not shown in the diagram on the left. of the simplicity, the NMR analytical technique may find use for the determinations of the enantiomeric purities of primary amines such as amino acids and their esters. 4. Chiral recognition mechanisms using 18-C-6-TA as a chiral selector CNR" R N 3 + C 2 R' C 2 In search for the origin of chiral discrimination between amino acid enantiomers and 18-C-6-TA, detailed studies on the elucidation of the chiral recognition interactions were performed [10,19]. According to our previous chiral selector studies using molecular dynamics calculations, NMR experiments, and QSPR models with capillary electrophoresis, it was reported that the ammonium moiety of phenylglycine was bound by three hydrogen bonds in a tripodal arrangement inside the chiral cavity of the 18-crown-6 ring of the CSP [10,19]. These hydrogen-bonding interactions were observed in not only the d-enantiomer/18-c-6-ta complex but also in the l-enantiomer/18-c-6-ta complex. In addition, another hydrogen bonding interaction between a carboxylic acid of 18-C- 6-TA and the carbonyl oxygen of the d-amino acid was observed to be essential in the d-enantiomer/18-c-6-ta complex, whereas the l-enantiomer/18-c-6-ta complex does not exhibit intermolecular hydrogen-bonding [19]. Based on the results obtained from NMR and molecular dynamics calculations, a chiral recognition mechanism for CSP 1 derived from (+)-18-C-6-TA in solution was proposed [17,19]. Fig. 5 shows not only a schematic representation of the proposed chiral recognition between (+)-18-C-6-TA derived CSP 1 and the second eluted enantiomer of the d-amino acid or its ester, but also shows the structure of (+)-18-C-6-TA/D-PG generated from NE data and molecular dynamics calculations. Another factor to consider is that the chiral selector of (+)-18-C-6-TA on CSP 1 is not allowed to move freely in PLC but is fixed to the aminopropyl silica gel in the stationary phase environment, unlike the free environment of the chiral selector (+)-18-C-6-TA in solution. This factor may lead to differences in chiral mechanisms as determined by PLC or NMR. The d-enantiomers of the resolved -amino acids and their esters were preferentially retained on the (+)-18-C-6-TA derived CSP 1 [17,18]. Unlike the other resolved -amino acids and their esters, the elution orders of serine, threonine and their methyl esters are reversed, with the (l)-enantiomers being preferentially retained on (+)-CSP 1 [12,18]. nce there is a hydroxy group on the -carbon of compounds such as serine and threonine, it is thought that this might directly influence chiral recognition on CSP 1. Therefore, we propose that there is a stronger intermolecular hydrogen-bonding interaction between the -hydroxy group of serine (or threonine) and the C of 18-C-6-TA, instead of CNR" Fig. 6. Proposed chiral recognition between (+)-18-C-6-TA derived CSP 1 and the second eluted enantiomer of l-serine (R ) [or l-threonine (R methyl)], showing intermolecular hydrogen bonding (dotted line) between the chiral selector of the CSP 1 and the oxygen of l-serine (or l-threonine) of the analyte. The tripodal hydrogen bonds inside the 18-crown-6 ring of the CSP are not shown. the intermolecular hydrogen-bonding interaction between the carbonyl oxygen of d-enantiomers and the C of 18-C-6-TA. Fig. 6 shows a schematic representation of the proposed chiral recognition between the CSP 1 derived from (+)-18-C-6-TA and the strongly eluted enantiomer of l-serine (or l-threonine). Data to support this hypothesis is currently being collected in our laboratories. In terms of spatial orientations during the formation of diastereomeric complex between l-serine (or l-threonine) and (+)-18-C-6-TA, it is expected that this intermolecular hydrogen-bonding interaction by the -hydroxy group of l-serine (or l-threonine) affords a favorable interaction, and that this results in a reversal of the PLC elution orders. 5. Conclusion Among chiral crown ethers, 18-C-6-TA was found to be one of the most effective chiral crown ethers used as a chiral selector. It has been widely used to resolve the enantiomers of chiral compounds containing a primary amine using capillary electrophoresis, highperformance liquid chromatography, NMR spectroscopy and so on. ere, chiral resolution techniques and their applications using the chiral selector of 18-C-6-TA were reviewed from the PLC and NMR chirotechnology perspective, and their chiral recognition mechanisms were described based on experimental studies and molecular dynamics calculations. Importantly, it was shown that the commercially available (+)- and ( )-CSP 1 based on (+)- and ( )-18-C-6-TA have been successfully applied for the resolution of various primary amines including amino acids. References [1] G. Subramanian (Ed.), Chiral Separation Techniques: A Practical Approach, Wiley-VC, Weinheim, 2001.

M.-J. Paik et al. / J. Chromatogr. A 1274 (2013) 1 5 5 [2] D.J. Cram, Angew. Chem. Int. Engl. 27 (1988) 1009. [3] X.-X. Zhang, J.S. Bradshaw, R.M. Izatt, Chem. Rev. 97 (1997) 3313. [4] F. Vögtle, E. Weber, ost Guest Complex Chemistry Macrocycles, Springer- Verlag, Berlin, 1985. [5] T. Shinbo, T. Yamaguchi, K. Nishimura, M. Sugiura, J. Chromatogr. A 405 (1987) 145. [6] Application Guide for Chiral PLC Selection, 3rd ed., Daicel Chemical Industries, Ltd., 2002. [7] R. Kuhn, F. Erni, T. Bereuter, J. ausler, Anal. Chem. 64 (1992) 2815. [8] R. Kuhn, J. Wagner, Y. Walbroehl, T. Bereuter, Electrophoresis 15 (1994) 828. [9] W. Lee, S. La, Y. Choi, K.-R. Kim, Bull. Korean Chem. Soc. 24 (2003) 1232. [10].-J. Park, Y. Choi, W. Lee, K.-R. Kim, Electrophoresis 25 (2004) 2755. [11] M.. yun, J.S. Jin, W. Lee, Bull. Korean Chem. Soc. 19 (1998) 819. [12] M.. yun, J.S. Jin, W. Lee, J. Chromatogr. A 822 (1998) 155. [13] M.. yun, J.S. Jin, W. Lee, J. Chromatogr. A 837 (1999) 75. [14] M.. yun, S.C. an, J.S. Jin, W. Lee, Chromatographia 52 (2000) 473. [15] M.. yun, S.C. an, Y.J. Cho, J.S. Jin, W. Lee, Biomed. Chromatogr. 16 (2002) 356. [16] W. Lee, C.-S. Baek, K. Lee, Bull. Korean Chem. Soc. 23 (2002) 1677. [17] W. Lee, J.Y. Jin, C.-S. Baek, Microchem. J. 80 (2005) 213. [18] J.Y. Jin, W. Lee, M.. yun, J. Liq. Chromatogr. Relat. Technol. 29 (2006) 841. [19] E. Bang, J.-W. Jung, W. Lee, D.W. Lee, W. Lee, J. Chem. Soc., Perkin Trans. 2 (2001) 1685. [20] W. Lee, E. Bang, W. Lee, Chromatographia 57 (2003) 457. [21] W. Lee, E. Bang, C.-S. Baek, W. Lee, Magn. Reson. Chem. 42 (2004) 389. [22] W. Lee, C.Y. ong, J. Chromatogr. A 879 (2000) 113. [23] Y. Machida,. Nishi, K. Nakamura,. Nakai, T. Sato, J. Chromatogr. A 805 (1998) 85. [24] Y. Machida,. Nishi, K. Nakamura, Chromatographia 49 (1999) 621. [25] M.. yun,.j. Koo, J.S. Jin, W. Lee, J. Liq. Chromatogr. Relat. Technol. 23 (2000) 2669. [26] M.. yun, J. Sep. Sci. 29 (2006) 750. [27] M.. yun, Y.J. Cho, J.A. Kim, J.S. Jin, J. Chromatogr. A 984 (2003) 163. [28] J.Y. Jin, W. Lee, Chirality 19 (2007) 120. [29] E. Bang, J.Y. Jin, J.. ong, J.S. Kang, W. Lee, W. Lee, Bull. Korean Chem. Soc. 33 (2012) 3481. [30] Y. Machida, M. Kagawa,. Nishi, J. Pharm. Biomed. Anal. 30 (2003) 1929. [31] Y. Machida,. Nishi, K. Nakamura, J. Chromatogr. A 810 (1998) 33. [32] T.J. Wenzel, J.E. Thurston, J. rg. Chem. 65 (2000) 1243. [33] T.J. Wenzel, J.E. Thurston, Tetrahedron Lett. 41 (2000) 3769. [34] T.J. Wenzel, J.D. Wilcox, Chirality 15 (2003) 256.