Accepted Manuscript Title: ‘Click’ preparation of a novel ‘native-phenylcarbamoylated’ bilayer cyclodextrin stationary phase for enhanced chiral differentiation Author: Jie Zhao Xiaohong Lu Yong Wang Jie Lv PII: DOI: Reference:
S0021-9673(15)00054-0 http://dx.doi.org/doi:10.1016/j.chroma.2015.01.008 CHROMA 356169
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
19-8-2014 12-12-2014 7-1-2015
Please cite this article as: J. Zhao, X. Lu, Y. Wang, J. Lv, ‘Click’ preparation of a novel ‘native-phenylcarbamoylated’ bilayer cyclodextrin stationary phase for enhanced chiral differentiation, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.01.008 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.
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‘Click’ preparation of a novel ‘native-phenylcarbamoylated’ bilayer
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cyclodextrin stationary phase for enhanced chiral differentiation
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Jie Zhaoa, Xiaohong Lua, Yong Wang*ab, Jie Lva
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300072
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(Tianjin), Tianjin 300072, China
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Corresponding author: Yong Wang
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Email: [email protected]
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Department of Chemistry, School of Science, Tianjin University, Tianjin
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Collaborative Innovation Center of Chemical Science and Engineering
Tel：+86 13502146137
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Building 7, Room 110, Tianjin University, No. 92 Weijin Road, Tianjin,
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China, 300072.
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Keywords: bilayer cyclodextrin, click, enantiorecognition, RP-HPLC.
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It is known that there is an increasing demand for enantiopure chiral drugs due to their different biological, pharmacological and toxicological effects from their enantiomers [1-5]. Chiral high performance liquid chromatography (CHPLC) has been proven to be a useful methodology not only for determining optical purity but also for obtaining pure enantiomers at preparative scale. The key point of CHPLC method is the development of effective chiral stationary phases (CSPs) [6-12].
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By far, cyclodextrin derivatives (CDs) have been used extensively in CSPs ascribing to their ability to accommodate guest molecules through the formation of inclusion complexes [13, 14]. Recent investigations on CD CSPs mainly focus on either new immobilization chemistry to afford stable and functional bridges, or CD rims functionalization to endow CD specific interactions [15-21]. Although there have emerged a series of CD CSPs developed via the above two ways, there still exist many challenges in this area. On one hand, development of stable and functional linkages is relatively difficult. On the other hand, CD rims modification usually enhances its enantioselectivity toward specific categories of enantiomers while diminish its original recognition ability to some enantiomers. Hence, it is a great challenge to explore novel approaches for the fabrication of more versatile CD CSPs.
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Binary chiral selector systems may provide an alternative to improve the separation and some successful applications have been performed using chiral mobile phase additives (CMP) in chiral capillary electrophoresis (CE) [22, 23]. In chromatography, binary CSP can be realized by incorporating two chiral selectors on same silica support or by direct mixing of two different CSPs. According to Levkin, Schurig and Lindner’s extensive studies, incorporating two selectors in a CSP or two CSPs in one column may extend the CSPs’ enantioselectivity profile, however it could hardly improve the enantioselectiviy compared to the CSPs with individual selector due to quasi pseudo-enantiomerical behaviors and negative effects from
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Abstract: This paper reports an effective approach for the fabrication of a novel hybrid bilayer cyclodextrin (CD) chiral stationary phase (CSP), where native and perphenylcarbamoylated-β-CD were successively immobilized onto silica surface via a two-step click approach to form a bilayer CD structure. By decorating the bulky phenylcarbamoylated CD onto the unmodified CD silica, the CSP can provide multiple interaction sites such as H-bonding (-OH, C=O, -NH-), steric effects, -, dipole-dipole and inclusion complexation interactions, which help to broaden the CSP’s enantioselectivity profile and enhance the enantioselectivity to some specific analytes. A group of enantiomer pairs such as isoxazolines, bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine, styrene oxide and dansyl amino acids can be baseline or partially separated on the current CSP under reversed phase high performance liquid chromatography (RP-HPLC). The selectivity and resolution of 4NPh-OPr reached 5.25 and 13.97, which is an exciting achievement for the enantioseparations by CD CSPs.
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Introduction
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In light of the above wisdom, the current work aims to develop novel CD CSPs by decorating native CD with functionalized CD moieties to introduce multiple interaction cites. A recent study by Tang et al reported efficient enantioseparation of twenty-six racemates on triazolyl linked phenylcarbamoylated CD CSP [32]. Although the bulky phenylcarbamoyl substitutions provide several interaction sites, the lack of original H-bonding sites (-OH) and inclusion complexation from native CD may diminish its selectivity to some analytes. Herein, via a smartly designed two-step click procedure, we construct a novel ‘native-perphenylcarbamoylated CD CSP material (denoted as DNPCDCSP) by introduction of native CD (down layer) and perphenylcarbamoylated CD (top layer) onto the silica surfaces.
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2.1 Chemicals and materials
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Sodium hydride (60%), ethyl acetate, petroleum ether, tetrahydrofuran and toluene were purchased from Jiangtian Chemical Reagents (Tianjin, China). Sodium azide and pyridine were purchased from Tianjin Chemical Regents (Tianjin, China). (3-propylchloride)triethoxysilane, γ-(2,3-epoxypropoxy)propytrimethoxysilane (KH-560), propargyl bromide, ethylene glycol, (3-glycidoxypropyl)trimethoxysilane and anhydrous N,N-Dimethylformamide (DMF) were purchased from HEOWNS (Tianjin, China). Phenylisocyanate was provided by Ouhe Technology (Beijing, China). HPLC-grade methanol (MeOH), acetonitrile (ACN), triethylamine (TEA) and acetic acid were purchased from Concord Technology (Tianjin, China). Ultra-pure water was prepared by Milli-Q water purification system (Billerca, MA, USA). Kromasil spherical silica gel (5 μm, 100 Å, surface area 300 m2·g-1) were purchased from Eka Chemicals (Bohus, Sweden). Isoxazoline racemates were provided by Tang’s group [33]. Acidic racemates were purchased from Sigma-Aldrich (Shanghai, China) and the other racemates were purchased from Energy-Chemical (Shanghai, China). The analyte structures are presented in Fig. S1 in the supporting information (SI).
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2.2 Instruments and measurements
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none-enantioselective interactions [24-26]. A good approach for enhancing the separation ability of CSPs is to incorporate more types of interaction sites and chiral centers on one selector skeleton such as brush-type Whelk-O1 CSPs (-donor and -acceptor), quinine CSPs and cinchona zwitterionic CSPs [3, 27-31].
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Experimental
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Fourier-transform infrared (FTIR) spectra were collected on an AVATR360 supplied by Thermo Nicolet (USA). 1H NMR were recorded on a Bruker ACF400 (400MHz) supplied by Bruker Biospin (Fällanden, Switzerland). Solid state 13C NMR was performed on a Varian infinityplus 300 NMR spectrometer (300MHz, 7.0T). Mass spectra data were collected on LCQ Deca XP MAX system (Thermo Fisher, USA). Thermal gravity analyses were conducted on a NETZSCH STA409PC TGA (Bavaria, Germany) in air atmosphere at heating rate of 10 oC·min-1. Elemental analyses were performed on a Var-ioMICRO CHNOS elemental analyzer (Elementar 3
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Analysensysteme, Hanau, Germany). Chromatographic analyses were performed on a Laballiance HPLC system with a diode array detection (DAD) system (State college, PA, USA). Samples were dissolved in MeOH/H2O (v/v=1:1) at a concentration of 1 mg·mL-1 and the injection volume was set as 10 μL. Triethylammonium acetate buffer (denoted as TEAA) was prepared by dissolving 1% (v/v) TEA in ultra-pure water and thereafter adjusted to the desired pH with acetate acid. Each solution was injected in triplicate and the average values were used. All the buffers and samples were filtered through 0.22 μm membranes before usage. The detection was performed at 220-300 nm. Calculations (capacity factor, k; selectivity, α and resolution, Rs) follow USP standards.
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2.3 Preparation of DNPCDCSP
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The synthetic route of DNPCDCSP is depicted in Fig. 1. Mono-6-deoxy-(p-tosylsulfonyl)-β-cyclodextrin (TsO-CD) 1 and Mono-6-deoxy-azido-β-cyclodextrin (N3-CD) 2 and Mono-6-deoxy-azido-perphenylcarbamoylated-β-cyclodextrin (N3-PhCD) 3 were synthesized and purified according to the reported approaches [34-36]. All the silica materials were characterized after vacuum drying (90 oC, 20 mmHg, 12 h). The detailed step by step synthetic procedures have been included in SI (Section 1 in SI).
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Insert Fig.1
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The prepared CSP were packed into stainless-steel column (150 mm × 4.6 mm I.D.) (Cherishtech, Beijing, China) using the typical slurry-packing technique with MeOH as the packing solvent (5000 psi, 30 min).
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3.1 Synthesis and Characterization of the DNPCDCSP
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Synthesis. Initially, we attempted to construct the hybrid CD structure by immobilization of N3-PhCD onto alkynyl-silica 6' (Fig. S2a in SI). However, the FITR spectrum (Fig. S3 in SI) of the resulting material did not show an adsorption around 1730 cm-1 (typical adsorption of carbonyl group), indicating that treatment of alkynyl-silica with N3-PhCD cannot lead to the formation of triazolyl. The reason might be due to the sterically restricted CD mouth of N3-PhCD, where the -N3 moiety was covered by phenylcarbamoyl groups, resulting in the hindrance of the short alkyne chain of alkynyl-CD to react with the -N3 moiety. Hence, it is necessary to make the spacer between the two CD layers longer to avoid the interference of steric hindrance.
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Accordingly, 1,2-bis(propargyloxy)ethane was synthesized, acting as an linker to connect N3CD-silica and N3-PhCD, where the alkynyl moieties of 1,2-bis(propargyloxy)ethane can readily insert into the bulky mouth of the N3-PhCD to react with the azido moiety. A two-step click strategy can be thereafter performed for the construction of desired DNPCDCSP (Fig. S2b). Initially, N3CD-silica was fabricated according to our previously reported method with slight changes
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Results and discussion
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(Regioselective reaction on the big CD mouth was justified via the reaction of -CD with methyl iodide using similar procedure, the 1H NMR and 13C NMR were included in Fig. S4) followed by reacting with 1,2-bis(propargyloxy)ethane to afford the alkynyl-CD silica 6 (Fig. 1) via the first click step. The desired DNPCDCSP was finally constructed by covering the alkynyl-CD with N3-PhCD through the second click step.
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Characterization. The prepared DNPCDCSP was characterized by FTIR, solid state 13C NMR, TGA and elemental analysis.
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The FTIR spectra of N3CD-silica, alkynyl-CD-silica and DNPCDCSP were depicted in Fig. S5 (SI). Successful immobilization of N3-CD onto silica surfaces was evidenced by the absorptions at 2110 cm-1 (–N3 moiety) and 2961 cm-1 (methylene moieties of CD). After the first click reaction step, the absorption at 2110 cm-1 was greatly depressed (nearly invisible), indicating that almost all the -N3 moieties have been exhausted by reacting with 1,2-bis(propargyloxy)ethane. For DNPCDCSP, there appeared typical adsorptions for the CD phenylcarbamoyl groups (1736 cm-1 corresponds to carbonyl groups; 1548 cm-1 and 1446 cm-1 are assigned to the phenyl rings).
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Solid state 13C NMR (Fig. 2) spectra provide more structure information of the obtained materials. Signals between 110 and 60 ppm of N3CD-silica confirms the successful immobilization of the down layer CD skeleton onto the silica surfaces. For DNPCDCSP, the signal around 55 ppm is assigned to the carbon atoms between the two triazole rings. The signals between 180 ppm and 115 ppm are assigned to the carbon atoms of phenylcarbamoyl groups and triazole rings. TGA weight losses (Fig. S5) of N3CD-silica and DNPCDCSP are 15.25% and 20.11% (w/w) from room temperature to 800 oC under air atmosphere at a heating rate of 10 oC·min-1. Elemental analysis (Table S1) shows that the carbon content increased from 8.53% to 11.22% after two-step click reaction.
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The FTIR, 13C NMR, and elemental analysis results affirm that the hybrid bilayer CD structure has been successfully fabricated on the silica surfaces.
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Insert Fig.2
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3.2 HPLC performance of DNPCDCSP
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Compared to the commercial column Cyclobond I 2000 (ASTEC) (4100 m-1; conditions: toluene as probe, flow rate = 0.6 mL/min, VMeOH:VH O = 50:50), the current column afforded relatively lower efficiency (2800 m-1). The reason might be attributed to the negative steric effect caused by squeezing such big CD-dimer molecules into 100 Å pores and the broken silica particles during the reaction as well as the low packing pressure may also result in the reduced efficiency. The enantioseparation abilities of DNPCDCSP were investigated by separation of racemic isoxazolines, flavonoids, Dns amino acids, bendroflumethiazide, atropine, indoprofen, diperodon, fenoterol, 4-chromanol, benzoin and styrene oxide in RP-HPLC using ACN and MeOH as organic modifiers. Preliminary optimized separation results and 2
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conditions are summarized in Table 1 and some representative chromatograms are shown in Fig. 3.
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3.2.1 Enantioseparation of isoxazoline derivatives
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Isoxazolines are important agrochemical agents, pharmaceuticals, and potential intermediates for the synthesis of natural compounds. Most of the investigated isoxazoline derivatives were neutral and the separations were carried out with water and organic modifiers (MeOH or ACN) as eluting phases [37]. As it is shown in Table 1, DNPCDCSP exhibits encouraging resolution ability towards most of the isoxazolines. The better resolution of Ar-OPr category (analytes bearing phenyl and oxopyrrolidin moieties) over 4NPh-Ph and 4NPh-Py might benefit from the carbonyl group of oxopyrrolidin, which is able to form H-bonding and dipole-dipole interactions with CD phenylcarbamoyl groups. Compared to 4NPh-Ph, the nitrogen atoms in pyridine ring of 4NPh-Py can form H-bonding with imino group, resulting in its higher enantioselectivity than 4NPh-Ph under the same condition.
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For Ar-OPr category, the phenyl rings on 3-position can be included into the down layer CD cavity and the oxopyrrolidone moiety can form hydrogen bonding and dipole-dipole interactions with phenylcarbamoyl groups to provide a well-established three-point model leading to better separation. Compared to Ph-OPr, better differentiation of MDOPh-OPr may profit from the ‘tight fit’ between the rigid MDOPh moiety and CD cavity. The relatively smaller retention and resolution of 4MOPh-OPr and 4MetPh-OPr are due to the steric hindrance of methyl group in forming the inclusion complex. The resolutions of chloride substituted Ph-OPr follow an order of Rs(3ClPh-OPr) > Rs(4ClPh-OPr) > Rs(2ClPh-OPr). This indicates that the substitution position may lead to different inclusion mechanism with the down-layer CD which further influences the dipole-dipole and H-bonding effects with the top-layer CD. It is encouraging that the resolution value of 4NPh-OPr reached as high as 13.97, presumably because the p–NO2 moiety and phenyl ring can form a large conjugated system which enables a more stable inclusion with CD cavity or strong - interactions with the phenylcarbamoyl. Poor resolution of 3FPh-OPr might be due to the weak host-guest complexation caused by the steric hindrance and strong electron-withdrawing ability of p-CF3 group.
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3.2.2 Enantioseparation of flavonoids and Dns amino acids
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Five pairs of flavonone enantiomers including those bearing –OCH3, and –OH groups on the bicyclic rings or –OH group on the single phenyl ring were partially separated with MeOH and water as the eluting phase. The unsubstituted flavonone registered better resolution than other analogs. Monohydroxyl-substitution contributed to smaller retention factors owing to the high polarity of the hydroxyl groups which is unfavorable for hydrophobic interaction. This suggests that the inclusion takes place between the benzopyran moiety and CD cavity. However, long retention does not always correlate with better resolution as observed for monohydroxyl flavonones.
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Nine Dns amino acids were partially resolved on DNPCDCSP with Dns-Aca exhibiting the longest retention but poorest resolution. This further indicates that strong retention does not necessarily result in good enantioseparation on CD CSPs. DNPCDCSP affords poorer separation towards Dns amino acids compared to our previously reported DCDCSP [38]. This might be due to the strong steric hindrance induced by the bulky phenylcarbamoyl moieties on the CD rims, which hinders the entrance of naphthalene into CD cavity to form the inclusion complex. The relatively short retention of Dns-Ser and Dns-Thr can be attributed to the high polarity of the –OH side chain.
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3.2.3 Enantioseparation of other aromatic enantiomer pairs
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The versatility and resolving ability of DNPCDCSP were further evaluated by enantioseparation of more analytes such as bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine, styrene oxide, 4-chromanol and benzoin. The enantioselectivity of the first four compounds were above 2.0 with that of indoprofen reaching 2.67. For these analytes, the two phenyl rings connected with long chains and other functional groups on their skeleton results in good inclusion and chiral differentiation. One phenyl rings can be included into the down layer CD cavity and the other phenyl ring as well as the functional substituents can form hydrogen bonding, π-π interaction or dipole-dipole interaction with phenylcarbamoyl groups on top layer CD to provide a well-established three-point model. It is worth mentioning that the selectivity of bendroflumethiazide obtained on DNPCDCSP was the highest among all the reported triazole bridged CD CSPs [34-36].
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3.3 Comparative studies of DNPCDCSP and DCDCSP
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DNPCDCSP described herein exhibits quite different chiral recognition properties compared to our previously reported unmodified bilayer DCDCSP [38]. DCDCSP was proven to be better at resolving Dns amino acids and aryl carboxylic acids which were only partially resolved using current CSP. However, DNPCDCSP afforded much better separation towards isoxazoline derivatives as shown in Table S2. DCDCSP can only partially resolve seven isoxazoline isomers while their selectivity and resolution were significantly enhanced on DNPCDCSP, especially for 4NPh-OPr and 3ClPh-OPr (Fig. 4). The enhanced enantioselectivity can be attributed to the introduction of phenylcarbamoylated CD which can provide strong -, dipole-dipole interactions as well as steric effects. Analytes such as bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine and styrene oxide, which were not able to be well-resolved on DCDCSP, can also be baseline or partially resolved using DNPCDCSP under the studied separation conditions. The above results indicate that the hybrid native-phenylcarbamoylated bilayer CD structure affords accentuated enantiorecognition ability toward a wider range of enantiomers than native CD bilayer structure due to the multiple types of interactions with analytes.
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3.4 Conclusions
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An effective surface-up two-step click approach was established for the fabrication of a novel hybrid bilayer CD CSP (DNPCDCSP) by anchoring perphenylcarbamoylated-β-CD onto N3-CD functionalized silica. The new CSP provides accentuated interactions with some guest molecules via both the interaction sites such as hydrogen bonding (C=O, -NH-), - dipole-dipole, steric effects from phenylcarbamolylated-β-CDs and the interaction sites like hydrogen bonding (-OH), inclusion complexation from native CDs. The enantionseparation abilities of bilayer CD CSPs could be tuned by endowing functionality to one of the CD layers. The proposed two-step click approach provides an encouraging way for the construction of multifunctional materials from molecules with bulky substituents. However, it is necessary to mention that the current CSP exhibits some unexpected features like the reduction of enantioselectivity toward some analytes compared to the individual native CD CSP. The detailed mechanism will be further investigated in our following study.
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A further study was conducted by investigating the separation difference between the single layer CDCSPs [N3CD-silica and PCDCSP (Fig.S7)] and DNPCDCSP (Table S3). It is found that DNPCDCSP exhibits the broadest separation profile and enhanced enantioselectivity toward some analytes due to the introduced interactions such as H-bonding (C=O, -NH-), steric effects, - and dipole-dipole interactions from phenylcarbamoylated-CD and triazolyl moieties. However, the selectivities of some analytes such as Dns amino acids and part of isoxazolines are significantly reduced compared to the single layer CDCSP, indicating there exist quasi pseudo-enantiomerical behaviors according to Levkin’s study stated in the Introduction section. This phenomena must be taken into consideration for the design of new CD CSPs.
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Acknowledgements
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We acknowledge the financial support from National Natural Science Foundation of China (No. 21205086), Tianjin Research Program of Application Foundation and Advanced Technology (13JCQNJC05400).
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Captions:
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Fig. 1 Synthetic pathway of DNPCDCSP. Fig. 2 Solid state 13C NMR of N3CD-silica and DNPCDCSP. Fig. 3 Representative chromatograms on DNPCDCSP. Separation conditions see Table 1. Fig. 4 Enantioseparation of 4NPh-OPr and 3ClPh-OPr on the two CD CSPs. Separation condition for DNPCDCSP：CH3OH/H2O = 60/40 (v/v), flow rate 0.5 mL·min-1; separation condition for DCDCSP：CH3OH/H2O = 40/60 (v/v), flow rate 0.6 mL·min-1.
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[21] D. Wolrab, P. Fruhauf, M. Kohout, W. Lindner, Click chemistry immobilization strategies in the development of strong cation exchanger chiral stationary phases for HPLC, J. Sep. Sci. 36 (2013)
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[22] A. Bielejewska, K. Duszczyk, A. Kwaterczak, D. Sybilska, Comparative study on the enantiomer separation of 1,1′-binaphthyl-2,2′diyl hydrogenphosphate and 1,1′-bi-2-naphthol by liquid
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chromatography and capillary electrophoresis using single and combined chiral selector systems, J. Chromatogr. A 977 (2002) 225-237.
[23] B.C. Valle, F.H. Billiot, S.A. Shamsi, X. Zhu, A.M. Powe, I.M. Warner, Combination of cyclodextrins
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and polymeric surfactants for chiral separations, Electrophoresis 25 (2004) 743-752. [24] P. Levkin, N.M. Maier, W. Lindner, V. Schurig, A practical method for the quantitative assessment of non-enantioselective versus enantioselective interactions encountered in liquid chromatography on brush-type chiral stationary phase, J. Chromatogr. A 1269 (2012) 270-278.
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[25] P.A. Levkin, N.M. Maier, V. Schurig, W. Lindner, Strong Detrimental Effect of a Minute Enantiomeric Impurity of a Chiral Selector on the Enantioselectivity Factor, Angew. Chem. Int. Ed. 49 (2010) 7742-7744.
[26] P.A. Levkin, V. Schurig, Apparent and true enantioselectivity of single- and binary-selector chiral stationary phases in gas chromatography, J. Chromatogr. A 1184 (2008) 309-322. [27] L. Asnin, F. Gritti, K. Kaczmarski, G. Guiochon, Features of the adsorption of Naproxen on the chiral stationary phase (S,S)-Whelk-O1 under reversed-phase conditions, J. Chromatogr. A 1217 (2010) 264-275. [28] J.M. Bobbitt, L. Li, D.D. Carlton Jr, M. Yasin, S. Bhawal, F.W. Foss Jr, S. Wernisch, R. Pell, W. Lindner, K.A. Schug, Diastereoselective discrimination of lysine–alanine–alanine peptides by zwitterionic cinchona alkaloid-based chiral selectors using electrospray ionization mass spectrometry, J. Chromatogr. A 1269 (2012) 308-315. [29] T. Zhang, E. Holder, P. Franco, W. Lindner, Method development and optimization on cinchona and chiral sulfonic acid–based zwitterionic stationary phases for enantiomer separations of free amino 12
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[30] C.F. Zhao, S. Diemert, N.M. Cann, Rational optimization of the Whelk-O1 chiral stationary phase using molecular dynamics simulations, J. Chromatogr. A 1216 (2009) 5968-5978. [31] R. Sardella, A. Carotti, A. Gioiello, A. Lisanti, F. Ianni, W. Lindner, B. Natalini, Chromatographic separation of free dafachronic acid epimers with a novel triazole click quinidine-based chiral stationary phase, J. Chromatogr. A 1339 (2014) 96-102. [32] L. Pang, J. Zhou, J. Tang, S.-C. Ng, W. Tang, Evaluation of perphenylcarbamated cyclodextrin clicked chiral stationary phase for enantioseparations in reversed phase high performance liquid
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chromatography, J. Chromatogr. A 1363 (2014) 119-127.
[33] Y. Gong, Y. Wang, W.-T. Zhao, X.-Y. Tang, Facile synthesis of 3-aryl-5-(2-oxopyrrolidin-1-yl)- and
5-(pyridin-4-yl)-4,5-dihydroisoxazoles via 1,3-dipolar cycloaddition under mild conditions, J. Chem. Res.
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[34] Y. Wang, T.-T. Ong, L.-S. Li, T.T.Y. Tan, S.-C. Ng, Enantioseparation of a novel “click” chemistry
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derived native β-cyclodextrin chiral stationary phase for high-performance liquid chromatography, J. Chromatogr. A 1216 (2009) 2388-2393.
[35] Y. Wang, D.J. Young, T.T.Y. Tan, S.-C. Ng, “Click” preparation of hindered cyclodextrin chiral Chromatogr. A 1217 (2010) 7878-7883.
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stationary phases and their efficient resolution in high performance liquid chromatography, J. [36] Y. Wang, D.J. Young, T.T.Y. Tan, S.-C. Ng, “Click” immobilized perphenylcarbamated and permethylated cyclodextrin stationary phases for chiral high-performance liquid chromatography
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application, J. Chromatogr. A 1217 (2010) 5103-5108.
[37] X. Yao, Y. Gong, R. Mamuti, W. Xing, H. Zheng, X. Tang, Y. Wang, Chiral differentiation of novel RSC Adv. 4 (2014) 30492-30499.
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isoxazoline derivatives on "clicked" thioether and triazole bridged cyclodextrin chiral stationary phases, [38] J. Zhao, X. Lu, Y. Wang, T.T.Y. Tan, Surface-up constructed tandem-inverted bilayer cyclodextrins for enhanced enantioseparation and adsorption, J. Chromatogr. A 1343 (2014) 101-108.
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acids by high-performance liquid chromatography, J. Chromatogr. A 1363 (2014) 191-199.
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465 466 467 468
The first native-phenylcarbamoylated bilayer CD CSP
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The CSP provides interactions like H-bonding, -, dipole-dipole, steric and
472 473 474
Phenylcarbamoylated CD serves as a moiety to enhance the separation ability of
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inclusion effects
native CD
An encouraging way for construction of functional materials from molecules with
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bulky moieties
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Figure1
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Figure2
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Figure3
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Figure4
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Table 1. Optimal enantioseparation results on DNPCDCSP. k1
k2
α
Rs
Ph-OPrI
1.19
1.24
1.89
3.86
4MOPh-OPrI
1.49
2.10
1.41
2.22
MDOPh-OPrI
2.23
5.02
2.25
6.84
4MetPh-OPrI
1.49
1.89
1.27
4ClPh-OPrI
2.48
3.74
1.51
3ClPh-OPrI
1.68
5.01
2.98
2ClPh-OPrII
5.15
5.61
1.09
3FPh-OPrII
5.77
6.41
4NPh-OPrI
2.52
13.21
3NPh-OPrI
1.72
3.23
4NPh-PhIII
3.96
4.41
4NPh-PyIII
3.66
5.00
Compounds
1.47
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3.78 7.77 0.83
1.11
1.04
5.25
13.97
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Flavonoids
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Isoxazolines
1.88
4.39
1.11
0.49
1.37
1.61
1.13
1.44
FlavanoneII
15.23
6-Methoxyflavanone
27.08II/6.06IV 30.72II/6.86IV 1.13II/1.13IV 1.35II/1.12IV
7-Methoxyflavanone
25.25II/5.56IV 27.84II/6.11IV 1.10II/1.10IV 0.90IV
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17.25
11.83
12.90
1.09
0.94
4’-HydroxyflavanoneII
11.13
12.34
1.11
1.09
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6-HydroxyflavanoneII
Dns amino acids Dns-NvaV
4.50
4.78
1.06
0.49
Dns-ValV
4.44
4.91
1.10
0.99
Dns-LeuV
5.05
5.41
1.07
0.63
Dns-AbaV
3.88
4.15
1.07
0.55
Dns-AcaV
8.47
8.82
1.04
0.25
Dns-PheV
6.73
7.13
1.06
0.57
Dns-SerV
2.83
3.02
1.07
0.40
Dns-NieV
5.09
5.34
1.05
0.28
Dns-ThrV
3.06
3.41
1.11
0.93
Other analytes
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8.25
2.22
5.60
AtropineVII
1.98
2.92
1.48
1.72
IndoprofenVIII
0.38
0.92
2.67
0.81
DiperodonIX
0.82
1.89
2.30
3.78
FenoterolIX
0.77
1.54
2.01
1.68
Styrene oxideIX
0.65
1.48
2.28
2.45
4-chromanolX
6.83
7.48
1.10
BenoinXI
6.78
7.64
1.10
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BendroflumethiazideVI 3.72
0.79
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0.98
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Conditions: I CH3OH/H2O = 60/40 (v/v), flow rate 0.5 mL·min-1; II CH3OH/H2O = 40/60 (v/v), flow rate 0.5 mL·min-1; III CH3OH/H2O = 70/30 (v/v), flow rate 0.6 mL·min-1; IV CH3OH/H2O = 55/45 (v/v), flow rate 0.6 mL·min-1; V ACN/1%TEAA pH 5.01 = 40/60 (v/v), flow rate 0.6 mL·min-1; VI CH3OH/H2O = 40/60 (v/v), flow rate 0.6 mL·min-1; VII CH3OH/1%TEAA pH 6.25 = 30/70 (v/v), flow rate 0.6 mL·min-1; VIII CH3OH/1%TEAA pH 5.01 = 60/40 (v/v), flow rate 0.6 mL·min-1; IX CH3OH/1%TEAA pH 5.01 = 40/60 (v/v), flow rate 0.6 mL·min-1; X CH3OH/H2O = 5/95 (v/v), flow rate 0.6 mL·min-1; XI CH3OH/H2O = 30/70 (v/v), flow rate 0.6 mL·min-1.
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S0021-9673(15)00054-0 http://dx.doi.org/doi:10.1016/j.chroma.2015.01.008 CHROMA 356169
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
19-8-2014 12-12-2014 7-1-2015
Please cite this article as: J. Zhao, X. Lu, Y. Wang, J. Lv, ‘Click’ preparation of a novel ‘native-phenylcarbamoylated’ bilayer cyclodextrin stationary phase for enhanced chiral differentiation, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.01.008 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.
1
‘Click’ preparation of a novel ‘native-phenylcarbamoylated’ bilayer
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cyclodextrin stationary phase for enhanced chiral differentiation
3
Jie Zhaoa, Xiaohong Lua, Yong Wang*ab, Jie Lva
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a
5
300072
6
b
7
(Tianjin), Tianjin 300072, China
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Corresponding author: Yong Wang
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Email: [email protected]
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Department of Chemistry, School of Science, Tianjin University, Tianjin
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Collaborative Innovation Center of Chemical Science and Engineering
Tel：+86 13502146137
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Building 7, Room 110, Tianjin University, No. 92 Weijin Road, Tianjin,
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China, 300072.
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Keywords: bilayer cyclodextrin, click, enantiorecognition, RP-HPLC.
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1
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It is known that there is an increasing demand for enantiopure chiral drugs due to their different biological, pharmacological and toxicological effects from their enantiomers [1-5]. Chiral high performance liquid chromatography (CHPLC) has been proven to be a useful methodology not only for determining optical purity but also for obtaining pure enantiomers at preparative scale. The key point of CHPLC method is the development of effective chiral stationary phases (CSPs) [6-12].
46 47 48 49 50 51 52 53 54 55 56
By far, cyclodextrin derivatives (CDs) have been used extensively in CSPs ascribing to their ability to accommodate guest molecules through the formation of inclusion complexes [13, 14]. Recent investigations on CD CSPs mainly focus on either new immobilization chemistry to afford stable and functional bridges, or CD rims functionalization to endow CD specific interactions [15-21]. Although there have emerged a series of CD CSPs developed via the above two ways, there still exist many challenges in this area. On one hand, development of stable and functional linkages is relatively difficult. On the other hand, CD rims modification usually enhances its enantioselectivity toward specific categories of enantiomers while diminish its original recognition ability to some enantiomers. Hence, it is a great challenge to explore novel approaches for the fabrication of more versatile CD CSPs.
57 58 59 60 61 62 63 64 65
Binary chiral selector systems may provide an alternative to improve the separation and some successful applications have been performed using chiral mobile phase additives (CMP) in chiral capillary electrophoresis (CE) [22, 23]. In chromatography, binary CSP can be realized by incorporating two chiral selectors on same silica support or by direct mixing of two different CSPs. According to Levkin, Schurig and Lindner’s extensive studies, incorporating two selectors in a CSP or two CSPs in one column may extend the CSPs’ enantioselectivity profile, however it could hardly improve the enantioselectiviy compared to the CSPs with individual selector due to quasi pseudo-enantiomerical behaviors and negative effects from
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Abstract: This paper reports an effective approach for the fabrication of a novel hybrid bilayer cyclodextrin (CD) chiral stationary phase (CSP), where native and perphenylcarbamoylated-β-CD were successively immobilized onto silica surface via a two-step click approach to form a bilayer CD structure. By decorating the bulky phenylcarbamoylated CD onto the unmodified CD silica, the CSP can provide multiple interaction sites such as H-bonding (-OH, C=O, -NH-), steric effects, -, dipole-dipole and inclusion complexation interactions, which help to broaden the CSP’s enantioselectivity profile and enhance the enantioselectivity to some specific analytes. A group of enantiomer pairs such as isoxazolines, bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine, styrene oxide and dansyl amino acids can be baseline or partially separated on the current CSP under reversed phase high performance liquid chromatography (RP-HPLC). The selectivity and resolution of 4NPh-OPr reached 5.25 and 13.97, which is an exciting achievement for the enantioseparations by CD CSPs.
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In light of the above wisdom, the current work aims to develop novel CD CSPs by decorating native CD with functionalized CD moieties to introduce multiple interaction cites. A recent study by Tang et al reported efficient enantioseparation of twenty-six racemates on triazolyl linked phenylcarbamoylated CD CSP [32]. Although the bulky phenylcarbamoyl substitutions provide several interaction sites, the lack of original H-bonding sites (-OH) and inclusion complexation from native CD may diminish its selectivity to some analytes. Herein, via a smartly designed two-step click procedure, we construct a novel ‘native-perphenylcarbamoylated CD CSP material (denoted as DNPCDCSP) by introduction of native CD (down layer) and perphenylcarbamoylated CD (top layer) onto the silica surfaces.
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2.1 Chemicals and materials
82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
Sodium hydride (60%), ethyl acetate, petroleum ether, tetrahydrofuran and toluene were purchased from Jiangtian Chemical Reagents (Tianjin, China). Sodium azide and pyridine were purchased from Tianjin Chemical Regents (Tianjin, China). (3-propylchloride)triethoxysilane, γ-(2,3-epoxypropoxy)propytrimethoxysilane (KH-560), propargyl bromide, ethylene glycol, (3-glycidoxypropyl)trimethoxysilane and anhydrous N,N-Dimethylformamide (DMF) were purchased from HEOWNS (Tianjin, China). Phenylisocyanate was provided by Ouhe Technology (Beijing, China). HPLC-grade methanol (MeOH), acetonitrile (ACN), triethylamine (TEA) and acetic acid were purchased from Concord Technology (Tianjin, China). Ultra-pure water was prepared by Milli-Q water purification system (Billerca, MA, USA). Kromasil spherical silica gel (5 μm, 100 Å, surface area 300 m2·g-1) were purchased from Eka Chemicals (Bohus, Sweden). Isoxazoline racemates were provided by Tang’s group [33]. Acidic racemates were purchased from Sigma-Aldrich (Shanghai, China) and the other racemates were purchased from Energy-Chemical (Shanghai, China). The analyte structures are presented in Fig. S1 in the supporting information (SI).
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2.2 Instruments and measurements
99 100 101 102 103 104 105 106
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none-enantioselective interactions [24-26]. A good approach for enhancing the separation ability of CSPs is to incorporate more types of interaction sites and chiral centers on one selector skeleton such as brush-type Whelk-O1 CSPs (-donor and -acceptor), quinine CSPs and cinchona zwitterionic CSPs [3, 27-31].
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Fourier-transform infrared (FTIR) spectra were collected on an AVATR360 supplied by Thermo Nicolet (USA). 1H NMR were recorded on a Bruker ACF400 (400MHz) supplied by Bruker Biospin (Fällanden, Switzerland). Solid state 13C NMR was performed on a Varian infinityplus 300 NMR spectrometer (300MHz, 7.0T). Mass spectra data were collected on LCQ Deca XP MAX system (Thermo Fisher, USA). Thermal gravity analyses were conducted on a NETZSCH STA409PC TGA (Bavaria, Germany) in air atmosphere at heating rate of 10 oC·min-1. Elemental analyses were performed on a Var-ioMICRO CHNOS elemental analyzer (Elementar 3
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Analysensysteme, Hanau, Germany). Chromatographic analyses were performed on a Laballiance HPLC system with a diode array detection (DAD) system (State college, PA, USA). Samples were dissolved in MeOH/H2O (v/v=1:1) at a concentration of 1 mg·mL-1 and the injection volume was set as 10 μL. Triethylammonium acetate buffer (denoted as TEAA) was prepared by dissolving 1% (v/v) TEA in ultra-pure water and thereafter adjusted to the desired pH with acetate acid. Each solution was injected in triplicate and the average values were used. All the buffers and samples were filtered through 0.22 μm membranes before usage. The detection was performed at 220-300 nm. Calculations (capacity factor, k; selectivity, α and resolution, Rs) follow USP standards.
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2.3 Preparation of DNPCDCSP
118 119 120 121 122 123 124
The synthetic route of DNPCDCSP is depicted in Fig. 1. Mono-6-deoxy-(p-tosylsulfonyl)-β-cyclodextrin (TsO-CD) 1 and Mono-6-deoxy-azido-β-cyclodextrin (N3-CD) 2 and Mono-6-deoxy-azido-perphenylcarbamoylated-β-cyclodextrin (N3-PhCD) 3 were synthesized and purified according to the reported approaches [34-36]. All the silica materials were characterized after vacuum drying (90 oC, 20 mmHg, 12 h). The detailed step by step synthetic procedures have been included in SI (Section 1 in SI).
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Insert Fig.1
126 127 128
The prepared CSP were packed into stainless-steel column (150 mm × 4.6 mm I.D.) (Cherishtech, Beijing, China) using the typical slurry-packing technique with MeOH as the packing solvent (5000 psi, 30 min).
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3
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3.1 Synthesis and Characterization of the DNPCDCSP
131 132 133 134 135 136 137 138 139 140
Synthesis. Initially, we attempted to construct the hybrid CD structure by immobilization of N3-PhCD onto alkynyl-silica 6' (Fig. S2a in SI). However, the FITR spectrum (Fig. S3 in SI) of the resulting material did not show an adsorption around 1730 cm-1 (typical adsorption of carbonyl group), indicating that treatment of alkynyl-silica with N3-PhCD cannot lead to the formation of triazolyl. The reason might be due to the sterically restricted CD mouth of N3-PhCD, where the -N3 moiety was covered by phenylcarbamoyl groups, resulting in the hindrance of the short alkyne chain of alkynyl-CD to react with the -N3 moiety. Hence, it is necessary to make the spacer between the two CD layers longer to avoid the interference of steric hindrance.
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Accordingly, 1,2-bis(propargyloxy)ethane was synthesized, acting as an linker to connect N3CD-silica and N3-PhCD, where the alkynyl moieties of 1,2-bis(propargyloxy)ethane can readily insert into the bulky mouth of the N3-PhCD to react with the azido moiety. A two-step click strategy can be thereafter performed for the construction of desired DNPCDCSP (Fig. S2b). Initially, N3CD-silica was fabricated according to our previously reported method with slight changes
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(Regioselective reaction on the big CD mouth was justified via the reaction of -CD with methyl iodide using similar procedure, the 1H NMR and 13C NMR were included in Fig. S4) followed by reacting with 1,2-bis(propargyloxy)ethane to afford the alkynyl-CD silica 6 (Fig. 1) via the first click step. The desired DNPCDCSP was finally constructed by covering the alkynyl-CD with N3-PhCD through the second click step.
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Characterization. The prepared DNPCDCSP was characterized by FTIR, solid state 13C NMR, TGA and elemental analysis.
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The FTIR spectra of N3CD-silica, alkynyl-CD-silica and DNPCDCSP were depicted in Fig. S5 (SI). Successful immobilization of N3-CD onto silica surfaces was evidenced by the absorptions at 2110 cm-1 (–N3 moiety) and 2961 cm-1 (methylene moieties of CD). After the first click reaction step, the absorption at 2110 cm-1 was greatly depressed (nearly invisible), indicating that almost all the -N3 moieties have been exhausted by reacting with 1,2-bis(propargyloxy)ethane. For DNPCDCSP, there appeared typical adsorptions for the CD phenylcarbamoyl groups (1736 cm-1 corresponds to carbonyl groups; 1548 cm-1 and 1446 cm-1 are assigned to the phenyl rings).
164 165 166 167 168 169 170 171 172 173
Solid state 13C NMR (Fig. 2) spectra provide more structure information of the obtained materials. Signals between 110 and 60 ppm of N3CD-silica confirms the successful immobilization of the down layer CD skeleton onto the silica surfaces. For DNPCDCSP, the signal around 55 ppm is assigned to the carbon atoms between the two triazole rings. The signals between 180 ppm and 115 ppm are assigned to the carbon atoms of phenylcarbamoyl groups and triazole rings. TGA weight losses (Fig. S5) of N3CD-silica and DNPCDCSP are 15.25% and 20.11% (w/w) from room temperature to 800 oC under air atmosphere at a heating rate of 10 oC·min-1. Elemental analysis (Table S1) shows that the carbon content increased from 8.53% to 11.22% after two-step click reaction.
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The FTIR, 13C NMR, and elemental analysis results affirm that the hybrid bilayer CD structure has been successfully fabricated on the silica surfaces.
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Insert Fig.2
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3.2 HPLC performance of DNPCDCSP
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Compared to the commercial column Cyclobond I 2000 (ASTEC) (4100 m-1; conditions: toluene as probe, flow rate = 0.6 mL/min, VMeOH:VH O = 50:50), the current column afforded relatively lower efficiency (2800 m-1). The reason might be attributed to the negative steric effect caused by squeezing such big CD-dimer molecules into 100 Å pores and the broken silica particles during the reaction as well as the low packing pressure may also result in the reduced efficiency. The enantioseparation abilities of DNPCDCSP were investigated by separation of racemic isoxazolines, flavonoids, Dns amino acids, bendroflumethiazide, atropine, indoprofen, diperodon, fenoterol, 4-chromanol, benzoin and styrene oxide in RP-HPLC using ACN and MeOH as organic modifiers. Preliminary optimized separation results and 2
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conditions are summarized in Table 1 and some representative chromatograms are shown in Fig. 3.
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Insert Table 1
191
3.2.1 Enantioseparation of isoxazoline derivatives
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Isoxazolines are important agrochemical agents, pharmaceuticals, and potential intermediates for the synthesis of natural compounds. Most of the investigated isoxazoline derivatives were neutral and the separations were carried out with water and organic modifiers (MeOH or ACN) as eluting phases [37]. As it is shown in Table 1, DNPCDCSP exhibits encouraging resolution ability towards most of the isoxazolines. The better resolution of Ar-OPr category (analytes bearing phenyl and oxopyrrolidin moieties) over 4NPh-Ph and 4NPh-Py might benefit from the carbonyl group of oxopyrrolidin, which is able to form H-bonding and dipole-dipole interactions with CD phenylcarbamoyl groups. Compared to 4NPh-Ph, the nitrogen atoms in pyridine ring of 4NPh-Py can form H-bonding with imino group, resulting in its higher enantioselectivity than 4NPh-Ph under the same condition.
203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219
For Ar-OPr category, the phenyl rings on 3-position can be included into the down layer CD cavity and the oxopyrrolidone moiety can form hydrogen bonding and dipole-dipole interactions with phenylcarbamoyl groups to provide a well-established three-point model leading to better separation. Compared to Ph-OPr, better differentiation of MDOPh-OPr may profit from the ‘tight fit’ between the rigid MDOPh moiety and CD cavity. The relatively smaller retention and resolution of 4MOPh-OPr and 4MetPh-OPr are due to the steric hindrance of methyl group in forming the inclusion complex. The resolutions of chloride substituted Ph-OPr follow an order of Rs(3ClPh-OPr) > Rs(4ClPh-OPr) > Rs(2ClPh-OPr). This indicates that the substitution position may lead to different inclusion mechanism with the down-layer CD which further influences the dipole-dipole and H-bonding effects with the top-layer CD. It is encouraging that the resolution value of 4NPh-OPr reached as high as 13.97, presumably because the p–NO2 moiety and phenyl ring can form a large conjugated system which enables a more stable inclusion with CD cavity or strong - interactions with the phenylcarbamoyl. Poor resolution of 3FPh-OPr might be due to the weak host-guest complexation caused by the steric hindrance and strong electron-withdrawing ability of p-CF3 group.
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3.2.2 Enantioseparation of flavonoids and Dns amino acids
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Five pairs of flavonone enantiomers including those bearing –OCH3, and –OH groups on the bicyclic rings or –OH group on the single phenyl ring were partially separated with MeOH and water as the eluting phase. The unsubstituted flavonone registered better resolution than other analogs. Monohydroxyl-substitution contributed to smaller retention factors owing to the high polarity of the hydroxyl groups which is unfavorable for hydrophobic interaction. This suggests that the inclusion takes place between the benzopyran moiety and CD cavity. However, long retention does not always correlate with better resolution as observed for monohydroxyl flavonones.
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Nine Dns amino acids were partially resolved on DNPCDCSP with Dns-Aca exhibiting the longest retention but poorest resolution. This further indicates that strong retention does not necessarily result in good enantioseparation on CD CSPs. DNPCDCSP affords poorer separation towards Dns amino acids compared to our previously reported DCDCSP [38]. This might be due to the strong steric hindrance induced by the bulky phenylcarbamoyl moieties on the CD rims, which hinders the entrance of naphthalene into CD cavity to form the inclusion complex. The relatively short retention of Dns-Ser and Dns-Thr can be attributed to the high polarity of the –OH side chain.
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3.2.3 Enantioseparation of other aromatic enantiomer pairs
239 240 241 242 243 244 245 246 247 248 249 250
The versatility and resolving ability of DNPCDCSP were further evaluated by enantioseparation of more analytes such as bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine, styrene oxide, 4-chromanol and benzoin. The enantioselectivity of the first four compounds were above 2.0 with that of indoprofen reaching 2.67. For these analytes, the two phenyl rings connected with long chains and other functional groups on their skeleton results in good inclusion and chiral differentiation. One phenyl rings can be included into the down layer CD cavity and the other phenyl ring as well as the functional substituents can form hydrogen bonding, π-π interaction or dipole-dipole interaction with phenylcarbamoyl groups on top layer CD to provide a well-established three-point model. It is worth mentioning that the selectivity of bendroflumethiazide obtained on DNPCDCSP was the highest among all the reported triazole bridged CD CSPs [34-36].
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Insert Fig.3
3.3 Comparative studies of DNPCDCSP and DCDCSP
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268
DNPCDCSP described herein exhibits quite different chiral recognition properties compared to our previously reported unmodified bilayer DCDCSP [38]. DCDCSP was proven to be better at resolving Dns amino acids and aryl carboxylic acids which were only partially resolved using current CSP. However, DNPCDCSP afforded much better separation towards isoxazoline derivatives as shown in Table S2. DCDCSP can only partially resolve seven isoxazoline isomers while their selectivity and resolution were significantly enhanced on DNPCDCSP, especially for 4NPh-OPr and 3ClPh-OPr (Fig. 4). The enhanced enantioselectivity can be attributed to the introduction of phenylcarbamoylated CD which can provide strong -, dipole-dipole interactions as well as steric effects. Analytes such as bendroflumethiazide, indoprofen, diperodon, fenoterol, atropine and styrene oxide, which were not able to be well-resolved on DCDCSP, can also be baseline or partially resolved using DNPCDCSP under the studied separation conditions. The above results indicate that the hybrid native-phenylcarbamoylated bilayer CD structure affords accentuated enantiorecognition ability toward a wider range of enantiomers than native CD bilayer structure due to the multiple types of interactions with analytes.
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Insert Fig. 4
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3.4 Conclusions
282 283 284 285
An effective surface-up two-step click approach was established for the fabrication of a novel hybrid bilayer CD CSP (DNPCDCSP) by anchoring perphenylcarbamoylated-β-CD onto N3-CD functionalized silica. The new CSP provides accentuated interactions with some guest molecules via both the interaction sites such as hydrogen bonding (C=O, -NH-), - dipole-dipole, steric effects from phenylcarbamolylated-β-CDs and the interaction sites like hydrogen bonding (-OH), inclusion complexation from native CDs. The enantionseparation abilities of bilayer CD CSPs could be tuned by endowing functionality to one of the CD layers. The proposed two-step click approach provides an encouraging way for the construction of multifunctional materials from molecules with bulky substituents. However, it is necessary to mention that the current CSP exhibits some unexpected features like the reduction of enantioselectivity toward some analytes compared to the individual native CD CSP. The detailed mechanism will be further investigated in our following study.
297 298 299 300 301
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A further study was conducted by investigating the separation difference between the single layer CDCSPs [N3CD-silica and PCDCSP (Fig.S7)] and DNPCDCSP (Table S3). It is found that DNPCDCSP exhibits the broadest separation profile and enhanced enantioselectivity toward some analytes due to the introduced interactions such as H-bonding (C=O, -NH-), steric effects, - and dipole-dipole interactions from phenylcarbamoylated-CD and triazolyl moieties. However, the selectivities of some analytes such as Dns amino acids and part of isoxazolines are significantly reduced compared to the single layer CDCSP, indicating there exist quasi pseudo-enantiomerical behaviors according to Levkin’s study stated in the Introduction section. This phenomena must be taken into consideration for the design of new CD CSPs.
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302 303 304 305 306 8
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Acknowledgements
308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337
We acknowledge the financial support from National Natural Science Foundation of China (No. 21205086), Tianjin Research Program of Application Foundation and Advanced Technology (13JCQNJC05400).
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Captions:
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Fig. 1 Synthetic pathway of DNPCDCSP. Fig. 2 Solid state 13C NMR of N3CD-silica and DNPCDCSP. Fig. 3 Representative chromatograms on DNPCDCSP. Separation conditions see Table 1. Fig. 4 Enantioseparation of 4NPh-OPr and 3ClPh-OPr on the two CD CSPs. Separation condition for DNPCDCSP：CH3OH/H2O = 60/40 (v/v), flow rate 0.5 mL·min-1; separation condition for DCDCSP：CH3OH/H2O = 40/60 (v/v), flow rate 0.6 mL·min-1.
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The first native-phenylcarbamoylated bilayer CD CSP
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The CSP provides interactions like H-bonding, -, dipole-dipole, steric and
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Phenylcarbamoylated CD serves as a moiety to enhance the separation ability of
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inclusion effects
native CD
An encouraging way for construction of functional materials from molecules with
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bulky moieties
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Figure1
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Figure2
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Table 1. Optimal enantioseparation results on DNPCDCSP. k1
k2
α
Rs
Ph-OPrI
1.19
1.24
1.89
3.86
4MOPh-OPrI
1.49
2.10
1.41
2.22
MDOPh-OPrI
2.23
5.02
2.25
6.84
4MetPh-OPrI
1.49
1.89
1.27
4ClPh-OPrI
2.48
3.74
1.51
3ClPh-OPrI
1.68
5.01
2.98
2ClPh-OPrII
5.15
5.61
1.09
3FPh-OPrII
5.77
6.41
4NPh-OPrI
2.52
13.21
3NPh-OPrI
1.72
3.23
4NPh-PhIII
3.96
4.41
4NPh-PyIII
3.66
5.00
Compounds
1.47
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3.78 7.77 0.83
1.11
1.04
5.25
13.97
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Flavonoids
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Isoxazolines
1.88
4.39
1.11
0.49
1.37
1.61
1.13
1.44
FlavanoneII
15.23
6-Methoxyflavanone
27.08II/6.06IV 30.72II/6.86IV 1.13II/1.13IV 1.35II/1.12IV
7-Methoxyflavanone
25.25II/5.56IV 27.84II/6.11IV 1.10II/1.10IV 0.90IV
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17.25
11.83
12.90
1.09
0.94
4’-HydroxyflavanoneII
11.13
12.34
1.11
1.09
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6-HydroxyflavanoneII
Dns amino acids Dns-NvaV
4.50
4.78
1.06
0.49
Dns-ValV
4.44
4.91
1.10
0.99
Dns-LeuV
5.05
5.41
1.07
0.63
Dns-AbaV
3.88
4.15
1.07
0.55
Dns-AcaV
8.47
8.82
1.04
0.25
Dns-PheV
6.73
7.13
1.06
0.57
Dns-SerV
2.83
3.02
1.07
0.40
Dns-NieV
5.09
5.34
1.05
0.28
Dns-ThrV
3.06
3.41
1.11
0.93
Other analytes
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8.25
2.22
5.60
AtropineVII
1.98
2.92
1.48
1.72
IndoprofenVIII
0.38
0.92
2.67
0.81
DiperodonIX
0.82
1.89
2.30
3.78
FenoterolIX
0.77
1.54
2.01
1.68
Styrene oxideIX
0.65
1.48
2.28
2.45
4-chromanolX
6.83
7.48
1.10
BenoinXI
6.78
7.64
1.10
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BendroflumethiazideVI 3.72
0.79
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0.98
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Conditions: I CH3OH/H2O = 60/40 (v/v), flow rate 0.5 mL·min-1; II CH3OH/H2O = 40/60 (v/v), flow rate 0.5 mL·min-1; III CH3OH/H2O = 70/30 (v/v), flow rate 0.6 mL·min-1; IV CH3OH/H2O = 55/45 (v/v), flow rate 0.6 mL·min-1; V ACN/1%TEAA pH 5.01 = 40/60 (v/v), flow rate 0.6 mL·min-1; VI CH3OH/H2O = 40/60 (v/v), flow rate 0.6 mL·min-1; VII CH3OH/1%TEAA pH 6.25 = 30/70 (v/v), flow rate 0.6 mL·min-1; VIII CH3OH/1%TEAA pH 5.01 = 60/40 (v/v), flow rate 0.6 mL·min-1; IX CH3OH/1%TEAA pH 5.01 = 40/60 (v/v), flow rate 0.6 mL·min-1; X CH3OH/H2O = 5/95 (v/v), flow rate 0.6 mL·min-1; XI CH3OH/H2O = 30/70 (v/v), flow rate 0.6 mL·min-1.
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