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Enantioseparation using ortho- or meta-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography
Journal of Chromatography A, 1286 (2013) 41–46
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Enantioseparation using ortho- or meta-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography Jun Shen a,∗ , Yongqiang Zhao a , Shinji Inagaki b , Chiyo Yamamoto b , Yue Shen a , Shuangyan Liu a , Yoshio Okamoto a,b a b
Polymer Materials Research Center, College of Material Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
a r t i c l e
i n f o
Article history: Received 26 November 2012 Received in revised form 5 February 2013 Accepted 6 February 2013 Available online 20 February 2013 Keywords: Enantioseparation Chiral recognition Phenylcarbamate Amylose Chiral stationary phase (CSPs) High-performance liquid chromatography (HPLC)
a b s t r a c t Six ortho- and six meta-substituted phenylcarbamate derivatives of amylose were prepared and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The substitution at the meta-position on the aromatic ring was more preferable than that at the ortho-position to obtain CSPs with a high chiral recognition ability, and the introduction of either an electron-withdrawing or electron-donating substituent can improve the chiral resolving power of the meta-substituted phenylcarbamates of amylose. The chiral recognition ability of the amylose phenylcarbamates and elution order of the enantiomers were significantly dependent on the position, nature and number of the substituents on the phenyl group. Correlations between the chiral recognition ability and the N–H frequencies in the IR spectra and the chemical shifts of the N–H protons in the 1 H NMR spectra of the carbamate moieties of the amylose derivatives were discussed. The structures of the amylose derivatives were also investigated by circular dichroism spectroscopy. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Direct enantioseparation by high-performance liquid chromatography (HPLC) has been one of the most efficient techniques for obtaining pure enantiomers of various chiral compounds including drugs, agrochemicals, food additives, etc. The key feature of this technique is the design and preparation of efficient chiral stationary phases (CSPs), and in recent decades, a significant variety of CSPs has been developed [1,2]. Among them, polysaccharide derivatives, especially phenylcarbamates and benzoates of cellulose and amylose, exhibit high chiral recognition abilities for a wide range of chiral compounds [3–10]. Intensive investigations of the chiral recognition mechanism of polysaccharide derivatives, especially those of the cellulose phenylcarbamates, revealed some correlations between the electronic and structural properties of the substituents on the phenyl group and their chiral recognition. It has been empirically established that their chiral recognition abilities significantly depend on the
∗ Corresponding author. Tel.: +81 52 753 7292; fax: +81 52 753 7292. E-mail addresses: [email protected] (J. Shen), [email protected] (Y. Okamoto). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.02.020
type and position of the substituents introduced on the aromatic moieties [11–20]. The introduction of either an electron-donating group, like a methyl, or an electron-withdrawing group, like a chloro, at the meta- or para-position on the phenyl group has a tendency to significantly improve the chiral recognition ability of the phenylcarbamate derivatives [11,15]. However, the orthosubstituted derivatives show a low chiral recognition. It has been proposed that the chiral recognition ability can be controlled by changing the polarity of the carbamate residues via the introduction of substituents on the phenyl group. Compared to the cellulose derivatives, the amylose derivatives with a mono-substituent have not been thoroughly studied although some para-substituted or disubstituted derivatives have been known to show a high chiral recognition [18,20–22]. Interestingly, contrary to the cellulose derivatives, amylose derivatives with two substituents including the ortho-position, such as 2methyl-5-chloro- or 2-methyl-5-fluoro-phenylcarbamates, exhibit a very high enantioseparation power for many racemates [20,21]. The difference in the helical conformation of cellulose (left-handed 3/2 helix) and amylose (left-handed 4/3 helix) is considered to be the possible reason for their difference in the substituent effect on the chiral recognition [23–25]. Among the amylose phenylcarbamate derivatives, the ortho- or meta-monosubstituted
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Fig. 1. Structures of amylose derivatives.
phenylcarbamates have not yet been investigated in detail and their chiral recognition mechanism still remains obscure. In order to get a better understanding of the effect of substituents at the ortho- or meta-position on the chiral recognition of the phenylcarbamate derivatives of amylose and to elucidate the relationship between the electronic and structural characteristics of substituents and the chiral recognition ability, in this study, six ortho- (1a–f) and six meta-substituted (2a–f) phenylcarbamate derivatives of amylose were prepared (Fig. 1) and their chiral recognition abilities were evaluated as CSPs by HPLC. The influence of the nature and position of the substituents on the chiral recognition and elution order of enantiomers was carefully studied. Correlations between the chiral recognition ability and the N–H frequencies in IR spectra and the chemical shifts of the N–H protons in the 1 H NMR spectra of the carbamate moieties of these derivatives were systematically examined. The structures of the amylose derivatives were also investigated by circular dichroism (CD) spectroscopy.
before use. The solvents used in the chromatographic experiments were of HPLC grade. The racemates were commercially available or prepared by the usual methods. 2.2. Synthesis of amylose phenylcarbamates bearing a substituent at the ortho- or meta-position The amylose phenylcarbamate derivatives (1a–f and 2a–f) bearing a substituent at the ortho- or meta-position, respectively, were synthesized as previously described by the reaction of amylose with an excess of the corresponding isocyanates in a mixture of DMAc, lithium chloride and dry pyridine at 80 ◦ C and isolated as methanolinsoluble fractions [11]. Elemental analysis (Table 1) and the 1 H NMR spectra showed that the hydroxy groups of amylose were almost quantitatively converted into the carbamate moieties. The relatively lower carbon and nitrogen contents and slightly higher hydrogen content were probably ascribed to the presence of small amount of water, as shown in Table 1.
2. Experimental 2.3. Preparation of chiral stationary phases (CSPs) 2.1. Chemicals Twelve amylose derivatives (1a–f, 2a–f) (0.35 g each) were first dissolved in THF (8 mL) and then coated on the aminopropyl silanized silica gel (1.40 g) according to a previous method [11]. The 1- and 2-coated silica gels were then packed into a stainless-steel tube (25 cm × 0.20 cm i.d.) by a slurry technique. The plate numbers of the packed columns were 1600–3200 for benzene using a hexane/2-propanol (90:10, v/v) mixture as the eluent at a flow rate of 0.1 mL/min. 1,3,5-Tri-t-butylbenzene was used as a nonretained compound to estimate the dead time (t0 ) [26].
Amylose (DP = 300) was a kind gift from the Daicel Corporation (Tokyo, Japan). N,N-dimethylacetamide (DMAc), lithium chloride, THF and pyridine were purchased from Kermel (Tianjin, China). The phenyl isocyanate derivatives were obtained from TCI (Tokyo, Japan) except for the 3-fluorophenyl, 2-methoxyphenyl and 2-isopropylphenyl isocyanates from Aldrich (USA). 3Isopropylaniline and triphosgene were purchased from TCI. 3Isopropylphenyl isocyanate was prepared from 3-isopropylaniline by the conventional method using triphosgene in toluene at ca. 80 ◦ C. Wide-pore silica gel (Daiso gel SP-1000) with a mean particle size of 7 m and a mean pore diameter of 100 nm, which was kindly supplied by Daiso Chemical (Osaka, Japan), was silanized using (3-aminopropyl)triethoxysilane in toluene at 80 ◦ C. All solvents used in the preparation of the amylose derivatives were of analytical reagent grade and dehydrated by fractional distillation
2.4. Apparatus and chromatography Chromatographic experiments were performed using a JASCO PU-2089 chromatograph equipped with UV–Vis (JASCO UV-2070) and circular dichroism (JASCO CD-2095) detectors at room temperature. A solution of a racemate (3 mg/mL) was injected into the
Table 1 Elemental analysis of amylose derivatives. Derivatives 1c 1d 2b 2c 2d 2f a
Calculated (%)a C
H
N
Found (%) C
H
N
52.07 64.16 56.55 52.07 64.16 59.11
3.56 5.56 3.87 3.56 5.56 5.13
6.75 7.48 7.33 6.75 7.48 6.89
51.76 61.65 55.14 51.73 62.78 58.43
3.57 5.67 3.99 3.63 5.45 5.13
6.55 6.78 7.01 6.65 7.12 6.73
Estimated based on a repeated glucose unit.
J. Shen et al. / J. Chromatogr. A 1286 (2013) 41–46
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Fig. 2. Structures of racemates 3–11.
Table 2 Resolution of racemates (3–11) on amylose derivatives (1a–f). Racemate
3 4 5 6 7 8 9 10 11
1a(2-NO2 )
1b(2-F)
1c(2-Cl)
1d(2-CHI )
1e(2-(CH3 )2 CH)
1f(2-CH3 O)
k1
␣
k1
␣
k1
␣
k1
␣
k1
␣
k1
␣
0.41 0.76 2.94 0.92 0.99 0.18(−) 1.90 1.62 7.51
1.0 1.0 1.0 1.0 1.0 ∼1 1.0 1.0 1.0
0.91 0.36(+) 3.46(+) 1.03(−) 1.37 2.36(−) 1.65(+) 0.22(−) 6.42(−)
1.0 1.28 ∼1 ∼1 1.0 ∼1 ∼1 ∼1 1.09
0.98 0.55(+) 3.22(+) 0.95(−) 1.70 1.22(−) 1.76(+) 0.20 12.1(−)
1.0 1.38 ∼1 ∼1 1.0 ∼1 1.04 1.0 1.05
0.63(+) 0.39(+) 2.74(+) 0.79(−) 1.18 0.97(+) 1.44 0.12 9.34(−)
1.09 1.72 1.06 ∼1 1.0 ∼1 1.0 1.0 ∼1
0.28(+) 0.27(+) 1.80(+) 0.64(−) 0.72(-) 0.27(+) 1.28(−) – –
1.50 1.56 1.21 ∼1 1.13 ∼1 ∼1 – –
0.59(+) 0.70(+) 4.22(+) 1.04(−) 3.01 0.20(+) 2.19 – –
∼1 1.48 1.09 ∼1 1.0 ∼1 1.0 – –
Column: 25 cm × 0.20 cm ID. Flow rate: 0.1 mL/min. Eluent: hexane/2-propanol (90/10, v/v). The signs in parentheses represent the circular dichroism detection at 254 nm of the first-eluted enantiomer.
chromatographic system through an intelligent sampler (JASCO AS2055). The IR analyses were carried out by a PE FT-IR spectrometer (Spectrum 100) using a KBr pellet. The 1 H NMR spectra (500 MHz) were recorded in DMSO-d6 at 80 ◦ C using a Bruker-500 spectrometer (Bruker, USA). The thermogravimetric analyses (TGA) were performed using a TGA Q 50 instrument (TA, USA).
3. Results and discussion
electron-donating substituent for racemates 3–5 and 7. CSPs 1a–c with an electron-withdrawing group seem to show slightly lower recognition abilities compared to 1d–f, and almost no racemic compound used in this study was recognized by CSP 1a with a nitro substituent at the ortho-position. The reason for this low ability is not clear at present, but it may be ascribed to the irregular structure of the amylose chain induced by the nitro group, which will be discussed later. These results indicate that the introduction of an electron-donating substituent is more preferable than an electron-withdrawing group for the amylose ortho-substituted
3.1. Chiral recognition of CSPs derived from ortho- and meta-substituted amylose phenylcarbamate derivatives by HPLC The chiral recognition abilities of the CSPs based on the orthoand meta-substituted amylose phenylcarbamates were evaluated by HPLC with nine racemates (3–11, Fig. 2). Fig. 3 shows a chromatogram of the resolution of the racemic trans-stilbene oxide (4) on the 1d-coated CSP. The enantiomers were eluted at retention times t1 and t2 , respectively. The dead time (t0 ) was estimated to be 9.88 min with 1,3,5-tri-tert-butylbenzene. The retention factors, k1 ((t1 –t0 )/t0 ) and k2 ((t2 –t0 )/t0 ), were estimated to be 0.39 and 0.67, respectively, which led to the separation factor ␣ (k2 /k1 ) 1.72. The baseline separation of racemate 4 was attained. The results of the enantioseparation of racemates 3–11 on the 1-coated CSPs bearing different ortho-substituted groups (1a–f) are summarized in Table 2. The derivatives from left to right are arranged in the order of increasing electron-donating effect of the substituents. As can be seen, in general, the ortho-substituted amylose phenylcarbamate derivatives showed relatively low chiral recognitions, which is analogous to the previous observation for the ortho-substituted phenylcarbamates of cellulose [11,15]. Efficient chiral recognition was exhibited by CSPs 1d–f bearing an
Fig. 3. Chromatogram for the resolution of rac-4 on 1d with hexane/2-propanol (90/10) as eluent (column: 25 cm× 0.20 cm (i.d.), flow rate: 0.1 mL/min).
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Table 3 Resolution of racemates (3–11) on amylose derivatives (2a, 2b, 2d-f)a and Chiralpak ID.b Racemate
3 4 5 6 7 8 9 10 11
ID(3-Cl)b
2a(3-NO2 )
2b(3-F)
k1
␣
k1
␣
k1
␣
2d(3-CH3 ) k1
␣
2e(3-(CH3 )2 CH) k1
␣
2f(3-CH3 O) k1
␣
2.06(+) 0.56(+) 6.81(+) 2.05 1.38 3.02(+) 2.89(+) 0.33 9.78(-)
∼1 ∼1 ∼1 1.0 1.0 ∼1 1.17 1.0 1.08
0.93(+) 0.50(+) 4.97(+) 1.31(−) 0.45(−) 1.34(+) 2.13(+) 0.31(+) 3.21
1.33 2.46 1.25 1.08 1.11 1.24 1.52 ∼1 1.0
1.03(+) 0.53(+) 6.30(+) 1.54(−) 0.48(−) 1.33(+) 2.43(+) 0.38(+) 2.74(−)
1.45 2.17 1.15 1.12 1.10 1.23 1.37 1.16 1.04
0.43(+) 0.30(+) 2.87(+) 0.45 0.79(+) 0.41 0.75(+) 0.25 5.79(+)
1.58 2.77 1.31 1.0 1.06 1.0 1.55 1.0 1.05
0.37(+) 0.22(+) 0.43 0.46(−) 1.08 0.18(+) 0.58 0.36 15.0(+)
1.49 2.05 1.0 ∼1 1.0 ∼1 1.0 1.0 ∼1
0.76(+) 0.44(+) 5.30(−) 0.99(−) 1.34(+) 1.10(+) 1.92(+) 0.34 5.63
1.50 2.07 1.20 ∼1 1.11 1.10 1.30 1.0 1.0
The signs in parentheses represent the circular dichroism detection at 254 nm of the first-eluted enantiomer. a Column: 25 cm × 0.20 cm ID. Flow rate: 0.1 mL/min. Eluent: hexane/2-propanol (90/10, v/v). b Daicel immobilized column, Chiralpak ID. Column: 25 cm × 0.46 cm ID. Flow rate: 0.5 mL/min. Eluent: hexane/2-propanol (90/10, v/v).
phenylcarbamates. Analogous results have been obtained for the amylose disubstituted phenylcarbamates with chloro and methyl groups [21]; namely, the 2-methyl derivatives showed a better ability than the 2-chloro derivatives. The resolution results of the nine racemates on CSPs 2a, 2b and 2d–f are shown in Table 3. CSP 2c with a 3-chloro group cannot be evaluated under the chromatographic conditions in the study due to its too high solubility. For comparison, the results of the chromatographic resolution on the commercial amylose-based chiral column, Chiralpak ID, containing immobilized amylose tris(3chlorophenylcarbamate) as the chiral selector, are also included. Much higher efficient chiral recognition abilities were exhibited by the meta-substituted amylose phenylcarbamates compared to the ortho-substituted ones (see Table 2) except for 2e, which is similar to the results for the cellulose phenylcarbamate derivatives [11]. CSPs 2a, 2b, 2d–f and Chiralpak ID bearing an electrondonating or electron-withdrawing group showed the characteristic chiral recognitions for the different racemic compounds. For example, Chiralpak ID with an electron-withdrawing group at the meta-position can separate all nine racemic compounds with a reasonable selectivity, and CSP 2d with an electron-donating group resolved racemate 3–5, and 9 with higher values. These results indicate that the introduction of either electron-donating or electron-withdrawing substituents can improve the chiral recognition ability of the amylose meta-substituted phenylcarbamate derivatives, and that the substitution at the meta-position is more preferable than that at the ortho-position. As already previously proposed, the intramolecular hydrogen bonding can probably contribute to maintaining a higher ordered secondary structure of the polysaccharide derivatives [15,21]. For the meta-substituted phenylcarbamates of amylose, the intramolecular hydrogen bonding is much stronger compared to that of the ortho-substituted derivatives, as will be shown later by the IR spectra, which probably led to the significant difference in the chiral recognition abilities of the derivatives and elution orders of some enantiomers. The chiral recognition ability of 2d based on the ␣ value is much higher than that of 1d and also the previously evaluated amylose tris (para-methylphenylcarbamate), the ␣ value of which for racemates 3, 4 and 5 were 1.0, 1.38 and 1.0, respectively [22], indicating that the meta-methyl derivative is the best among the three monomethyl phenylcarbamate derivatives of amylose. On the other hand, various amylose disubstituted phenylcarbamates, which contain both the electron-donating methyl and electron-withdrawing chloro or fluoro groups, have also previously been used as CSPs [20,21]; the 2-methyl-5-chloro- and 2-methyl5-fluoro-phenylcarbamates exhibited the higher enantioselectivity for most of the racemates than the 2-methyl derivative 1d, while the former disubstituted carbamate can resolve all the racemates.
However, the 3-methyl-4-chloro and 3-methyl-4-fluoro derivatives show slightly lower enantioselectivities than the 3-methyl derivative 2d. As previously discussed [11,15,21], the methyl group can enhance the intramolecular hydrogen bond of the polysaccharide phenylcarbamates, and the halogen groups can enhance the acidity of the NH group. An explanation for the high chiral recognition ability of the amylose 2-methyl-5-chlorophenylcarbamate has been proposed based on a balance between the combined effects from both substituents [21], although the total reason is still not fully known. Interestingly, the effects from both the electrondonating methyl and electron-withdrawing fluoro appeared to be “reciprocal”. On one hand, the introduction of the 2-methyl group helps to maintain a higher order structure for the derivative through intramolecular hydrogen bonding accompanied by a decrease in its adsorbing power to the solutes capable of interacting with the NH groups of the carbamate moieties. In contrast, the introduction of the 5-chloro and fluoro leads to a disordered structure of the derivative while simultaneously enhancing its adsorbing power. Besides this, considering the structure of the 2,5-disubstituted phenylcarbamate (Fig. 4(A)) [27], a larger dipole moment may be directionally induced by the introduction of both the 2-methyl and 5-halogen onto the aromatic group [28], which may also be involved in the construction of a regular structure for the amylose phenylcarbamate derivative. This effect of the dipole moment may contribute to the superiority of the 2-methyl-5halogen phenylcarbamates over the 2-methyl derivative. However, the introduction of the 3-methyl and 4-chloro or fluoro onto the phenyl ring cannot generate this effect. These results imply that the chiral resolving power of the amylose phenylcarbamates can be significantly influenced by the position, nature and number of the substituents.
Fig. 4. (A) Structure of 2,5-disubstituted phenylcarbamate, (B) hydrogen bond between NH and NO2 groups of the phenylcarbamate of 1a.
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Fig. 6. IR spectra of N–H region of amylose phenylcarbamate derivatives (A: 1a–d, 1f, B: 2a–d, 2f).
Fig. 5. 1 H NMR spectra (500 MHz) of N–H region of amylose phenylcarbamate derivatives at 80 ◦ C (A: 1a-d, 1f in DMSO-d6 , B: 2a–d, 2f in DMSO-d6 , C: 2d, 2e in pyridine-d5 ).
3.2. 1 H NMR, IR and CD spectra of amylose phenylcarbamate derivatives Fig. 5(A) and (B) show the 1 H NMR spectra of the N–H region of the ortho- and meta-substituted phenylcarbamate derivatives of amylose in DMSO-d6 . The 1 H NMR of 2e with 3-isopropyl was measured in pyridine-d5 due to its poor solubility in DMSO, and the NMR spectrum of its N–H region is shown in Fig. 5(C), together with that of 2d with 3-methyl in pyridine-d5 . Three resonances corresponding to the N–H protons of the carbamate moieties at the 2-, 3- and 6-positions of the glucose units were observed between 7.5 and 10 ppm, and for 1d and 2a, the two peaks overlapped. The N–H resonance in the lowest field can be assigned to the N–H proton at the 6-position of the glucose unit [29]. The chemical shifts of the N–H resonances significantly depend on the acidity of the N–H protons and will shift downfield with an increase in
the acidity of the N–H groups of the carbamate residues [11]. The specific association of acidic NH with DMSO, a typical hydrogen bonding solvent, also cannot be ignored in this case [30,31]. The N–H protons of 2a–d and 2f show obvious downfield resonances compared to those of 1a–d and 1f, implying that the N–H protons of the meta-substituted amylose phenylcarbamates are more acidic than those of the ortho-substituted derivatives. This may be due to more steric hindrance of the substituents at the ortho-position than that at the meta-position, which may disturb the planar structure of the phenylcarbamate residues, weaken the deshielding effect of a phenyl group and decrease the association constant with DMSO. The N–H protons of 1b with a fluoro group resonated highly downfield to that of 1c having a chloro group although the two derivatives had similar electron-withdrawing substituents at the same orthoposition. This may be attributed to the fact that the planar structure of the phenylcarbamate residue is easier for 1b with a smaller fluoro group. The NH protons of 2d with the 3-methyl and 2e with the 3-isopropyl show almost identical chemical shifts due to similar electron-donation by the two alkyl groups, as shown in Fig. 5(C). The different proton accepting abilities and anisotropic effect between pyridine and DMSO are also reflected in Fig. 5(C), leading to a much downfield shift in pyridine compared to that in DMSO (Fig. 5(B)) for the NH protons of 2d [31,32]. On the other hand, the more acidic N–H proton will probably have a much stronger interaction with some racemic compounds through hydrogen bonding. The longer retention time of the enantiomers of 3–6, 8 and 9 on CSPs 2b and 2c may be due to this effect. The extreme downfield shift of 1a
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wavelength range of 255–325 nm. The rather stiff structure of 1a shown in Fig. 4(B) may induce the exciton coupling between the two phenylcarbamate groups. 4. Conclusions
Fig. 7. UV and CD spectra of amylose ortho-substituted phenylcarbamates (1a–d, 1f) in THF.
seems to be mostly ascribed to the hydrogen bond between the NH and NO2 groups of the phenylcarbamate group shown in Fig. 4(B). The IR spectra of the ortho- and meta-substituted amylose phenylcarbamate derivatives are depicted in Fig. 6. Two N–H peaks can be observed in the IR spectra for most of the amylose derivatives. One peak in the higher wavenumber region is assigned to a free N–H bond and the other peak in the lower wavenumber region to an N–H group involved in the intramolecular hydrogen bonding between the carbamate residues of neighboring glucose units [11]. Three ortho-substituted amylose derivatives, 1b, 1c and 1f, possess a much higher amount of free N–H than the metasubstituted derivatives, 2a–d and 2f. The much higher amount of the hydrogen bonded N–H of the meta-substituted derivatives may be caused by the intramolecular hydrogen bond between the carbamate groups of adjacent glucose units [11]. This hydrogen bond must lead to the formation of a regular structure, which contributes to their higher chiral recognition. As shown in Fig. 6, because of the efficient hydrogen bond formation of 1a, no free N–H exists. For 2a, the hydrogen bond may occur between the nitro and NH groups on a different carbamate residue. The CD spectra of 1 and 2 are shown in Figs. 7 and 8, respectively. In both figures, the derivatives with electron-withdrawing fluoro or chloro groups exhibit peaks with higher intensities except for 1a with the 2-nitro group, and the peak intensities of 2b–d, and 2f were higher than those of 1b–d, and 1f with the same substituent. It is worth noting that the derivative 1a bearing a 2-nitro group shows a rather different CD pattern from that of 2a with the 3-nitro group and those of the other derivatives shown in both figures within the
Fig. 8. UV and CD spectra of amylose meta-substituted phenylcarbamates (2a–d, 2f) in THF.
The ortho- and meta-substituted phenylcarbamates of amylose were prepared and their chiral recognition abilities were evaluated by HPLC. A better resolution for most racemates can be achieved on the meta-substituted amylose phenylcarbamate derivatives compared to those on the ortho-substituted derivatives, indicating that a conformational alteration of the phenylcarbamates of amylose may depend on the positions of the substituents on the phenyl group. The introduction of an electron-withdrawing or electrondonating substituent could improve the chiral recognition ability of the amylose meta-substituted phenylcarbamates. Some correlations were observed between the chiral resolving power of the amylose phenylcarbamate derivatives and the IR frequencies of the N–H groups and 1 H NMR chemical shifts of the N–H protons of the carbamate moieties. It is proposed that the chiral recognition of the amylose phenylcarbamate derivatives significantly depends on the position, nature and number of substituents of the carbamate residues, which may influence the higher order structure of the amylose derivatives mainly via intramolecular hydrogen bonding. Acknowledgements JS acknowledges the support of the National Natural Science Foundation of China (No. 51073046), the Natural Science Foundation of Heilongjiang Province (No. E201012), the Special Fund for Scientific and Technological Innovative Talents in Harbin City (2011RFLXG009) and the Fundamental Research Funds for the Central Universities (HEUCF201310003). This study was also partially supported by the Daicel Corporation (Tokyo, Japan). References [1] E. Francotte, W. Lindner (Eds.), Chirality in Drug Research, Wiley-VCH, Weinheim, 2006. [2] G. Subramanian (Ed.), Chiral Separation Techniques: Practical Approach, 3rd ed., Wiley-VCH, Weinheim, 2007. [3] T. Ikai, Y. Okamoto, Chem. Rev. 109 (2009) 6077. [4] Y. Okamoto, J. Polym. Sci. A 47 (2009) 1731. [5] Y. Okamoto, T. Ikai, Chem. Soc. Rev. 37 (2008) 2593. [6] E. Francotte, J. Chromatogr. A 906 (2001) 379. [7] Y. Okamoto, E. Yashima, Angew. Chem. Int. Ed. 37 (1998) 1020. [8] Y. Okamoto, R. Aburatani, K. Hatada, J. Chromatogr. 389 (1987) 95. [9] J. Shen, T. Ikai, Y. Okamoto, J. Chromatogr. A 1217 (2010) 1041. [10] J. Shen, S. Liu, P. Li, X. Shen, Y. Okamoto, J. Chromatogr. A 1246 (2012) 137. [11] Y. Okamoto, M. Kawashima, K. Hatada, J. Chromatogr. 363 (1986) 173. [12] Y. Okamoto, Y. Kaida, H. Hayashida, K. Hatada, Chem. Lett. (1990) 909. [13] Y. Kaida, Y. Okamoto, J. Chromatogr. 641 (1993) 267. [14] B. Chankvetadze, E. Yashima, Y. Okamoto, Chem. Lett. (1993) 617. [15] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39. [16] T. Ikai, C. Yamamoto, M. Kamigaito, Y. Okamoto, Chirality 17 (2005) 299. [17] B. Chankvetadze, L. Chankvetadze, S. Sidamonidze, E. Kasashima, E. Yashima, Y. Okamoto, J. Chromatogr. A 787 (1997) 67. [18] Y. Okamoto, T. Ohashi, Y. Kaida, E. Yashima, Chirality 5 (1993) 616. [19] C. Yamamoto, S. Inagaki, Y. Okamoto, J. Sep. Sci. 29 (2006) 915. [20] E. Yashima, C. Yamamoto, Y. Okamoto, Polym J. 27 (1995) 856. [21] B. Chankvetadze, E. Yashima, Y.Y. Okamoto, J. Chromatogr. A 694 (1995) 101. [22] Y. Okamoto, K. Hatano, R. Aburatani, K. Hatada, Chem. Lett. (1989) 715. [23] H. Steinmeier, P. Zugenmaier, Carbohydr. Res. 164 (1987) 97. [24] U. Vogt, P. Zugenmaier, Ber. Bunsen-Ges. Phys. Chem. 89 (1985) 1217. [25] U. Vogt, P. Zugenmaier, Presented at the European Science Foundation Workshop on Specific Interaction in Polysaccharide Systems, Uppsala, Sweden, 1983. [26] H. Koller, K.-H. Rimböck, A. Mannschreck, J. Chromatogr. 282 (1983) 89. [27] B.T. Loughrey, M.L. Williams, P.C. Healy, Acta Cryst. E67 (2011), m1231. [28] J.J. Conradi, N.C. Li, J. Am. Chem. Soc. 75 (1953) 1785. [29] C. Yamamoto, E. Yashima, Y. Okamoto, J. Am. Chem. Soc. 124 (2002) 12583. [30] R.J. Abraham, J.J. Byrne, L. Griffiths, M. Perez, Magn. Reson. Chem. 44 (2006) 491. [31] J.S. Lomas, A. Adenier, J. Chem. Soc. Perkin Trans. 2 (2001) 1051. [32] J.S. Lomas, J. Phys. Org. Chem. 25 (2012) 620.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Enantioseparation using ortho- or meta-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography Jun Shen a,∗ , Yongqiang Zhao a , Shinji Inagaki b , Chiyo Yamamoto b , Yue Shen a , Shuangyan Liu a , Yoshio Okamoto a,b a b
Polymer Materials Research Center, College of Material Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
a r t i c l e
i n f o
Article history: Received 26 November 2012 Received in revised form 5 February 2013 Accepted 6 February 2013 Available online 20 February 2013 Keywords: Enantioseparation Chiral recognition Phenylcarbamate Amylose Chiral stationary phase (CSPs) High-performance liquid chromatography (HPLC)
a b s t r a c t Six ortho- and six meta-substituted phenylcarbamate derivatives of amylose were prepared and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The substitution at the meta-position on the aromatic ring was more preferable than that at the ortho-position to obtain CSPs with a high chiral recognition ability, and the introduction of either an electron-withdrawing or electron-donating substituent can improve the chiral resolving power of the meta-substituted phenylcarbamates of amylose. The chiral recognition ability of the amylose phenylcarbamates and elution order of the enantiomers were significantly dependent on the position, nature and number of the substituents on the phenyl group. Correlations between the chiral recognition ability and the N–H frequencies in the IR spectra and the chemical shifts of the N–H protons in the 1 H NMR spectra of the carbamate moieties of the amylose derivatives were discussed. The structures of the amylose derivatives were also investigated by circular dichroism spectroscopy. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Direct enantioseparation by high-performance liquid chromatography (HPLC) has been one of the most efficient techniques for obtaining pure enantiomers of various chiral compounds including drugs, agrochemicals, food additives, etc. The key feature of this technique is the design and preparation of efficient chiral stationary phases (CSPs), and in recent decades, a significant variety of CSPs has been developed [1,2]. Among them, polysaccharide derivatives, especially phenylcarbamates and benzoates of cellulose and amylose, exhibit high chiral recognition abilities for a wide range of chiral compounds [3–10]. Intensive investigations of the chiral recognition mechanism of polysaccharide derivatives, especially those of the cellulose phenylcarbamates, revealed some correlations between the electronic and structural properties of the substituents on the phenyl group and their chiral recognition. It has been empirically established that their chiral recognition abilities significantly depend on the
∗ Corresponding author. Tel.: +81 52 753 7292; fax: +81 52 753 7292. E-mail addresses: [email protected] (J. Shen), [email protected] (Y. Okamoto). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.02.020
type and position of the substituents introduced on the aromatic moieties [11–20]. The introduction of either an electron-donating group, like a methyl, or an electron-withdrawing group, like a chloro, at the meta- or para-position on the phenyl group has a tendency to significantly improve the chiral recognition ability of the phenylcarbamate derivatives [11,15]. However, the orthosubstituted derivatives show a low chiral recognition. It has been proposed that the chiral recognition ability can be controlled by changing the polarity of the carbamate residues via the introduction of substituents on the phenyl group. Compared to the cellulose derivatives, the amylose derivatives with a mono-substituent have not been thoroughly studied although some para-substituted or disubstituted derivatives have been known to show a high chiral recognition [18,20–22]. Interestingly, contrary to the cellulose derivatives, amylose derivatives with two substituents including the ortho-position, such as 2methyl-5-chloro- or 2-methyl-5-fluoro-phenylcarbamates, exhibit a very high enantioseparation power for many racemates [20,21]. The difference in the helical conformation of cellulose (left-handed 3/2 helix) and amylose (left-handed 4/3 helix) is considered to be the possible reason for their difference in the substituent effect on the chiral recognition [23–25]. Among the amylose phenylcarbamate derivatives, the ortho- or meta-monosubstituted
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Fig. 1. Structures of amylose derivatives.
phenylcarbamates have not yet been investigated in detail and their chiral recognition mechanism still remains obscure. In order to get a better understanding of the effect of substituents at the ortho- or meta-position on the chiral recognition of the phenylcarbamate derivatives of amylose and to elucidate the relationship between the electronic and structural characteristics of substituents and the chiral recognition ability, in this study, six ortho- (1a–f) and six meta-substituted (2a–f) phenylcarbamate derivatives of amylose were prepared (Fig. 1) and their chiral recognition abilities were evaluated as CSPs by HPLC. The influence of the nature and position of the substituents on the chiral recognition and elution order of enantiomers was carefully studied. Correlations between the chiral recognition ability and the N–H frequencies in IR spectra and the chemical shifts of the N–H protons in the 1 H NMR spectra of the carbamate moieties of these derivatives were systematically examined. The structures of the amylose derivatives were also investigated by circular dichroism (CD) spectroscopy.
before use. The solvents used in the chromatographic experiments were of HPLC grade. The racemates were commercially available or prepared by the usual methods. 2.2. Synthesis of amylose phenylcarbamates bearing a substituent at the ortho- or meta-position The amylose phenylcarbamate derivatives (1a–f and 2a–f) bearing a substituent at the ortho- or meta-position, respectively, were synthesized as previously described by the reaction of amylose with an excess of the corresponding isocyanates in a mixture of DMAc, lithium chloride and dry pyridine at 80 ◦ C and isolated as methanolinsoluble fractions [11]. Elemental analysis (Table 1) and the 1 H NMR spectra showed that the hydroxy groups of amylose were almost quantitatively converted into the carbamate moieties. The relatively lower carbon and nitrogen contents and slightly higher hydrogen content were probably ascribed to the presence of small amount of water, as shown in Table 1.
2. Experimental 2.3. Preparation of chiral stationary phases (CSPs) 2.1. Chemicals Twelve amylose derivatives (1a–f, 2a–f) (0.35 g each) were first dissolved in THF (8 mL) and then coated on the aminopropyl silanized silica gel (1.40 g) according to a previous method [11]. The 1- and 2-coated silica gels were then packed into a stainless-steel tube (25 cm × 0.20 cm i.d.) by a slurry technique. The plate numbers of the packed columns were 1600–3200 for benzene using a hexane/2-propanol (90:10, v/v) mixture as the eluent at a flow rate of 0.1 mL/min. 1,3,5-Tri-t-butylbenzene was used as a nonretained compound to estimate the dead time (t0 ) [26].
Amylose (DP = 300) was a kind gift from the Daicel Corporation (Tokyo, Japan). N,N-dimethylacetamide (DMAc), lithium chloride, THF and pyridine were purchased from Kermel (Tianjin, China). The phenyl isocyanate derivatives were obtained from TCI (Tokyo, Japan) except for the 3-fluorophenyl, 2-methoxyphenyl and 2-isopropylphenyl isocyanates from Aldrich (USA). 3Isopropylaniline and triphosgene were purchased from TCI. 3Isopropylphenyl isocyanate was prepared from 3-isopropylaniline by the conventional method using triphosgene in toluene at ca. 80 ◦ C. Wide-pore silica gel (Daiso gel SP-1000) with a mean particle size of 7 m and a mean pore diameter of 100 nm, which was kindly supplied by Daiso Chemical (Osaka, Japan), was silanized using (3-aminopropyl)triethoxysilane in toluene at 80 ◦ C. All solvents used in the preparation of the amylose derivatives were of analytical reagent grade and dehydrated by fractional distillation
2.4. Apparatus and chromatography Chromatographic experiments were performed using a JASCO PU-2089 chromatograph equipped with UV–Vis (JASCO UV-2070) and circular dichroism (JASCO CD-2095) detectors at room temperature. A solution of a racemate (3 mg/mL) was injected into the
Table 1 Elemental analysis of amylose derivatives. Derivatives 1c 1d 2b 2c 2d 2f a
Calculated (%)a C
H
N
Found (%) C
H
N
52.07 64.16 56.55 52.07 64.16 59.11
3.56 5.56 3.87 3.56 5.56 5.13
6.75 7.48 7.33 6.75 7.48 6.89
51.76 61.65 55.14 51.73 62.78 58.43
3.57 5.67 3.99 3.63 5.45 5.13
6.55 6.78 7.01 6.65 7.12 6.73
Estimated based on a repeated glucose unit.
J. Shen et al. / J. Chromatogr. A 1286 (2013) 41–46
43
Fig. 2. Structures of racemates 3–11.
Table 2 Resolution of racemates (3–11) on amylose derivatives (1a–f). Racemate
3 4 5 6 7 8 9 10 11
1a(2-NO2 )
1b(2-F)
1c(2-Cl)
1d(2-CHI )
1e(2-(CH3 )2 CH)
1f(2-CH3 O)
k1
␣
k1
␣
k1
␣
k1
␣
k1
␣
k1
␣
0.41 0.76 2.94 0.92 0.99 0.18(−) 1.90 1.62 7.51
1.0 1.0 1.0 1.0 1.0 ∼1 1.0 1.0 1.0
0.91 0.36(+) 3.46(+) 1.03(−) 1.37 2.36(−) 1.65(+) 0.22(−) 6.42(−)
1.0 1.28 ∼1 ∼1 1.0 ∼1 ∼1 ∼1 1.09
0.98 0.55(+) 3.22(+) 0.95(−) 1.70 1.22(−) 1.76(+) 0.20 12.1(−)
1.0 1.38 ∼1 ∼1 1.0 ∼1 1.04 1.0 1.05
0.63(+) 0.39(+) 2.74(+) 0.79(−) 1.18 0.97(+) 1.44 0.12 9.34(−)
1.09 1.72 1.06 ∼1 1.0 ∼1 1.0 1.0 ∼1
0.28(+) 0.27(+) 1.80(+) 0.64(−) 0.72(-) 0.27(+) 1.28(−) – –
1.50 1.56 1.21 ∼1 1.13 ∼1 ∼1 – –
0.59(+) 0.70(+) 4.22(+) 1.04(−) 3.01 0.20(+) 2.19 – –
∼1 1.48 1.09 ∼1 1.0 ∼1 1.0 – –
Column: 25 cm × 0.20 cm ID. Flow rate: 0.1 mL/min. Eluent: hexane/2-propanol (90/10, v/v). The signs in parentheses represent the circular dichroism detection at 254 nm of the first-eluted enantiomer.
chromatographic system through an intelligent sampler (JASCO AS2055). The IR analyses were carried out by a PE FT-IR spectrometer (Spectrum 100) using a KBr pellet. The 1 H NMR spectra (500 MHz) were recorded in DMSO-d6 at 80 ◦ C using a Bruker-500 spectrometer (Bruker, USA). The thermogravimetric analyses (TGA) were performed using a TGA Q 50 instrument (TA, USA).
3. Results and discussion
electron-donating substituent for racemates 3–5 and 7. CSPs 1a–c with an electron-withdrawing group seem to show slightly lower recognition abilities compared to 1d–f, and almost no racemic compound used in this study was recognized by CSP 1a with a nitro substituent at the ortho-position. The reason for this low ability is not clear at present, but it may be ascribed to the irregular structure of the amylose chain induced by the nitro group, which will be discussed later. These results indicate that the introduction of an electron-donating substituent is more preferable than an electron-withdrawing group for the amylose ortho-substituted
3.1. Chiral recognition of CSPs derived from ortho- and meta-substituted amylose phenylcarbamate derivatives by HPLC The chiral recognition abilities of the CSPs based on the orthoand meta-substituted amylose phenylcarbamates were evaluated by HPLC with nine racemates (3–11, Fig. 2). Fig. 3 shows a chromatogram of the resolution of the racemic trans-stilbene oxide (4) on the 1d-coated CSP. The enantiomers were eluted at retention times t1 and t2 , respectively. The dead time (t0 ) was estimated to be 9.88 min with 1,3,5-tri-tert-butylbenzene. The retention factors, k1 ((t1 –t0 )/t0 ) and k2 ((t2 –t0 )/t0 ), were estimated to be 0.39 and 0.67, respectively, which led to the separation factor ␣ (k2 /k1 ) 1.72. The baseline separation of racemate 4 was attained. The results of the enantioseparation of racemates 3–11 on the 1-coated CSPs bearing different ortho-substituted groups (1a–f) are summarized in Table 2. The derivatives from left to right are arranged in the order of increasing electron-donating effect of the substituents. As can be seen, in general, the ortho-substituted amylose phenylcarbamate derivatives showed relatively low chiral recognitions, which is analogous to the previous observation for the ortho-substituted phenylcarbamates of cellulose [11,15]. Efficient chiral recognition was exhibited by CSPs 1d–f bearing an
Fig. 3. Chromatogram for the resolution of rac-4 on 1d with hexane/2-propanol (90/10) as eluent (column: 25 cm× 0.20 cm (i.d.), flow rate: 0.1 mL/min).
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Table 3 Resolution of racemates (3–11) on amylose derivatives (2a, 2b, 2d-f)a and Chiralpak ID.b Racemate
3 4 5 6 7 8 9 10 11
ID(3-Cl)b
2a(3-NO2 )
2b(3-F)
k1
␣
k1
␣
k1
␣
2d(3-CH3 ) k1
␣
2e(3-(CH3 )2 CH) k1
␣
2f(3-CH3 O) k1
␣
2.06(+) 0.56(+) 6.81(+) 2.05 1.38 3.02(+) 2.89(+) 0.33 9.78(-)
∼1 ∼1 ∼1 1.0 1.0 ∼1 1.17 1.0 1.08
0.93(+) 0.50(+) 4.97(+) 1.31(−) 0.45(−) 1.34(+) 2.13(+) 0.31(+) 3.21
1.33 2.46 1.25 1.08 1.11 1.24 1.52 ∼1 1.0
1.03(+) 0.53(+) 6.30(+) 1.54(−) 0.48(−) 1.33(+) 2.43(+) 0.38(+) 2.74(−)
1.45 2.17 1.15 1.12 1.10 1.23 1.37 1.16 1.04
0.43(+) 0.30(+) 2.87(+) 0.45 0.79(+) 0.41 0.75(+) 0.25 5.79(+)
1.58 2.77 1.31 1.0 1.06 1.0 1.55 1.0 1.05
0.37(+) 0.22(+) 0.43 0.46(−) 1.08 0.18(+) 0.58 0.36 15.0(+)
1.49 2.05 1.0 ∼1 1.0 ∼1 1.0 1.0 ∼1
0.76(+) 0.44(+) 5.30(−) 0.99(−) 1.34(+) 1.10(+) 1.92(+) 0.34 5.63
1.50 2.07 1.20 ∼1 1.11 1.10 1.30 1.0 1.0
The signs in parentheses represent the circular dichroism detection at 254 nm of the first-eluted enantiomer. a Column: 25 cm × 0.20 cm ID. Flow rate: 0.1 mL/min. Eluent: hexane/2-propanol (90/10, v/v). b Daicel immobilized column, Chiralpak ID. Column: 25 cm × 0.46 cm ID. Flow rate: 0.5 mL/min. Eluent: hexane/2-propanol (90/10, v/v).
phenylcarbamates. Analogous results have been obtained for the amylose disubstituted phenylcarbamates with chloro and methyl groups [21]; namely, the 2-methyl derivatives showed a better ability than the 2-chloro derivatives. The resolution results of the nine racemates on CSPs 2a, 2b and 2d–f are shown in Table 3. CSP 2c with a 3-chloro group cannot be evaluated under the chromatographic conditions in the study due to its too high solubility. For comparison, the results of the chromatographic resolution on the commercial amylose-based chiral column, Chiralpak ID, containing immobilized amylose tris(3chlorophenylcarbamate) as the chiral selector, are also included. Much higher efficient chiral recognition abilities were exhibited by the meta-substituted amylose phenylcarbamates compared to the ortho-substituted ones (see Table 2) except for 2e, which is similar to the results for the cellulose phenylcarbamate derivatives [11]. CSPs 2a, 2b, 2d–f and Chiralpak ID bearing an electrondonating or electron-withdrawing group showed the characteristic chiral recognitions for the different racemic compounds. For example, Chiralpak ID with an electron-withdrawing group at the meta-position can separate all nine racemic compounds with a reasonable selectivity, and CSP 2d with an electron-donating group resolved racemate 3–5, and 9 with higher values. These results indicate that the introduction of either electron-donating or electron-withdrawing substituents can improve the chiral recognition ability of the amylose meta-substituted phenylcarbamate derivatives, and that the substitution at the meta-position is more preferable than that at the ortho-position. As already previously proposed, the intramolecular hydrogen bonding can probably contribute to maintaining a higher ordered secondary structure of the polysaccharide derivatives [15,21]. For the meta-substituted phenylcarbamates of amylose, the intramolecular hydrogen bonding is much stronger compared to that of the ortho-substituted derivatives, as will be shown later by the IR spectra, which probably led to the significant difference in the chiral recognition abilities of the derivatives and elution orders of some enantiomers. The chiral recognition ability of 2d based on the ␣ value is much higher than that of 1d and also the previously evaluated amylose tris (para-methylphenylcarbamate), the ␣ value of which for racemates 3, 4 and 5 were 1.0, 1.38 and 1.0, respectively [22], indicating that the meta-methyl derivative is the best among the three monomethyl phenylcarbamate derivatives of amylose. On the other hand, various amylose disubstituted phenylcarbamates, which contain both the electron-donating methyl and electron-withdrawing chloro or fluoro groups, have also previously been used as CSPs [20,21]; the 2-methyl-5-chloro- and 2-methyl5-fluoro-phenylcarbamates exhibited the higher enantioselectivity for most of the racemates than the 2-methyl derivative 1d, while the former disubstituted carbamate can resolve all the racemates.
However, the 3-methyl-4-chloro and 3-methyl-4-fluoro derivatives show slightly lower enantioselectivities than the 3-methyl derivative 2d. As previously discussed [11,15,21], the methyl group can enhance the intramolecular hydrogen bond of the polysaccharide phenylcarbamates, and the halogen groups can enhance the acidity of the NH group. An explanation for the high chiral recognition ability of the amylose 2-methyl-5-chlorophenylcarbamate has been proposed based on a balance between the combined effects from both substituents [21], although the total reason is still not fully known. Interestingly, the effects from both the electrondonating methyl and electron-withdrawing fluoro appeared to be “reciprocal”. On one hand, the introduction of the 2-methyl group helps to maintain a higher order structure for the derivative through intramolecular hydrogen bonding accompanied by a decrease in its adsorbing power to the solutes capable of interacting with the NH groups of the carbamate moieties. In contrast, the introduction of the 5-chloro and fluoro leads to a disordered structure of the derivative while simultaneously enhancing its adsorbing power. Besides this, considering the structure of the 2,5-disubstituted phenylcarbamate (Fig. 4(A)) [27], a larger dipole moment may be directionally induced by the introduction of both the 2-methyl and 5-halogen onto the aromatic group [28], which may also be involved in the construction of a regular structure for the amylose phenylcarbamate derivative. This effect of the dipole moment may contribute to the superiority of the 2-methyl-5halogen phenylcarbamates over the 2-methyl derivative. However, the introduction of the 3-methyl and 4-chloro or fluoro onto the phenyl ring cannot generate this effect. These results imply that the chiral resolving power of the amylose phenylcarbamates can be significantly influenced by the position, nature and number of the substituents.
Fig. 4. (A) Structure of 2,5-disubstituted phenylcarbamate, (B) hydrogen bond between NH and NO2 groups of the phenylcarbamate of 1a.
J. Shen et al. / J. Chromatogr. A 1286 (2013) 41–46
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Fig. 6. IR spectra of N–H region of amylose phenylcarbamate derivatives (A: 1a–d, 1f, B: 2a–d, 2f).
Fig. 5. 1 H NMR spectra (500 MHz) of N–H region of amylose phenylcarbamate derivatives at 80 ◦ C (A: 1a-d, 1f in DMSO-d6 , B: 2a–d, 2f in DMSO-d6 , C: 2d, 2e in pyridine-d5 ).
3.2. 1 H NMR, IR and CD spectra of amylose phenylcarbamate derivatives Fig. 5(A) and (B) show the 1 H NMR spectra of the N–H region of the ortho- and meta-substituted phenylcarbamate derivatives of amylose in DMSO-d6 . The 1 H NMR of 2e with 3-isopropyl was measured in pyridine-d5 due to its poor solubility in DMSO, and the NMR spectrum of its N–H region is shown in Fig. 5(C), together with that of 2d with 3-methyl in pyridine-d5 . Three resonances corresponding to the N–H protons of the carbamate moieties at the 2-, 3- and 6-positions of the glucose units were observed between 7.5 and 10 ppm, and for 1d and 2a, the two peaks overlapped. The N–H resonance in the lowest field can be assigned to the N–H proton at the 6-position of the glucose unit [29]. The chemical shifts of the N–H resonances significantly depend on the acidity of the N–H protons and will shift downfield with an increase in
the acidity of the N–H groups of the carbamate residues [11]. The specific association of acidic NH with DMSO, a typical hydrogen bonding solvent, also cannot be ignored in this case [30,31]. The N–H protons of 2a–d and 2f show obvious downfield resonances compared to those of 1a–d and 1f, implying that the N–H protons of the meta-substituted amylose phenylcarbamates are more acidic than those of the ortho-substituted derivatives. This may be due to more steric hindrance of the substituents at the ortho-position than that at the meta-position, which may disturb the planar structure of the phenylcarbamate residues, weaken the deshielding effect of a phenyl group and decrease the association constant with DMSO. The N–H protons of 1b with a fluoro group resonated highly downfield to that of 1c having a chloro group although the two derivatives had similar electron-withdrawing substituents at the same orthoposition. This may be attributed to the fact that the planar structure of the phenylcarbamate residue is easier for 1b with a smaller fluoro group. The NH protons of 2d with the 3-methyl and 2e with the 3-isopropyl show almost identical chemical shifts due to similar electron-donation by the two alkyl groups, as shown in Fig. 5(C). The different proton accepting abilities and anisotropic effect between pyridine and DMSO are also reflected in Fig. 5(C), leading to a much downfield shift in pyridine compared to that in DMSO (Fig. 5(B)) for the NH protons of 2d [31,32]. On the other hand, the more acidic N–H proton will probably have a much stronger interaction with some racemic compounds through hydrogen bonding. The longer retention time of the enantiomers of 3–6, 8 and 9 on CSPs 2b and 2c may be due to this effect. The extreme downfield shift of 1a
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wavelength range of 255–325 nm. The rather stiff structure of 1a shown in Fig. 4(B) may induce the exciton coupling between the two phenylcarbamate groups. 4. Conclusions
Fig. 7. UV and CD spectra of amylose ortho-substituted phenylcarbamates (1a–d, 1f) in THF.
seems to be mostly ascribed to the hydrogen bond between the NH and NO2 groups of the phenylcarbamate group shown in Fig. 4(B). The IR spectra of the ortho- and meta-substituted amylose phenylcarbamate derivatives are depicted in Fig. 6. Two N–H peaks can be observed in the IR spectra for most of the amylose derivatives. One peak in the higher wavenumber region is assigned to a free N–H bond and the other peak in the lower wavenumber region to an N–H group involved in the intramolecular hydrogen bonding between the carbamate residues of neighboring glucose units [11]. Three ortho-substituted amylose derivatives, 1b, 1c and 1f, possess a much higher amount of free N–H than the metasubstituted derivatives, 2a–d and 2f. The much higher amount of the hydrogen bonded N–H of the meta-substituted derivatives may be caused by the intramolecular hydrogen bond between the carbamate groups of adjacent glucose units [11]. This hydrogen bond must lead to the formation of a regular structure, which contributes to their higher chiral recognition. As shown in Fig. 6, because of the efficient hydrogen bond formation of 1a, no free N–H exists. For 2a, the hydrogen bond may occur between the nitro and NH groups on a different carbamate residue. The CD spectra of 1 and 2 are shown in Figs. 7 and 8, respectively. In both figures, the derivatives with electron-withdrawing fluoro or chloro groups exhibit peaks with higher intensities except for 1a with the 2-nitro group, and the peak intensities of 2b–d, and 2f were higher than those of 1b–d, and 1f with the same substituent. It is worth noting that the derivative 1a bearing a 2-nitro group shows a rather different CD pattern from that of 2a with the 3-nitro group and those of the other derivatives shown in both figures within the
Fig. 8. UV and CD spectra of amylose meta-substituted phenylcarbamates (2a–d, 2f) in THF.
The ortho- and meta-substituted phenylcarbamates of amylose were prepared and their chiral recognition abilities were evaluated by HPLC. A better resolution for most racemates can be achieved on the meta-substituted amylose phenylcarbamate derivatives compared to those on the ortho-substituted derivatives, indicating that a conformational alteration of the phenylcarbamates of amylose may depend on the positions of the substituents on the phenyl group. The introduction of an electron-withdrawing or electrondonating substituent could improve the chiral recognition ability of the amylose meta-substituted phenylcarbamates. Some correlations were observed between the chiral resolving power of the amylose phenylcarbamate derivatives and the IR frequencies of the N–H groups and 1 H NMR chemical shifts of the N–H protons of the carbamate moieties. It is proposed that the chiral recognition of the amylose phenylcarbamate derivatives significantly depends on the position, nature and number of substituents of the carbamate residues, which may influence the higher order structure of the amylose derivatives mainly via intramolecular hydrogen bonding. Acknowledgements JS acknowledges the support of the National Natural Science Foundation of China (No. 51073046), the Natural Science Foundation of Heilongjiang Province (No. E201012), the Special Fund for Scientific and Technological Innovative Talents in Harbin City (2011RFLXG009) and the Fundamental Research Funds for the Central Universities (HEUCF201310003). This study was also partially supported by the Daicel Corporation (Tokyo, Japan). References [1] E. Francotte, W. Lindner (Eds.), Chirality in Drug Research, Wiley-VCH, Weinheim, 2006. [2] G. Subramanian (Ed.), Chiral Separation Techniques: Practical Approach, 3rd ed., Wiley-VCH, Weinheim, 2007. [3] T. Ikai, Y. Okamoto, Chem. Rev. 109 (2009) 6077. [4] Y. Okamoto, J. Polym. Sci. A 47 (2009) 1731. [5] Y. Okamoto, T. Ikai, Chem. Soc. Rev. 37 (2008) 2593. [6] E. Francotte, J. Chromatogr. A 906 (2001) 379. [7] Y. Okamoto, E. Yashima, Angew. Chem. Int. Ed. 37 (1998) 1020. [8] Y. Okamoto, R. Aburatani, K. Hatada, J. Chromatogr. 389 (1987) 95. [9] J. Shen, T. Ikai, Y. Okamoto, J. Chromatogr. A 1217 (2010) 1041. [10] J. Shen, S. Liu, P. Li, X. Shen, Y. Okamoto, J. Chromatogr. A 1246 (2012) 137. [11] Y. Okamoto, M. Kawashima, K. Hatada, J. Chromatogr. 363 (1986) 173. [12] Y. Okamoto, Y. Kaida, H. Hayashida, K. Hatada, Chem. Lett. (1990) 909. [13] Y. Kaida, Y. Okamoto, J. Chromatogr. 641 (1993) 267. [14] B. Chankvetadze, E. Yashima, Y. Okamoto, Chem. Lett. (1993) 617. [15] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39. [16] T. Ikai, C. Yamamoto, M. Kamigaito, Y. Okamoto, Chirality 17 (2005) 299. [17] B. Chankvetadze, L. Chankvetadze, S. Sidamonidze, E. Kasashima, E. Yashima, Y. Okamoto, J. Chromatogr. A 787 (1997) 67. [18] Y. Okamoto, T. Ohashi, Y. Kaida, E. Yashima, Chirality 5 (1993) 616. [19] C. Yamamoto, S. Inagaki, Y. Okamoto, J. Sep. Sci. 29 (2006) 915. [20] E. Yashima, C. Yamamoto, Y. Okamoto, Polym J. 27 (1995) 856. [21] B. Chankvetadze, E. Yashima, Y.Y. Okamoto, J. Chromatogr. A 694 (1995) 101. [22] Y. Okamoto, K. Hatano, R. Aburatani, K. Hatada, Chem. Lett. (1989) 715. [23] H. Steinmeier, P. Zugenmaier, Carbohydr. Res. 164 (1987) 97. [24] U. Vogt, P. Zugenmaier, Ber. Bunsen-Ges. Phys. Chem. 89 (1985) 1217. [25] U. Vogt, P. Zugenmaier, Presented at the European Science Foundation Workshop on Specific Interaction in Polysaccharide Systems, Uppsala, Sweden, 1983. [26] H. Koller, K.-H. Rimböck, A. Mannschreck, J. Chromatogr. 282 (1983) 89. [27] B.T. Loughrey, M.L. Williams, P.C. Healy, Acta Cryst. E67 (2011), m1231. [28] J.J. Conradi, N.C. Li, J. Am. Chem. Soc. 75 (1953) 1785. [29] C. Yamamoto, E. Yashima, Y. Okamoto, J. Am. Chem. Soc. 124 (2002) 12583. [30] R.J. Abraham, J.J. Byrne, L. Griffiths, M. Perez, Magn. Reson. Chem. 44 (2006) 491. [31] J.S. Lomas, A. Adenier, J. Chem. Soc. Perkin Trans. 2 (2001) 1051. [32] J.S. Lomas, J. Phys. Org. Chem. 25 (2012) 620.