Preparation of Silica Gel-Bonded Amylose through Enzyme-Catalyzed

Anal. Chem. 1996, 68, 2798-2804

Preparation of Silica Gel-Bonded Amylose through Enzyme-Catalyzed Polymerization and Chiral Recognition Ability of Its Phenylcarbamate Derivative in HPLC Naoki Enomoto,† Sachiko Furukawa,† Yasushi Ogasawara,† Hirofumi Akano,† Yoshiya Kawamura,† Eiji Yashima,‡ and Yoshio Okamoto*,‡

Nakano Vinegar Co., Ltd., Handa, Aichi 475, Japan, and Department of Applied Chemistry, School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

Amylose was prepared by enzymatic polymerization of r-Dglucose 1-phosphate dipotassium catalyzed by a phosphorylase using two kinds of the primers derived from maltopentaose, and then it was chemically bonded to silica gel to be used as a chiral stationary phase (CSP) in high-performance liquid chromatography. In method I, maltopentaose was first lactonized and allowed to react with (3-aminopropyl)triethoxysilane to form an amide bond. Amylose chains with a desired chain length and a narrow molecular weight distribution were then constructed by the enzymatic polymerization. The resulting amylose bearing a trialkoxysilyl group at the terminal was allowed to react with silica gel for immobilization. In method II, maltopentaose was first oxidized to form a potassium gluconate at the reducing terminal. After the enzymatic polymerization was performed with the potassium gluconate, the amylose end was lactonized to be immobilized to 3-aminopropyl-silanized silica gel through amide bond formation. Two amylose-conjugated silica gels thus obtained were treated with a large excess of 3,5dimethylphenyl isocyanate to convert hydroxy groups of amylose to corresponding carbamate residues. The CSP derived through method II was superior in chiral recognition to the CSP derived from method I and showed better resolving power and higher durability against solvents such as tetrahydrofuran compared with a coated-type CSP. Influences of degree of polymerization of amylose, the spacer length between amylose and silica gel, and mobile phase compositions on chiral recognition were investigated. Chromatographic enantioseparations, particularly resolution by high-performance liquid chromatography (HPLC), have advanced considerably in the past 15 years and have become a practically useful method for determining optical purity and for obtaining optical isomers.1-7 Particularly, in the pharmaceutical industry, †

Nakano Vinegar Co. Ltd. Nagoya University. (1) Armstrong, D. W. Anal. Chem. 1987, 59, 84A-91A. (2) Okamoto, Y. Chemtech 1987, 176-181. (3) Allenmark, S. G. Chromatographic Enantioseparation; Ellis Horwood: Chichester, 1988. (4) Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989, 89, 347-362. (5) Chiral Separations by Liquid Chromatography; Ahuja, S., Ed.; ACS Symposium Series 471; American Chemical Society: Washington, DC, 1991. (6) Taylor, D. R.; Maher, K. J. Chromatogr. Sci. 1992, 30, 67-85. ‡

2798 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

Figure 1. Structures of tris(3,5-dimethylphenylcarbamate)s of amylose (ADMPC, 1) and cellulose (2).

the chiral HPLC is essential for research and development of chiral drugs.8,9 Phenylcarbamate derivatives of cellulose and amylose are known to show high chiral recognition as chiral stationary phases (CSPs) in HPLC.10-18 Particularly, tris(3,5-dimethylphenylcarbamate) derivatives of amylose (ADMPC, 1) and cellulose (2) (Figure 1) have been widely used to separate a broad range of racemates, including drugs.14-16 The CSPs are usually prepared by coating or adsorbing the polysaccharide derivatives on macroporous silica gel; therefore, the solvents such as tetrahydrofuran (THF) and chloroform which dissolve or swell the polysaccharides cannot be used as main mobile phases. To overcome this defect, we prepared CSPs in which the polysac(7) A Practical Approach to Chiral Separations by Liquid Chromatography; Subramanian, G., Ed.; VCH: New York, 1994. (8) Mutton, I. M. In A Practical Approach to Chiral Separations by Liquid Chromatography; Subramanian, G., Ed.; VCH: New York, 1994; Chapter 11. (9) Stinson, S. C. Chem. Eng. News 1994, 72 (Sept 19), 38-72. (10) Okamoto, Y.; Kawashima, M.; Hatada, K. J. Am. Chem. Soc. 1984, 106, 5357-5359. (11) Okamoto, Y.; Kawashima, M.; Hatada, K. J. Chromatogr. 1986, 363, 173186. (12) Shibata, T.; Okamoto, I.; Ishii, K. J. Liq. Chromatogr. 1986, 9, 313-340. (13) Okamoto, Y.; Aburatani, R.; Hatano, K.; Hatada, K. J. Liq. Chromatogr. 1988, 11, 2147-2163. (14) Okamoto, Y.; Kaida, Y. J. High Resolut. Chromatogr. 1990, 13, 708-712. (15) Okamoto, Y.; Kaida, Y.; Aburatani, R.; Hatada, K. In Chiral Separations by Liquid Chromatography; Ahuja, S., Ed.; ACS Symposium Series 471; American Chemical Society: Washington, DC, 1991; pp 101-113. (16) (a) Okamoto, Y.; Kaida, Y. J. Chromatogr. A 1994, 666, 403-419. (b) Yashima, E.; Okamoto, Y. Bull. Chem. Soc. Jpn. 1995, 68, 3289-3307. (17) Dingene, J. In A Practical Approach to Chiral Separations by Liquid Chromatography; Subramanian, G., Ed.; VCH: New York, 1994; Chapter 6. (18) Oguni, K.; Oda, H.; Ichida, A. J. Chromatogr. A 1995, 694, 91-100. S0003-2700(96)00002-9 CCC: $12.00

© 1996 American Chemical Society

charide phenylcarbamates were chemically bonded randomly19 and regioselectively20 to silica gel through hydroxy groups of the polysaccharides. These bonded CSPs, however, showed lower chiral recognition ability, particularly at a high concentration of the chemical bond, than the corresponding coated-type CSPs. The introduction of the above-mentioned chemical bond may disturb the formation of regular higher order structure of the polysaccharides, which is essential for high chiral recognition.20 In the present study, we prepared ADMPC CSP in which amylose was chemically bonded to silica gel only at the reducing terminal residue. The amylose chain, with a desired chain length and a narrow molecular weight distribution, was constructed by polymerization of R-D-glucose 1-phosphate dipotassium with a functionalized maltooligosaccharide catalyzed by an enzyme, phosphorylase isolated from potato. This amylose synthesis using a primer was first investigated by Cori and Cori21 and developed by Pfannemu¨ller et al.22,23 and has recently been utilized for grafting amylose chains onto poly(dimethylsiloxane).24 The new ADMPC CSP showed high resolving power and can be used with solvents such as THF and chloroform which cannot be used for the coated-type CSPs. EXPERIMENTAL SECTION Reagents. Porous spherical silica gel with a mean particle size of 7 µm and a mean pore diameter of 100 nm (Daiso gel SP1000) was a gift from Daiso Chemical (Osaka, Japan), and silica gels with a mean particle size of 5 µm and mean pore diameters of 12 and 50 nm were obtained from YMC (Kyoto, Japan) and Fuji Silysia Chemical (Aichi, Japan), respectively. Triphosgene was from Tokyo Kasei (Tokyo, Japan). (3-Aminopropyl)triethoxysilane and [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane were purchased from Chisso (Chiba, Japan). [N-[N′-(2-Aminoethyl)-2aminoethyl]-3-aminopropyl]trimethoxysilane was obtained from Shinetsu Chemical (Tokyo, Japan). 3,5-Dimethylphenyl isocyanate was prepared from 3,5-dimethylaniline by the conventional method using triphosgene.11 Maltopentaose and maltoheptaose were purchased from Nacalai Tesque (Kyoto, Japan). R-D-Glucose 1-phosphate dipotassium was from Fluka (Buchs, Switzerland). Phosphorylase (∼6000 units) was obtained from potato tubers (10 kg) and purified according to the method of Kamogawa et al.25 Enzymatic activity was determined to be 22.3-24.0 units/mg of protein by detection of liberated phosphate, which was quantitatively measured according to the method of Fiske and Sabbarow.26 In this work, 1 unit is defined as the amount of enzyme that liberates 1 µmol of phosphate per minute from R-D-glucose 1-phosphate under the assay condition.25 The total protein values were determined by Bio-Rad protein assay with bovine serum albumin as standard. The enzyme was found to be free from other carbohydrase activities. All solvents used in the preparation of CSPs were of analytical reagent grade, carefully dried, and distilled before use. Solvents used in the chromatographic experiments were of HPLC grade. Racemates were commercially available or were prepared by the usual method.10,11 (19) Okamoto, Y.; Aburatani, R.; Miura, S.; Hatada, K. J. Liq. Chromatogr. 1987, 10, 1613-1628. (20) Yashima, E.; Fukaya, H.; Okamoto, Y. J. Chromatogr. A 1994, 677, 11-19. (21) Cori, G. T.; Cori, C. F. J. Biol. Chem. 1940, 135, 733-756. (22) Pfannemu ¨ ller, B.; Burchard, W. Makromol. Chem. 1969, 121, 1-17. (23) Kitamura, S.; Yunokawa, H.; Mitsuie, S.; Kuge, T. Polym. J. (Tokyo) 1982, 14, 93-99. (24) Braunmu ¨ hl, V.; Jonas, G.; Stadler, R. Macromolecules 1995, 28, 17-24. (25) Kamogawa, A.; Fukui, T.; Nikuni, Z. J. Biochem. 1968, 63, 361-369. (26) Fiske, C. H.; Sabbarow, Y. J. Biol. Chem. 1925, 66, 375.

Figure 2. Scheme of synthetic route for ADMPC-bonded silica gel.

Methods. Preparation of CSPs. Amylose was prepared by enzymatic polymerization of R-D-glucose 1-phosphate dipotassium catalyzed by the phosphorylase from potato using two kinds of the primers derived from maltopentaose (see method I in Figure 2 and method II in Figure 3). The polymerization of R-D-glucose 1-phosphate dipotassium with phosphorylase was performed according to the method described by Kitamura et al.23 with a slight modification. Oxidation of oligosaccharides was carried out according to the reported methods.27,28 The preparation schemes for methods I and II are described in Figures 2 and 3, respectively. Method I. To maltopentaose (6.0 g) dissolved in distilled water (9 mL) was added iodine (4.0 g) in methanol (30 mL) under stirring. The solution was then heated to 40 °C, and 4% potassium hydroxide solution in methanol (100 mL) was added. The reaction was continued until the color from iodine disappeared. The solution was cooled in an ice-water bath. Crystals precipitated were collected by filtration and washed with cold methanol and ethanol. The crystals were dissolved in water (100 mL), and the solution was treated with charcoal powder. After the charcoal powder was separated by filtration, the filtrate was lyophilized to (27) Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym. J. (Tokyo) 1985, 17, 567-575. (28) Kobayashi, K.; Sumitomo, H.; Itoigawa, T. Macromolecules 1987, 20, 906908.

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2799

Figure 3. Scheme of synthetic route for ADMPC-bonded silica gel.

afford potassium [O-R-D-glucopyranosyl-(1f4)]4-D-gluconate (G4gluconate). The resulting potassium G4-gluconate was dissolved in water again and was purified by chromatography using a column packed with Amberlite IR-120B (H+ type). The lactonized product was collected by evaporating the solvent, and then the concentrated solution was lyophilized to give 5.2 g of [O-R-Dglucopyranosyl-(1f4)]4-D-gluconolactone (G4-gluconolactone, 87% yield). Maltoheptaose was also oxidized and lactonized to give [O-R-D-glucopyranosyl-(1f4)]6-D-gluconolactone (G6-gluconolactone) by a similar method (69% yield). G4-Gluconolactone (4.0 g) was allowed to react with (3aminopropyl)triethoxysilane (1.2 g) in dry dimethyl sulfoxide (DMSO, 30 mL) at 70 °C for 5 h. After the reaction mixture was cooled to room temperature, the mixture was poured into dry acetone under a nitrogen atmosphere. The precipitates were collected by filtration, washed with dry acetone and dry hexane, and dried in vacuo at 60 °C for 2 h to give 4.3 g of N-(3triethoxysilylpropyl)-[O-R-D-glucopyranosyl-(1f4)4-D-gluconamide (SiG4-gluconamide, 85% yield). The 13C NMR spectrum of SiG4-gluconamide showed clear resonances due to the N-(3triethoxysilylpropyl)-D-gluconamide residue besides the resonances of the glucopyranosyl carbons. The IR spectrum also exhibited absorptions at 1640 and 1540 cm-1, which were ascribed to the amide bond. N-(3-Triethoxysilylpropyl)-[O-R-D-glucopyranosyl-(1f4)6-D-gluconamide (SiG6-gluconamide) was also prepared 2800

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from G6-gluconolactone by the same method used for the synthesis of SiG4-gluconamide (96% yield). The 13C NMR spectrum of SiG4-gluconamide is available as supporting information. The polymerization of R-D-glucose 1-phosphate dipotassium by potato phosphorylase was performed using SiG4-gluconamide as a primer. R-D-Glucose 1-phosphate dipotassium (20 g) was dissolved in distilled water (900 mL), and the pH of the solution was adjusted to 6.0 with HCl. To this solution was added SiG4-gluconamide (2.0 g) and phosphorylase (300 units), and the mixture was left at 30 °C for 2.25 h. The reaction flask was then placed in a hot water bath to stop the polymerization reaction. The sediments were filtered, washed with 50% ethanol in water, ethanol, and ether, and dried in vacuo at 60 °C for 2 h to give 3.6 g (35% yield) of amylose derivative bearing a 3-triethoxysilylpropyl group at the terminal (SiG34-gluconamide). The weight-average molecular weight (Mw) and its distribution (Mw/Mn) of the amylose derivative were estimated to be 5500 and 1.12, respectively, by gel permeation chromatography (GPC). This corresponds to a degree of polymerization (DP, the average number of repeating glucose units) of 34, if the silanizing reagent at the terminal is not taken into consideration. The 1H NMR spectrum of the amylose derivative showed signals due to the terminal silanizing reagent residue. 1H NMR (DMSO-d6, 60 °C, c 5 wt %): δ 0.54 (br, SiCH2), 1.15 (t, SiOCH2CH3), 1.53 (m, SiCH2CH2). The resonances from SiOCH2CH3 and NCH2 were not assigned due to overlapping with glucose resonances. The amylose derivative with DP ) 93 and Mw/Mn ) 1.10 (SiG93-gluconamide, 2.2 g, 52% yield) was also prepared by the same enzymatic polymerization of R-Dglucose 1-phosphate dipotassium (12 g) using SiG4-gluconamide as a primer (0.3 g) with phosphorylase (210 units) at 30 °C for 4 h. SiG34-gluconamide (1.0 g) was allowed to react with silica gel (3.0 g, 7 µm particle size and 100 nm pore) in a mixture of dry DMSO (14 mL) and dry pyridine (4 mL) at 90 °C for 12 h. The silica gel-bonded amylose through amide bond formation was filtered, washed with DMSO, methanol, acetone, and hexane, and dried in vacuo at 60 °C for 2 h. After the material was dispersed in a mixture of dry DMSO (8 mL) and dry pyridine (3 mL), an excess of 3,5-dimethylphenyl isocyanate (1.5 mL) was added to the suspended solution, and the mixture was stirred at 80 °C for 5 h. The silica gel was recovered by filtration and washed with THF. The silica gel was dispersed again in a mixture of dry DMSO (8 mL) and dry pyridine (3 mL), and an excess of trimethylsilyl chloride (3.0 mL) was added to end-cap silanol groups on the silica surface at 60 °C. After 12 h, ADMPC (CSP I (Ia-34)) chemically bonded to silica gel was separated by filtration, washed with THF, methanol, acetone, and hexane, and dried in vacuo at 60 °C for 2 h. Similarly, CSPs derived from maltooligosaccharide (DP ) 7; Ia-7) and amylose (DP ) 93; Ia93) were prepared from SiG7-gluconamide and SiG93-gluconamide using silica gels (5 µm particle size and 12 nm pore silica, and 7 µm particle size and 100 nm pore silica, respectively). The contents of 3,5-dimethylphenylcarbamoylated amylose on silica surface were calculated on the basis of the elemental analyses of the CSPs prior to end-capping with trimethylsilyl chloride. A control experiment was carried out by treating silica gel (7 µm, 100 nm) with amylose (DP ) 100) having no triethoxysilyl group at the terminal which had been prepared by enzymatic polymerization with maltopentaose as a primer. However, amylose was not immobilized to the silica gel judging from the elemental analysis (see Table 1).

Table 1. Elemental Analysis of CSPs silica gel

label

particle pore size size DP of (µm) (nm) amylose

Ia-7 IIa-7

5 5

12 12

7 7

Ia-34 IIa-37

7 7

100 100

34 37

Ia-93 IIa-120

7 7

100 100

93 120

IIa-120b

7

100

120

IIa-120c

7

100

120

IIa-120s

5

50

120

silica gelc

7

100

elemental analysisa (%) C H N 16.06 21.49 (4.67) 4.47 7.09 (0.57) 7.15 9.14 (0.57) 9.00 (0.86) 8.90 (1.11) 14.36 (1.14) 0.06

1.98 2.56 (1.22) 0.49 0.69 (0.10) 0.70 0.88 (0.10) 0.90 (0.17) 0.90 (0.21) 1.57 (0.40) 0.26

1.55 2.77 (1.30) 0.20 0.64 (0.01) 0.41 0.86 (0.01) 1.01 (0.18) 1.08 (0.27) 1.74 (0.43) 0.00

amount of ADMPCb (wt %) 25.3 27.1 6.8 8.3 10.8 11.4 10.6 10.5 17.2

a The value in parentheses represents the percentage for aminofunctionalized silica gels. b Calculated from C%. c Silica gel was allowed to react with amylose having no functional group at the terminal (see Experimental Section).

Method II. Maltopentaose (18.9 g) was oxidized with iodine (13.0 g) in a similar way as in method I to obtain potassium G4gluconate (19.7 g, 96% yield). The enzymatic polymerization of R-D-glucose 1-phosphate dipotassium (32 g) using potassium G4gluconate (2.1 g) as a primer was carried out in water (800 mL) at pH 6.0 adjusted with HCl using phosphorylase (240 units) at 45 °C for 2 h. After the enzyme was inactivated by heating at a high temperature (90 °C) and was filtered off, the filtrate was adjusted to pH 1.0 with hydrochloric acid to covert the potassium gluconate into free acid, which was then lactonized. The amylose derivative was precipitated by the addition of ethanol (800 mL), collected by filtration, washed with 50% ethanol in water, ethanol, and ether, and dried in vacuo at 60 °C for 2 h, yielding 1.0 g (7%) of lactonized amylose derivative with DP ) 37 and Mw/Mn ) 1.10 (LG37-gluconolactone). The low yield of this material was due to high solubility of the product in the 50% ethanol used as the solvent for precipitation. The amylose derivative with DP ) 120 and Mw/Mn ) 1.07 (LG120-gluconolactone) (2.5 g, 43% yield) was also prepared by similar enzymatic polymerization of R-D-glucose 1-phosphate dipotassium (16 g) using G4-gluconate as a primer (0.27 g) with phosphorylase (550 units) at 40 °C for 5 h. LG37-gluconolactone (1.0 g) was allowed to react with aminofunctionalized silica gel (3.0 g, 7 µm particle size and 100 nm pore), which had been obtained by treatment with a large excess of a silane coupling reagent, (3-aminopropyl)triethoxysilane, in dry DMSO (8 mL) at 80 °C for 12 h. The silica gel-bonded amylose obtained through amide bond formation was recovered by filtration, washed with DMSO, THF, methanol, acetone, and hexane to remove unreacted LG37-gluconolactone, and dried in vacuo at 60 °C for 2 h. The silica gel was then dispersed in the mixture of dry N,N-dimethylacetamide (8 mL) and dry pyridine (3 mL) containing anhydrous lithium chloride (0.6 g), and an excess of 3,5-dimethylphenyl isocyanate (1.5 mL) was added to the mixture. The reaction mixture was left at 80 °C for 5 h. The suspended silica gel (CSP II (IIa-37)) was then collected by filtration, washed with THF, methanol, acetone, and hexane, successfully, and dried in vacuo at 60 °C for 2 h. In method II, the amino groups

remaining on silica surface may be converted to 3,5-dimethylphenyl urethane residues. The silica surface of the CSP in method II was different from that in method I, and this difference may affect the chromatographic separation. Similarly, CSPs derived from maltooligosaccharide (DP ) 7; IIa-7) and amylose (DP ) 120; IIa-120) were prepared from LG7gluconolactone (0.4 g) and LG120-gluconolactone (1.0 g) with the amino-functionalized silica gels (3.0 g, 5 µm particle size and 12 nm pore silica, and 7 µm particle size and 100 nm pore silica, respectively). To investigate the effect of the spacer between the reducing terminal of amylose and silica gel on chiral recognition, silica gels were treated with [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane or [N-[N′-(2-aminoethyl)-2-aminoethyl]-3-aminopropyl]trimethoxysilane, and these amino-functionalized silica gels (3.0 g, 7 µm particle size and 100 nm pore) were allowed to react with LG120-gluconolactone (1.0 g), followed by reaction with 3,5dimethylphenyl isocyanate to give CSPs (IIa-120b and IIa-120c). Other functionalized silica gel (5 µm particle size and 50 nm pore) with [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane was also used as a support for immobilization of the 3,5-dimethylphenylcarbamoylated amylose (DP ) 120; IIa-120s). The contents of 3,5-dimethylphenylcarbamoylated amylose on silica surface were calculated on the basis of the elemental analyses of the CSPs (Table 1). Preparation of Chiral Columns. Each column packing material was packed into a stainless-steel tube (25 cm × 0.46 (i.d.) cm) by conventional high-pressure slurry packing technique using a Model CCP-085 Econo packer pump (Chemco, Osaka, Japan).11 The plate numbers of the columns were 4500-6500 for benzene with hexane-2-propanol (90:10) as the eluent at a flow rate of 0.5 mL/min. 1,3,5-Tri-tert-butylbenzene was used as a nonretained compound for estimating the dead time (t0).11 Instruments and Chromatography. IR spectra were measured on a Jasco IR-810 spectrometer with sample as a KBr pellet. 1H and 13C NMR spectra were taken in DMSO-d with a JEOL 6 GX-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) using tetramethylsilane (TMS) as the internal standard. Chromatographic experiments were performed on an HPLC system consisting of an HPLC pump (Waters 510), an automated gradient controller (Waters 680), and UV (Waters 480) and polarimetric (Shodex OR-1) detectors at room temperature. A solution of a racemate (1-10 µL) was injected into the chromatographic system with a Rheodyne Model 7125 injector. A mixture of hexane-2-propanol (90:10) was used as the eluent unless specified otherwise at a flow rate of 0.5 mL/min. GPC was performed using a Waters HPLC system equipped with an RI detector (SIC Chromatocorder 12). GPC columns, Tosoh G6000 PWXL and G3000 PWXL (30 cm × 0.78 (i.d.) cm), were connected in series, and 0.25 M potassium acetate was used as the eluent at a flow rate of 0.7 mL/min at 55 °C. The molecular weight calibration curve was obtained with standard amylose (Mw/Mn < 1.1; Nakano Vinegar, Aichi, Japan), which was prepared by enzymatic polymerization of R-D-glucose 1-phosphate dipotassium using maltopentaose as a primer with phosphorylase; its Mw had been determined by light scattering, sedimentation equilibrium, and viscosity measurements.29 (29) Nakanishi, Y.; Norisue, T.; Teramoto, A.; Kitamura, S. Macromolecules 1993, 26, 4220-4225.

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Figure 4. Separation of (()-trans-stilbene oxide (3) on a CSP (IIa120). Eluent, hexane-2-propanol (90:10), flow rate, 0.5 mL/min.

Figure 5. Structures of racemates 3-12.

RESULTS AND DISCUSSION Preparation of CSPs. The amylose bonded to silica gel only at its terminal residue was successfully prepared by the enzymatic polymerization of R-D-glucose 1-phosphate dipotassium catalyzed by potato phosphorylase using two kinds of the primers derived from maltopentaose. The methods are illustrated as methods I and II in Figures 1 and 2, respectively. In method I, the primer was synthesized from maltopentaose, which was first lactonized, followed by reaction with (3-aminopropyl)triethoxysilane to form an amide bond. In method II, the potassium gluconate prepared by oxidation of maltopentaose at the reducing terminal was used as the primer. After amylose chains with a desired chain length and a narrow molecular weight distribution were constructed by enzymatic polymerization of R-D-glucose 1-phosphate dipotassium using phosphorylase, the resulting amylose, bearing a trialkoxysilyl residue at the terminal, was allowed to react with silica gel for immobilization in method I. In method II, the potassium gluconate end was lactonized to be immobilized on three different amino-functionalized silica gels through amide bond formation. The amylose-conjugated silica gels were treated with a large excess of 3,5-dimethylphenyl isocyanate to convert hydroxy groups of amylose to the corresponding carbamate residues. The advantages of the present methods for immobilization of amylose on silica gel compared with the previous methods using a cross-linking agent such as diisocyanates are that (1) a desired chain length of amylose with a narrow molecular weight distribution (Mw/Mn < 1.15) can be readily prepared without difficulty and (2) the immobilization to silica gel through an appropriate spacer only at the reducing terminal residue of amylose can be achieved. These features, particularly the latter, are important for avoiding a structural alternation of ADMPC during chemical bond formation between hydroxy groups of amylose derivatives and silica gel, as seen in the chemical bonding of polysaccharide 2802

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derivatives with diisocyanates as a cross-linker. The structure change of ADMPC will reduce chiral recognition ability compared with the corresponding coated-type CSPs.20 The immobilization of ADMPC on silica surface was confirmed from the IR spectra; two peaks due to the carbamoyl and phenyl groups were observed for all CSPs at around 1620 and 1720 cm-1, respectively. The contents of ADMPC of the packing materials estimated by elemental analyses were 7-27 wt %, depending on DP of amylose immobilized (Table 1). The CSPs (Ia-7 and IIa-7) contained a larger amount of ADMPC than other CSPs. The amount of ADMPC bonded to silica gel by the present methods is comparable to those prepared by immobilization of amylose using diisocyanates. The difference in the amount of ADMPC immobilized on silica surface was negligible in the two methods. The only difference between the two types of CSPs prepared by methods I and II may be attributed to the silica surface; the former silica surface was end-capped with trimethylsilyl chloride, while the latter was end-capped with 3,5-dimethylphenyl isocyanate. Accordingly, methods I and II seem suitable for the immobilization of amylose only at the reducing terminal residue. Chromatographic Enantioseparation. Figure 4 shows a chromatogram of the resolution of racemic trans-stilbene oxide (3) on a CSP (IIa-120). The enantiomers eluted at retention times of t1 and t2 showing complete baseline separation. Capacity factors, k1′ [) (t1 - t0)/t0] and k2′ [) (t2 - t0)/t0], were 0.23 and 0.59, respectively. Separation factor R [) k2′/k1′] and resolution factor Rs [) 2(t2 - t1)/(w1 + w2)] were found to be 2.56 and 4.80, respectively. The results of the enantioseparation of 10 racemates (Figure 5) bearing a various functional groupss3, Tro¨ger base (4), flavanone (5), 2-phenylcyclohexanone (6), cobalt(III) tris(acetylacetonate) (7), benzoin (8), trans-cyclopropanedicarboxylic acid dianilide (9), 1,2,2,2-tetraphenylethanol (10), and 2,2′-dihydroxy6,6′-dimethyl-1,1′-biphenyl (11)son the six ADMPC phases shown in Table 1 are summarized in Table 2. For comparison, the resolution results on the coated-type ADMPC phase,16 which was prepared by physical adsorption of ADMPC on amino-functionalized silica gel, and the ADMPC phase regioselectively bonded at the six-position to silica gel using a small amount of 4,4′diphenylmethane diisocyanate as a cross-linker20 are also shown. The results indicate that the chiral discrimination ability depends on both the DP of amylose and the methods used for immobilization. The resolving power tends to increase as the DP of amylose increases, and the ADMPC phases prepared by method II (IIa) seem to exhibit a higher resolving power than the corresponding CSPs by method I (Ia) when the DPs of amylose are comparable. The difference in chiral recognition ability of these two phases may be due partly to the difference in the silica surface as reported previously;30 in method I, trimethylsilyl chloride was used for endcapping, while 3,5-dimethylphenyl isocyanate was used in method II. It should be noted that the chiral recognition abilities of IIa120 are higher than those of the ADMPC phase regioselectively bonded at the six-position to silica gel except for 8 and 11 and are comparable to those of the coated-type ADMPC phase. Moreover, the compounds 5, 9, and 10 were resolved better on IIa-120 than on the coated-type ADMPC phase, although the amount of ADMPC on IIa-120 was less than one-half compared with the coated-type ADMPC phase. Hence, the chemical bonding (30) Grieb, S. J.; Matlin, S. A.; Belenguer, A. M.; Ritchie, H. J. J. Chromatogr. A 1995, 697, 271-278.

Table 2. Resolution Results for Racemates 3-11 on ADMPC-Bonded Phasesa Ia-7 k1′ 3 4 5 6 7 8 9 10 11

b

R

0.41(+) 1.79 2.63(+) 0.85(-) 1.67(-) 1.74(+) 0.86(+) 0.82(+) 0.99(-)

1.15 ∼1 1.06 ∼1 ∼1 1.13 1.09 1.23 1.13

IIa-7 Rs

k1′

0.71

0.48(+) 0.89(+) 3.45(+) 0.94(-) 1.40(-) 2.70(+) 1.39(+) 1.17(+) 1.72(-)

0.67e 1.32 2.13 1.63

b

R

Ia-93

3 4 5 6 7 8 9 10 11

k1′ b

R

0.21(+) 1.37(+) 0.57(+) 0.39(-) 0.20(-) 1.50(-) 1.28(+) 0.81(+) 0.88(-)

2.03 1.10 1.10 ∼1 ∼1 1.08 1.44 1.81 1.83

1.19 1.10 1.06 ∼1 ∼1 1.10 1.22 1.31 1.06

Ia-34 Rs

k1′

1.00

0.12(+) 0.29(+) 0.34(+) 0.24(-) 0.21(-) 0.88(-) 0.72(+) 0.41(+) 0.47(-)

0.53e 0.81 0.74 2.22

k1′ b

R

3.37

0.23(+) 0.40(+) 0.62(+) 0.45(-) 0.64(+) 1.83(-) 1.31(+) 1.06(+) 1.16(-)

2.56 1.56 1.33 ∼1 ∼1 1.07 2.15 2.18 1.68

4.34 0.91 2.08 4.22 4.89

R 1.85 1.19 1.08 ∼1 ∼1 ∼1 1.24 1.76 1.61

IIa-37 Rs

k1′

2.20

0.17(+) 0.29(+) 0.45(+) 0.33(-) 0.45(+) 1.25(-) 0.79(+) 0.59(+) 0.77(-)

3.68 2.14

b

R

ADMPC-bondedc

IIa-120 Rs

b

Rs

k1′ b

R

4.80 2.43 1.76

0.14(+) 0.20(+) 0.34(+) 0.65

0.79 6.47 8.24 5.70

1.04(-) 0.84(+) 0.70(+) 0.67(-)

1.96 1.44 1.23 ∼1 ∼1 ∼1 1.33 2.01 1.50

Rs 2.10 2.52 1.25

1.60 5.41 2.97

coated-typed Rs

k1′ b

R

2.53 1.37 ∼1 1.0

2.27 0.62

1.09 1.75 1.94 2.10

0.61 1.81 4.12 4.42

0.42(+) 0.53(+) 0.93(+) 0.61(+) 0.25(-) 3.14(-) 3.25(+) 2.65(+) 2.46(-)

3.04 1.58 1.12 ∼1 ∼1 1.21 2.01 1.98 2.11

Rs 6.67 2.30 0.77 2.07 2.01 5.48 6.38

a Eluent, hexane-2-propanol (90:10); flow rate, 0.5 mL/min. b The sign in parentheses represents optical rotation of the first-eluting enantiomer. Bonded at six-position to silica gel. Data are taken from ref 20. d Data are taken from ref 16. e Eluent, hexane-2-propanol (99:1); flow rate, 0.5 mL/min.

c

Table 3. Effect of Silane Coupling Reagents on Resolution of Racemates 3-11 Using ADMPC-Bonded Phasesa IIa-120b k1′ 3 4 5 6 7 8 9 10 11

e

0.23(+) 0.40)+) 0.62(+) 0.45(-) 0.64(+) 1.83(-) 1.31(+) 1.06(+) 1.16(-)

R 2.56 1.56 1.33 ∼1 ∼1 1.07 2.15 2.18 1.68

IIa-120bc Rs

k 1′

4.80 2.43 1.76

0.27(+) 0.33(+) 0.55(+) 0.38(-) 0.15(-) 1.75(-) 1.67(+) 1.34(+) 1.40(-)

0.79 6.47 8.24 5.70

e

R 2.94 1.50 1.07 ∼1 ∼1 1.21 3.35 2.28 2.22

IIa-120cd Rs

k1′

7.14 2.26

0.24(+) 0.30(+) 0.50(+) 0.34(-) 0.15(-) 1.57(-) 1.48(+) 1.21(+) 1.27(-)

2.17 9.90 10.11 12.75

e

R 2.94 1.49 1.06 ∼1 ∼1 1.20 3.50 2.28 2.20

IIa-120sc Rs

k1′

6.49 1.76

0.57(+) 0.74(+) 1.22(+) 0.77(-) 0.32(-) 3.71(-) 4.08(+) 2.74(+) 2.80(-)

1.93 8.37 8.70 9.20

e

R 2.69 1.47 1.12 1.06 ∼1 1.14 3.47 2.11 2.16

Rs 7.42 2.28 1.03 1.70 8.47 9.18 7.14

a Eluent, hexane-2-propanol (90:10); flow rate, 0.5 mL/min. b NH -(CH ) -Si-(OCH CH ) was used as silane coupling reagent. c NH 2 2 3 2 3 3 2 (CH2)2NH(CH2)3-Si-(OCH3)3 was used. d NH2-(CH2)2NH(CH2)2NH(CH2)3-Si-(OCH3)3 was used. e The sign in parentheses represents optical rotation of the first-eluting enaniomer.

to silica gel only at the reducing terminal residue of amylose must be preferable in order to prepare the ADMPC-based phase that can maintain a higher order structure of ADMPC on the silica surface to show an excellent chiral recognition. Effect of Spacer Length on Chiral Recognition. The influence of the spacer length between the reducing terminal of amylose and silica gel on chiral recognition was investigated. The resolution results on the ADMPC phases prepared with three different amine-containing silica gels are shown in Table 3. Method II was used for the immobilization of the reducing terminal of amylose (DP ) 120) to the silica gels. The results indicate that IIa-120b and IIa-120c, bearing a longer spacer, exhibited higher chiral recognition than IIa-120 with the aminopropyl residue, except for the racemates 4 and 5. CSP IIa-120s prepared with a small-particle silica gel (5 µm and 50 nm pore) showed as high chiral recognition as IIa-120b and IIa-120c columns. Consequently, the high molecular weight ADMPC bonded to silica gel at the reducing terminal of amylose through a longer spacer by using method II afforded the efficient and

practically useful CSP showing high chiral discrimination ability for many racemates. Effect of Mobile Phase Composition on Enantioseparation. The chemically bonded-type CSPs prepared in this work can be used with polar solvents such as THF and chloroform which cannot be used for the coated-type ADMPC because of solubility. The capacity factor (k1′) and enantioselectivity (R) of Tro¨ger base (4) were unaltered on IIa-120 column, even after THF had been used continuously as the eluent for more than 800 h. Enantioseparation of some racemates using chloroform, THF, and N,N-dimethylacetamide as a component was also studied on the IIa-120s column (Table 4). Tro¨ger base (4) and flavanone (5) were more effectively resolved, with separation factors R ) 1.79 and 1.72 for 4 and 1.80 and 1.67 for 5, when hexane-chloroformmethanol (95:5:1) and hexane-chloroform-THF (95:5:2) were used as the eluents, respectively. These values were larger than those (R ) 1.47 and 1.12 for 4 and 5) under a typical normal phase condition with hexane-2-propanol (90:10). Moreover, 7 was completely resolved (R ) 1.33) on the column when hexaneAnalytical Chemistry, Vol. 68, No. 17, September 1, 1996

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phase must be important for achieving efficient resolution. Obviously, more experiments should be done to investigate the effect of solvents on the chiral discrimination, and the present CSPs can be used for this purpose.

Table 4. Effect of Eluents A-D on Resolution of Racemates 4-8 on IIa-120s Columna A k1′ b

B R

k1′ b

C R

k 1′ b

D R

k1′ b

R

0.70(+) ∼1 1.21(-) 1.13 1.17(-) ∼1 0.37(-) ∼1 3.36(-) 1.21

ACKNOWLEDGMENT This work was partially supported by Grant-in-Aids for Scientific Research No. 05559009 and on Priority Areas No. 06242101 from the Ministry of Education, Science, Sports and Culture, Japan.

a Eluent, hexane-2-propanol (90:10) (A), hexane-CHCl -methanol 3 (95:5:1) (B), hexane-CHCl3-THF (95:5:2) (C), and hexane-CHCl3b N,N-dimethylacetamide (95:5:2) (D). The sign in parentheses represents optical rotation of the first-eluting enantiomer. c Eluent, hexaneCHCl3-THF (95:4:1).

SUPPORTING INFORMATION AVAILABLE 13C NMR spectrum of SiG -gluconamide (1 page). See any 4 current masthead page for ordering information.

4 5 6 7 8

0.74(+) 1.47 1.22(+) 1.12 0.77(-) 1.06 0.32(-) ∼1 3.71(-) 1.14

1.12(+) 1.79 2.22(+) 2.54(+) 1.80 4.71(+) 1.31(+) 1.04 3.20(+)c 0.54(+) ∼1 0.61(-) 7.09(-) ∼1 12.5(+)

1.72 1.67 1.05 1.33 1.17

Received for review January 3, 1996. Accepted May 23, 1996.X chloroform-THF (95:5:2) was used as the eluent system, while the coated-type ADMPC could not resolve it (R ≈ 1) with hexane2-propanol (90:10). The selection of the composition of the mobile

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Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

AC960002V X

Abstract published in Advance ACS Abstracts, July 1, 1996.