npgrj_nprot_2007-479 3195..3200

PROTOCOL

Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals Weihua Tang1 & Siu-Choon Ng2 1Department of Chemistry, National University of Singapore, Singapore 117543, Singapore. 2Division of Chemical and Biomolecular Engineering, College of Engineering,

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Nanyang Technological University, Singapore 627833, Singapore. Correspondence should be addressed to W.T. ([email protected]) or S.-C.N. ([email protected]). Published online 6 December 2007; doi:10.1038/nprot.2007.479

We describe a protocol for the synthesis of mono-6A-(1-butyl-3-imidazolium)-6A-deoxy-b-cyclodextrin chloride (BIMCD), a cationic, water-soluble cyclodextrin used in the chiral separation of amino acids and anionic pharmaceuticals by capillary electrophoresis. Starting from commercially available chemicals, BIMCD is synthesized in five steps. The first step involves a nucleophilic substitution between p-toluenesulfonyl chloride and imidazole to afford 1-(p-toluenesulfonyl)imidazole (A). In the second step, a nucleophilic substitution between b-cyclodextrin and A affords mono-6A-(p-toluenesulfonyl)-6A-deoxy-b-cyclodextrin (B). In the third step, a nucleophilic substitution between 1-bromobutane and imidazole affords 1-butylimidazole (C). In the fourth step, a nucleophilic addition between A and C affords BIMCD tosylate. In the final step, anion exchange using an ion-exchange resin yields BIMCD as a highly water-soluble solid. Each step takes up to 2 d, including the time required for product purification. The overall protocol requires approximately 6 d.

INTRODUCTION Chiral separation has attracted considerable attention in academics have been developed5–7. Until now, a large spectrum of negatively and industry, especially in biological, pharmaceutical and agro- charged single-isomer CDs has been developed5,8,9. In comparison, chemical fields1. This interest can be attributed largely to a positively charged single-isomer CDs are less reported, with only a heightened awareness that many compounds of biological and few monosubstituted (one primary hydroxy group substituted)5,10 pharmaceutical interest are chiral, and that their enantiomers and persubstituted ones (only on the primary rim of CD)5,11–13 often exhibit different biological properties, in terms of, for having been synthesized. Recently, we developed the facile synthetic methodologies example, protein binding, pharmacodynamics, pharmacokinetics for novel cationic single-isomer CDs by replacing a CD’s primary and toxicity2. The demand for single enantiomers in developing chiral drugs has led to an outburst of new strategies toward hydroxy group with alkylimidazolium, alkylpyridinium, alkylamasymmetric synthesis on the one hand and new methods of chiral monium or ammonium cations14–17. The CDs have demonresolution on the other3. As such, various chromatographic and strated good resolution abilities toward a large pool of anionic electromigration techniques have been developed to meet the need racemates and amino acids14–19. BIMCD is one such example. The for characterizing chiral compounds to greater extents and with key step in the synthesis of BIMCD involves the nucleophilic greater accuracy and precision. As an ever-increasingly popular addition between B and C (see Figs. 1 and 2), which yields technique for enantioseparation, capillary electrophoresis (CE) is advantageous in – offering fast analysis with high efficiency. O O Cl H N + N i Cyclodextrins (CDs) are the most widely + H3C S Cl H3C N S N N H + H N used chiral selectors for CE4. For solubility O O and separation concerns, charged CDs are A (precipitate) gaining much interest because of their abilities to perform rapid separations at low HO (OH)6 O TsO (OH)6 N concentrations and achieve better resolving ii N H3C S + 5 power to oppositely charged analytes . O O Although randomly multisubstituted, (OH)14 (OH)14 S Ts = H3C charged CDs may provide higher enantioO B selectivity, their practical applications are iii + C4H9Br greatly affected by the irreproducibility of N N N N H C4 H 9 the synthesis from batch to batch and C separation efficiency from run to run. To counter these problems, structurally well- Figure 1 | Synthesis of intermediate compounds B and C. Reaction conditions are as follows: (i) CH Cl , 2 2 defined CDs (either monosubstituted or ice-water bath, overnight to room temperature; (ii) water, 4 h, room temperature, then 20% (wt/vol) persubstituted), that is, single-isomer CDs, NaOH solution, 10 min and (iii) sodium ethoxide, ethanol, reflux. NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3195

PROTOCOL BIMCD tosylate. In the following step, treatment of BIMCD tosylate with chloride ion-exchange resin yields the target cationic CD, as a highly water-soluble solid (see Fig. 2). This protocol


MATERIALS REAGENTS . Imidazole (99%; Merck, cat. no. 436151) . p-Toluenesulfonyl chloride (99%; Fluka, cat. no. 89730) . b-Cyclodextrin (495%; TCI, cat. no. C0900) . Sodium hydroxide (NaOH, 97%; Sigma-Aldrich, cat. no. 138701) . Ammonium chloride (Z99.5%; Fluka, cat. no. 09725) . Sodium ethoxide (96%; Merck, cat. no. 156248) . Sodium acetate (Z99%; Sigma-Aldrich, cat. no. S3272) . 1-Bromobutane (98%; Fluka, cat. no. 19681) . Amberlite IRA-900 ion-exchange resin (Sigma-Aldrich, cat. no. 216585) . Calcium chloride (Z97%; Fluka, cat. no. 21074) . Hexane (98.5%, ACS reagent; Sigma-Aldrich, cat. no. 178918) . Ethyl acetate (99.5%, ACS reagent; Sigma-Aldrich, cat. no. 141786) . Acetone (99.5%, ACS reagent; Sigma-Aldrich, cat. no. 179124) . Ethanol (99.5%, ACS reagent; Merck, cat. no. 459844) . Dichloromethane (CH2Cl2, 99.6%, ACS reagent; Sigma-Aldrich, cat. no. 443484) . N,N-Dimethylformamide (DMF, 99.8%, ACS reagent; Sigma-Aldrich, cat. no. 319937) . Dansyl-DL-glutamic acid bis(cyclohexylammonium) salt (Sigma, cat. no. D8756) . Dansyl-DL-phenylalanine cyclohexylammonium salt (Sigma, cat. no. D9506) . N-Dansyl-DL-threonine cyclohexylammonium salt (Sigma, cat. no. D0881) . Dansyl-DL-valine cyclohexylammonium salt (Sigma, cat. no. D1131) EQUIPMENT . Magnetic stirrer with thermal and speed controller (Heidolph) . Rotary evaporator (Bu¨chi, R205)

describes the synthesis of BIMCD. This methodology can also be applied for the synthesis of other imidazolium-, ammonium-based single-isomer cationic CDs14–17.

. Vacuum pump . Balance . Round-bottomed flask . Conical flask . Allihn/Liebig condenser . Vigreux condenser . Reflux condenser . Teflon-coated magnetic stirring bars . NMR spectrometer (300 MHz; Bruker, ACF300) . Pressure-equalizing addition funnel . Bu¨chner funnel . Drying tube . Dewar dish . Glass and plastic syringes (polypropylene) . Disposable hypodermic syringe needles . Beckman P/ACE MDQ CE unit (Fullerton) with on-line photodiode array (PDA, 190–300 nm) detector and high-voltage (up to 30 kV) power supply

. Uncoated fused-silica capillary (59.2 cm in length, 50 mm in inner diameter; Polymicro Technologies)

. Syringe type filter membrane (0.45 mm pore size; Millipore) EQUIPMENT SETUP CE operating procedure Turn on the Beckman CE system and equilibrate the capillary by flushing sequentially with 0.1 M NaOH, 1 M NaOH and deionized water for 30 min each. Between sample injections, flush the capillary again with 0.1 M NaOH, 1 M NaOH, deionized water and acetate buffer for 2 min each. The sample is injected by 0.5 p.s.i. pressure of nitrogen for 10 s.

PROCEDURE Synthesis of compound A 1| Fit a 250-ml three-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum, an Allihn condenser and a 100 ml pressure-equalizing addition funnel. Fit the condenser with a drying tube (filled with calcium chloride) to prevent the ingress of water. 2| Weigh out 10 g (52.5 mmol) p-toluenesulfonyl chloride into the flask under the protection of nitrogen. m CRITICAL STEP p-Toluenesulfonyl chloride is very smelly and highly corrosive. It is recommended that it be weighed in a glovebox and transferred in a sealed bottle. Please refer to the MSD sheet of this compound for safety information. 3| Transfer 50 ml dry CH2Cl2 into the flask with a glass syringe fitted with a 20-gauge hypodermic needle. ? TROUBLESHOOTING

HO

3196 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

H3C

N

N

S O

(OH)14

(OH)14 B

(OH)6

TsO

5| Fit a 100-ml two-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum. Degas the flask by applying two freeze–pump–thaw cycles and fill the flask with nitrogen.

7| Transfer the solution from Step 6 to the addition funnel in Step 1 with a glass syringe. ! CAUTION It is essential to perform this reaction with exclusion of water, as the presence of water will reduce the yield.

O +

4| Cool the flask to 0 1C in ice-water bath (Dewar dish), under stirring.

6| Under nitrogen protection, add 8 g (118 mmol) imidazole and 50 ml dry CH2Cl2 into the flask. Dissolve imidazole in CH2Cl2 under stirring. ? TROUBLESHOOTING

(OH)6

TsO

(OH)6

+

N

R

i

N

(OH)14 B

C –

OTs R

N

+

N

R

(OH)6

N

+

Cl

–

N

(OH)6

ii

(OH)14 R = C4H9 BIMCD tosylate

(OH)14 R = C4H9 BIMCD

Figure 2 | Synthesis of BIMCD. Reaction conditions are as follows: (i) DMF, 90 1C, 48 h and (ii) Amberlite IRA-900 ion-exchange resin.

PROTOCOL 8| Gradually add the imidazole solution to the reaction flask over 1.5 h. Maintain the temperature and stirring as in Step 4. 9| Turn on the water for the condenser to control the reflux of CH2Cl2. 10| Allow the reaction to warm to room temperature (25 1C). Continue vigorous stirring for an additional 2 h. ’ PAUSE POINT Can be left overnight at room temperature. 11| Filter off the insoluble solid. Collect the filtrate. 12| Concentrate the filtrate to B20 ml using rotary evaporator at 40 1C. Precipitate the product using hexane (150 ml).


13| Collect the solid by filtration. Wash the solid with ethyl acetate (50 ml 3). 14| Dry the solid overnight at reduced pressure, to yield A as a white solid (10.5 g, 90% yield). ’ PAUSE POINT Compound A can be stored in nitrogen at room temperature for several months. Synthesis of compound B 15| Mix b-cyclodextrin (35.0 g, 30.84 mmol), A (8.9 g, 40.0 mmol) and 350 ml deionized water in a 1-liter conical flask containing a stir bar. ? TROUBLESHOOTING 16| Allow the reaction to proceed for 4 h at room temperature under vigorous stirring. 17| Gradually add aqueous NaOH solution (20% (wt/vol), 50 ml) to the mixture and continue stirring for an additional 10 min. 18| Filter off the insoluble solid and collect the filtrate. 19| Neutralize the filtrate to pH 7 with NH4Cl (B24.2 g, 0.45 mol) to induce precipitation. Check the pH of the solution with pH paper. 20| Collect the precipitate by filtration. Wash the solid residue with cold water (100 ml 3) and acetone (100 ml 2). 21| Dry the solid in a drying oven at 60 1C under vacuum (10 mm Hg) overnight to yield B as a white solid (16.7 g, 42% yield). ’ PAUSE POINT Compound B can be stored in nitrogen at room temperature for several months. Synthesis of compound C 22| Fit a 250-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a Liebig condenser and a 50 ml dropping funnel. 23| Degas the flask by applying two freeze–pump–thaw cycles. Fill the flask with nitrogen. 24| Under the protection of nitrogen, add 15 g (220 mmol) imidazole and 75 ml ethanol into the flask. Turn on the magnetic stirrer. 25| Add sodium ethoxide (16.5 g, 243 mmol) into the flask in one portion. Allow the resulting warm solution to mix for 30 min. m CRITICAL STEP Sodium ethoxide is moisture sensitive; thus, it is recommended to perform the weighing and transferring steps quickly. ? TROUBLESHOOTING 26| Gradually add 26 ml anhydrous 1-bromobutane (64.0 g, 243 mmol) into the reaction flask with the aid of a dropping funnel over 1 h. 27| Reflux the reaction mixture for 30 min before cooling down to room temperature. 28| Filter off the solid and wash with CH2Cl2. Concentrate the pooled filtrate with rotary evaporator at 40 1C. 29| Distill the residual liquid over a 10 cm Vigreux condenser under 3.5 Mbar pressure. Collect the distillate at temperature between 84 and 86 1C to yield C as a colorless liquid (15.8 g, 58% yield). m CRITICAL STEP Distillation should be carried out in a fume hood using a cold trap filled with dry ice and acetone to minimize the smell of 1-bromobutane. ’ PAUSE POINT Compound C can be stored in a refrigerator at 4 1C for several months. Synthesis of BIMCD tosylate 30| Set up a reaction by mixing B (12.9 g, 10 mmol), C (3.7 g, 30 mmol) and DMF (25 ml) in a 100-ml one-neck, round-bottomed flask, equipped with Liebig condenser and a stir bar. Fit the Liebig condenser with a septum. NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3197

PROTOCOL 31| Degas the flask and fill it with nitrogen as in Step 23. 32| Allow the reaction to proceed at 90 1C for 48 h under reflux. Cool down the reaction mixture to room temperature. ? TROUBLESHOOTING 33| Precipitate out the product using acetone (150 ml). 34| Collect the precipitate by filtration. Wash the solid with acetone.


35| Dry the solid overnight at 60 1C in a vacuum oven (10 mm Hg) to yield BIMCD tosylate as a white solid (12.3 g, 87% yield). ’ PAUSE POINT BIMCD tosylate can be stored in nitrogen at room temperature for several months. Synthesis of BIMCD 36| Dissolve the BIMCD tosylate (1.4 g, 1 mmol) in 50 ml deionized water. 37| Fill an addition funnel (100 ml) with Amberlite IRA-900 ion-exchange resin to one-half its original height. 38| Transfer the solution from Step 36 into the addition funnel, and let it stand for 1 h. Subsequently, collect the eluent. 39| Distill off water under reduced pressure using a vacuum pump. 40| Dry the solid residue overnight at 60 1C in a vacuum oven (10 mm Hg) to yield BIMCD as a light yellow solid (1.2 g, 94% yield). ’ PAUSE POINT BIMCD can be stored in nitrogen at room temperature for several months. ? TROUBLESHOOTING

TIMING Synthesis of A: Steps 1–4, 2 h; Steps 5–7, 30 min; Steps 8 and 9, 2 h; Step 10, 2 h; Steps 11–13, 1 h; Step 14, 12 h Synthesis of B: Steps 15–18, 5 h; Steps 19 and 20, 1 h; Step 21, 12 h Synthesis of C: Steps 22–25, 1 h; Step 26, 1 h; Steps 27 and 28, 2 h; Step 29, 4 h Synthesis of BIMCD tosylate: Steps 30 and 31, 1 h; Step 32, 48 h; Steps 33 and 34, 2 h; Step 35, 12 h Synthesis of BIMCD: Steps 36 and 37, 30 min; Step 38, 2 h; Step 39, 2 h; Step 40, 12 h ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 | Troubleshooting table. Problem Low yield of compound A

Possible reasons Solvent (CH2Cl2) is of poor quality; moisture intervention (Steps 3 and 6)

Solution Store CH2Cl2 over calcium hydride and distill it from calcium hydride immediately before use. Ensure that the reaction is carried out under nitrogen protection

Low yield of compound B

b-Cyclodextrin contains high content of H2O. Large particles of A present (Step 15)

Dry b-cyclodextrin overnight at 60 1C in a vacuum oven. Grind the large particles of A with mortar and pestle before adding to the reaction

Low yield of compound C

Moisture absorbed by sodium ethoxide in the course of weighing and transferring into the flask (Step 25)

Weigh sodium ethoxide quickly and store it in a sealed bottle before adding into the flask in one portion

Low yield of BIMCD tosylate

Reaction conditions are not guaranteed, that is, time is shorter than 48 h and temperature below 85 1C (Step 32)

Ensure that the reaction proceeds under the required conditions, with nitrogen protection

Low yield of BIMCD

Amberlite IRA-900 ion-exchange resin is of poor quality; moisture intervention; short time for ion exchange

Repeat the reaction with fresh ion-exchange resin. Ensure complete ion exchange by equilibrating the ion-exchange bed for 1 h

ANTICIPATED RESULTS Typical yield The overall yield of the product is approximately 31%. Analytical data NMR spectroscopy data of A, B, C, BIMCD tosylate and BIMCD, along with mass spectroscopy and elemental analysis data for B, BIMCD tosylate and BIMCD are given below. 3198 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

PROTOCOL


Compound A: 1H NMR (300 MHz, DMSO-d6): d 8.35 (s, 1H), 7.96 (d, J ¼ 7.2 Hz, 2H), 7.71 (s, 1H), 7.50 (d, J ¼ 7.2 Hz, 2H), 7.11 (s, 1H), 2.39 (s, 3H). Compound B: 1H NMR (300 MHz, DMSO-d6): d 7.72 (d, J ¼ 8.4 Hz, 2H), 7.41 (d, J ¼ 8.4 Hz, 2H), 5.60–5.89 (m, 14H), 4.75–4.81 (m, 7H), 4.15–4.62 (m, 6H), 3.45–3.72 (m, 28H), 3.15–3.47 (m, overlapping with HDO, m, 24H), 2.41 (s, 3H). 13C NMR (75 MHz, DMSO-d ): d 145.3, 133.1, 130.4, 128.0, 6 102.4, 82.0, 81.2, 73.5, 73.2, 72.9, 72.5, 70.2, 69.4, 60.4, 21.7. ESI-MS (m/z): calculated, 1,288.4 for C49H76O37S; found, 1,311.5 for [M + Na]+. Elemental analysis: calculated, C 45.67, H 5.91, S 2.50; found, C 42.71, H 5.79, S 2.29.

Abs (AU)

Dns-Val Dns-Phe Dns-Thr

Dns-Glu EOF

4

8

12

16

20

24

28

32

36

Migration time (min)

Compound C: 1H NMR (300 MHz, CDCl3): d 7.42 (s, 1H), 7.01 (s, 1H), 6.86 (s, 1H), 3.89 (t, J ¼ 7.2 Hz, 2H), 1.70 (q, J ¼ 7.2 Hz, J ¼ 7.5 Hz, 2H), 1.28 (m, 2H), 0.89 (t, J ¼ 7.2 Hz, 3H).

Figure 3 | Enantiomeric separation of a mixture containing four Dns-amino acids with 10 mM BIMCD. Dns, dansyl; Phe, phenylalanine; Val, valine; Thr, threonine; Glu, glutamic acid; EOF, electroosmotic flow.

BIMCD tosylate: 1H NMR (300 MHz, DMSO-d6): d 9.09 (s, 1H), 7.78 (s, 1H), 7.72 (s, 1H), 7.48 (d, J ¼ 7.6 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 5.98 (d, J ¼ 5.8 Hz, 1H), 5.63–5.84 (m, 13H), 4.97 (d, J ¼ 3.6 Hz, 1H), 4.85 (d, J ¼ 3.2 Hz, 6H), 4.54 (t, J ¼ 5.2 Hz, 3H), 4.47 (t, J ¼ 5.2 Hz, 2H), 4.30 (t, J ¼ 8.8 Hz, 1H), 4.14 (t, J ¼ 6.6 Hz, 2H), 4.00 (t, J ¼ 10.5 Hz, 1H), 3.83 (t, J ¼ 7.0 Hz, 2H), 3.54–3.64 (m, 25H), 3.22–3.38 (overlap with HDO, m, 12H), 3.04–3.07 (m, 1H), 2.83–2.89 (m, 1H), 2.29 (s, 3H), 1.78 (q, J ¼ 7.0 Hz, 2H), 1.27 (m, 2H), 0.90 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): d 146.7, 137.5, 136.6, 127.9, 126.4, 123.0, 122.2, 101.9, 83.2, 81.5, 73.2, 72.3, 72.0, 69.6, 60.0, 49.9, 48.6, 31.0, 30.7, 20.7, 18.8, 13.2. Elemental analysis: calculated for C56H88N2S1O37 2H2O (1,499.38), C 46.40, H 6.40, N 1.93, S 2.21; found, C 46.46, H 6.59, N 2.16, S 1.92. ESI-MS (m/z): calculated for [M+], 1,241.60; found, 1,241.61; calculated for [OTs], 171.19; found, 171.5. BIMCD: 1H NMR (300 MHz, DMSO-d6): d 9.18 (s, 1H), 7.80 (s, 1H), 7.73 (s, 1H), 5.95 (d, J ¼ 6.0 Hz, 1H), 5.63–5.82 (m, 13H), 4.96 (d, J ¼ 3.6 Hz, 1H), 4.84 (d, J ¼ 3.2 Hz, 6H), 4.56 (t, J ¼ 5.2 Hz, 3H), 4.41–4.32 (m, 3H), 4.29 (t, J ¼ 8.8 Hz, 1H), 4.13 (t, J ¼ 6.6 Hz, 2H), 3.81 (t, J ¼ 7.0 Hz, 2H), 3.52–3.65 (m, 25H), 3.22–3.46 (m, 12H), 3.07 (m, 1H), 2.83 (m, 1H), 1.76–1.80 (q, J ¼ 7.0 Hz, 2H), 1.22–1.29 (m, 2H), 0.74 (t, J ¼ 7.2 Hz, 3H). Elemental analysis, calculated for C49H81N2Cl1O34 (1,277.64), C 46.06, H 6.39, N 2.19, Cl 2.77; found, C 44.92, H 6.49, N 1.83, Cl 3.06. ESI-MS (m/z): calculated for [M+], 1,242.19; found, 1,242.60. CE enantiomeric separation data Typical enantiomeric separation via CE is demonstrated in Figure 3. A mixture of four dansyl-DL-amino acids was efficiently separated in a single run within 36 min by using 10 mM BIMCD in 50 mM acetate buffer (pH 6.0) at 25 1C. The stock mixture solution (50 mg ml1) was prepared with 50:50 (vol/vol) methanol/water mixture solution, filtered with 0.45 mm syringe type Millipore membrane. Acetate buffer was prepared by dissolving a desirable amount of sodium acetate in water, with its pH (6.0) adjusted with acetic acid.

ACKNOWLEDGMENTS We are grateful to the National University of Singapore and the Institute of Chemical and Engineering Sciences, Singapore, for their financial support. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Ahuja, S Chiral Separations by Chromatography (ACS, New York, USA, 2000). 2. Ikai, T., Yamamoto, C., Kamigaito, M. & Okamoto, Y. Immobilized polysaccharide derivatives: chiral packing materials for efficient HPLC resolution. Chem. Rec. 7, 91–103 (2007). 3. Agranat, I., Caner, H. & Cadwell, J. Putting chirality to work: the strategy of chiral switches. Nat. Rev. Drug Discov. 1, 753–768 (2002). 4. Ward, T.J. Chiral separation. Anal. Chem. 78, 3947–3956 (2006). 5. de Boer, T., de Zeeuw, R.A., de Jong, G.J. & Ensing, K. Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis. Electrophoresis 21, 3220–3239 (2000).

6. Nair, U.B. & Armstrong, D.W. Evaluation of two amine-functionalized cyclodextrins as chiral selectors in CE: comparisons to vancomycin. Microchem. J. 57, 199–217 (1997). 7. Chankvetadze, B. & Blaschke, G. Enantioseparations in capillary electromigration techniques: recent developments and future trends. J. Chromatogr. A 906, 309–363 (2001). 8. Zhu, W. & Vigh, G. A family of single-isomer, sulfated g-cyclodextrin chiral resolving agents for capillary electrophoresis: octa(6-o-sulfo)-g-cyclodextrin. Electrophoresis 24, 130–138 (2003). 9. Kirby, D.M. & Vigh, G. Heptakis(2-o-methyl-3,6-di-o-sulfo)-b-cyclodextrin: a single-isomer, 14-sulfated–b-cyclodextrin for use as a chiral resolving agent in capillary electrophoresis. Electrophoresis 22, 3152–3162 (2001). 10. Iva´nyi, R., Jicsinszky, L., Juvancz, Z., Roos, N., Otta, K. & Szejtli, J. Influence of (hydroxy)alkylamino substituents on enantioseparation ability of single-isomer amino-b-cyclodextrin derivatives in chiral capillary electrophoresis. Electrophoresis 25, 2675–2686 (2004). 11. Lee, D. & Shamsi, S.A. Chiral separation of anionic and neutral compounds using a hepta-substituted cationic b-cyclodextrin as a chiral selector in capillary electrophoresis. Electrophoresis 23, 1314–1319 (2002).

NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3199

PROTOCOL


12. Iva´nyi, R., Jicsinszky, L. & Juvancz, Z. Chiral separation of pyrethroic acids with single isomer permethyl monoamino b-cyclodextrin selector. Electrophoresis 22, 3232–3241 (2001). 13. Budanova, N., Shapovalova, E., Lopatin, S., Varlamov, V. & Shpigun, O. Heptakis(6-amino-6-deoxy)-b-cyclodextrin as a chiral selector for the separation of anionic analyte enantiomers by capillary electrophoresis. Electrophoresis 25, 2795–2802 (2004). 14. Tang, W.H., Ong, T.T., Muderawan, I.W., Ng, S.C. & Chan, H.S.O. Synthesis and applications of single-isomer 6-mono(alkylimidazolium)-b-cyclodextrins as chiral selectors in chiral capillary electrophoresis. Electrophoresis 26, 3839–3848 (2005). 15. Tang, W.H., Muderawan, I.W., Ong, T.T. & Ng, S.C. Facile synthesis and enantioseparation performance of positively charged single-isomers of a- and g-cyclodextrin. Tetrahedron Asymmetry 18, 1548–1553 (2007).

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16. Tang, W.H., Muderawan, I.W., Ong, T.T., Ng, S.C. & Chan, H.S.O. Synthesis and application of mono-6-ammonium-6-deoxy-b-cyclodextrin chloride as chiral selector for capillary electrophoresis. J. Chromatogr. A 1094, 187–191 (2005). 17. Tang, W.H., Muderawan, I.W., Ong, T.T. & Ng, S.C. Enantioseparation of acidic enantiomers in capillary electrophoresis using a novel single-isomer positively charged b-cyclodextrin: mono-6A-N-pentylammonium-6A-deoxy-b-cyclodextrin chloride. J. Chromatogr. A 1091, 152–157 (2005). 18. Tang, W.H., Ong, T.T., Muderawan, I.W. & Ng, S.C. Effect of alkylimidazolium substituents on enantioseparation ability of single-isomer alkylimidazoliumb-cyclodextrin derivatives in capillary electrophoresis. Anal. Chim. Acta 585, 227–233 (2007). 19. Tang, W.H., Ong, T.T. & Ng, S.C. Chiral separation of dansyl amino acids in capillary electrophoresis using mono-(3-methyl-imidazolium)-b-cyclodextrin chloride as selector. J. Sep. Sci. 30, 1343–1349 (2007).