Journal of Chromatographic Science Advance Access published December 3, 2014 Journal of Chromatographic Science 2014;1– 4 doi:10.1093/chromsci/bmu158

Article

Enantioseparation of Three Important Intermediates of Tanikolide with Immobilized Cellulose Chiral Stationary Phase Zhou Jie1,2, Du Qiuzheng1, Zhao Suzhen1, Sun Fang1, Li Xinyu1 and Zhang Zhenzhong1,2* 1 School of Pharmacy, Zhengzhou University, Zhengzhou, Henan 450001, PR China, and 2Key Laboratory of State Ministry of Education for Pharmaceutical Technology, Zhengzhou, Henan 450001, PR China

*Author to whom correspondence should be addressed. Email: [email protected] Received 11 September 2013; revised 8 October 2014

Three stereoselective HPLC methods have been developed for the chiral separation of the enantiomers of three intermediates in the preparation of (1)-tanikolide. The enantiomers were separated on Chiralpak IC (250 3 4.6 mm, 5 mm) in normal phase HPLC. Three intermediates were all baseline separated (RS 5 2.84, 2.58 and 3.58, respectively). By comparing the chromatograms of racemates and single enantiomers of the three intermediates, the e.e. values of the single enantiomers were determined and calculated.

area of two isomers, the formula as follows: e:e: ð%Þ ¼ jASiso  ARiso j=ðASiso þ ARiso Þ  100% (A is peak area, S-iso and R-iso are S- and R-isomer, respectively). In this study, the e.e. values of the single intermediates (as shown in Figure 2) of (þ)-tanikolide were determined by normal phase HPLC (NP-HPLC) analysis on chiralpak IC. Herein we report the results of our studies concerning the effect of variation of experimental conditions and optimization of the chiral separation of the enantiomers of the three intermediates. The e.e. values of the single intermediates were also determined.

Introduction (þ)-Tanikolide (as shown in Figure 1) is an antifungal marine metabolite, isolated from the lipid extract of the marine cyanobacterium Lyngbya majuscule, collected from Tanikeli Island, Madagascar (1). It exhibited antifungal activity against Candida albicans, along with LD50 of 3.6 mg/mL against brine shrimp and 9.0 mg/mL against the snail (2). Because of its structural feature and the unique bioactivity, (þ)-tanikolide has become an attractive target for organic synthesis (3 – 6). (þ)-Tanikolide is a toxic and antifungal agent, whereas (2)-tanikolide has no pharmaceutical activities, and the purity of the intermediates affect the purity of (þ)-tanilolide, So it is very important to separate the racemates and determine the e.e. values of the single intermediates in the preparation of (þ)-tanikolide. The cellulosic-based chiral stationary phases (CSPs) have been one of the most widely utilized materials for enantiomeric resolution by HPLC. The first generation is coated on silica gels and has mobile phase limitations because certain solvents will dissolve the coating. The second generation is chemically immobilized and overcomes the limitations of the coated CSPs with a number of advantages, such as new selectivity profile, enhancement of sample solubility in the mobile phase, etc. (7). In this study, chiralpak IC, which is made by immobilizing cellulosic tris (3,5-dichlorophenylcarbamate) on silica gels, was used to separate the three racemate intermediates of (þ)-tanikolide for the first time. Relative to similar columns, chiralpak IC might possess advantages in terms of robustness and the range of mobile phase solvents that can be utilized (8 – 10). The most versatile mobile phases on chiralpak IC are hexane-isopropanol, hexaneethanol, methyl t-butyl ether-based and dichloromethane-based eluents. The e.e. values refer to the enantiomeric excess of one enantiomer relative to the other. There are numerous methods to determine the e.e. values, for example, polarimetry, HPLC, GC and NMR. In HPLC, the e.e. values are calculated by the peak

Experimental Apparatus The HPLC instrument used in this study was an Agilent 1100 series apparatus (Palo Alto, CA, USA). It was equipped with a quaternary pump, a vacuum degasser, a column oven, a multiple wavelength UV detector, an auto-sampler and HP Chemstation software (Rev. B. 04. 01 SP1 [647]). The analysis was carried out on chiralpak IC (250  4.6 mm, 5 mm; Daicel, Japan).

Reagents HPLC-grade n-hexane, ethanol and isopropanol were obtained from T&J Kermel Reagent Company. The racemates of the three intermediates were obtained from Zhengzhou Chuangsheng Biological Engineering Company (Zhengzhou, China). The single enantiomer of the three intermediates was synthesized by Yang Hua (School of Pharmacy, Zhengzhou University) who devoted himself in asymmetric synthesis of (þ)-tanikolide with optically active amino alcohol as raw material. The synthetic scheme of (þ)-tanikolide can refer to Ref. (11).

Sample preparation 1.0 mg racemic compounds (1), (2) and (3) were dissolved in 1.0 mL ethanol, respectively. The solutions were all filtered (0.22 mm) to prepare sample solution.

Chromatographic condition The basic solvent of mobile phase was n-hexane, ethanol or isopropanol was chosen as mobile phase modifier. Mobile phase was filtered with 0.45 mm solvent filter and ultrasonically degassed. Separations were performed on chiralpak IC at 15 –358C with a

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flow rate of 0.4 – 1.2 mL min21. The detection wavelengths of compounds (1) and (2) were all set at 206 nm, compound (3) was set at 230 nm. The volume of sample injected was 10 mL. The dead time was determined by injecting 1,3,5-tert-butyl benzene as a non-retained marker.

in Table II). But in this study, it was found that the resolution of compound might be increased by increasing temperature (e.g., compound (3) in Table II). Parameters were calculated by the equation (16) as below:

ln a ¼  4R;S 4 H W =RT þ 4R;S 4 S W =R: Results and Discussion Effect of alcohols modifier The effect that the content of mobile phase modifiers had on the enantioselectivity of the three racemic compounds was studied. The results were shown in Table I. Hydrogen bonding, p –p, dipole– dipole stacking and steric interactions, hydrophobic interactions between analytes and CSPs played an important role in the mechanism of enantiorecognition (12). In this study, the interactions between the three intermediates and CSPs were hydrogen bonding, p – p and steric interactions. By increasing the alcohols content of the mobile phase, its eluting strength increased, the hydrophobic interactions between analytes and CSPs were weakened, and the resolution was decreased. The logical approach to improving chiral resolution was to allow for longer retention. However, in cases when retention time were longer, further decreasing the organic modifier content in the mobile phase might produce baseline separation only with very long retention time or might not work at all. This latter phenomenon could be explained by an excessive increase in peak widths noticed at long retention time occasionally counterbalancing any benefit in regards of selectivity (13). By mobile phase modified with ethanol, the retention time was shorter than with isopropanol, but the resolution was decreased at the same time.

Here, a ¼ k20 =k10 , a is separation factor, R is the gas constant and T is the temperature in K, 4 R,S 4H W and 4 R,S 4S W are enthalpy change and entropy change of two enantiomers from the mobile phase to the stationary phase during the distribution process, respectively. Van 0 t Hoff plots were drawn for logarithm of a (ln a) versus inverted temperature (1/T) for two isomers. The regression equations of the three racemic compounds were shown in Table III. The values of 4 R,S 4H W and 4 R,S 4S W were obtained from slope and intercept of the straight lines, respectively (as reported in Table III). Over the temperature ranges of 288 – 308 K, for compounds (1) and (2), j4 R,S 4H Wj.jT4 R,S 4S W j, for compound (3), j4 R,S 4H Wj,jT4 R,S 4S Wj. Therefore, the separation process of compounds (1) and (2) were controlled by enthalpy, compound 3 was controlled by entropy. The results indicated that the temperature should be carefully controlled for optimum chiral separation of enantiomers retention. Due to 258C closer to room temperature, the other parameters were optimized at 258C.

Table I Effect of Content of Alcohols on the Enantioselectivity of the Three Racemic Compounds Compound

Effect of column temperature The effect that the temperature had on the retention and resolution of the three racemic compounds was studied in the range of 15 –358C. The separation data were shown in Table II. Several authors have reported improved resolution on polysaccharide-based CSPs when the column was operated at sub-ambient temperature (14, 15) (e.g., compounds (1) and (2)

tR1 (1)

(2)

(3)

Figure 1. The structure of (þ)-tanikolide.

Figure 2. The structure of the single intermediates.

2 Jie et al.

Isopropanol (modifier) tR2

48.43 73.50 15.69 23.46 7.20 9.34 5.50 6.59 4.87 5.51 23.71 29.01 11.64 13.39 6.82 7.36 5.64 5.11 .100 34.16 51.20 15.35 20.57 10.83 13.55 9.13 10.90

Ethanol (modifier)

a

RS

%

tR1

tR2

1.55 1.62 1.53 1.45 1.36 1.26 1.21 1.15 – – – 1.55 1.43 1.35 1.30

4.95 4.31 2.84 1.74 1.10 2.58 1.47 0.81 – – – 8.24 5.98 4.57 3.58

5 10 20 30 40 5 10 20 30 40 5 10 20 30 40

19.04 20.99 8.93 9.64 5.76 5.93 4.92 4.58 13.34 14.28 7.89 8.17 5.72 4.94 4.69 26.03 29.09 13.42 14.30 8.40 8.66 6.77 6.04

a

RS

%

1.12 1.12 1.07 – – 1.09 1.06 – – – 1.13 1.09 1.05 – –

1.44 1.24 0.39 – – 1.03 0.57 – – – 2.54 1.50 0.63 – –

5 10 20 30 40 5 10 20 30 40 5 10 20 30 40

tR1, tR2, retention times; a, separation factor; RS, resolution factor; “– ”, retention time is too long (.100 min). Chromatographic conditions: the basic solvent of mobile phase was n-hexane, the column temperature was at 258C with a flow rate of 0.8 mL min21.

Effect of flow rate In the present study, the effect of the flow rate on enantioresolution of the three racemic compounds was studied, resolution values were recorded in Table IV.

Table II Effect of Temperature on the Enantioselectivity of the Three Racemic Compounds Compound

tR1

tR2

a

RS

Temperature (8C)

(1)

7.65 7.35 7.20 7.00 6.89 27.64 25.04 23.71 22.74 22.02 12.05 9.89 9.13 8.65 7.96

10.36 9.66 9.34 8.89 8.59 34.55 31.02 29.01 27.67 26.44 14.18 11.78 10.90 10.23 9.38

1.60 1.55 1.53 1.48 1.45 1.28 1.27 1.26 1.25 1.24 1.25 1.28 1.30 1.32 1.34

3.26 3.15 2.84 2.66 2.47 2.92 2.80 2.58 2.44 2.25 2.71 3.41 3.58 3.68 3.75

15 20 25 30 35 15 20 25 30 35 15 20 25 30 35

(2)

(3)

With increased the flow rate of the mobile phase, there was observed decreased resolution. According to the Van Deemter rate theory, the longitudinal diffusion played a major role at lower flow rate, and the mass transfer resistance played a major role at higher rate. In order to reduce the analytical time and to get better separation, the flow rate of 0.8 mL min21 was used. The chromatograms of the three compounds were shown in Figure 3. Determination of the e.e. values The e.e. values of the single enantiomers of the three intermediates were shown in Figure 4. The results showed that the first peak of compound (3) was determined as R-isomer, and the after peak as S-isomer. In addition, the e.e. values of the single enantiomers of the three

Table IV Effect of Flow Rate on the Enantioselectivity of the Three Racemic Compounds Compound

tR1

tR2

a

RS

Flow rate (mL min21)

(1)

14.21 9.51 7.20 5.75 4.78 45.12 30.58 23.71 18.74 15.99 18.33 12.25 9.13 7.31 6.07

18.33 12.26 9.34 7.41 6.16 55.00 37.25 29.01 22.84 19.52 21.79 14.56 10.90 8.68 7.20

1.52 1.51 1.51 1.51 1.51 1.25 1.25 1.26 1.25 1.25 1.29 1.29 1.29 1.28 1.28

2.93 2.90 2.84 2.74 2.67 2.66 2.60 2.58 2.49 2.40 4.18 3.86 3.58 3.35 3.15

0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2

Chromatographic conditions: compound (1) was n-hexane-isopropanol (80/20, v/v), compound (2) was n-hexane-isopropanol (95/5, v/v) and compound (3) was n-hexane-isopropanol (60/40, v/v). The flow rate was 0.8 mL min21. (2)

Table III The Thermodynamic Data of the Three Racemic Compounds

(3)

Compound

(1)

(2)

(3)

4 R,S 4H W (kJ/mol) 4 R,S 4S W (J/mol K) The regression equation

20.441 21.064 ln a ¼ 442.98/ T 2 1.0681 (R ¼ 0.9961)

20.144 20.254 ln a ¼ 144.40/ T 2 0.2539 (R ¼ 0.9995)

0.308 1.294 ln a ¼ 309.17/ T þ 1.2987 (R ¼ 0.9962)

Chromatographic conditions: compound (1) was n-hexane-isopropanol (80/20, v/v), compound (2) was n-hexane-isopropanol (95/5, v/v) and compound (3) was n-hexane-isopropanol (60/40, v/v). The column temperature was at 258C.

Figure 3. HPLC chromatograms of the three racemic compounds. Chromatographic conditions: compound (1) was n-hexane-isopropanol (80/20, v/v), compound (2) was n-hexane-isopropanol (95/5, v/v) and compound (3) was n-hexane-isopropanol (60/40, v/v). The column temperature was at 258C with a flow rate of 0.8 mL min21.

Enantioseparation of Three Important Intermediates of Tanikolide 3

Figure 4. Chromatograms of the single enantiomers of the three single compounds. Chromatographic conditions: compound (1) was n-hexane-isopropanol (80/20, v/v), compound (2) was n-hexane-isopropanol (95/5, v/v) and compound (3) was n-hexane-isopropanol (60/40, v/v). The column temperature was at 258C with a flow rate of 0.8 mL min21.

intermediates were also determined (e.e. 98.6%, e.e. 95.4% and e.e. 93.1%, respectively).

7.

Conclusion The enantiomers of the three intermediates in the preparation of (þ)-tanikolide were first separated on chiralpak IC in NP-HPLC. The optimum chromatographic conditions of the three compounds are as follows: compound (1): n-hexane-isopropanol (80/ 20, v/v), RS ¼ 2.84; compound (2): n-hexane-isopropanol (95/5, v/v), RS ¼ 2.58; compound (3): n-hexane-isopropanol (60/40, v/v), RS ¼ 3.58. The column temperature was at 258C with a flow rate of 0.8 mL min21. The volume of sample injected was 10 mL. The e.e. values of the three single intermediates of (þ)tanikolide were also determined by NP-HPLC on chiralpak IC. References 1. Singh, I.P., Milligan, K.E., Gewick, W.H.; Tanikolide, a toxic and antifungal lactone from the marine cyanobacterium Lyngbya majuscula; Journal of Natural Products, (1999); 62(9): 1333– 1335. 2. Zhang, C.X., Hosoda, N., Asami, M.; Concise asymmetric synthesis of (R)-(þ)-tanikolide; Tetrahedron: Asymmetry, (2007); 18: 2185–2189. 3. Vichare, P., Chattopadhyay, A.; Organometallation of (R)-2,3-cyclohexylideneglyceraldehyde derived ketones: a simple and stereoselective strategy for the synthesis of (þ)-tanikolide; Tetrahedron: Asymmetry, (2008); 19: 598–602. 4. Doran, R., Duggan, L., Singh, S., Duffy, C.D., Guiry, P.J.; Asymmetric synthesis of (þ)-tanikolide and the ss-methyl-substituted analogues of (þ)-tanikolide and (2)-malyngolide; European Journal of Organic Chemistry, (2011); 35: 7097–7106. 5. Chen, Q.S., Deng, H.B., Zhao, J.R., Lu, Y., He, M.Y., Zhai, H.B.; Two efficient four-step routes to marine toxin tanikolide; Tetrahedron, (2005); 61: 8390–8393. 6. Tadaaki, O., Shigeru, N.; Total synthesis of (þ)-tanikolide, a toxic and antifungal d-lactone, utilizing bromoalkene intermediates

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