Mayaka
Maeno
a,
Etsuko
Tokunaga
a,
Takeshi
Yamamoto
a,
Toshiya
Suzuki
b,
Yoshiyuki
Ogino
b,
Emi
Ito
a,
Motoo
Shiro
c,
Toru
Asahi
*b and
Norio
Shibata
*a
aDepartment of Nanopharmaceutical Sciences and Department of Frontier Materials, Nagoya Institute of Technology, Gokiso, Showa-ku, Nagoya 466-8555, Japan. E-mail: nozshiba@nitech.ac.jp; Fax: +81-52-735-7543
bDepartment of Life Science and Medical Bioscience, Waseda University (TWIns), Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan. E-mail: tasahi@waseda.jp
cConsolidated Research Institute for Advanced Science and Medical Care, Waseda University (ASMeW), Waseda-tsurumaki-cho 513, Shinjuku-ku, Tokyo 162-0041, Japan
First published on 30th October 2014
We report on the self-disproportionation of enantiomers (SDE) of non-racemic thalidomide (1) and 3′-fluorothalidomide (2) under the conditions of gravity-driven achiral silica-gel chromatography. The presence of a fluorine atom on the chiral center dramatically alters the structure and polarity of 1 and 2, resulting in the opposite SDE profile on silica-gel.
Self-disproportionation of enantiomers (SDE) was coined by Soloshonok in 2006 (ref. 6) to describe a process by which enantiomerically enriched compounds are separated into fractions of a different proportion of enantiomers (enantiomerically enriched and depleted), compared to the original sample, without the assistance of any external chiral sources.7 This phenomenon is fundamentally general and can be expected for any chiral compound being subjected to achiral chromatography,8 sublimation9 or distillation.10 While the phenomenon itself might not be surprising the SDE phenomenon has never been systematically studied and therefore is still unpredictable in terms of the relationship between the observed magnitude of SDE and compound structures.11
During our research on thalidomide and its derivatives,12 we came across the unique behavior of non-racemic 1 and fluorinated analogue 2 under the conditions of a commonly used gravity-driven achiral chromatography. In this paper, we disclose that both non-racemic 1 and 2 show high magnitude of SDE, but their SDE profiles are completely opposite. Thus, achiral chromatography of non-racemic 1 (35.5% ee) resulted in isolation of enantiomerically enriched 1 (87% ee) in the first fraction while enantiomerically depleted 1 (21% ee) was observed in the last fraction. On the other hand, 2 with a highest ee of 71%, was eluted in the last fraction under similar achiral chromatographic conditions, while 2 with at lowest 30% ee was found in the first fraction, different from the original ee of 2, 34%. X-ray crystallographic analysis and computations of 1 and 2 revealed that the introduction of a single fluorine atom in the chiral center of 1 dramatically altered the monomeric and dimeric structures, and logP values of 1. The opposite behaviors of 1 and 2 on SDE can be explained by the difference of aggregations and polarities of chiral, non-racemic 1 and 2 and racemic 1 and 2 (Fig. 1).
An experiment was conducted to examine whether SDE occurs for 1 under conventional chromatographic conditions with regular silica-gel on an achiral stationary phase. Partially enantioenriched (R)-1 (ca. 40% ee) served as the loading substrate. Table 1 shows the data for the experiment involving the SDE of 1 during achiral silica-gel chromatography. We first attempted to separate 1 on a glass column of 10 mm diameter and 50 mm length filled with regular silica-gel (KANTO CHEMICAL CO., INC., Silica Gel 60N, spherical, neutral, 63–210 μm) as the stationary phase at atmospheric pressure using DMSO as the loading solvent. The difference between minimum and maximum ees for the chromatographic fractions is shown as an evaluation value of this phenomenon. The SDE was not observed under dichloromethane/methanol (95/5) eluent (run 1). SDE was pronounced when hexane/ethyl acetate (5/5) was used as the eluent, and good Δee was obtained using hexane/ethyl acetate (7/3) (runs 2 and 3). The use of DMF or dioxane for loading decreased the Δee values (runs 4 and 5).
Runa | Loading solvent | Eluent | % ee minb | % ee maxb | Δeec |
---|---|---|---|---|---|
a Regular silica-gel packed in a glass column (10 × 50 mm) was used under atmospheric pressure. b ee was determined by HPLC using a CHIRALCEL OJ-H with ethanol as the eluate. c Δee = (% ee max) – (% ee min). | |||||
1 | DMSO | DCM/MeOH = 95/5 | — | — | — |
2 | DMSO | H/A = 5/5 | 21.2 | 57.8 | 36.6 |
3 | DMSO | H/A = 7/3 | 20.3 | 71.3 | 51.0 |
4 | DMF | H/A = 7/3 | 25.6 | 66.5 | 40.9 |
5 | Dioxane | H/A = 7/3 | 26.2 | 64.9 | 38.7 |
Next, separation was attempted using various silica-gels as column packing materials with different column lengths under hexane/ethyl acetate (7/3) conditions (Table 2). The Δee observed by flash silica-gel (KANTO CHEMICAL CO., INC., Silica Gel 60N, spherical, neutral, 40–50 μm) was better than that by regular silica-gel pre-treated with water (10 wt%) (runs 1 and 2). The highest Δee for 1 was 66.2% on a column filled with mesoporous silica-gel (run 3). The Δee improved on a longer column (runs 4 and 5). It should be noted that the phenomenon of SDE is quite general for 1 under ubiquitous purification conditions such as silica-gel/ethyl acetate–hexane. When we attempted separation of 1 using Al2O3, the SDE effect was not significant and a fluctuating performance was observed (run 6).
Runa | Starting ee of (R)-1 (%) | Silica-gel | % ee minb | % ee maxb | Δeec |
---|---|---|---|---|---|
a Achiral silica-gel packed in a glass column (10 × 50 mm) was used under atmospheric pressure. DMSO was used as the solvent for loading. b ee was determined by HPLC using a CHIRALCEL OJ-H with ethanol as the eluate. c Δee = (% ee max) – (% ee min). d Silica-gel was wetted with 10 wt% water. e A 10 × 80 mm column was used. f A 10 × 110 mm column was used. | |||||
1 | 35.5 | Regulard | 27.4 | 80.9 | 53.5 |
2 | 41.6 | Flash | 23.8 | 83.1 | 59.2 |
3 | 35.5 | Mesoporous | 20.7 | 86.9 | 66.2 |
4e | 41.6 | Flash | 17.7 | 80.1 | 62.4 |
5f | 36.2 | Flash | 15.0 | 80.0 | 65.0 |
6 | 31.1 | Al2O3 | 21.6 | 34.5 | 12.9 |
SDE was also observed for the 3′-fluorinated analogue of thalidomide, 2 (Table 3). When we attempted to separate 2 using a glass column filled with regular silica-gel, the SDE effect was not significant and 2 was partially decomposed during purification (run 1). When mesoporous silica-gel was used instead, a low Δee value was obtained without decomposition of 2 (run 2). Separation was next performed on silica-gel pre-treated with water to prevent decomposition. Although the SDE effect was unsuccessful using regular silica-gel pre-treated with 5 wt% water (run 3), a moderate Δee value was obtained on regular silica-gel with 10 wt% water or flash silica-gels with 5 and 10 wt% water (runs 4–6). When we attempted separation of 2 using Al2O3, the SDE effect was not significant and a fluctuating performance was again observed (run 7).
Runa | Starting ee of 2 (%) | Silica-gel | % ee minb | % ee maxb | Δeec |
---|---|---|---|---|---|
a Achiral silica-gel packed in a glass column (10 × 50 mm) was used under atmospheric pressure. DMSO was used as the solvent for loading. b ee was determined by HPLC using a CHIRALCEL OJ-H with ethanol as the eluate. c Δee = (% ee max) – (% ee min). d Silica-gel was wetted with 5 wt% water. e Silica-gel was wetted with 10 wt% water. | |||||
1 | 25.0 | Regular | 14.7 | 26.7 | 12.0 |
2 | 34.2 | Mesoporous | 32.2 | 56.1 | 23.9 |
3 | 34.2 | Regulard | 31.9 | 38.6 | 6.7 |
4 | 34.2 | Regulare | 30.0 | 70.6 | 40.6 |
5 | 27.3 | Flashd | 14.4 | 52.5 | 38.1 |
6 | 27.3 | Flashe | 9.4 | 50.4 | 41.0 |
7 | 37.2 | Al2O3 | 27.4 | 38.6 | 11.2 |
With these results in hand, we investigated the relationship between the ee value and mass of each fraction, which was estimated based on the peak area of 1 and 2 after HPLC analysis since the total recoveries of 1 and 2 were quantitative at the end of the chromatographic separation for each experiment. Fig. 2 shows the details of chromatography of 1 with an ee value of 36.3% using a 10 × 50 mm column filled with flash silica-gel (Fig. 2a), and 2 (32.0% ee) using a 10 × 50 mm column filled with regular silica-gel over wetted 10 wt% water (Fig. 2b). In the case of 1, the first fraction has the highest ee value and the ee values decreased gradually as the fractions increased. The last ee value converged to a lower ee than that of the loading sample. On the other hand, in the case of fluorinated 2, the first fraction had the lowest ee value and the ee values increased as the fraction number increased. The highest ee of 2 was observed in the last fraction. The masses are described by a parabola-like curve in both cases.
The basic mechanism for the phenomenon of SDE has been proposed to involve homochiral vs. heterochiral high-order species with different molecular weights such as monomers, dimers or oligomers, allowing their separation under the condition of achiral chromatography.8 We therefore considered the potential formation of heterochiral or homochiral dimers in the intermolecular interactions between the enantiomers of 1 in solution leading to the manifestation of the SDE. In our previous report of the X-ray crystal structure analysis of 1, the racemic mixture, (R/S)-1 forms symmetrical (R/S)-heterochiral dimers, and (S)-1 is found as asymmetrical (S/S)-homochiral dimers in the crystals.13 The crystals were taken from MeOH–water. The X-ray crystal structures show the differences in the hydrogen-bonded lengths between heterochiral and homochiral dimers. The hydrogen bonds in (R/S)-heterochiral dimers are slightly shorter than those of asymmetrical (S/S)-homochiral dimers (Fig. 3). In addition, the heterochiral dimer was estimated to be approximately 1 kcal mol−1 more stable than the homochiral dimer by theoretical calculations.13
Fig. 3 X-Ray crystallographic structures of racemic 1 (monoclinic, ESI†) and (S)-1 (monoclinic, ESI†).10 |
An understanding of the conformational changes and aggregation states of thalidomide by fluorine-replacement leads to additional insight into the mechanisms of SDE. X-Ray crystal structures of racemic 2, and (S)-2 were next investigated (crystallized from ethanol). To our great astonishment, both structures of 2 were very different from the parent, non-fluorinated thalidomide (1) despite their sterically isosteric relationship. While racemic 2 shows the structure of a (R/S)-heterochiral dimer, (S)-2 exists as a monomer without any hydrogen bonding between enantiomers. Even more interestingly, in racemic 2, the hydrogen bonding system between (R)-2 and (S)-2 is entirely different from that of the (R/S)-heterochiral dimer of racemic 1 (Fig. 4). These significant differences are likely to be attributed to a significant conformational change, as compared to original thalidomide, induced by the presence of the fluorine atom. In thalidomide (1), a sterically demanding phthalimido group occupies the equatorial position. On the other hand, fluorine is located at the equator and the phthalimido moiety at the axial position, despite its steric bulkiness (Fig. 5). Although the reason for the fluorine effect on the conformational change is not clear, it could be explained by the electrostatic repulsion between the fluorine and the two carbonyls of the piperidine-2,6-dione ring, and/or a strong dipole induced by the fluorine atom.14 Namely, the equatorial-fluorine conformation of 2 is presumably preferable, since the fluorine exists on the same plane as the two carbonyls of piperidine-2,6-dione in 2. On the other hand, the fluorine is almost perpendicular to the two carbonyls in the axial-fluorine conformation of 2, resulting in less-stabilization. The computations (DFT, B3LYP/6-311+G(d,p)) also support these results that the phthalimide moiety of 1 occupies an equatorial place while the fluorine occupies the equatorial position in 2 (Fig. 6).
Fig. 4 X-Ray crystallographic structures of racemic 2 (monoclinic, ESI†) and (S)-2 (monoclinic, ESI†). |
Crystal structures of racemic thalidomide have been investigated since 1971.5c The existence of polymorphism of racemic 1 has been suggested with the relationship between its different physical forms and dissolution behavior.5d,5h,5i We thus re-attempted to grow crystals of fluorinated thalidomide 2 using different solvents. In this attempt, (R)-2 was taken from chloroform and acetonitrile solutions. Similar to the case of (S)-2, i.e., crystals from ethanol (Fig. 4b), the unsolvated monomeric structures were revealed without detecting dimerization structures from both chloroform and acetonitrile solutions. Interestingly, while crystals of (R)-2 (α-form, monoclinic, Fig. 7a, ESI†) obtained from acetonitrile are the same as (S)-2 from ethanol (Fig. 4b), an alternate arrangement of (R)-2 was obtained from chloroform solution (β-form, orthorhombic, Fig. 7b, ESI†), with an infinite hydrogen bonded chain in (R)-2 (Fig. 7b). It should be mentioned that optically pure 2 is always obtained as a “monomer” independent of crystal solvents, while all attempts for the crystallization of racemic 2 gave the same crystal system of monoclinic.
Fig. 7 X-Ray crystallographic structures of unsolvated crystals of (R)-2; (a) α-form (monoclinic, ESI†); (b) β-form (orthorhombic, ESI†) with infinite hydrogen bonded chain. |
Starting with the X-ray crystallographic structures, we further estimated the logP values of (R)-1, (R/S)-heterochiral dimer 1, (R)-2, and (R/S)-heterochiral dimer 2 using DFT computations (B3LYP/6-31+G(d,p)) to be able to discuss the SDE on achiral silica-gel, since the holding time of substrates during silica-gel column chromatography are likely to be intricately related with the polarity of the substrates. The calculated logP values are: −0.15 for (R)-1; −0.30 for (R/S)-heterochiral dimer 1; 0.53 for (R)-2; 1.07 for (R/S)-heterochiral dimer 2. The computations indicated that thalidomide (1) changes to become more hydrophilic by the formation of its dimer, while fluorinated thalidomide (2) becomes more hydrophobic with dimerization.
Structural differences, aggregation states, and logP values of 1, 2 and their enantiomers suggest the supposed mechanisms of SDE of 1 and 2. Enantioenriched (R)-1 exists as a mixture of (R)-enantiomer 1 and racemate 1. Both (R)-enantiomer 1 and racemic 1 form dimers. However, the (R/R)-homochiral dimer from (R)-1 is less stable than the (R/S)-heterochiral dimer from racemic 1 based on the calculations. (R)-Enantiomer 1 becomes a monomer on silica-gel during elution while racemic 1 tends to stay as a dimer. Hence, enantioenriched (R)-1 was eluted first as a monomer while racemic 1 was eluted in the last fraction as a dimer, due to the difference in logP values ((R)-1: −0.15; (R/S)-heterochiral dimer 1: −0.30). In the case of fluorinated thalidomide (2), (R)-2 exists as a monomer independent of solvent while racemic 2 forms a dimer. The logP values of the monomer and dimer show an opposite tendency to non-fluorinated thalidomide ((R)-2: 0.53 vs. (R/S)-heterochiral dimer 2: 1.07). Consequently, racemic 2 (a dimer form) was observed in the first fraction while (R)-2 (a monomer form) was observed in the final fraction (Fig. 8).
Fig. 8 Proposed mechanisms for the opposite behaviors of SDE of (a) enantioenriched 1 and (b) enantioenriched 2. |
Footnote |
† Electronic supplementary information (ESI) available. CCDC 1009504, 1009505, 1009506, 1009507, 1009508, 1009509. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4sc03047h |
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