Chiral Separation of a Racemic Compound Induced by Transformation of Racemic Crystal Structures: DL-Glutamic Acid Masaaki Yokota,* Norihito Doki, and Kenji Shimizu Department of Chemical Engineering, Iwate UniVersity, 4-3-5 Ueda, Morioka 020-8551, Japan
CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 7 1588-1590
ReceiVed NoVember 1, 2005; ReVised Manuscript ReceiVed February 5, 2006
ABSTRACT: We found that an appropriate additive (L-Arg) induced the crystallization of DL-glutamic acid (DL-Glu) as a mixture of enantiomerically pure crystals, even under conditions where racemic crystals (both enantiomers in the unit cell) normally crystallize from the pure system. In addition, the additive successfully delayed the crystallization of one of the enantiomers (L-Glu). On the basis of these results, kinetic resolution of racemic crystals into their constituent enantiomers by a chiral additive is proposed. 1. Introduction Using ordinary chiral molecular synthetic methods in the absence of an asymmetric catalyst, equimolar mixtures of two enantiomers (racemate) that are mirror images of one another are generally obtained. Separation of racemic molecules is extremely important in view of the fact that often for chiral compounds one enantiomer forms an effective drug, while the molecule of the opposite chirality exhibits toxicity. Crystallization is a useful separation method to resolve the constituents of a racemate, as demonstrated by Louis Pasteur’s classic experiment.1 When racemic sodium ammonium tartrate is crystallized from solution, two groups of crystals, which are mirror images of one another (but with symmetrical morphologies), are formed. The two enantiomers can be separated from the crystals’ morphology differences. The most important and useful resolution method by crystallization was developed by a research group at the Weizmann Institute.2 If crystals are deposited from a racemic (D + L) solution in the presence of other chiral molecules (for example, D′), L-crystals are preferentially crystallized. Using this method, called asymmetric crystallization, separation of chiral molecules can be achieved, even on an industrial scale. Two main types of crystalline states of a racemate exist. One is a physical mixture of enantiomerically pure crystals (racemic mixture or conglomerate). In the other type of crystal, equal amounts of both enantiomers are packed in the unit cell (racemic compound). Chiral organic compounds that form conglomerates are rare, and the majority form racemic compounds. Many studies regarding the resolution of a conglomerate by crystallization have been carried out; however, it is considered difficult to resolve racemic compounds by crystallization methods. We consider “conglomerate” and “racemic compound” as a kind of crystal polymorphism (correctly speaking, this is not polymorphism based on its crystallographic definition). There must be a system in which an unstable conglomerate is initially formed that is subsequently transformed into the stable racemic compound. In such a system, the method of employing a tailormade additive, as proposed by the Weizmann Institute group, can be applied for dual purposes. One is to delay the transition from a conglomerate into a racemic compound. The other is to delay the crystallization of one of the enantiomers. We propose an enantio-separation method of a racemic compound using DL-Glu as a model compound. Below room * To whom correspondence should be addressed. Phone and Fax: 81-19-621-6342. E-mail:
[email protected].
temperature, DL-Glu crystallized as a racemic compound from the pure system. However, a suitable chiral additive (L-Arg) deposits the conglomerate even below room temperature. The chiral additive retards the crystallization of only L-Glu. No crystallization retardation is found for D-Glu. On the basis of this method, the asymmetric crystallization of a racemic compound is proposed. 2. Experimental Section 2.1. Materials. DL-Glu and the additives L-Met and L-Lys were purchased from Tokyo Kasei Kogyo Co., Ltd. (Japan). Additives L-Val, L-Leu, L-Ile, L-Phe, L-Tyr, L-Trp, L-Ser, L-Thr, L-Cys, L-Asn, L-Gln, l-Pro, L-Asp, L-Arg, and L-His were purchased from Kanto Chemical Co Inc. (Japan). All the reagents listed above were of analytical reagent grade and used as received without further purification. 2.2. Equipment. The experiments were carried out in a cylindrical tank with a dished bottom and an internal volume of approximately 500 mL. The impeller used was a disk turbine with six blades at a speed of 600 rpm. 2.3. Procedure. 2.3.1. Selection of the Additives. For the kinetic resolution of DL-Glu, appropriate additives that exhibit a growth retardation (or inhibition) effect for one of the chiral crystals only needed to be found. We attempted this experimentally (screening test). The additive was dissolved in an aqueous solution of L-Glu (3.0 g of L-Glu/100 g of H2O, additive/L-Glu ) 10 mol %) and the solution was quickly (about 5 min) cooled to 298 K to commence crystallization isothermally. The time required for nucleation (the appearance of crystals) to occur was first determined by the naked eye. After 24 h, the crystals were separated from the solution by filtration, and the morphology of the crystals was observed using an optical microscope (SZ60 Olympus, Japan). Powder X-ray diffraction (PXRD) patterns of the sampled crystals were also measured to examine the structure of the crystals (RINT-2000, Rigaku, Japan: CuKR, 20 mA, 40 kV). The same experiment was carried out for D-Glu, and the results were compared with the L-Glu data. 2.3.2. Suitable Crystallization Temperature. At this point, it is important to know under which crystallization conditions a racemic compound could form in the pure system. DL-Glu is known to form both the conglomerate and the racemic compound, depending on the temperature. Unfortunately, detailed temperature data were not given. Thus, we attempted to find the temperature experimentally. DL-Glu was dissolved in water (4.0 g/100 g of H2O) at 338 K. The resulting solution was then cooled to the desired temperature to commence crystallization. After 24 h of isothermal crystallization, crystals were recovered and PXRD patterns of the sampled crystals were measured to examine their structure (RINT-2000, Rigaku, Japan). 2.3.3. Optical Resolution by Asymmetric Crystallization. An additive (additive/DL-Glu ) 20 mol %) was dissolved in a DL-Glu aqueous solution (4.0 g/100 g of H2O). The solution was cooled to a temperature determined from the preliminary experiments, and the crystallization was then carried out isothermally. The composition (optical purity) of the deposited crystals and the mother liquor were
10.1021/cg050579i CCC: $33.50 © 2006 American Chemical Society Published on Web 06/08/2006
Chiral Separation of a Racemic Compound
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Table 1. Effect of Various Amino Acids on Waiting Time for Nucleation of D- or L-Glua waiting time for nucleation [h] additive: side chain nonadditive neutral amino acids L-Val: CH(CH3)2 L-Leu: CH2CH(CH3) 2 L-Ile: CH (CH3)CH2CH3 L-Phe: CH2 (C6H5) L-Tyr: CH2 (C6H4)OH L-Trp: CH2 (C8H6N) L-Ser: CH2OH L-Thr: CH(CH3)OH L-Cys: CH2SH L-Met: CH2CH2SCH3 L-Asn: CH2CONH2 L-Gln: CH2CH2CONH2 L-Pro: (C4H8N)COOH acidic amino acids L-Asp: CH2COOH basic amino acids L-Arg: (CH2)3NHC(dNH)NH2 L-His: (C3H3N2)CH2 L-Lys: (CH2) 4NH2
D-Glu
L-Glu
0.5 (β)
0.5 (β)
0.5 (β) 1.0 (β) 0.5 (β) 0.5 (β) 0.5 (β) 0.5 (β) 1.0 (β + R) 0.5 (β) 0.5 (β) 1.0 (β + R) 0.5 (β) 0.5 (β) 1.0 (β + R)
1.0 (R) 2.0 (R) 1.0 (R) 2.0 (R) 4.0 (R + β) 4.0 (R) 1.0 (R) 0.5 (R) 4.0 (R) 2.0 (β) 1.0 (R) 1.0 (R) 1.0 (R)
1.0 (β)
1.0 (R)
0.5 (β) 0.5 (β) 1.0 (β)
15.0 (R) 7.0 (R) 4.0 (R)
Figure 1. Powder X-ray diffraction pattern of Glu crystals. Measured: Glu racemic compound formed in the absence of an additive. Calculated: XRD patterns calculated using single-crystal structure data of DL-Glu monohydrate (racemic compound), L-Glu R-form crystal, L-Glu β-form crystal. If Glu is crystallized as a conglomerate, their XRD patterns must agree with the L-Glu R- and/or β-form pattern(s).
a The symbol between parentheses denotes the crystal polymorphism after 24 h crystallization.
analyzed over time using HPLC (Mobile phase: aqueous HClO4 with pH 1.5, column: Crownpak CR+, detector: UV (200 nm)). PXRD patterns of the sampled crystals were also measured to examine their structure (RINT-2000, Rigaku, Japan). In some experiments, seed crystals of D-Glu were added to improve the enantio-separation efficiency.
3. Results and Discussion 3.1. Selection of Additives. Table 1 shows results of the screening tests, using a total of 17 L-amino acid additives. In the case of L-Glu crystallization (right-end column, Table 1), some additives, especially basic amino acids, delayed nucleation of L-Glu. In addition to the effect of the additives on the nucleation time, it was interesting to demonstrate their effect on crystal polymorphism. It is well-known that L-Glu forms two types of crystal structures. An unstable R-form transforms into a stable β-form by a solvent-mediated transformation mechanism. The symbol between parentheses in Table 1 denotes the crystal polymorphism after 24 h crystallization. For L-Glu, almost all the additives stopped (or strongly delayed) the R to β transformation and only R-form crystals remained. Among the R-form crystals, different types of crystal morphologies such as pyramid (L-Arg, L-Lys) were observed, depending on the additive. The mechanism has been investigated using MD simulations, details of which will be published in the near future. On the other hand, no effect of the additives on the time required for D-Glu nucleation was observed (central column, Table 1). With some exceptions, the additives basically had no influence on the morphology and crystal structure of D-Glu. On the basis of the results of the screening test, basic amino acids are candidates as appropriate kinetic resolving agents. Among the three kinds of basic amino acids employed as additives, the L-Arg effect was the most prominent. Thus, we decided to use L-Arg in the asymmetric crystallization of DLGlu described below. 3.2. Optical Resolution by Asymmetric Crystallization. Prior to the asymmetric crystallization experiment, we confirmed whether the racemic compound deposited in the absence of additives (pure system). As a result of repeated experiments
Figure 2. Change in optical purity of the formed crystals (PDC ) D/(D+L)) over time, D- and L-Glu concentration in the solution ([D]s, [L]s), with the progress of crystallization in the presence of L-Arg (nonseeded).
(more than 5 times at one temperature), 288 K was determined to be a reliable temperature at which the racemic compound was formed in our experimental system. The PXRD pattern is shown in Figure 1 (upper). When compared with calculated patterns, formation of the racemic compound (DL-Glu H2O) in the pure system was verified. Surprisingly, only the conglomerate was deposited in the presence of L-Arg, while the racemic compound was deposited in the absence of the additive. In Figure 2, the optical purity of the crystals (PDC ) D/D+L) and curves showing the decrease in the concentration of D- and L-Glu in solution with time are shown. By using L-Arg as the additive, we found it was possible to crystallize only D-Glu (asymmetric crystallization), at least for the first 15 h. However, before crystallization of D-Glu was completed (reached to a constant level), L-Glu also started to crystallize, causing the purity drop. The seed crystals loading technique is known as an efficient way to improve (batch) crystallization properties such as CSDs3 and productivity. The seed loading technique can be effective for chiral separation because it may accelerate crystallization of the enantiomer that has the same chirality as the seed crystals. Thus, we decided to load D-Glu seed crystals. D-Glu β-form crystals (0.9 g, average particle size 212 µm) were added as seed and crystallization at 288 K was started. The reason we selected β-form as seed crystals is as follows. If R-form crystals are introduced, they must dissolve soon because of solventmediated transformation. Thus, stable β-form crystals are suitable as seed crystals for successful asymmetric crystallization. The results are shown in Figure 3. By loading D-Glu seed
1590 Crystal Growth & Design, Vol. 6, No. 7, 2006
Yokota et al. Scheme 1. Proposed Scheme for the Chiral Separation of a Racemic Compound (DL-Glu) by an Additive (L-Arg)
Figure 3. Change in optical purity of the formed crystals (PDC ) D/(D+L)) over time, D- and L-Glu concentration in the solution ([D]s, [L]s), with the progressing crystallization in the presence of L-Arg (D-Glu seed crystals were loaded).
crystals must be D- and L-Glu, respectively. The granular and needle crystals were separated manually under an optical microscope, and their optical purity was analyzed using HPLC. The granular R-form crystals were >98% L-Glu, while the needle β-form crystals were 92% D-Glu, respectively. On the basis of the experimental results described above, we found that the appropriate additive (L-Arg) transforms the racemic compound (DL-Glu) into a racemic mixture (D-Glu/LGlu). Consequently, we propose that D-Glu and L-Glu can be separated using this method (Scheme 1). 4. Conclusions
Figure 4. Photographic image of Glu conglomerate after 100 h crystallization in the presence of L-Arg. Granular and needle crystals are R-form and β-form, respectively. Scale bar ) 1 mm.
crystals, the crystallization of D-Glu quickly progressed compared with Figure 2 (nonseeding). Almost all D-Glu molecules were deposited before L-Glu started to crystallize (20 h). After 20 h, only L-Glu was deposited (very slowly), and the total optical purity (PDC) began to drop. As shown in Figure 3, although the L-Glu concentration in the solution did not lower to a level equal to D-Glu, the concentration of the L-Glu continued to lower even after 100 h, and equilibrium was attained in about 250 h. Photographs (Figure 4) of the crystals obtained after 100 h of crystallization showed two types of crystal morphologies (needle and granular). On the basis of PXRD measurements, they were a mixture of R- and β-forms (conglomerate). The needle β-form crystals and granular R-form
We found addition of L-Arg caused crystallization of DL-Glu, which originally forms racemic compound, as a racemic mixture. In addition, L-Arg delayed the crystallization of only L-Glu. No effect was found for D-Glu. On the basis of this method, the asymmetric crystallization of a racemic compound is proposed. References (1) Pasteur, L. Ann. Chim. Phys. 1848, 24, 442-459. (2) (a) Addadi, L.; Berkovitch-Yellin, Z.; Domb, N.; Gati, E.; Lahav, M.; Leiserowitz, L. Nature 1982, 296, 21-26. (b) Addadi, L.; Gati, E.; Lahav, M. J. Am. Chem. Soc. 1981, 103, 1251-1252. (c) Addadi, L.; van Mil, J.; Lahav, M. J. Am. Chem. Soc. 1981, 103, 12491251. (d) Addadi, L.; Weinstein, S.; Gati, E.; Weissbuch, I.; Lahav, M. J. Am. Chem. Soc. 1982, 104, 4610-4617. (e) Weissbuch, I.; Addadi, L.; Lahav, M.; Leiserowitz, L. Science 1991, 253, 637645. (3) Doki, N.; Kubota, N.; Sato, A.; Yokota, M.; Hamada, O.; Masumi, F. AIChE J. 1999, 45, 2527-2533.
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