Application of preferential crystallization to resolve racemic compounds in a hybrid process

The application of preferential crystallization is at present limited to conglomerate forming systems, which cover only a minor part of chiral substances. In this paper, a hybrid process is proposed that extends the applicability of the preferential crystallization principle to the more common racemic compound forming systems. It comprises a preliminary (e.g., chromatographic) enantiomeric enrichment step and preferential crystallization to finally produce the desired pure enantiomer(s). The applicability of preferential crystallization to racemic compounds is demonstrated on the example of mandelic acid as a model system. Direct monitoring of the separation progress is performed using combined online polarimetry and online density measurements. A cyclic crystallization process, which provides alternating the pure mandelic acid enantiomer and the racemic compound, is feasible and allows the resolution of rac-mandelic acid as part of the proposed hybrid approach.     

Kinetics of chiral resolution in stirred crystallization of D/L‐glutamic acid

Stirred crystallization of racemic (D/L)‐glutamic acid (Glu) in the presence of small amounts of L‐ or D‐lysine (Lys) was studied for the effect of transient chiral resolution by monitoring the time evolution of optical rotation and the concentration of the solution. The presence of a small amount of L‐ or D‐Lys retards the crystallization rate of the corresponding enantiomer of Glu in a chirally selective manner, giving rise to transient optical resolution of racemic Glu during crystallization. The optical rotation of the Glu solution was found to increase from zero to a value corresponding to an enantiomeric excess (ee) of 22–35% and subsequently decreases to zero over a period of many hours. During this process, the ee of the crystallized Gu is nearly 100% during the first 35 min and then it decreases slowly to zero. Our results indicate that the time at which the ee of the solution reaches its maximum and the maximum value of the ee show a nonlinear dependence on the initial mole fraction of the chiral impurity. The effect of the impurity is highly chirally selective, indicating “molecular recognition.” Chirality 11:343–348, 1999. © 1999 Wiley‐Liss, Inc.     

Enzyme‐assisted physicochemical enantioseparation processes—Part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter‐enantiomer. Here we present first results of dynamic kinetic resolution of L ‐asparagine (L ‐Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L ‐Asn monohydrate could be crystallized, before crystal formation of d ‐Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L ‐Asn (198 mg L ‐Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling. Biotechnol. Bioeng. 2009; 104: 1235–1239. © 2009 Wiley Periodicals, Inc.     

Enantiomeric resolution of p-toluenesulfonate of valine benzyl ester by preferential crystallizaion

Preferential crystallization of amino acid derivatives by seeding a pure enantiomer into racemic amino acid solutions has been studied for many years. However, few examples of valine derivatives have been reported so far. Although there have been some reports using valine hydrogen chloride with preferential crystallization, it is difficult to obtain optical isomers for valine derivatives using preferential crystallization. In this study, repeated preferential crystallization of p-toluenesulfonate valine benzyl ester with a 20% e.e. in 2-propanol gave a 94% e.e. on sonication. Sonication accelerated crystallization rate, but there was not a big difference in e.e. between with and without sonication. However, this research demonstrates the first preferential crystallization of p-toluenesulfonate of valine benzyl esters with an acceleration of crystallization using sonication.     

Potential of different techniques of preferential crystallization for enantioseparation of racemic compound forming systems

Recently the feasibility of preferential crystallization for enantioseparation of racemic compound forming systems has been demonstrated (Lorenz et al., Application of preferential crystallization to resolve racemic compounds in a hybrid process. Chirality 2006;18:828-840; Polenske et al., Separation of the propranolol hydrochloride enantiomers by preferential crystallization: thermodynamic basis and experimental verification. Cryst Growth Des 2007;7:1628-1634). Here, the development and the potential of an efficient separation process operated via two different techniques of preferential crystallization are studied: (1) seeded isothermal preferential crystallization and (2) auto-seeded polythermal preferential crystallization. Both techniques were investigated in the batch and in the cyclic operation mode. On the example of mandelic acid as a typical racemic compound forming system, it is demonstrated that a cyclic auto-seeded polythermal process is feasible and significantly more efficient than the seeded isothermal one.     

Optical resolution by preferential crystallization of DL-methionine hydrochloride

DL -Methionine hydrochloride (DL -Met·HCl) was found to exist as a conglomerate, based on the infrared spectrum, solubility, and melting point. The optical resolution of DL -Met·HCl was successfully achieved by preferential crystallization into D -and L -Met·HCl. Treatment of the purified D - and L -Met·HCl with triethylamine gave D - and L -methionine of 100% optical purities. Chirality, 9;48–51, 1997. © 1997 Wiley-Liss, Inc.     

Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients

Important progress has been made in recent years toward developing a molecular-level understanding of protein phase behavior in terms of the osmotic second virial coefficient, a thermodynamic parameter that characterizes pairwise protein interactions. Yet there has been little practical application of this knowledge to the field of protein crystallization, largely because of the difficult and time-consuming nature of traditional techniques for characterizing protein interactions. Self-interaction chromatography has recently been proposed as a highly efficient method for measuring the osmotic second virial coefficient. The utility of the technique is examined in this work by characterizing virial coefficients for ribonuclease A under 59 solution conditions using several crystallization additives, including PEG, sodium chloride, ammonium sulfate, and propanol. The virial coefficient measurements show some counterintuitive trends and shed light on the previous difficulties in crystallizing ribonuclease A. Crystallization experiments at the corresponding solution conditions were conducted by using ultracentrifugal crystallization. Using this methodology, ribonuclease A crystals were obtained under conditions for which the virial coefficients fell within the "crystallization slot." Crystallographic characterization showed that the crystals diffract to high resolution. Metastable crystals were also obtained for conditions outside, but near, the "crystallization slot," and they could also be frozen and used to collect structural information.     

Optical resolution by preferential crystallization of (RS)-2-amino-3-chloropropanoic acid hydrochloride

The racemic structures of (RS)-2-amino-3-chloropropanoic acid [(RS)-ACP] and (RS)-2-amino-3-chloropropanoic acid hydrochloride [(RS-ACP·HCl] were examined to obtain (R)- and (S)-ACP via optical resolution by preferential crystallization. The melting point, infrared spectrum, solubility, and ternary solubility diagram suggested that (RS)-ACP·HCl exists as a conglomerate and that (RS)-ACP forms a racemic compound. Optical resolution by preferential crystallization of (RS)-ACP·HCl was successfully achieved to yield (R)- and (S)-ACP·HCl. Optically pure (R)- and (S)-ACP were obtained from the purified (R)-and (S)-ACP·HCl, respectively. © 1996 Wiley-Liss, Inc.     

Amplification of optical activity by crystallization in the presence of tailor-made additives. The “inversion rule”

Experiments are described in which enantiomerically pure dimers, trimers and oligomers, generated from non chiral monomers by crystallization and topochemical reaction in suitably designed chiral crystals, induce preferential crystallization of the sarent monomer in the enantiomorphous chiral phase of opposite absolutute configuration. (We coin the term inversion rule for this effect). A general mechanism of amplification of optical activity by crystallization is proposed on the basis of these results, involving selective adsorption of resolved impurities on the surface of chiral crystals of similar stereochemistry, resulting in a decrease of the growth rate of the affected enantiomer and consequent preferential crystallization of the antipode. The implications of this mechanism to the generation and amplification of chiralily in a closed system are discussed.This paper was originally published in the Proceedings of the VIth International Conference on the Origins of Life, Jerusalem, June 1980, Ed. Y. Wolman, Reidel Dordrecht, pp. 355–364.     

Nonlinearity in optical resolution via distillation applying mixtures of resolving agents

During an optical resolution it is the resolving agent that has the strongest influence on the outcome of the process. Applying a mixture of resolving agents can result either in antagonism or in synergy. We found that using mixtures of tartaric acid and its derivatives chiral selectivity is at least the same, but in several cases markedly better (synergistic effect), than the sum of the effect of the individual resolving agents. Thus, the "Dutch method," reported for the crystallization method, also works for distillation. A calculation method is applied for measuring the synergistic effect. Interestingly, an individually inactive resolving agent can be a useful contributor to the mixture of the resolving agents.     

A hybrid process for chiral separation of compound‐forming systems

The resolution of chiral compound‐forming systems using hybrid processes was discussed recently. The concept is of large relevance as these systems form the majority of chiral substances. In this study, a novel hybrid process is presented, which combines pertraction and subsequent preferential crystallization and is applicable for the resolution of such systems. A supported liquid membrane applied in a pertraction process provides enantiomeric enrichment. This membrane contains a solution of a chiral compound acting as a selective carrier for one of the enantiomers. Screening of a large number of liquid membranes and potential carriers using the conductor‐like screening model for realistic solvation method led to the identification of several promising carriers, which were tested experimentally in several pertraction runs aiming to yield enriched (+)‐(S)‐mandelic acid (MA) solutions from racemic feed solutions. The most promising system consisted of tetrahydronaphthalene as liquid membrane and hydroquinine‐4‐methyl‐2‐quinolylether (HMQ) as chiral carrier achieving enantiomeric excesses of 15% in average. The successful production of (+)‐(S)‐MA with a purity above 96% from enriched solutions by subsequent preferential crystallization proved the applicability of the hybrid process. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Optical resolution by preferential crystallization of 4-methylpiperidinium hydrogen (RS)-phenylsuccinate

Gibbs energy of racemate formation, binary melting point diagram, and ternary solubility diagram suggested that 4-piperidinium hydrogen (RS)-phenylsuccinate [(RS)-4-MP salt] exists in a conglomerate. Appropriate conditions were explored on the basis of free energy of critical nucleation in a supersaturated solution to resolve efficiently (RS)-4-MP salt by preferential crystallization. Successive preferential crystallization of (RS)-4-MP salt in ethanol at 20°C gave (R)- and (S)-4-MP salts of 90–94% optical purities. Optically pure (R)- and (S)-phenylsuccinic acids were obtained by recrystallization of the (R)- and (S)-4-MP salts, followed by treatment of the salts purified with hydrochloric acid. © 1994 Wiley-Liss, Inc.     

Immobilization of an amino acid racemase for application in crystallization‐based chiral resolutions of asparagine monohydrate

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization‐based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalent‐binding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg‐enzyme/g‐support showed highest specific activity (approx. 500 U/g‐support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.     

Unexpected Differences between D- and L- Tyrosine Lead to Chiral Enhancement in Racemic Mixtures Dedicated to the memory of Prof. Shneior Lifson – A great liberal thinker.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 °C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

Unexpected differences between D- and L- tyrosine lead to chiral enhancement in racemic mixtures.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 degrees C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

Preferential Enrichment of DL‐Leucine Using Cocrystal Formation With Oxalic Acid Under Nonequilibrium Crystallization Conditions

By utilizing the preferential enrichment (PE) technique, we achieved an improved enantiomeric resolution of DL ‐leucine (Leu) using a 1:1 cocrystal (DL ‐ 1 ) of DL ‐Leu and oxalic acid. The crystal structure analysis of DL ‐ 1 indicated the occurrence of a novel type of phase transition and subsequent preferential redissolution of one enantiomer from the resulting crystals into solution. Chirality 27:405–410 2015. © 2015 Wiley Periodicals, Inc.     

Mass-transfer limitations for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor

A mathematical model has been developed for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor in which the enzyme-catalyzed reaction (the irreversible uni-uni competitive Michaelis-Menten kinetics is chosen as an example) was coupled with intraparticle diffusion, external mass transfer, and axial dispersion. The effects of mass-transfer limitations, competitive inhibition of substrates, deactivation on the enzyme effective enantioselectivity, and the optical purity and yield of the desired product are examined quantitatively over a wide range of parameters using the orthogonal collocation method. For a first-order reaction, an analytical solution is derived from the mathematical model for slab-, cylindrical-, and spherical-enzyme supports. Based on the analytical solution for the steady-state resolution process, a new concise formulation is presented to predict quantitatively the mass-transfer limitations on enzyme effective enantioselectivity and optical purity and yield of the desired product for a continuous steady-state kinetic resolution process in a fixed-bed reactor.     

A Facile Method of Preparing l-α-Methyl DOPA (l-3-(3,4-Dihydroxyphenyl)-2-methylalanine)

To develop a practical method for production of l-α-methyl DOPA, the optical resolution of its precursor, dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine, was studied. The monohydrochloride of dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine was resolved by a preferential crystallization procedure. Optically pure l-α-methyl DOPA was obtained in good yield. Industrial production of l-α-methyl DOPA by the present simple method is considered to be very promising.     

合成苏氨酸的研究（Ⅳ）──DL－苏氨酸的光学拆分

本文研究了用优先结晶法拆分ＤＬ－苏氨酸的有关问题,诸如（Ｌ－Ｔｈｒ＋Ｄ－Ｔｈｒ＋Ｈ２Ｏ）三元体系溶解度数据、“平液”及“贫液”拆分问题、拆分速度对晶种的依存、过饱和度的选择、连续拆分设想及间歇拆分示例等,为工业化过程提供了重要的实验依据。     

Preparation of enantiomeric gossypol by crystallization

Large enantiomorphic crystals of gossypol-acetone (1:3) were grown from acetone solutions of rac-gossypol-acetic acid (1:1) at 4 degrees C. By controlling the initial gossypol concentration, crystallization time, and solution volume, single crystals were grown that weighed >50 mg, equivalent to >37 mg of enantiomeric gossypol. Even larger crystals were possible, but it was difficult to produce these reliably without contamination of the antipode. Essentially all of the acetone within the crystal form was removed by storing the crystals under vacuum for 3-4 days. By employing these techniques, gram quantities of enantiomeric gossypol were prepared in high chemical and optical purity. Based on measured and reported optical rotations, the optical purity of samples prepared by crystallization was greater than the optical purity of samples prepared by chromatographic separation of gossypol-amine diastereomers. The principal limitation of crystallization as a preparative method is the need to determine the chirality and purity of each product crystal. Nevertheless, the method competes favorably with preparative-scale chromatographic procedures.