Analysis and Comparison of Commonly Used Acidic Resolving Agents

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Analysis and Comparison of Commonly Used Acidic Resolving Agents in Diastereomeric Salt Resolution Examples for DL-Serine Venkata S. Sistla,† J. von Langermann,† H. Lorenz,*,† and A. Seidel-Morgenstern†,‡ † ‡

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany Otto von Guericke University Magdeburg, Chair of Chemical and Process Engineering, Magdeburg, Germany

bS Supporting Information ABSTRACT: Diastereomeric salt resolution is still the most relevant technique in chiral resolution processes, e.g. for pharmaceutical relevant substances. For this purpose the choice of a suitable corresponding resolving agent is very important in respect of optimal yield, chemical purity and diastereomeric excess. Thus the solid phase behavior of diastereomeric salts is a crucial parameter and should be investigated prior to any resolution experiment. Within this contribution 3 commonly used acid resolving agents, 2,3-dibenzoyl-L-tartaric acid, L-(þ)mandelic acid, and L-(þ)-tartaric acid, are compared for the resolution of the model compound DL-serine. The behavior of each diastereomeric salt pair was analyzed experimentally and is discussed with the help of phase diagrams.

1. INTRODUCTION Crystallization belongs to the most relevant techniques to purify target compounds from byproducts, untreated excess reactants, etc.1 Herein, the differences in the physical-chemical properties of the substances are used to obtain the desired compound in its pure form. Its wide application in different chemical industries also covers the enantioseparation of racemic substances.15 For this purpose, the investigation of solid phase behavior is relevant for the design of an effective separation process. Three different categories are known for chiral substances; racemic compounds, conglomerates and solid solutions.2 However, only conglomerates can be separated via preferential crystallization directly from the racemic mixture. Racemic compound-forming substances, which represent 9095% of all chiral substances, and solid solutions (99.9% was purchased from VWR Prolabo (Darmstadt, Germany). Diethyl ether and D-/L-serine benzyl ester benzene sulfonic acid were obtained from Sigma-Aldrich GmbH (Steinheim, Germany) with a purity of >99.9%. 2,3-Dibenzoyl-Dtartaric acid, L-(þ)-mandelic acid, and L-(þ)-tartaric acid were supplied from Merck KGaA (Darmstadt, Germany) and Sigma-Aldrich (Steinheim, Germany) with purities of g99%. Deionized water was used throughout the studies. All substances were used without further purification. 2.2. Synthesis Procedure. The synthesis of all diastereomeric salts was initiated according to the procedure proposed by V.S. Sistla et al.12 In the final synthesis step D-/L-serine benzyl ester was converted with the different resolving agents in methanol. The final reactions are shown in Scheme 3, referring to the complete structures given in Scheme 2. All six salts were synthesized individually and purified via repeated recrystallization from methanol. They were characterized via 1H NMR, XRPD and melting point analysis. As these pairs of diastereomeric salt split into ions in the DMSO-d6 solvent, each pair of salts shows the same 1 H NMR spectrum (given below). 1 L-L or D-L salt: H NMR (400 MHz, DMSO-d6) δ 3.623.72 (m, 4H), 3.81 (t, 2H), 5.15 (q, 4H), 5.62 (s, 2H), 7.317.39 (m, 10H), 7.46 (t, 4H), 7.61 (m, 2H), 7.91 (d, 4H). 1 L-LM or D-LM salt: H NMR (400 MHz, DMSO-d6) δ 3.653.74 (m, 2H), 3.79 (t, 1H), 4.77 (s, 1H), 5.19 (t, 2H), 7.207.40 (m, 10H). 1 L-LT or D-LT salt: H NMR (400 MHz, DMSO-d6) δ 3.613.74 (m, 6H), 3.90 (s, 2H), 5.17 (t, 4H), 7.317.38 (m, 10H). 2.3. Binary Melting Behavior. To ensure mixing at a molecular level the pure diastereomeric salts at different compositions were mixed in a mortar and dissolved in methanol. The solvent was then completely evaporated and the remaining salt mixture crushed into powder. 10 mg of the mixture was taken into an aluminum crucible and the melting behavior was studied in a differential scanning calorimeter (DSC-131, Setaram, France) using a heating rate of 2 K/min. A continuous purge of He gas was used in order to maintain an inert atmosphere. 2.4. Solubility Measurements. Solubilities of diastereomeric salts in pure form or in mixtures were measured via an isothermal method in

Scheme 1. Chemical Structures of DL-Serine and DL-Serine Benzylester

Figure 1. Schematic principle of diastereomeric salt formation; aexcept rotation of plane-polarized light into opposite directions.

Figure 2. Differentiation of diastereomeric salt behavior via the ternary solubility phase diagram: a, simple eutectic; b, double salt; c, solid solutions (exemplary with solubility maximum). p- and n-salt represent the corresponding diastereomeric salts. 3762

dx.doi.org/10.1021/cg2001128 |Cryst. Growth Des. 2011, 11, 3761–3768

Crystal Growth & Design

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Scheme 2. Chemical Structures of the Three Diastereomeric Salts Pairs (1) D-L, L-L salts, (2) D-LT, L-LT salts, and (3) D-LM, L-LM salts

Scheme 3. Diastereomeric Salt Formation of D-/L-Serine Benzyl Ester with Three Acidic Resolving Agents

the respective solvent (methanol, acetone or water).13 The choice of the solvent is based on a reasonable solubility for crystallization purposes. Samples at different known compositions were introduced into a glass vessel and kept under constant temperatures in jacketed vessels for 48 h to obtain equilibrium conditions. The homogeneity in solution was maintained with the help of a magnetic stirrer. When equilibrium conditions were attained, the solutions were filtered and samples taken for concentration and composition measurements. Concentrations were measured via a gravimetric method, i.e. by evaporating the solvent and weighing the sample before and after evaporation. Subsequently, the content of each diastereomer in the sample was measured by high performance liquid chromatography (HPLC). Hereby the liquid phases were diluted with methanol and analyzed by HPLC. A Crownpak CR 150 4.6 mm column and a mobile phase of 1.63 g perchloric acid in 1 L of water at pH = 2 was applied. The flow rate was 0.3 mL/min. The column generated a pressure drop of 46 bar. All solid phases were studied by X-ray Powder Diffraction (XRPD), using an X'Pert Pro diffractometer (PANalytical GmbH, Germany) with Cu KR radiation. Small amounts of samples were prepared on Si (background-free) sample holders and scanned between 3 and 40° 2Theta with a step size of 0.017°. The measurements were used to identify the type of species present and also to check if solvates or polymorphs occurred in the crystal.

3. RESULTS AND DISCUSSION Numerous parameters are relevant for the design of an effective (classical) resolution process, as pointed out above. However, the knowledge of the basic phase diagrams is still the most crucial parameter to understand and control the separation process yielding in high productivities, purities and diastereomeric excesses. This includes, among other things, information concerning the type of solid phase behavior (as discussed above), position of eutectic (s), and solubility limits within the corresponding solvent. To show and discuss this concept 3 commonly used resolving agents will be compared for the resolution of a given exemplary

compound in respect of their solid phase behavior and finally discussed regarding feasible separation strategies. Fortunately, secondary effects like polymorphism and the existence of solvates were not present, which eased the investigations. The results are presented separately below for each case of diastereomeric salt behavior. Examples are given for all 3 cases: (a) simple eutectic behavior (L-L and D-L salts), (b) double salts behavior (L-LT and D-LT salts), and (c) solid solution behavior (L-LM and D-LM salts). 3.1. Simple Eutectic Behavior (L-L- and D-L-salts). The preferred option for a resolution process is naturally the direct start from a racemic mixture, as these substances are usually obtained from chemical synthesis. For a thermodynamically controlled process this is only accessible for substances with a simple eutectic behavior, which illustrates the significance of these solid phase properties. Starting with L-L- and D-L-salts the solid phase analysis was executed via XRPD, as shown for different mixtures in Figure 3. Distinct XRPD patterns were found for both pure diastereomeric salts. According to the 1H NMR results, these two salts also possess no solvates in the solvent used. The 50:50 mixture of both D-L- and L-L-salt include just the XRPD peaks that are present in the corresponding individual pure salts. No additional peaks were found. Not only the 50:50 mixture, but also the mixtures at other compositions (enriched with one of the diastereomeric salts) show patterns that possess peaks from both pure D-L- and L-L-salts without the appearance of any new peaks. This nature strongly supports the concept of a simple eutectic behavior in the binary mixtures, as no additional solid phases are found. Additionally, the melting temperatures were checked for both L-L- and D-L-salts to 148 °C for the L-L-salt and 152.5 °C for D-L-salt with decomposition upon melting, which decreases the accuracy of calorimetric measurements and subsequently makes it impossible to determine the binary phase diagram. However, even on the basis of the different melting temperatures of both salts, different solubilities can be anticipated. The expected simple eutectic behavior was therefore checked by measuring the ternary solubility phase diagram in methanol (Figure 4). At the temperature of 35 °C, the solubility of D-L-salt is 3.5 wt %, while the solubility of L-L salt is significantly different at 23 wt %. This difference of ∼20 wt % in the individual pure salt solubilities is a good indication for a useful separation process via 3763



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Figure 3. XRPD patterns of pure D-L- and L-L-salts and mixtures of different composition.

Figure 4. Ternary solubility phase diagram for D-L- and L-L-salts in methanol at 35 °C (D-L, L-L, D-L þ L-L: existence regions of the respective salts in the phase diagram).

diastereomeric salt crystallization. Consequently, as indicated also by the results from XRPD, no special solid state behavior (like mixed crystals and double salts) was found in the ternary system. The phase diagram also clearly shows that the solubility is increasing with increasing composition of the other salt up to a certain composition for both L -L - and D - L -salts. The maximum solubility was observed at around 52% diastereomeric excess (d.e.) at a composition of 76:24 L-L/D-Lsalts, which hereby represents the eutectic composition in the diastereomeric system. Only one eutectic point is observed in this ternary system, which again proves the simple eutectic behavior and suggests the separation of both salts via crystallization is feasible.

The separation strategy from a racemic mixture of DL-serine and accordingly a 50:50 diastereomeric salt mixture is the crystallization within the two phase region of D-L salt (solid D-L þ saturated liquid). Fortunately, this phase region is extremely wide compared to the two phase region of L-L salt (solid L-L þ saturated liquid). This is due to the pronounced difference in solubility of the pure salts and the position of the eutectic close to the side of L-L-salt, which facilitates a wide scope for crystallization of the D-L salt. From this it follows that the crystallization of L-L-salt is not likely, which will result in high yield at high diastereomeric purities for the D-L-salt. The unnatural form D-serine can eventually be obtained from the pure D-L-salt via basic extraction. Noteworthy, L-L-salt might be obtained within the three phase region via preferential (cooling) crystallization conditions, which are not discussed herein. 3.2. Double Salt Behavior (L-LT- and D-LT-salts). The change of the resolving agent from dibenzoyltartaric acid to tartaric acid, even with the given structural similarities, yields in a significantly different solid phase behavior. Again, the absence of different polymorphic forms and solvates was assured and D-LT, L-LT and mixtures thereof were characterized by different XRPD patterns (Figure 5). Again, the XRPD patterns of pure L-LT- and D-LT-salts are totally dissimilar. Next to this behavior, the XRPD pattern for the 50:50 mixture of D-LT and L-LT salt was also dissimilar to the pure salts. However, mixtures at other compositions of both salts showed XRPD patterns with peaks of both individual salts as well as the peaks of the 50:50 mixture. This supports the hypothesis that these diastereomeric salts form a double salt close to the 50:50 composition. This assumption was subsequently supported by DSC measurements (see also Supporting Information). For both pure D-LT and L-LT salts sharp melting was observed with a melting temperature for the D-LT salt of 154.7 °C, and for the L-LT salt of 143.3 °C. The melting behavior of mixtures of both salts included two merged peaks with different eutectic melting temperatures. Decomposition during melting was also observed for this salt pair, which prevented more accurate quantitative analysis. 3764



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Figure 5. XRPD patterns for both D-LT and L-LT salts and their mixtures (arrows indicate significant reflections).

Figure 6. Solubility phase diagram for D-LT and L-LT salts in water for 25 °C (For clarity purposes just the upper 20% of the phase diagram is shown, therefore phase boundary lines are just schematic representation to illustrate the conditions in the system).

The double salt behavior was proven again with the help of the ternary solubility phase diagram, which was determined at 25 °C in water (Figure 6). A difference of 4 wt % in the solubility of the pure diastereomeric salts was observed, in comparison to D-L- and L-L-salts a

notable lower difference in solubilities. It can also be seen that the salt solubility of the mixture is increasing from both pure salt solubilities as the fraction of the other salt is increasing. Two local maximum solubilities were observed at two points on both sides of the 50:50 mixture. Maximum solubilities were observed at the compositions 79:21 and 5:95 of D - L T/ L - L T-salts respectively (i.e., 58% diastereomeric excess of D-LT and 90% diastereomeric excess of L-LT-salt, respectively). In the figure line segments are indicated to separate the phase regions. The regions 1, 2, 3 in the phase diagram represent two phase regions (one solid and one liquid phase), while the regions 4, 5 represent three phase regions (two solid and one liquid phase). After determination of the phase diagram it is obvious that a separation process directly from the 50:50 mixture of the diastereomeric salts is not feasible. A preliminary enrichment would be necessary, which is not practical and in particular not the intention of a diastereomeric salt resolution. 3.3. Solid Solution Behavior (L-LM- and D-LM-salts). In comparison to the tartrates salt shown above mandelic acid is a monoacid resolving agent, which will form naturally a different stoichiometry with the monobasic serine benzyl ester. Furthermore, mandelic acid is regularly used in laboratory and industrial relevant scale. Again, D-LM-and L-LM-salts and mixtures of both salts were characterized by XRPD patterns, showing an obvious similarity of the main peaks (Figure 7). The signals are slightly shifted against each other (e.g., peaks at 17.4°/17.5°, 28°/28.1° and 33°/34° for L-LM/D-LM), which is a strong indication for solid solution behavior. 3765



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Figure 7. XRPD patterns for pure D-LM and L-LM salts and mixtures of both (arrows indicate significant reflections).

Figure 8. DSC melting curves for pure D-LM and L-LM salts and two mixtures.

The assumption of solid solution behavior is further strengthened by the DSC melting curves (Figure 8) and ternary solubility phase diagram (Figure 9). Pure D-LM and L-LM salts comprise lone sharp melting peaks at similar melting temperatures of 134.9 and 135.6 °C, respectively. Significant variation is obtained in the melting enthalpies of both salts (D-LM, 15.6 kJ/mol; L-LM,

41.5 kJ/mol), which could lead to an asymmetry in the phase diagrams. Single sharp peaks were observed for any mixture and no peak for a eutectic melting was observed. The solubility phase diagram for the diastereomeric pair L-LM and D-LM in acetone is shown in Figure 9. Isotherms at 5 and 15 °C are presented. Both pure L-LM and D-LM salts possess 3766



Figure 9. Ternary solubility phase diagram for D-LM and L-LM salts in acetone at 5 °C and 15 °C.

almost an identical solubility with a slightly higher value for D-LM salt, which is consistent with the slightly lower melting temperature of D-LM. As the fraction of the other salt is increasing, the solubility of the mixture in acetone is steadily decreasing on both sides of the phase diagram. The lowest solubility is observed somewhere around the 50:50 mixture of both salts for the two temperatures, which is related to a solubility reducing effect of one salt on the other salt in solution. Furthermore, no eutectic compositions were observed in the phase diagram for both isotherms. Deviations in the data of the 15 °C isotherm can be attributed to the high volatility of acetone. The solubility phase diagram also supports the concept of formation of mixed crystals within the whole system, as also indicated by XRPD and DSC melting curves. As crystallization processes are mainly based on the binary and ternary phase behavior of the salts, this type of phase diagram hinders the separation and will cause very low yield if high purity is required. From the results obtained for the L-LM and D-LM salts, it can be concluded that the most abundantly available and most frequently used resolving agent L-(þ)-mandelic acid is not suitable to separate the enantiomers of serine.

4. CONCLUSIONS The synthesis of three pairs of diastereomeric salts with different resolving agents was successful as proven by the analytical techniques used (1H NMR, DSC and XRPD). The salt pair D-L and L-L (resolving agent 2,3-dibenzoyl-L-tartaric acid) showed a simple eutectic behavior both in XRPD and in the ternary solubility phase diagram. Chiral separation via a simple crystallization-based separation process is feasible in this case. The application of L-(þ)-tartaric acid, one of the most abundantly used resolving agents lead to a salt pair (D-LT and L-LT), which creates a double salt. This situation is not optimal for resolution processes, as an additional enrichment step would be necessary to obtain pure salts via crystallization. Third, the solid state behavior of salt pair L-LM and D-LM (resolving agent L-(þ)-mandelic acid) is characterized by formation of mixed crystals (solid solutions), which

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was verified by both melt and solution studies. Thus, only the diastereomeric salt pair D-L and L-L could be selected for the development of a separation process for the resolution of DL-serine. The results of the present work approve the fact that the separation of a chiral compound-forming substance should be tested with a number of different resolving agents. Each pair of salts that is formed should be analyzed with different analytical techniques such as 1H NMR, DSC, and XRPD to check for the purity and the possibility of polymorphism or solvate formation, which have a strong effect on the feasibility and the final yield of crystallization processes. Measurements of all physical-chemical properties like melting points, solubilities of pure salts are necessary to find the differences needed for the design of an effective separation process. Later the binary melting and ternary solubility phase diagrams of the salts should be generated more precisely, so that the behavior of the system (either simple eutectic, double salt or mixed crystals) can be identified. If the system is of simple eutectic type, the composition of the eutectic in the diastereomeric system plays a very important role. Supposing the eutectic composition is very close to one of the pure salts, the other salt can be separated with high yields, otherwise the yield will be reduced significantly. In the above study in the comparison of resolving agents for the separation of DL-serine, the L-enantiomer of the resolving agent 2,3-dibenzoyl-L-tartaric acid yielded the D-L-salt to crystallize first, thus the unnatural amino acid D-serine can be achieved in pure form. If L-serine is to be produced via classical resolution then the D-enantiomer of the resolving agent would be suitable. In general, basic thermodynamic information and kinetic data are absolutely necessary for the quantitative and optimized design of a crystallization separation process. In the end, the ease in the recovery of the pure enantiomers and the resolving agent has also a considerable impact on the process. The present study also partially resembles the Dutch resolution process14,15 and tries to understand the effect of resolving agents belonging to the same family like 2,3-dibenzoyl-L-tartaric acid and L-(þ)-tartaric acid. Strong differences were observed with these two resolving agents during the investigations. Thus, a combined usage of both resolving agents might yield reduced purities and yields. Further work will concentrate on the optimization of the corresponding separation process.

’ ASSOCIATED CONTENT

bS

Supporting Information. A selection of representative DSC-measurements for the binary melting behavior of the double salt (L-LT and D-LT-salts). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (0049) 391 6110 293. Fax: (0049) 391 6110 524. E-mail: [email protected].

’ ACKNOWLEDGMENT The authors would like to thank Dr. L. Hilfert, Otto von Guericke University, Magdeburg and J. Kaufmann, L. Borchert, Max Planck Institute, Magdeburg, Germany for their support in analytical work. 3767



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’ REFERENCES (1) Mullin, J. W. Crystallization, 3rd ed.; Butterworth-Heinemann: Oxford, U.K., 1997. (2) Jacques, J.; Collet, A.; Wilen, S. H. Enantiomers, Racemates, and Resolutions; Krieger Publishing, FL, 1994. (3) Collet, A. Enantiomer 1999, 4, 157 –172. (4) Subramanian, C. V. Chiral Separation Techniques. A Practical Approach; Wiley-VCH; Weinheim, Germany, 2001. (5) Kozma, D. Optical Resolutions via Diastereomeric Salt Formation; CRC Press LLC, London, 2002. (6) Leusen, F. J. J.; Rationalization of Racemate Resolution. PhD Thesis. Katholieke University Nijmegen, 1993. (7) Mersmann, A. Crystallization Technology; Marcel Dekker Inc., New York, 2001. (8) Eger, D., Ed. Handbook of Chiral Chemicals, CRC press LLC, London, 2005. (9) Kleemann, A.; Engel, J.; Kutscher, B.; Reichert, D. Pharmaceutical Substances: Synthesis, Patents and Applications of the Most Relevant Apis; Thieme, Stuttgart, Germany, 2009. (10) Klussmann, M.; Iwamura, H.; Mathew, S. P.; Wells, D. H., Jr; Pandya, U.; Armstrong, A.; Blackmond, D. G. Nature 2006, 441, 621–623. (11) Losse, G.; Augustin, M. Chem. Ber 1958, 91, 157–159. (12) Sistla, V. S.; von Langermann, J.; Lorenz, H.; Seidel-Morgenstern, A. Chem. Eng. Technol. 2010, 33, 780–786. (13) Hefter., G. T.; Tomkins, R. P. T. The Experimental Determination of Solubilities; J. Wiley & Sons. Ltd, New York, 2003. (14) Vries, T.; Wynberg, H.; van Echten, E.; Koek, J.; Ten Hoeve, W.; Kellogg, R. M.; Broxterman, Q. B.; Minnaard, A.; Kaptein, B.; van der Sluis, S.; Hulshof, L. A.; Kooistra, J. Angew. Chem., Int. Ed. 1998, 37, 2349–2354. (15) Broxterman, Q. B.; van Echten, E.; Hulshof, L. A.; Kaptein, B.; Kellogg, R. M.; Minnaard, A. J.; Vries, T. R.; Wynberg, H. Chem. Today 1998, 129, 4278.

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Analysis and Comparison of Commonly Used Acidic Resolving Agents

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