Characterization and Crystallization of Chiral Aromatic Amino Acids by Yan Li, M. Eng. A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Engineering Faculty of Engineering and Applied Science Memorial University of Newfoundland November 2014 St. John's Newfoundland and Labrador Canada Abstract The aromatic amino acids play a vital role in both biology and pharmacology areas. On one hand, they are the essential building blocks of protein, which can carry out many crucial bodily functions. On the other hand, these substances are recognized as chiral drugs containing a racemate and two enantiomers. In addition, the special aromatic ring adds a complex structure to this kind of amino acids. As a result, the investigation of the aromatic amino acids gains great significance and can provide people some practical ideas in the researching works about protein. This thesis presents a comprehensive study on the characterization and crystallization of aromatic amino acids. In the first part, diverse recognition methods were applied to characterize the L-tryptophan (L-Try), such as X-ray single crystal and powder diffraction analyses, differential scanning calorimetry, as well as measurement of solid- liquid equilibrium in water/isopropanol solution. The absence of eutectic point was demonstrated by the binary melting point diagram and the ternary phase diagram, which indicated the existence of a pseudoracemate (i.e., solid solution). The racemate, enantiomers, and the mixtures of them gave the identical powder X-ray diffraction patterns, and the X-ray single crystal analysis showed that the racemate and enantiomers of tryptophan gained very similar solid-state packing geometries, which confirmed the conclusion above. As a result, all the thermodynamic and crystallographic analyses have supported the conclusion that the tryptophan crystallized as a pseudoracemate. The second part of this thesis mainly focused on the study of the effect of tailor-made i impurity on the crystallization process. The influences of L-tyrosine (L-Tyr) on the solubility, crystallization kinetics, and polymorphism of L-phenylalanine (L-Phe) were respectively investigated. The results indicated that the presence of L-Tyr could increase the solubility and the crystal nucleation rate of L-Phe in water, however, the growth of L- Phe on a certain direction would be suppressed and thus the formation of polymorphs became unavoidable. Specifically, the addition of L-Tyr could make the anhydrate form of L-Phe more stable at 24 °C. ii Co-Authorship Statement Chapter 3: A version of chapter 3 was published as:  Li Y., Y.M. Zhao, Y. Zhang. Solid tryptophan as a pseudoracemate: physicochemical and crystallographic characterization. Chirality 2014; doi: 10.1002/chir.22399. The experiments were designed by Dr. Y. Zhang. The data were collected by the author. The manuscript was written by the author and revised by Dr. Y. Zhang and Dr. Y.M. Zhao. iii Acknowledgement First and foremost, I am heartily grateful to my supervisor, Dr. Yan Zhang, a respectable, responsible, and resourceful scholar. Her encouragement, support, and guidance for my research make me confident and motivated to continue my research in chemical and process engineering. I also appreciate the financial support from Natural Sciences and Engineering Council of Canada through Discovery Grant and Memorial University of Newfoundland. I could not finish this research without their support. I shall extend my gratitude to Dr. Yuming Zhao, who has provided me with valuable guidance in preparing my first paper. I would also like to thank Ms. Julie Collins at C-CART, Memorial University of Newfoundland for helpful discussions on the single crystal structure of DL-Try. My gratitude also goes to Mr. Xiong, and Mdm. Mao, for all their kindness and help. Last but not least, I'd like to thank all my friends for their encouragement and support. iv Table of contents Abstract i Acknowledgement iv Table of contents v List of Figures viii List of Tables xii List of Abbreviations and Symbols xiii Chapter 1 Introduction 1 1.1 Problem Statement 2 1.2 Objective and Scope of Study 4 1.3 Thesis Outline 5 Chapter 2 Literature Review 7 2.1 Amino Acids 7 2.2 Crystallization of Amino Acids 10 2.2.1 Crystallization from solution 10 2.2.2 Factors affecting the crystallization of amino acids 12 2.2.2.1 Chirality of amino acids 12 2.2.2.2 Polymorphism of amino acids 16 2.3 Characterization of Chiral Modifications of Amino Acids 18 2.4 Effect of Foreign Molecules on the Crystallization of Amino Acids 23 2.4.1 Effect on the solubility 23 2.4.2 Effect on the nucleation 25 2.4.3 Effect on the crystal growth 27 2.4.4 Effect on the polymorphism 29 Chapter 3 Solid Tryptophan as a Pseudoracemate: Physicochemical and Crystallographic Characterization 33 v 3.1 Introduction 33 3.2 Materials and Methods 38 3.2.1 Materials 38 3.2.2 Differential scanning calorimetry 38 3.2.3 Solubility measurement 39 3.2.4 Powder X-ray diffraction 40 3.2.5 Single-crystal growth and structure determination 40 3.3 Results and Discussion 41 3.3.1 Absence of eutectic melting 41 3.3.2 Ternary solid-liquid equilibria 44 3.3.3 Powder X-ray diffraction 47 3.3.4 Single crystal structures of DL- and L-Try 48 3.4 Conclusions 54 Chapter 4 Effects of Structure Similar Additive on the Crystallization of L- Phenylalanine from Aqueous Solution 56 4.1 Introduction 56 4.2 Material and Methods 59 4.2.1 Material 59 4.2.2 Preparation of L-Phe anhydrate and monohydrate 59 4.2.3 Solubility and metastable limit measurement 60 4.2.4 Seeded batch crystallization of L-Phe in the absence or presence of L-Tyr 61 4.2.5 Characterization methods 62 4.3 Results and Discussion 63 4.3.1 Characterization of L-Phe anhydrate and monohydrate 63 4.3.2 Effect of L-Tyr on the solubility of L-Phe 65 4.3.3 Effect of L-Tyr on primary nucleation of L-Phe 67 4.3.4 Effect of L-Tyr on the growth and morphology of L-Phe 69 4.4 Conclusion 75 Chapter 5 Conclusions and Recommendations 77 vi 5.1 Conclusions 77 5.1.1 Characterization of L-Try 77 5.1.2 The effect of L-Tyr on the crystallization of L-Phe 78 5.2 Recommendations 78 References 80 vii List of Figures Figure 2.1 Structure of amino acids 7 Figure 2.2 The formation of proteins 7 Figure 2.3 Structural diagrams of phenylalanine (a); tryptophan (b); and tyrosine (c) 9 Figure 2.4 Classification of nucleation 11 Figure 2.5 Classification of the chiral system 13 Figure 2.6 Binary phase diagrams of (a) conglomerate; (b) racemic compound; (c) solid solution: 1, ideal; 2, with a maximum; or 3, with a minimum 14 Figure 2.7 PXRD spectra of the (a) α-glycine and (b) γ-glycine crystals (Srinivasan et al., 2011) 17 Figure 2.8 DSC curves of the (a) α-glycine and (b) γ-glycine crystals (Srinivasan et al., 2011) 18 Figure 2.9 Photographs of the glycine polymorphs (Srinivasan et al., 2011) 18 Figure 2.10 Binary melting point phase diagram of the mandelic acid (Lorenz et al., 2002) 20 Figure 2.11 Ternary (solubility) phase diagram of mandelic acid (Lorenz et al., 2002) 20 Figure 2.12 Powder XRD patterns of (R)-3-ClMA and (RS)-3-ClMA (Zhang et al., 2009) 22 Figure 2.13 Mole fraction solubility of L-phenylalanine anhydrate from 293.00 to 308.00 K and monohydrate from 312.00 to 328.00 K in pure water and viii aqueous solution in the presence of four salts: ◆,pure water; ▲, potassium chloride; ■, sodium sulfate; ●,ammonium sulfate; −, sodium chloride (Wang et al., 2014) 24 Figure 2.14 Electron micrographs of L-Glu crystallized with L-Phe: (a) no additive, (b,c) 1×10-2 M; (d) 2×10-2 M. (L-Glu= 0.3 M) (Cashell et al., 2005) 31 Figure 2.15 PMG crystallized from pure solution (a) and from impurity-doped solutions (1.0 wt% on PMG): (b) IMPA, (c) AMPA, and (d) PIDA (Poornachary et al., 2008) 31 Figure 2.16 Four different positions for the imposter L-Alanine to replace glycine molecules and form dimers within the unit cell (Kuvadia et al., 2013) 32 Figure 3.1 Melting point phase diagrams for pseudoracemates: Type I-ideal solution; Type II-solution with maximum melting temperature; Type III- solution with minimum melting temperature 35 Figure 3.2 Molecular structures of D- and L-tryptophan 37 Figure 3.3 DSC curves of tryptophan with various mass fraction of L-Try 42 Figure 3.4 DSC curves of enantiomeric mixtures with mass fraction of L-Try identical or close to the calculated eutectic composition 43 Figure 3.5 Binary melting point diagram of tryptophan system 44 Figure 3.6 Ternary phase diagram of tryptophan system in H O/IPA 46 2 Figure 3.7 Powder XRD patterns of tryptophan with various mass fraction of L-Try 48 Figure 3.8 (a) ORTEP plot of D-Try at 50% probability ellipsoids with atomic numbering. (b) Four molecules in the unit cell of DL-Try 50 Figure 3.9 Hydrogen bonding packing of (a) DL-Try and (b) L-Try (The numbers identify three types of hydrogen bonds) 52 ix