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Effect of Additives on the Crystal Morphology of Amino Acids: A Theoretical and Experimental Study Edward N Constance, Muzaffer Mohammed, Adeolu Mojibola, Micah Egiefameh, Oluseyi Daodu, Travis Clement, Taiwo O Ogundolie, Chinenye Nwawulu, and Kadir Aslan J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b04868 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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The Journal of Physical Chemistry

Effect of Additives on the Crystal Morphology of Amino Acids: A Theoretical and Experimental Study Edward N. Constance, Muzaffer Mohammed, Adeolu Mojibola, Micah Egiefameh, Oluseyi Daodu, Travis Clement, Taiwo Ogundolie, Chinenye Nwawulu and Kadir Aslan* Morgan State University, Department of Chemistry, Baltimore, MD 21251 *Corresponding author: Telephone: 1 443 885 4257, E-mail: [email protected] ABSTRACT Crystal morphology of amino acids can be altered in a controlled manner through inclusion of tailor-made additives in their structure, in-order to widen their scope for applications in drug design and targeted delivery. In this study, the effect of multiadditive combinations of hydrophobic and hydrophilic amino acids on the growth and morphology of L-alanine was investigated. Theoretical calculations were performed using two crystal growth models in Material Studio software™: 1) Build-in model 2) Surface Docking model. Crystallization experiments were carried out using MetalAssisted and Microwave Accelerated Evaporative Crystallization (MA-MAEC) technique with multiple hydrophobic and hydrophilic amino acids added in stoichiometric amounts to L-alanine solution. The crystal morphology was established and compared with predicted crystal morphology. The use of hydrophilic and hydrophobic additives predicted to have significant changes in the morphology of L-alanine crystals. Multiadditive combinations with hydrophobic amino acids resulted in elongation of L-alanine crystals through (120) face. Experimental data corroborates with the theoretical predictions in relation to the morphological changes due to additives, indicating the accuracy of theoretical models in predicting the impact of additives in crystal growth. 1 ACS Paragon Plus Environment


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INTRODUCTION The understanding of the intermolecular interactions involved in crystal packing and the implementation of this knowledge in the design of molecular solids are fundamental aspects of crystal engineering. In this field, it is essential to understand crystal structure in terms of intermolecular interactions as this understanding is integral to the discovery of methods by which we can reliably predict crystal morphology, and thus target desired crystal properties.1 Crystal morphology is a key factor in the development of crystals for production in pharmaceuticals,2 optoelectronics,3 and explosives4. Furthermore, the influence of impurities on the shape of a crystal is of major interest since they are known to directly impact the crystal growth. Molecules occurring as an impurity tend to adsorb onto certain crystal surfaces, hindering the advancement of step layers and leading to growth retardation normal to the surfaces.5 Crystal shape is determined by the relative growth rates of the crystallographic facets that bound the crystal. The slowest growing faces dominate the crystal habit; however, in the presence of trace impurities, the crystal habit can undergo significant changes.6,7 Consequently, by selection of additive molecules with specific chemical properties, crystallization can be directed and crystal morphology can be controlled.8 Tailor-made additives are molecules that closely resemble the structure of the host molecule, as a result they are considered to be appropriate for use in modifying crystal morphology. Klug and Van Mil observed significant reduction in the incorporation of impurities, when small amounts (1%) of caproic acid were crystallized with adipic acid leading to improved flow properties.9 Impurities in the crystallization of adipic acid preferentially adsorb on to the (100) faces, resulting in plate-like morphology. However, 2 ACS Paragon Plus Environment

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in the presence of caproic acid, the (100) face is inhibited. This results in the (001) face dominating the crystal habit while the growth of the (100) face is drastically reduced, causing flat need-like morphology. L-alanine crystals have shown promising properties for potential use in nonlinear optics applications.10 Previous studies on the crystallization of L-alanine with additives like L-valine11 and L-leucine12 were found to affect the crystal morphology. These additives were crystallized together with L-alanine using the Metal-Assisted and Microwave-Accelerated Evaporative Crystallization technique (MA-MAEC) resulting in an increase in crystal size and needle-like morphology. Razzetti et al.

13, 14

observed

that the (011) and (120) facets dominate the crystal habit of pure L-alanine crystals, indicating that these faces have high morphological importance. Therefore, it is critical to examine the effect of additive molecules on these faces as a method of determining the changes in morphology. Certain hydrophobic amino acid additives selectively bind to and inhibit the (120) face of L-alanine crystals. Previous research shows that nonpolar additives preferentially adsorb onto L-alanine crystals along the hydrophobic chains found on the (120) face, while adsorption on the (011) face occurs to a much lesser extent.6 The interplay between impurities, whether competitive or not, during crystallization could potentially be a decisive factor in crystal growth. Nonetheless, comprehensive studies that demonstrate the effect of multiple additives interacting simultaneously with crystals growing from solution are required to efficiently predict the crystal morphology that would be obtained through different additives. In this regard, there exists a need for reliable methods to predict the crystal morphologies of the various crystalline solids under investigation.

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In this work, a detailed theoretical and experimental study of the effects of various hydrophobic and hydrophilic amino acids on L-alanine crystals, including various combinations of these additives is presented. The importance of this study arises due to critical role that growth morphology plays in the effectiveness of crystalline solids in many industries. L-alanine was crystallized in the presence of multi-additive combinations using the MA-MAEC technique, which affords for rapid crystallization of amino acids.12, 15-16 Combinations of additives were defined as hydrophobic, hydrophilic and mixed (combinations of hydrophobic and hydrophilic). Concurrently, theoretical models: i) Build-in model ii) Surface docking model, were used to predict the crystal morphologies and these results were correlated with the observed morphologies. The build-in model involves the substitution of L-alanine molecules in the unit cell at the surface of the crystal. We investigated the degree of incorporation of additives into L-alanine surfaces as a method of assessing the molecular compatibility of the additives with molecules in the crystal bulk. The build-in method was employed to determine the impurity effects of amino acid additives within the surface of L-alanine faces. The surface docking model considers the interaction of additive molecules placed directly on the crystal surface.17 It uses molecular dynamics to compute and compare the surface binding energies of additive and host molecule. Both models were employed to simulate the effect of additives on the morphologically important faces of L-alanine crystals. Using theoretical data, crystal morphology was predicted and compared with experimental data to identify the accuracy of theoretical calculations.


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MATERIALS AND METHODS Materials. Amino acids (L-alanine, L-valine, L- Leucine, L-proline, L-arginine, Lhistidine, and L-glutamic acid) were purchased from Sigma Aldrich Inc., USA. PMMA discs (5 cm diameter) were purchased from McMaster-Carr Inc., USA. 21-well silicone isolators, EMS 150 RS sputter coating system and silver target were purchased from Electron Microscopy Sciences Inc., USA. Harrick plasma PDC 32G and optical microscope was purchased from Thermo-Fisher Inc., USA. 900 W conventional microwave was purchased from Walmart Inc. Material Studio 8 was obtained from Accelrys Inc., USA. Methods Preparation of circular crystallization platforms. PMMA discs were subjected to plasma cleaning in Harrick plasma PDC 32G for 2 min. A 21 well polypropylene mask was placed on the disc and 1 nm Ag was deposited using plasma sputter coater. A 21well silicone isolator was then attached onto the disc. Preparation of amino acid solutions. L-alanine stock solutions were made to 2.4 g/10 mL solvent for each additive combination (single, double and triple). Different additive solutions were made for each additive set (hydrophobic, hydrophilic and mixed). A 0.2 g of each amino acid additive was dissolved separately in 10 mL deionized water and kept at 40 °C. Additives were introduced in varying quantities into L-alanine solution, according to the additive combination. A 1 mL of each L-alanine stock was replaced with 1 mL of additive solution (for single additives) to obtain 0.93 g additive/100 g Lalanine. The additive to L-alanine mass ratio was maintained for each combination attempted. For double-additive mixtures, 0.5 mL of each of the two additive solutions 5 ACS Paragon Plus Environment


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replaced 1 mL of L-alanine stock solution. For triple-additive mixtures, 0.33 mL of each of the three additive solutions replaced 1 mL of L-alanine stock solution. Crystallization experiments using the MA-MAEC technique. A 20 µL of solution is pipetted into each well of circular crystallization platforms and left at room temperature under optical microscope. For the MA-MAEC technique, the circular crystallization platform is subjected to microwave heating in 900 W microwave at power level (PL) 1 until entire solution evaporates. Platforms are stored in stability chambers until further use. Characterization of crystals. Crystals were characterized using Powder X-Ray Diffraction technique (Rigaku Miniflex). Data was analyzed to identify characteristic peaks associated with additives. Raw data was used in Material Studio 8 to compare with theoretical predictions of crystal morphology. Theoretical simulations. L-alanine crystallizes in an orthorhombic lattice with space group P212121 and there are four molecules in the unit cell linked by a three-dimensional network of hydrogen bonds. The unit cell dimensions (a=6.032, b=12.343, c=5.784; α=β=γ=90°) were obtained from the Cambridge Structural Database (CSD) and the calculations were carried out using Biovia Materials Studio 8. Build-in method. The build-in approach seeks to measure the degree of incorporation of additives into the unit cell of L-alanine crystals. Through this method, incorporation energies of additives and the host molecule can be compared to investigate the effects of the additives. This method is comparable to the one outlined by Yang et. al.18


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The model assumes that the additive is substituted within the surface by replacement of an L-alanine molecule and the conformation of lowest energy reduces the effects of repulsive forces due to the foreign molecule. Theoretically, the stability of the additive within the surface of L-alanine crystals is predicted by calculating the total surface energies following the additive substitution. As the additive is incorporated within the surface, it interferes with hydrogen bonding and nucleation resulting in inhibition. The more stable an additive is within the surface, the greater the ease of incorporation. Based on an optimized unit cell, the (011) and (120) faces of L-alanine were cleaved to a depth of 30 Å. The cleaved structure was then extended into threedimensional vacuum slabs with the vacuum thickness set to 50 Å to ensure that nonbond interactions reach their asymptotic values. Additives were substituted for each of the four orientations of L-alanine within its unit cell at the topmost layer. Each additive was substituted only once to maintain a one to one ratio among the respective additives. For single additives, it was observed that four different orientations were possible for substitution into the surface. However, for double and triple additive substitution, 12 different orientations were used. Subsequently, the periodical structure was subjected to geometry optimization with CVFF force-field and force-field assigned charges within the Forcite module. Other optimization parameters applied included SMART algorithm and Ewald summation to compute long-range interactions. The incorporation energy of L-alanine (i.e., energy of pure bulk) was calculated as control for comparison. Surface docking method. In this model, impurity effects are measured by calculating the binding energies of additive molecules directly bound to the flat surface of L-alanine 7 ACS Paragon Plus Environment


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crystals. This method uses molecular dynamics to simulate the effect of impurity molecules on a cleaved surface of a crystal and computes the surface binding energy of an additive and compares it to that of the host molecule. Surface binding energy measures the affinity of an additive molecule to a specific crystal face where the larger the absolute value of the binding energy, the greater the affinity of the additive to the crystal face.19 The underlying theory in the model is that additives adsorb on to specific crystal faces and disrupt or interfere with crystal growth, thereby affecting the growth rate of those faces. Additives that demonstrate a strong affinity for a particular face will reduce the growth rate of that face by lowering its attachment energy.17 Through molecular dynamics simulations, the interface interaction of L-alanine with the given amino acid additives is modeled and the corresponding surface binding energies are calculated. In the case of multiple additives, modifications were made to the calculations by incorporating the mean of the additive energies and subtracting from the mean total energy of the additives with the crystal surface, which helps in an assessment of the effect of the different molecules that can be computed as the mean binding energy. Subsequently, the inhibition effect (∆ܾ௛௞௟ ) can be estimated by the difference between the binding energy of the respective amino acid combination and the binding energy of L-alanine. The larger the absolute value of the inhibition, the lower is the attachment energy, and therefore the lower the growth rate. The optimized unit cell was used to cleave the (011) and (120) faces to a thickness of over 10 Å and a supercell structure was created with appropriate dimensions (011: U=5, V=2; 120: U=4, V=2). The supercell was then extended into a vacuum slab with vacuum thickness of over 30 Å to avoid non-bond interactions from periodic boundary


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conditions. The Cartesian coordinates of the L-alanine molecules in the superstructure were constrained in order to precisely reproduce the intermolecular interactions within the bulk crystal. The additive molecules are docked in a consistent manner at the center of the surfaces followed by optimization to identify the lowest energy conformation of the additives. Then molecular dynamics simulations were performed using CVFF force-field, force-field assigned charges, and Ewald summation as implemented within the Forcite module. The simulations were carried out with an NVT ensemble at a time step of 0.1 fs for a duration of 50 ps. A target temperature of 298 K was set and the temperature during simulation was controlled using the Nosé-Hoover-Langevin thermostat with a q ratio of 1. L-alanine molecule was also docked onto the crystal surface and its binding ௔௟௔௡௜௡௘ energy (‫ܧ‬௕,௛௞௟ ) was computed in order to conduct a comparative binding energy

analysis. Crystal Morphology. The crystal morphology module within Material Studio was used to predict the morphological effects of modifying the growth rate of the (011) and (120) faces. To investigate the effects of additives on the shape of L-alanine crystals, modified attachment energies were used to generate a crystal habit. Modified attachment energies were computed using ௣௨௥௘

௠௢ௗ ‫ܧ‬௔௧௧,௛௞௟ = ‫ܧ‬௔௧௧,௛௞௟ ቆ1 −

∆ܾ௛௞௟

௔௟௔௡௜௡௘ ‫ܧ‬௕,௛௞௟

ቇ

A simulated crystal habit of pure L-alanine was created and later modified attachment ௠௢ௗ energies (‫ܧ‬௔௧௧,௛௞௟ ) were substituted for the original values (pure attachment energy,



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௣௨௥௘

‫ܧ‬௔௧௧,௛௞௟ ) followed by a recalculation of the crystal habit. The CVFF force-field and Ewald summations were used in all calculations of the crystal morphology. RESULTS AND DISCUSSION Theoretical simulations were performed to study the effects of various combinations of amino acid additives on the morphologically important faces of Lalanine crystals, namely (011) and (120). These additive combinations were classified as hydrophobic, hydrophilic or mixed (both hydrophobic and hydrophilic). The build-in and surface docking models were used to determine the impurity effects of additives on L-alanine crystal faces using molecular dynamics. In the build-in model, the least energy positions were identified as the most favorable adsorption sites. In the surface docking model, the binding energy of additives were calculated to investigate the effect of additives on the attachment energy of the crystal surface. These results were used to generate the crystal morphology of L-alanine crystals under the influence of various additives. Table 1 shows the surface energies calculated by build in and docking models after the introduction of hydrophobic molecules (L-leucine, L-proline, L-valine individually, {L-leucine + L-valine}, {L-leucine + L-proline}, {L-proline + L-valine}, and {Lleucine + L-proline + L-valine}) in to L-alanine. There was an overall increase in surface energy for hydrophobic additives in both the (011) and (120) faces, which is an indicative of low molecular compatibility with either face. For example, for the addition of a hydrophobic amino acid (L-leucine), the change in surface energies (∆‫ܧ‬௦௨௥௙ ) were lower on the (120) face compared to (011) in all orientations, which implies a higher


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degree of incorporation (i.e., inhibition) of L-leucine into the (120) face of L-alanine and a smaller relative growth rate. The {L-leucine + L-valine} and {L-leucine + L-proline} combinations of additives also show a stronger inhibitory effect on the (120) face, which was marked by lower ∆‫ܧ‬௦௨௥௙ values. The {L-leucine + L-valine + L-proline} combination

(Figure 1) had the highest ∆‫ܧ‬௦௨௥௙ values for both faces. L-proline and combinations involving L-proline also exhibit high ∆‫ܧ‬௦௨௥௙ values for both surfaces, implying a lesser

ease of incorporation into either face. With respect to the hydrophobicity of amino acids, the intermediate value of the Kyte-Doolittle hydropathy index (Table S1, Supporting Information) for L-proline could be a factor in its limited adsorption onto L-alanine faces.20 Notably, position 9 on the (011) face, where the additives were placed adjacent to each other, seems favorable for double additive hydrophobic combinations (Supporting information, Figures S1-S3). A reverse trend was predicted for hydrophilic additives with an overall decrease in the surface energies except for single additive (L-histidine) and double additives (Lhistidine + L-glutamic acid), as compared to the hydrophobic additives (Table 1). This prediction suggests that hydrophilic additives exhibit a high degree of molecular compatibility with both the (011) and (120) faces of L-alanine. In the cases of L-histidine and {L-histidine + L-glutamic acid} additives, there was an increase in the surface energies particularly on the (011) face, which implies a lower compatibility or difficulty in incorporation of these additives in to the L-alanine crystal face. Also, a reduction in surface energies of both faces in the presence of the strongly hydrophilic additive, Larginine, was predicted. Combinations of additives involving L-arginine are predicted to enhance the incorporation of these additives into both faces of L-alanine, especially in



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the (120) face as reflected by the more negative ∆‫ܧ‬௦௨௥௙ values. These predictions are contrary to the expected behavior due to the hydrophobic and hydrophilic nature of the (120) and (011) faces, respectively. Interestingly, L-glutamic acid (kd index = -3.5) seems to exhibit less inhibitory effect than that of L-histidine (kd index = -3.2) and larger than that of L-arginine (kd index = -4.5), as revealed by the ∆‫ܧ‬௦௨௥௙ values. Table 2 also reveals that position 2 on the (011) face of L-alanine is predicted to be favorable for single hydrophilic additives (Supporting information-S4-S6). Mixtures of hydrophobic and hydrophilic amino acid additives show varying degrees of incorporation on both the (011) and (120) surfaces, as shown in Table 1. Combinations involving L-proline and L-histidine exhibit an increase in the surface energies indicating lower molecular compatibility. Whereas, combinations with Larginine and L-leucine lower the surface energies particularly on the (120) face signifying larger incorporation. The {L-arginine + L-leucine} combination resulted in the lowest energy for both faces. However, the {L-proline + L-glutamic acid + L-valine} combination produced the highest energy for both faces. Given that L-arginine is the most hydrophilic amino acid according to its hydropathy score, its higher incorporation on the hydrophobic face (120) is unexpected. In this regard, the total surface energies are a good indicator of the impurity effects of these amino acid additives; however, the build-in model does not accurately reflect the host-guest interactions that occur at the surface of L-alanine crystals. Hence, the surface docking model was employed to investigate the binding process of additives on the flat interfaces of L-alanine crystals. The morphology of L-alanine crystals in the presence of the amino acid additives were predicted using the modified attachment energies obtained from the surface


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docking model. According to the periodic bond chain (PBC) theory, the nature of a crystal face depends on the strong periodic chains of molecules parallel to the corresponding face. The PBC theory distinguishes flat faces as being parallel to at least two nonparallel intersecting periodic bond chains.21 These types of faces are stable and are the slowest growing faces. The facets that dominate the crystal structure are the slowest growing faces and thus have the greatest morphological importance. The growth morphology method, which is used to calculate the crystal habit, assumes that the attachment energy of a crystal face is proportional to its growth rate. Hence, the attachment energy is inversely related to the morphological importance of a given crystal surface. The modified attachment energies obtained from the surface docking model are used to calculate the center-to-plane distances for the respective faces, which is used to deduce the crystal morphology. According to the surface docking model employed for this study, the absolute ∆ܾ௛௞௟ values were predicted to be larger (i.e., more negative) on the (120) face for hydrophobic additives, which implies preferential adsorption onto that face. These predictions can be attributed to the hydrophobic nature of the (120) face owing to the presence of methyl groups projecting from the surface. In addition, the (011) face is hydrophilic due to the presence of carboxylic and amino groups.6 The characterization of (120) face as hydrophobic and (011) face as hydrophilic was validated on the basis of a simulated density profile of water over the surfaces, which showed water molecules to be closer to the (011) surface, therefore hydrophobic additives would have minimal effect in that location.22 Table 1 shows that the inhibition effect of L-proline was considerably small on the (120) face and has a non-inhibiting effect on the (011) face,



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which implies that this amino acid has little impact on the morphology of L-alanine crystals. On the other hand, multi-additive combinations, such as {L-leucine + L-valine + L-proline} demonstrate strong inhibition on the (120) face, as evidenced by the reduction in absolute attachment energies. In order to further understand these predictions, we investigated the dynamics of binding of hydrophobic additives (i.e., {Lleucine + L-valine + L-proline}) on to the (120) face of L-alanine, as shown in Figure 2. In the beginning (Figure 2a) of the binding interactions, the additives were found to be farther apart with bound to the surface via hydrogen bonds. When the potential energy becomes more negative, the additive molecules were reoriented to have their side chains located away from the surface but their charged functional groups positioned to interact with the surface (Figure 2b). As the simulation proceeds, additional hydrogen bonds were formed as the molecules continue to reposition themselves (Figure 2c). Eventually, the additives were oriented such that the side chains were closer to the surface (Figure 2d). This type of interaction reflects the hydrophobic character of the (120) face as it involves non-polar molecules interacting favorably with the surface. Thus, this multi-additive combination is predicted to be adsorbed onto the (120) face, thereby slowing the growth rate. These simulation results concur with the data obtained from the build-in model indicating the preferential inhibition of (120) face by hydrophobic additives. In the case of hydrophilic additives in the docking model, significant inhibition of both faces was predicted with the exception of L-histidine and {L-histidine + L-arginine}, which exhibit notably smaller effects on either face. L-histidine is predicted to have a significantly low impact on either face as evidenced by the relatively small ∆ܾ௛௞௟ values 14 ACS Paragon Plus Environment

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in Table 1. The modified attachment energy, as a result of this additive, was relatively close to the pure attachment energy, which implies the small impact of L-histidine on both surfaces. The dynamics simulations predict less frequent interaction (van der Waals and hydrogen bonding) of L-histidine with either face, particularly with regard to the imidazole side-chain. Similarly, the ∆ܾ௛௞௟ values for the {L-histidine + L-arginine} combination are relatively small due to the limited interaction of L-histidine. In addition, the inhibition effects were similar on the (011) face, however more pronounced for multiadditive combinations on the (120) face. These results align with our build-in model where the overall effect of hydrophilic additives involves significant interaction with both faces, especially the (120) face. Surface docking model for the mixed additives yielded similar predictions made for the other groups of additives, where the (120) face of L-alanine is predicted to be inhibited by the mixed additives. Table 1 also shows the results of the surface docking model using mixed additives. From the previous simulations, we observed that both Lproline and L-histidine each had little effect (low ∆ܾ௛௞௟ values) on either face of Lalanine. Hence, the relatively low absolute ∆ܾ௛௞௟ values associated with the {L-proline + L-histidine} combination is consistent with the observed trends. Materials Studio also affords for the visualization of predicted crystal morphology of L-alanine in the absence and the presence of other amino acids. Figure 3 shows the predicted changes in the morphology of L-alanine crystals in the presence of a multiadditive hydrophobic combination of {L-Leucine + L-Proline + L-Valine}. Elongation of the crystals can be attributed to the inhibition of the (120) face resulting in an increase in



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the relative growth rate of the (011) face. Inhibition of the (120) face reduced its perpendicular growth while the faster growing (011) face extended along the c-axis. In order to assess whether the predicted morphologies of L-alanine crystals in the presence of additives can be realized experimentally, we have employed the MAMAEC technique. The predicted morphologies of L-alanine crystals in the presence of additives are given with the optical images of L-alanine crystals grown using the MAMAEC technique in Figures 4-6. For the sake of brevity, we presented the data in three groups: L-alanine with hydrophobic additives, L-alanine with hydrophilic additives and Lalanine with mixed additives. Figure 4 shows that predicted morphologies of L-alanine crystals in the presence of hydrophobic additives were in good agreement with the experimental results. For example, the combinations {L-leucine + L-valine} and {Lleucine + L-valine + L-proline}, both involving the more hydrophobic amino acids, resulted in needle-like morphology. It should be noted that the morphology in the presence of L-proline additive is similar to that of pure L-alanine, which can be attributed to the poor interaction of the additive with the crystal faces. Predicted morphologies of L-alanine crystals in the presence of hydrophilic additives were not in complete agreement when multiple additives were used at a time. The calculated morphologies for multi-additive hydrophilic combinations ({L-arginine + L-glutamic acid}, {L-arginine + L-histidine}, {L-glutamic acid + L-histidine} predicted short crystals (Figure 5) in contrast to the needle-like crystals grown experimentally. Single additives were found to have close correlation between predicted and observed morphologies. Calculations performed for L-histidine had the largest growth along the b-axis. L-arginine and Lglutamic acid predictions had limited growth along the b-axis on account of inhibition


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effects. For mixed additives, the predicted morphologies resembled the experimental morphologies for {L-valine + L-glutamic acid}, {L-proline + L-histidine} and {L-valine + Lglutamic acid + L-histidine} (Figure 6). It was also observed that the habit predictions in the presence of the additives, L-proline and L-histidine, which have the least interaction with L-alanine surfaces concur with the experimental results. It is important to note that the simulations performed in this study were modeled without taking into account the effect of solvent-additive and solvent-solute interactions. Hence, the lack of non-solvated interfaces might explain the irregularities observed in the prediction of the crystal morphology of L-alanine in the presence of hydrophilic additives. In the presence of a solvent, these additives would have readily attracted solvent molecules that might have a significant impact on their interaction with L-alanine surfaces. Moreover, improved mathematical models are required to better predict the changes in attachment energy at the crystal surface. It is also important to comment on the experimental observations reported in this study. Using the MA-MAEC technique, the crystallization of well-developed crystals of L-alanine in the absence and presence of the amino acid additives occurred in 45 min or less. Pure L-alanine crystals were fully grown after 45 min on circular crystallization platforms during microwave heating. Well-developed crystals of L-alanine with hydrophobic additives were observed over a range of 25 to 40 mins with a mean average of 27 ± 6 min. In the presence of hydrophilic additives, full grown crystals were formed over a range of 25 to 35 min with a mean average of 29 ± 5 min. Crystals of Lalanine in the presence of mixed additives were fully formed over a range of 25 to 40 min with a mean average of 36 ± 5 min. The differences in crystallization times of the



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solutions can be attributed to variation in solubility and the degree of inhibition by additives. Hydrophobic additives appear to lower the solubility of L-alanine by disrupting solute-solvent interactions, which causes crystallization to occur more readily. Furthermore, slower crystallization of hydrophilic and mixed additives relative to the hydrophobic additives can be attributed to significant interference with nucleation which coincides with higher inhibition on L-alanine surfaces. Additionally, the hydrophilic additives studied were much larger in size than their hydrophobic counterparts; therefore, these molecules have greater surface coverage of L-alanine surfaces, which further impedes the addition of new layers. Hydrophobic combinations involving Lleucine resulted in relatively longer crystals. Whereas, L-proline additive resulted in shorter, bulkier crystals resembling those of pure L-alanine. With L-arginine present, hydrophilic combinations resulted in longer crystals; however, histidine additive resulted in larger, bulkier crystals. Mixed additives showed varying shapes and sizes; notably, the {L-leucine + L-arginine} combination produced longer crystals. It is also important to comment on the applicability of the current approach presented here to more complex molecules, such as peptides. As it relates to the theoretical aspect of this study, the methods outlined could be applied to peptides in a similar manner by using the total energy of the peptides under consideration for a comparative binding analysis with the host molecule. In this instance, if using a specific peptide, there would be no requirement for the mean binding energy since the peptide may be treated as a single additive. However, given a variety of peptide additives, the average binding energy would be computed to reflect the inhibition effect of a group of molecules on a particular crystal surface. With regard to experimental methods,


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considerations should be made for the impact of several factors such as hydrophobicity, solubility and peptide denaturability on the crystallization of peptides in the presence of other additives. Future studies will include development of models that consider the effects of solvent on the crystallization of L-alanine with additives and crystallization of peptides in the presence of amino acid and peptide additives. The effect of polymers of amino acid additives, in which the functional groups of the amino acids are involved in peptide linkages, will also be studied on the important facets of L-alanine crystals and will be reported in due course. CONCLUSIONS The predicted morphologies of L-alanine crystals under the influence of hydrophobic and hydrophilic amino acid additives were calculated based on build in and surface docking models by means of molecular dynamics. Our results also show that the strong hydrophilic additives appear to inhibit L-alanine surfaces, with a greater effect on the (120) face. L-leucine and L-arginine show a significant interaction resulting in inhibition of L-alanine surfaces. On the contrary, L-proline additive showed little interaction with L-alanine surfaces. Interestingly, L-valine and L-glutamic acid display intermediate effects. The importance of molecular simulations is recognized in determining the impact of impurity molecules on crystal growth. Moreover, the possibility of significantly manipulating crystal morphology through addition of suitable additives is established through this study. In addition, to investigate whether theoretical calculations can be verified by experimental results, we have grown L-alanine crystals in the presence of amino acid additives using the MA-MAEC technique, where a close agreement between both methods were observed. This study clearly demonstrates that



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the desired crystal morphology of L-alanine can be created by the combined used of theoretical calculations and the experimental MA-MAEC technique. SUPPORTING INFORMATION. Additional information related to simulations of Lalanine crystals in the presence of additives not shown in the main text are provided. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENTS Research reported in this publication was partially supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number R41TR001275. REFERENCES 1.

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Table 1. Incorporation Energies and Attachment Energies Induced by additives in the Build-in and Surface Docking Models, Respectively The reported positions indicate the least energy orientations of the additives within the ௣௨௥௘ surface of L-alanine crystals. Docking model: Pure Attachment Energy, ‫ܧ‬଴ଵଵ = -147.8 ௣௨௥௘ kcal/mol, ‫ܧ‬ଵଶ଴ = -65.7 kcal/mol.



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Figure 1. Build-in model simulations on L-alanine surfaces. Left: (011) and Right: (120) substituted by L-valine, L-proline, and L-leucine in 12 symmetry positions in the build-in model. L-valine, L-proline, and L-leucine molecules are colored in yellow, green, and red, respectively. Positions are read in ascending order (1-12) from left to right.


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Figure 2. Time-scaled snapshots of L-leucine (dark red), L-proline (green) and Lvaline (yellow) adsorption on (120) surface of L-alanine



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Figure 3. Predicted modification of crystal habit due to influence of {L-leucine + Lproline + L-valine} additives.


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Figure 4. Experimental state and predicted growth morphology of L-alanine crystals as influenced by hydrophobic additives.



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Figure 5. Experimental state and predicted growth morphology of L-alanine crystals as influenced by hydrophilic additives.


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Figure 6. Experimental state and predicted growth morphology of L-alanine crystals as influenced by hydrophobic and hydrophilic additives.



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