Chiral Resolution of Aspartic Acid Racemate by Single Crystallization With Phenylalanine as Additive

ABSTRACT

The effective chiral resolution of amino acid racemates has important significance in fields such as medicine and pharmaceuticals. By using phenylalanine as additive, the chiral resolution of aspartic acid racemate was investigated through crystallization method. It reveals that the enantiomeric excess of the crystalline product is affected by the seeding temperature, crystallization temperature, crystallization time, seed dosage, phenylalanine dosage, and the relative supersaturation. Under optimized condition, the average enantiomeric excess could reach 85% after a single crystallization in water. The excess enantiomer of the crystalline product is determined by the configuration of phenylalanine. The enantiomeric excess and the excess enantiomer were confirmed by chiral high performance liquid chromatography. The powder X‐ray diffraction and Fourier transform infrared spectral results show that conglomerate was formed in the crystalline product. Seed and additive both play great role in the chiral resolution. The findings could give inspirations for efficient resolution of aspartic acid racemate into enantiopure compounds.

Chiral resolution of racemic aspartic acid was explored by using seed and phenylalanine. The enantiomeric excess of the crystalline product could reach 85% after a single crystallization in water. XRD and FT‐IR spectra indicate the formation of conglomerate, which induces the chiral resolution.

1. Introduction

The configuration of chiral drugs plays an extremely important role in the field of medicine and pharmaceuticals, since one configuration enantiomer might show a positive therapeutic effect and the other one might exhibit no therapeutic effect or even side effects and adverse reactions [1, 2]. Considering that chemical synthesis frequently gives racemic compounds, the enantiomeric separation of the racemic mixtures, especially for the active pharmaceutical ingredients (APIs), into enantiopure compounds has been paid much attention both in theoretical research and industrial production [3, 4, 5].

Amino acid is one kind of important compound applied in pharmaceuticals. Since most of the amino acids have two configurations D‐ and L‐, the acquisition of pure enantiomer is essential. Much effort has been paid for resolving amino acids to obtain highly pure enantiomer [6, 7, 8], because enantiomeric mixtures are often obtained through chemical synthesis, although great progress has been made in asymmetric synthesis and enzymatic catalysis. Crystallization is an efficient strategy for achieving enantiopure compounds, particularly in the medicine owing to the fact that this method is better suited for large‐scale production [9]. In the early 1970s, optical resolution of DL‐amino acids by preferential crystallization was carried out for alanine, leucine, lysine, serine, and tryptophan by forming aromatic sulfonates of amino acids [10]. Resolution of DL‐glutamic acid can be performed in two crystallization chambers separated by an inorganic aluminum oxide membrane by adding seed crystals in the chambers appropriately [11]. The enantiomeric excess (ee) of crystals from two chambers ranges from 94.1% to 98.4%. The combination of crystallization with membrane was explored. By using continuous membrane ultrafiltration and crystallization, chiral resolution of racemic phenylalanine, alanine, and valine in aqueous media was investigated [12]. The chiral selector bovine serum albumin (BSA) plays a great role by binding preferentially to the L‐amino acid enantiomers. After recycling membrane ultrafiltration and selective crystallization, crystals of pure D‐enantiomers can be obtained. If a conglomerate is formed for the racemic solution, preferential crystallization of one enantiomer from enantiomeric mixtures could occur [13]. The application of chiral additive is one of the effective strategies for enantioseparation and the preferential crystallization of one enantiomer from solution is a kinetically controlled process [14, 15]. The mechanism of such kinetic‐based resolution has been systematically studied since the early 1980s. Racemic glutamic acid hydrochloride (Glu·HCI), threonine (Thr), and asparagine hydrate (Asn·H₂0) were resolved by amino acid additives [16]. The opposite enantiomer with respect to the chiral additive is preferentially crystallized and this is so called the “rule of reversal.” It should be noted that the additive as impurity would not exist in the bulk of crystals with the opposite configuration insuring the purity of the crystal product. A chiral surfactant (16PC‐DPHE) synthesized from D‐phenylalanine and palmitoyl chloride showed selective chiral recognition to L‐alanine and could enhance ee values from 0% to 99% of L product via crystallization of the racemic alanine [17]. In this case, the stereoselective recognition mechanism of the direct crystallization process is different from the “rule of reversal.” Polymers were also applied as additives for the enantioseparation of racemic amino acids by stereoselective crystallization and ee could reach over 99% [18]. Employment of seeding crystal and appropriate additive can facilitate the preferential enrichment [19, 20].

Aspartic acid is one of the proteinogenic amino acids [21]. At an early stage, Harada has done a series of work on the resolution of racemic aspartic acid; however, post‐processing procedures are prerequisite to obtain free aspartic acid [22, 23]. Polymer [24, 25], self‐assembled monolayer [26], membranes [27], and nanorods [28] were explored to resolve enantiomeric mixtures of aspartic acid. Deracemization as an effective way for acquiring pure enantiomers was also applied [29]. Sato reported the resolution of racemic aspartic acid (Asp) by crystallization with asparagine as a tailor‐made additive [30]. Seed and additive amount are 1 and 40 mol%, respectively. When D‐asparagine was used as the tailor‐made additive, the predominant configuration in the crystalline product of the first crop was L‐aspartic acid, with an ee of 8.5%; the predominant configuration in the crystalline product of the second crop was D‐aspartic acid, with an ee of 40.8%. Since the amount of D‐asparagine is up to 40 mol%, the crystalline product consists of 8.5 and 5.7 mol% of additive for the first and second crop, respectively. Liu reported that the enantiomeric excess of the product crystals from racemic aspartic acid solution (V_EtOH:V_water = 1.5:1) with 10 mol% L‐phenylalanine (Phe) as additive is 17.3% after a single crystallization [31]. In addition, no additive was detected by high‐performance liquid chromatography (HPLC) for the crystalline product. Based on the results from Sato and Liu, the resolution of racemic aspartic acid in water by crystallization with amino acid additive has considerable potential.

Herein, phenylalanine is used as the additive with the aim at the resolution of aspartic acid racemate in water by a single crystallization. Seed crystal was also induced [19]. The impact of seeding and crystallization temperature, crystallization time, dosage of seed and additive, and relative supersaturation on the ee of the crystalline product has been investigated. By using L‐phenylalanine as an additive and D‐aspartic acid as seed, the excess enantiomer of the crystalline product is D‐aspartic acid with an average ee of up to 85.4%. The average yield of the crystalline product is 12.8%. The present resolution process is simple and a high ee value of the product crystal can be reached after a single crystallization.

2. Materials and Methods

2.1. Materials

DL‐Aspartic acid (99%, MREDA), D‐aspartic acid (99%, MREDA), L‐aspartic acid (99%, MREDA), D‐phenylalanine (99%, Aladdin), and L‐phenylalanine (99%, Aladdin) were purchased.

2.2. Chiral Resolution and Characterization

The chiral resolution of aspartic acid racemate was performed by a single crystallization in deionized water with phenylalanine as a tailor‐made additive. The process is as follows: DL‐aspartic acid (200 mg), phenylalanine, and water (10 mL) were added in a glass vial (20 mL) with a temperature sensor. The glass vial was heated to 70°C with magnetic agitation (300 rpm) in a water bath to obtain a clear aqueous solution. The solution was allowed to stand and cool naturally to 50°C or 55°C. The seed crystal was added, and the vial was transferred to an incubator for the crystallization at 25°C. After a certain time, the suspension was filtered by suction to collect the solid product, which was further dried in vacuo at 65°C for 8 h. The amount of DL‐aspartic acid, the configuration and amount of additive, and the configuration and amount of seed were varied depending on experimental motivation. Phenylalanine is completely dissolved in all experiments [32]. For each condition, at least three parallel experiments were performed. The enantiomeric excess of the crystalline product was calculated by specific rotation and chiral high‐performance liquid chromatography (chiral HPLC). The specific rotation was obtained from a WZZ‐2S digital automatic polarimeter (Shanghai Physical Optics Instrument Co., LTD), [α]_D = −23.5°~−25.5° and [α]_L = +23.9°~+25.3° (c = 1 g 100 cm⁻³ in 5‐M HCl), the temperature was 24°C ± 0.5°C. The HPLC experiment was performed by a Shimadzu LC 40D equipment (column CROWNPAK CR‐I(+), column size 3.0 mm I.D. × 150 mm L × 5 μm, 3.0 mg L⁻¹ in pH 1.0 HClO₄, mobile phase pH 1.0 HClO₄/methanol = 70/30 (v/v), temperature 20°C, flow rate 0.1 mL min⁻¹, wavelength UV 205 nm).

In this work, the relative supersaturation ($\sigma$) of solutions at 25°C was defined as

$$\sigma =\frac{c-c^{*}}{c^{*}}$$ (1)

where $c^{*}$ is the concentration of saturated solution [33] and $c$ is the concentration of supersaturated solution in experiments.

The yield (Y%) of the crystalline product was defined as follows:

$$Y\%=\frac{\text{total mass of crystalline product}}{\text{mass of}\mathit{\text{initial racemic}}\mathit{Asp}}\times 100\%$$ (2)

The enantiomeric excess (ee) of the crystalline product was calculated as follows:

$$\mathit{ee}=\frac{{\left[\alpha \right]}_{\mathit{\text{crystalline product}}}}{{\left[\alpha \right]}_{D-\mathit{Asp}\mathit{\text{or}}L-\mathit{Asp}}}\times 100\%$$ (3)

or

$$\mathit{ee}=\left|\frac{A{\%}_{D-\mathit{Asp}}-A{\%}_{L-\mathit{Asp}}}{A{\%}_{D-\mathit{Asp}}+A{\%}_{L-\mathit{Asp}}}\right|\times 100\%$$ (4)

In formula (3), ${\left[\alpha \right]}_{\mathit{\text{crystalline product}}}$ denotes the specific rotation of the crystalline product, and ${\left[\alpha \right]}_{D-\mathit{Asp}\mathit{\text{or}}L-\mathit{Asp}}$ stands for the specific rotation of pure D‐Asp or L‐Asp. In formula (4), $A{\%}_{D-\mathit{Asp}}$ and $A{\%}_{L-\mathit{Asp}}$ represent the peak area percentage of D‐Asp and L‐Asp, respectively.

¹H NMR spectra (D₂O) were recorded on a Bruker AVANCE NEO 400‐MHz spectrometer. The powder X‐ray diffraction (XRD) was performed on a Bruker‐D8 instrument. Scanning rate is 10° min⁻¹. ${\lambda }_{\mathit{Cu}\mathit{K\alpha }}=0.154\mathrm{nm}$. Fourier transform infrared (FT‐IR) spectrum was obtained by the KBr pellet technique on a Bruker VERTEX 70 apparatus. It was recorded with a resolution of 4 cm⁻¹ and 32 scans. Scanning electron microscopy (SEM) was performed on a Hitachi Regulus 8100 field‐emission scanning electron microscope with a voltage of 8 kV. A small amount of the crystalline powder was dispersed onto conductive tape surface, which is attached on the specimen stub. The stub was subsequently inverted and gently blown with a rubber bulb. Prior to SEM imaging, the sample was sputter‐coated with a thin platinum (Pt) film to improve the conductivity.

3. Results and Discussion

3.1. Seeding and Crystallization Temperature

The single crystallization was carried out at different temperatures for 20 h in the presence of 5 mol% L‐phenylalanine as an additive and 2 mol% D‐aspartic acid as seeds. In addition, two different seeding temperatures, 50°C and 55°C, were detected as well. The concentration of racemic aspartic acid was close to but below the solubility at the two seeding temperatures [33]. The ee values of crystalline products after a single crystallization were evaluated and the statistical results are shown in Figure 1. For all crystalline products, the excess enantiomer is D‐aspartic acid.

FIGURE 1.

The enantiomeric excess of the crystalline product crystallized at different temperatures. Bars with different colors show the two different seeding temperatures.

The chirality of the excess enantiomer matches that of the seed and is opposite to that of the additive. It can be seen that ee values increase and then decrease with increasing crystallization temperature when seeding both at 50°C and 55°C. Regardless of the crystallization temperature, the enantiomeric excess of the crystalline products obtained with a seeding temperature of 55°C is typically higher than that observed with a seeding temperature of 50°C. At both seeding temperatures, the highest enantiomeric excess is achieved at a crystallization temperature of 25°C and reaches up to 89.3% with a crystal yield of 12.6% (Table S1) when seeding at 55°C. The above results indicate that the optimal seeding temperature is 55°C and the optimal crystallization temperature is 25°C. Notably, the enantiomeric excess achieved in this work was substantially improved with the same additive after a single crystallization [31].

3.2. Crystallization Time

The crystallization time is often directly impacting the enantiomeric excess of crystals. The crystallization time effect was inspected on the ee values of crystals, and the experimental results are in Figure 2. The crystal yield increased over crystallization time (Table S2). The enantiomeric excess in favor of the D‐enantiomer increased initially but decreased with increasing crystallization time. Although the ee values for 10, 15, and 20 h were high (79.0%, 76.7%, and 85.6%), prolonged crystallization to 25 and 30 h resulted in a marked decrease (43.4% and 37.3%), suggesting that the resolution process is kinetically controlled and extended crystallization time is not conducive to a high resolution. The above findings suggest that the favorable crystallization time is 20 h with 85.6% ee and 13.2% yield.

FIGURE 2.

The enantiomeric excess of the crystalline product crystallized at 25°C with different crystallization times. The seeding temperature is 55°C.

3.3. Additive Dosage

The influence of additive dosage impact on the enantiomeric excess was investigated with L‐phenylalanine from 1 to 10 mol%, as shown in Figure 3. A decrease in crystal yield was observed with increasing additive amount (Table S3). The enantiomeric excess is significantly enhanced as the dosage of additive increases from 1 to 5 mol%. However, 10 mol% of L‐phenylalanine led to a decline in ee.

FIGURE 3.

The enantiomeric excess of the crystalline product crystallized at 25°C for 20 h from solutions with diverse dosages of L‐phenylalanine.

It has been adequately explained that the proper amount of additive can facilitate the crystallization of enantiomers with opposite configuration. The promotion of enantioselectivity is due to the preferential adsorption of the additive on specific crystal facets of the growing crystals with the same configuration due to the structural analogy [16, 34]. Importantly, an excessive amount of additive can disturb the enantioselective nucleation and growth, thereby preventing racemates from resolution. The present observation is in accordance with previous reports. Herein, the variation trend of ee demonstrates that 5 mol% is the preferred option to attain a high ee.

3.4. Amount of Seed and Additive

Based on the above exploration, molar quantities were varied with a molar ratio of 1:2.5 (seed: L‐phenylalanine) to conclude the effect of diverse quantities of seed and additive on the enantiomeric excess (Figure 4). When the additive dosage was elevated from 2.5 to 10 mol%, the enantiomeric excess of the obtained crystals with respect to the D‐aspartic acid firstly was enhanced and then reduced. The experimental results established that a lower dosage of seed and additive is not beneficial for efficient resolution of aspartic acid racemate, although the crystal yield is the highest (Table S4). For a high dosage of additive (10 mol%), even with an increased amount of seed (4 mol%), the enantiomeric excess is suboptimal. The dosage of seed and L‐phenylalanine was confirmed to be 2 and 5 mol%, respectively, for effective resolution of aspartic acid racemate by a single crystallization.

FIGURE 4.

The enantiomeric excess of the crystalline product crystalized at 25°C for 20 h. The molar ratio of seed to L‐phenylalanine is 1:2.5 with varied molar quantities.

3.5. Relative Supersaturation

As well known, the supersaturation plays a critical role in determining the enantiomeric excess in chiral resolution. Excessively high supersaturation tends to induce rapid, non‐selective nucleation, often leading to the simultaneous formation of both enantiomorphs and a subsequent decline in ee; conversely, insufficient supersaturation fails to provide adequate driving force for preferential nucleation, resulting in unsatisfactory resolution efficiency. Under optimal conditions discussed above, the relative supersaturation of the racemic aspartic acid on the efficiency of chiral resolution was then studied. The enantiomeric excesses of the acquired crystals from solutions with relative supersaturations of 1.2–2.0 are shown in Figure 5. D‐Aspartic acid is always the favorable enantiomer. The enantiomeric excess is promoted on changing the relative supersaturation from 1.2 to 1.55, but dramatically decreased and eventually to 0% despite giving the highest crystal yield (Table S5) on further increasing the relative supersaturation from 1.6 to 2.0. The optimal relative supersaturation is 1.55, and L‐phenylalanine can effectively adsorb onto the crystal faces of L‐aspartic acid, thereby inhibiting its nucleation and growth under this condition. Lower or higher supersaturation compromises the chiral discrimination ability of L‐phenylalanine, ultimately reducing the ee of the crystalline aspartic acid product.

FIGURE 5.

The enantiomeric excess of the crystalline product crystalized at 25°C for 20 h from solutions with different relative supersaturations.

3.6. Enantiomeric Excess

Under the optimal conditions, 14 batches of parallel single crystallizations were carried out to gain reliable statistical results of ee (Figure 6). The seed and additive are D‐aspartic acid and L‐phenylalanine, respectively. D‐Aspartic acid is consistently the predominant enantiomer. Among the data from 14 batches, 80.3% and 94.4% are the lowest and highest ee, respectively. The average enantiomeric excess of the crystalline products is 85.4%, and the average yield of the crystalline products is 12.8%.

FIGURE 6.

The enantiomeric excess of the crystalline product of 14 batches of parallel experiments.

Crystallizations using L‐aspartic acid as seed and D‐phenylalanine as additive were performed as well (Table 1). L‐Aspartic acid as seed and D‐phenylalanine as additive give an 84.6% ee in favor of L‐aspartic acid. The average yield of the crystalline products is 12.4%. The values are within experimental error in contrast with the results from the crystallization using D‐aspartic acid as seed and L‐phenylalanine as additive. Chiral HPLC was conducted for the crystals from one batch of a single crystallization. The enantiomeric excess is 89.0% calculated from the specific rotation and 89.7% analyzed from chiral HPLC (Figure S1). The HPLC data confirm the reliability of ee derived from the specific rotation.

TABLE 1.

The chiral resolution of racemic aspartic acid with seed and phenylalanine.

Asp a	Phe b	ee (%)	Yield (%)	Predominant configuration c
		— d	—	—
D		0.0	28.3 ± 0.7	DL
	L	—	—	—
D	L	85.4 ± 4.9	12.8 ± 1.8	D
D	D	52.5 ± 4.8	15.5 ± 1.5	L
L	L	57.1 ± 6.9	14.4 ± 3.7	D
L	D	84.6 ± 3.7	12.4 ± 0.7	L

Note: The blank cells indicate that no seed or additive was used.

^a The configuration of seed.

^b The configuration of phenylalanine.

^c The configuration of excess aspartic acid enantiomer.

^d — indicates that no crystalline product was obtained.

Then the seed and additive with the same configuration (D‐aspartic acid and D‐phenylalanine; L‐aspartic acid and L‐phenylalanine) for the single crystallization were also investigated. In addition, experiments in the absence of seed and/or phenylalanine were carried out, respectively. The corresponding results are summarized in Table 1 as well.

No crystalline product was obtained for crystallizations in the absence of seed and phenylalanine, and seed. If only seed was applied, crystals were obtained but the enantiomeric excess is 0, indicating that no resolution occurred. This observation is understandable, because a racemic compound is formed in water for racemic aspartic acid. The enantioenrichment (52.5% ee and 57.1% ee) was accomplished for the combinations with the same configuration for seed and additive, although the enantiomeric excesses are much lower than that from the combinations with opposite configuration for seed and additive (85.4% and 84.6%). In comparison with previous reports using asparagine or phenylalanine as an additive for the resolution of aspartic acid racemate [30, 31], the enantiomeric excess of the crystalline product accomplished via a single crystallization in the present contribution is greatly enhanced.

Based on the above discussions, enantioenrichment of the crystal product is realized after a single crystallization under the condition that seed and additive are both added. Obviously, the opposite configuration of seed and additive can ensure the high performance of chiral resolution. Notably, the chirality of the excess enantiomer is always opposite to that of the additive, regardless of the configuration of seed. So, the configuration of the additive determines the chirality of the excess enantiomer. For example, L‐aspartic acid and D‐phenylalanine afford crystals with excess L‐aspartic acid. For this case, D‐phenylalanine prevents D‐aspartic acid from nucleation and growth, and at the same time the seeded L‐aspartic acid crystals facilitate the nucleation and growth of L‐aspartic acid. The synergistic interaction of seeded L‐aspartic acid and D‐phenylalanine plays a great importance for the effective chiral resolution of aspartic acid racemates.

For the case of D‐aspartic acid and D‐phenylalanine, D‐phenylalanine is beneficial for the nucleation and growth of L‐aspartic acid, while the seeded D‐aspartic acid crystals induce the nucleation and growth of D‐aspartic acid. The opposite interaction of D‐aspartic acid seed and D‐phenylalanine leads to resolution but with relatively low enantiomeric excess for the crystals. The excess enantiomer is L‐aspartic acid, indicating that the additive D‐phenylalanine plays a predominant role in the enantioselective nucleation and growth.

¹H NMR spectra were recorded in D₂O (Figures S2–S5). The chemical shifts (δ) of α‐H for aspartic acid and phenylalanine were selected to clarify the interactions between the two kinds of amino acids in water solution. Due to the overlapping of signals, the chemical shifts were determined as the center of gravity of the multiplet using the Multiplet Analysis tool in MestReNova. The α‐H shifted by 0.02 ppm (from 4.01 to 3.99) for D‐aspartic acid and by 0.01 ppm (from 4.01 to 4.00) for L‐aspartic acid. The ΔΔδ of 0.01 ppm implies that the interactions between D‐aspartic acid and L‐phenylalanine is slightly preferred in water solution. In our experiments, D‐aspartic acid was enriched in crystals when L‐phenylalanine was used as additive. It can be deduced that L‐phenylalanine with a bulky benzyl group of acts as a surface‐specific inhibitor, selectively adsorbing onto the L‐aspartic acid crystal surface and blocking its growth, while the less‐inhibited D‐aspartic acid was enriched in crystals. The different enantioselectivities between in water solution and at the crystal surface could be due to the steric effect, especially there is also a bulky benzyl group of phenylalanine.

3.7. Analysis of Crystalline Product

The crystalline product with an ee of 89.7% (L‐Asp) was analyzed by powder XRD (Figures 7 and S6). Two intense peaks at 11.9° and 23.8° demonstrate that the crystalline product is in α form, which is thermodynamically stable. The peak at 19.4° suggests the existence of the racemic compound, and that at 11.9°, 25.6°, and 28.3° indicates the formation of the conglomerate [35, 36]. The intensity of the peak at 19.4° is very weak, illustrating the low weight percent of the racemic compound. FT‐IR spectrum was also recorded (Figure S7). The absorptions at 658, 873, 1047, and 1250 cm⁻¹ are ascribed to the conglomerate, and the weak absorption at 1081 cm⁻¹ is from the racemic compound [35]. The results of FT‐IR are in accordance with those of XRD analysis. Since the racemic compound instead of the conglomerate is normally crystallized from aspartic acid racemate in water [13], it can be concluded that the addition of phenylalanine induces the formation of a metastable conglomerate [37], which is essential for chiral resolution through crystallization.

FIGURE 7.

XRD of the crystalline product.

Plates were mainly observed for obtained crystals from SEM image (Figure S8). Based on the data from Figure 7 and simulated XRD pattern (Figure S9) [38], the obtained crystal exhibits a dominant peak at 2θ = 23.8°, which corresponds to the (200) plane. So, the crystal growth in the $a$‐axis direction was inhibited. From the packing arrangement of L‐aspartic acid viewed along the $b$‐axis (Figure S10), there are $-\text{COOH}$ groups pointing out and pits on plane (100). Intermolecular $\mathrm{O}-\mathrm{H}\cdots \mathrm{O}$ hydrogen bonds can be formed between L‐aspartic acid and D‐phenylalanine. The benzyl group of D‐phenylalanine might accommodate into the pits due to the hydrophobic interaction. Apart from that, there might be electrostatic interactions. On the synergetic interactions of all weak interactions, D‐phenylalanine prefers to adsorb onto the (100) face of L‐aspartic acid crystal, thereby inhibiting the crystal growth along the $a$‐axis direction. The face‐selective adsorption contributes to the formation of plates. On the other hand, the strong enantioselective interaction between D‐phenylalanine and D‐aspartic acid could inhibit the nucleation and growth of D‐aspartic acid, accounting for the predominant content of L‐aspartic acid in crystalline product.

It is proposed that the additive phenylalanine not only prevents the nucleation and growth of aspartic acid with the same configuration but also contributes to the formation of conglomerates for the racemic aspartic acid. In addition, the seed induces the nucleation and growth of aspartic acid. The combination of seed and additive with opposite configuration gives rise to high performance of chiral resolution of racemic aspartic acid after a single crystallization in water. A schematic diagram for the chiral resolution is illustrated in Scheme 1.

SCHEME 1.

Schematic diagram of chiral resolution through a single crystallization.

4. Conclusions

Herein, phenylalanine is used as the additive for the resolution of aspartic acid racemate in water by a single crystallization. Under optimized conditions of crystallization, the average ee can reach 85.4% (D‐Asp) and 84.6% (L‐Asp). The excess enantiomer of the crystalline product has the opposite chirality as phenylalanine. The additive phenylalanine induces the formation of conglomerates during the crystallization, thus initiating the chiral resolution of aspartic acid racemate in water. Seed with opposite configuration of phenylalanine is essential for high performance of resolution. The present strategy shows great potential for the chiral resolution of aspartic acid racemate by crystallization.

Supporting information

Table S1: Yields of the crystalline products from different seeding temperatures and crystallization temperatures.

Table S2: Yields of the crystalline products from different crystallization times.

Table S3: Yields of the crystalline products from different additive dosages.

Table S4: Yields of the crystalline products from different amounts of seed and additive with a molar ratio 1:2.5.

Table S5: Yields of the crystalline products from different relative supersaturations of DL‐aspartic acid.

Figure S1: HPLC chromatogram of the crystalline product with an ee of 89.7% (L‐Asp).

Figure S2: ¹H NMR spectrum of DL‐Asp in D₂O.

Figure S3: ¹H NMR spectrum of L‐Phe in D₂O.

Figure S4: ¹H NMR spectrum of D‐Asp with L‐Phe in a 1:1 M ratio in D₂O.

Figure S5: ¹H NMR spectrum of L‐Asp with L‐Phe in a 1:1 M ratio in D₂O.

Figure S6: XRD spectrum of the crystalline product with an ee of 89.7% (L‐Asp).

Figure S7: FT‐IR spectrum of the crystalline product with an ee of 89.7% (L‐Asp).

Figure S8: SEM image of crystals obtained under optimized crystallization conditions.

Figure S9: Simulated XRD pattern of L‐aspartic acid (CSD refcode: LASPRT) generated using Mercury (2026.1.1).

Figure S10: The packing arrangement of L‐Asp viewed along the $b$‐axis.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.