ARTICLE pubs.acs.org/crystal
Thermally-Induced Homogeneous Racemization, Polymorphism, and Crystallization of Pyroglutamic Acid Han Wu*,† and Anthony R West*,‡ † ‡
Department of Chemical Engineering, University College London, London, U.K., WC1E 7JE Department of Materials Science and Engineering, University of Sheffield, Sheffield, U.K., S1 3JD
bS Supporting Information ABSTRACT: Pyroglutamic acid (P) racemized spontaneously when heated above its melting temperature, ∼162 °C. No catalysts or solvents were involved in the racemization, and the rate was very temperaturedependent, requiring, for example, > 24.5 h at 160 °C but ∼1.5 h at 210 °C. The degree of racemization was studied indirectly by both X-ray powder diffraction and IR spectroscopy on samples that had been crystallized after melting for various times; the occurrence of racemization was also confirmed by HPLC analysis. The time spent in the molten state controlled the enantiomeric composition of the liquid and this had a major effect on the subsequent composition-dependent crystallization kinetics. Thus, crystallization of P from undercooled melt was particularly slow if the enantiomeric compositions of the melt and the crystallizing phases were significantly different; crystallization then required long-range counter-diffusion of P molecules in the viscous, hydrogen-bonded melt. On crystallization of P from melt, evidence was obtained for new metastable L, D, and DL polymorphs, labeled as γ. The binary phase diagram LDLD for P has been determined experimentally and compares well with a calculated phase diagram.
1. INTRODUCTION Chirality, or more specifically, enantiomeric purity, is a major concern in the pharmaceutical industry because two enantiomers may exhibit remarkably different pharmacological effects in biological systems.1,2 Consequently, manufacturers strive to market enantiopure pharmaceuticals to minimize possible side effects from undesired (i.e., inactive or toxic) enantiomers.2 Although racemization of a pure enantiomer is usually undesirable, a key step in enantiomeric resolution can be to racemize the unwanted enantiomer and subsequently separate the preferred enantiomer from the racemic mixture3,4 to increase the yield from 50% to 100%. Knowledge of racemization is a fundamental issue to be considered in the design and development of new homochiral drugs. Racemization occurs not only in solution during formulation or crystallization, but also may take place unpredictably in either the dry or liquid states during downstream processes, i.e. granulation, tableting, transport and storage. For these reasons, the conditions, and mechanisms, under which molecules racemize is of both scientific and commercial interest. The racemization of amino acids has been studied extensively in different media57 and a number of factors are known to influence its rate. These include amino acid structure,8 pH,9,10 temperature,11 ionic strength,8,12 and the presence of water, catalyst13,14 and metal cations.15 The generally accepted mechanism for racemization in aqueous solution invokes a resonancestabilized planar anion intermediate1618 formed by removal of the methine proton. This was first proposed by Neuberger19 in 1948; racemization involves readdition of a proton to the negatively charged carbonion, resulting in a racemic mixture. The rate of racemization has been used in dating biological materials r 2011 American Chemical Society
such as tooth enamel of mammals,20 bovine bones21 and marine sediments.22 Despite extensive investigations covering the mechanism and kinetics of amino acid racemization, the reported cases are all solution-mediated or occur in the presence of suitable media/catalysts; to the best of our knowledge, the homogeneous racemization of an amino acid in the molten state, as reported here, is not a previously recognized phenomenon. L-Pyroglutamic acid (L-P), Figure 1, a lactam formed by cyclodehydration of L-Glutamic acid (L-Glu),23 is an important active pharmaceutical ingredient (API) with a wide range of pharmaceutical applications associated with brain-enhancing effects,24 age-associated memory decline,25 dermal penetration enhancer26 and hair growth agents.27 Its polymorphism, thermal stability and possible thermal racemization are significant in food and pharmaceutical industries. Our earlier studies28 showed that L-P exists in three polymorphs with the transformation sequence ∼ 126 °C
∼68 °C
∼162 °C
R0 rsf R rsf β rsf liquid The crystal structures of R0 and R polymorphs differ in the nature of the hydrogen bonding network that links the molecules into a 3D array.2830 The transformation sequence on heating was characterized by X-ray diffraction, XRD, and laser Raman spectroscopy. An unusual observation, by hot stage optical microscopy, was that crystals jumped around the microscope slide on passing through the R/β transformation on both heating and cooling Received: November 25, 2010 Revised: May 20, 2011 Published: May 23, 2011 3366
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Figure 1. Molecular structure of pyroglutamic acid, P.
cycles; this was attributed to dimensional changes in the crystals leading to a spring effect.28,31,32 The polymorphic transformations were reversible and readily studied by differential scanning calorimetry, DSC. However, crystallization of P from melt was found to be difficult leading to complex DSC heatcool cycles. Investigation of this behavior in more detail has revealed that racemization of L-P and D-P molecules occurs above the melting temperature, without the need for a catalyst and at a rate that is very temperature-dependent. The present work applied four complementary techniques, XRD, infrared spectroscopy, IR, high-performance liquid chromatography, HPLC, and DSC to characterise this racemization process. It is supported by determination of the LDLD phase diagram for P and the discovery of metastable polymorphs of P labeled γ L, γ DL, and γ D.
Figure 2. DSC (using Perkin-Elmer DSC 1 coupled with liquid N2 cooling system) curves on cooling and heating D-P between 75 and 150 °C. Heating rate 10 °C/min, cooling rate 30 °C/min between 75 and 100 °C, 5 °C/min between 100 and 150 °C.
2. EXPERIMENTAL SECTION 2.1. Materials. All experiments were performed with L-P, D-P, and DL-P obtained from Sigma-Aldrich (purity reported to be g99%), with no further purification. However, a small amount (∼1.4%) of racemic DL-P was found to be present in the commercial L-P sample. Reference materials used for calibration of the DSC were cyclohexane (purity 99.98%) from Fisher Scientific, indium (purity 99.999%) and zinc (purity 99.999%) certified by Perkin-Elmer. 2.2. Preparation of P Binary Mixtures. For construction of the phase diagram, weighed mixtures of enantiomer and racemate with different enantiomeric composition were gently ground by mortar and pestle prior to DSC measurement. Consistent DSC curves demonstrated that the physical mixing method used to prepare samples was satisfactory and gave reliable results. 2.3. Thermal Analysis. The solidliquid equilibrium of P mixtures with different enantiomeric composition and the polymorphic transformations of P were examined by DSC using two different instruments, a Perkin-Elmer DSC 7 under dry Ar and a Perkin-Elmer DSC 1 equipped with liquid N2 cooling system under dry He. DSC 1 was calibrated for temperature with pure In (MPt = 156.6 °C) and cyclohexane (MPt = 6.5 °C); DSC 7 was calibrated with pure In and Zn (MPt = 419.5 °C). The heat flow for both instruments was calibrated using pure In (ΔH = 28.45 J/g). Pyris 7.0 software was used for data analysis. Approximately 810 mg sample was weighed into an Al pan fitted with a perforated lid, and heated/cooled between 155 and 200 °C at rates 2 and 10 °C/min. The liquidus and solidus curves were estimated by measuring the temperature at the beginning (onset temperature of the eutectic endotherm) and the end (peak maximum temperature) of fusion of the mixture. The weight loss during heating DL-P was quantified by Thermogravimetric Analysis, TGA, using a Perkin-Elmer Pyris 1 TG system. Samples were heated from 60 to 190 at 10 °C/min in dry N2. 2.4. In-House X-ray Powder Diffraction. Routine phase analysis of P was performed using two STOE STADI P X-ray powder diffractometers (Darmstadt, Germany), with different radiation and detector systems and a Ge (111) monochromator in both. A Mo KR1, λ = 0.70926 Å, system equipped with a linear position sensitive detector (PSD), over the 2θ range 4.5 to 24.0°, in DebyeScherrer geometry was used for indexing and unit cell determination using Stoe WinXPow software version 1.10 and least-squares refinement. A Cu KR1, λ = 1.54051 Å, system with an image plate (IP) PSD
covering the 2θ range 6 to 125° was used for phase identification in transmission geometry. Samples were prepared either in 0.9 mm borosilicate glass capillaries (Mo KR1 radiation) or as thin deposits on transparent tape (Cu KR1 radiation) and placed in rotating sample holders. 2.5. Synchrotron X-ray Powder Diffraction. Because of the possible disordered structure of γ polymorphs and uncertainty of indexing lab data, fast-acquisition, high-resolution synchrotron X-ray data of γ forms (samples from the same batch as used for inhouse analysis) were collected at beamline I11 at the Diamond Light Source, U.K. Simultaneous measurement of 45 powder patterns by 45-crystal multi-analyzing crystal (MAC) detector was carried out throughout 2θ 0150°, at 0.001° binned step size, with radiation of wavelength 0.825572(2) Å, based on calibration with a silicon standard. The beamline design and technical details were described elsewhere.33 Samples were packed into 0.7 mm borosilicate glass capillaries mounted on brass holders and placed on the magnetic spinner of the diffractometer by Robot arm. Sample damage under high-energy synchrotron radiation is very common and this was also examined and confirmed. For this reason, 6 min data collection time on 10 different positions along the capillary were summed to give 1 h total data collection per sample. 2.6. Fourier Transform Infrared Spectroscopy. The infrared spectra were recorded on a Perkin-Elmer Spectrum One FTIR spectrometer in the range 4000 to 650 cm1 at a resolution of 4 cm1. Twelve scans were accumulated for each spectrum to increase signal-to-noise ratio. Perkin-Elmer Spectrum software was used for data analysis. 2.7. High-Performance Liquid Chromatography. The enantiomeric composition of recrystallized samples from undercooled melt was examined by Daicel Chiral Technologies Co. Ltd. (China), using a CHIRALPAK AS-H column (4.6 mm ID 250 mm L), mobile phase hexane:IPA:TFA = 60:40:0.1, flow rate 0.9 mL/min, with a 214 nm UV detector SPD-20A. Twenty microliters of the solution was injected. The retention time for L-P and D-P was ∼12.5 and 17.5 min, respectively.
2.8. Construction of Calculated Binary Phase Diagram. The theoretical phase diagram was calculated from the temperature of melting and enthalpy of fusion of pure enantiomers (L-P, D-P) and the racemic compound (DL-P). The liquidus curve for the portion between the two eutectics and the pure enantiomers was obtained 3367
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Figure 3. (a) TGA curve on heating DL-P from 60 to 190 at 10 °C/min. (b) DSC (using PerkinElmer DSC 7) curves on heatcoolheat cycles of DL-P between 60 and 190 °C. Heating rate 10 °C/min from 60 to 160 °C, then at 2 °C/min to 190 °C, cooling rate 10 °C/min. from the simplified SchroderVan Laar equation,2 ! ΔHAf 1 1 ln x ¼ R T fA T f
ð1Þ
Figure 4. Selection of DSC (using Perkin-Elmer DSC 7) patterns from heating L/DL mixtures with various compositions (x = 0, 0.025, 0.05, 0.085, 0.15, 0.2, 0.3, 0.4, 0.475, 0.5) from 145 to 190 °C (heating rate 2 °C/min), which are used for the phase diagram construction. *The commercial sample of nominally pure L-P was found to contain 1.4% DL-P, that is, x = 0.007, from DSC analysis, Supporting Information Figure S1. The impurity was confirmed by HPLC analysis, Table 3. Therefore a small peak at ∼153 °C corresponding to eutectic melting is shown for pure L-P sample.
The liquidus curve between the two eutectics and including melting of the racemic compound was predicted using the PrigogineDefay equation2 ! 2ΔHRf 1 1 ð2Þ ln 4xð1 xÞ ¼ R T fR T f where x is the molar fraction of one enantiomer in the mixture, ΔHfA and TfA are, respectively, the enthalpy of fusion in joules per mole and melting point in kelvin of the pure enantiomers, ΔHfR and TfR are similar data for the racemic compound, and R is the gas constant, 8.31 J 3 mol1 3 K1.
3. RESULTS 3.1. Thermal Analysis. Previous studies on the polymorphism of L-P used DSC to determine transition temperatures. To confirm that the properties of the D enantiomer are very similar or identical to those of the corresponding L enantiomer, DSC data for D-P were recorded and are shown in Figure 2; the same sequence of transitions occurs and at very similar temperatures. Particularly noteworthy is the R to R0 transition, which has martensitic character on cooling,28 as shown by the sawtooth nature of the DSC peak. The R f β transition is observed on heating as an endotherm peaking at ∼67 °C with some hysteresis on cooling that depends on cooling rate (e.g., observed as an exotherm peaking at ∼52 °C at a cooling rate 30 min/°C), Figure 2. The melting and subsequent crystallization of D-P (and L-P) was also studied by DSC and these results are presented later. DSC data for DL-P over the temperature range 60 to 200 °C are shown in Figure 3b; no solidsolid polymorphic transformation of DL-P was detected over this range nor for temperatures down to 180 °C (not shown). A melting endotherm is observed on heating at ∼185 °C but subsequent crystallization is sluggish; it
Figure 5. Calculated binary phase diagram of LD pyroglutamic acid using simplified SchroderVan Laar and PrigogineDefay equations. Experimental points measured by DSC (using Perkin-Elmer DSC 7) are indicated by blue dots.
was not observed on the first cooling cycle but was seen as a broad exotherm peaking at ∼92 °C on reheating. TGA data, Figure 3a, show no weight loss below 170 °C but a small, gradual weight loss of 12% at higher temperature. This may be associated with volatilisation of DL-P molecules; there is therefore no evidence of significant sample decomposition on melting. 3.2. Binary Phase Diagram. The melting behavior of mixtures of L-, DL- and D-P with different enantiomeric compositions was determined by DSC on heating. A selection of results for L/DL mixtures is shown in Figure 4; similar results were obtained for 3368
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Crystal Growth & Design mixtures. For most compositions, the first melting event occurs at ∼153 °C, which is attributed to the eutectic on the LDL phase diagram. A Tamman plot2 of fusion enthalpy versus composition, Supporting Information Figure S1, shows the enthalpy of this melting peak passes through a maximum at composition ∼10% D-P, 90% L-P, which therefore provides one estimate of the eutectic composition. On further heating, Figure 4, a second broad endotherm is seen whose temperature is very composition-dependent. The termination of this peak is taken to indicate completion of melting; for convenience in constructing the phase diagram, the peak maximum temperature was taken as the liquidus temperature. From these results, the phase diagram, Figure 5, was constructed; data points indicate the temperatures of the two DSC endotherms; solid curves represent the calculated phase diagram which shows close agreement with the experimental data. The calculated liquidus curves intersect at x1 = 0.108, Te1 = 152.3 °C and x2 = 0.897, Te2 = 151.2 °C, which is in good agreement with the experimentally determined values x1 = 0.105, Te1 = 152.7 °C and x2 = 0.899, Te2 = 151.3 °C. Therefore, the equilibrium phase diagram of the P system is determined. The phase diagram, confirmed to be that of a racemic compound,2 shows one congruently melting composition for DL-P and may therefore be divided into two very similar simple eutectic systems. The possibility exists that DL may form a limited range of equilibrium solid solution extending to compositions to either side of the DL stoichiometry by up to 23%. Evidence for this was provided by the Tamman plot, Figure S1: the composition-dependence of the enthalpy of the melting onset at the eutectic temperature extrapolated to zero enthalpy at 96% DL-P, 4% L-P (when x = 0.48) for the LDL part of the phase diagram. There was no evidence of any significant compositional range for crystalline L-P, however and indeed, the commercial sample of L-P used for these experiments, which was claimed to be g99% pure, showed a small endotherm at the eutectic temperature indicating the presence of a small amount (∼1.4%) of DL-P, corresponding to composition x = 0.007, in the commercial L-P sample. As discussed next, liquid-state racemization of D-P and L-P occurred and therefore, would have commenced during the DSC heatcool cycles leading to a gradual variation in the enantiomeric composition, x, of the samples during the DSC experiments. The experimental data points used in Figure 5 represent the starting composition. No attempt is made to determine error bars representing possible compositional drift during the DSC experiments since this cannot be quantified and anyway, is probably small, especially during the early stages of heating. We have also not corrected the composition axis in Figure 5 for the small amount of D-P impurity in the L-P starting material. 3.3. Crystallization Behavior of Melt with Different Enantiomeric Composition. The melting data obtained on the DSC heating cycles were generally reproducible and varied systematically with composition. The behavior on cooling, however, was found to depend on the temperature and time for which the sample was held in the liquid state prior to cooling by DSC. A selection of data showing three typical but different kinds of behavior is shown in Figure 6. Response a is for a sample of D-P that was cooled from just above the melting temperature, 175 °C without any hold period at that temperature. An exotherm corresponding to crystallization is observed on cooling with significant hysteresis
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D/DL
Figure 6. Three typical types of recrystallization behavior of D-P after melting and holding at 175 °C for different annealing times represented by DSC (using Perkin-Elmer DSC 7) curves on (a) heating and cooling D-P between 25 and 175 °C without annealing (b) heatcoolheat cycle of D-P with annealing at 175 °C for 20 min prior to cooling (c) similar to b but annealing at 175 °C for 60 min prior to cooling. Heating/cooling rate 10 °C/min. The effect of a second and subsequent heat on sample a, giving a small increase in time in the liquid state, is shown in Figure 11. 3369
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compared to the melting endotherm on heating. At lower temperatures, the β to R transition also occurs as an exothermic event (peaking at ∼54 °C at cooling rate of 10 °C/min) and shows hysteresis compared to the endotherm temperature on heating. Response b is for a sample that was annealed at 175 °C for 20 min prior to cooling by DSC. In this case, no crystallization exotherm is observed on cooling but is seen on subsequent reheating. It is also noted that the subsequent melting endotherm is displaced to lower temperature by a few degrees. The third type of response, c, for a sample that was held at 175 °C for 60 min shows no exothermic peaks on either cooling or subsequent reheating. These three patterns of behavior may be taken as indicating an increase in difficulty of nucleation of crystalline P on increasing the time of holding the sample in the liquid state. Thus, crystallization, appears not to occur at all in case c. 3.4. Homogenous Racemization of L- and D-P. XRD Analysis. XRD was used to identify the crystallization products that formed, either during the DSC experiments or in samples that were heated isothermally in an oven, removed, quenched to room temperature and then crushed. In many cases, uncrushed samples were found to be amorphous to X-rays and therefore, had not crystallized. The crushing served to produce nucleation sites for subsequent crystallization. The XRD patterns of D-P samples held for short times in the liquid state at 175 °C prior to cooling and crystallization showed a general similarity to that of D-P but with some significant line shifts that appeared to depend on annealing conditions. These patterns may represent a new, metastable polymorph and are labeled γ. A selection of XRD traces illustrating the difference between R and γ patterns is shown in Figure 7. Similar behavior was seen for L-P in
which small XRD line shifts were seen in samples that crystallized from the liquid state. The XRD data for γ L-P were indexed on a similar orthorhombic unit cell to that of R L-P with results summarized in Table 1. The patterns of γ do not correspond to any of the R0 , R and β polymorphs of L-P, Supporting Information Figure S2, and instead, are believed to represent a metastable crystalline polymorph that forms as a first product of crystallization of liquid P. As discussed later, we believe that liquid state racemization of L-P (and D-P) occurs gradually and spontaneously, at a rate that is very temperature-dependent; it is possible that the γ polymorphs represent a solid solution of L (or D) in which a significant number of the L (D) molecules are replaced in the crystal structure by molecules in the D (L) configuration. There is also evidence for a metastable polymorph of DL-P labeled γ DL-P, which is obtained either by crystallization of liquid DL-P during the DSC heatcoolheat cycle or by isothermal heatcoolgrinding experiments. XRD data for R and γ DL-P are shown in Figure 8; again, the XRD pattern can be indexed on a similar monoclinic unit cell to that of R DL-P but with small shifts in lattice parameters, as summarized in Table 2. Fully indexed X-ray powder diffraction data for γ L-P and γ DL-P will be submitted to the International Centre for Diffraction Data (ICDD) for inclusion in the Powder Diffraction File. On annealing, and subsequently crystallizing, samples of L-P (or D-P) for relatively short times, the XRD patterns corresponded to the γ L-P (or D-P) polymorph. With longer times, however, the appearance of γ DL-P peaks in the XRD patterns was
Figure 7. Selection of XRD (Cu KR1 radiation, IP-PSD) traces illustrating differences between R and γ patterns of D-P.
Figure 8. Selection of XRD (Cu KR1 radiation, IP-PSD) patterns showing differences between R and γ DL-P.
Table 1. Lattice Parameters of γ Form of LP Determined from Synchrotron Powder XRD at Room Temperature, Space Group P212121, in Comparison with Reported R0 , R, and β Data2830 R0
30 L
R L29
R0
L
28
R L28
β L28
γL
temperature
154 °c
room temperature
180 °c
25 °C
80 °C
method
single crystal
single crystal
powder
powder
powder
powder
in-house
in-house
in-house
synchrotron 9.005 (1) 13.441 (3)
X-ray source
room temperature
a (Å) b (Å)
8.197 (1) 14.340 (2)
9.018 (8) 13.495 (8)
8.146 (2) 14.269 (4)
8.95 (1) 13.35 (1)
9.086 (6) 13.140 (6)
c (Å)
14.666 (2)
14.662 (4)
14.610 (5)
14.52 (2)
14.586 (7)
14.649 (2)
v (Å3)
1724.1 (4)
1784.33
1698.2 (6)
1735 (3)
1741 (1)
1773.0 (4)
3370
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Table 2. Lattice Parameters of γ Form of DL-P Determined from Synchrotron Powder XRD at Room Temperature, Monoclinic Unit Cell, in Comparison with Reported r form Data34,35
method
R DL34
R DL35
single crystal
single crystal
X-ray source
γ DLa powder synchrotron
space group
P21/c
P21/a
P21/a
a (Å)
8.14(2)
9.194(2)
9.372(2)
b (Å)
8.86 (2)
8.744 (1)
8.695 (1)
c (Å)
9.32 (2)
7.976 (2)
8.045 (1)
β (deg) v (Å3)
116.5 (2) 601.3
116.29 (2) 574.9
116.87 (1) 584.8 (1)
a γ form can be indexed on alternative unit cells: (a) space group P21/n, a = 9.187(1), b = 8.695(1), c = 8.045(1), β = 114.49(1)°, V = 584.8(1) Å3 or (b) space group P21/a, a = 9.372(1), b = 8.695(1), c = 9.187(1), β = 128,63(1)°, V = 584.8(1) Å3. It is not possible to choose the correct unit cell for γ form without crystal structure information. Thus, the proposed unit cell and indexing was chosen to relate to the known R form.35
Figure 10. IR spectra of a selection of subsequently crystallized products obtained after heating L-P at 210 °C for various time, confirming phase transition of R to γ L-P followed by racemization of L- to DL-P.
Figure 9. XRD (Cu KR1 radiation, IP-PSD) patterns of D-P heated at 200 °C for 0.54 h, cooled, crushed, and analyzed at room temperature, showing phase transition of R to γ D-P, followed by racemization of D-P to DL-P.
increasingly observed and after sufficiently long heating periods, samples of X-ray-pure γ DL-P were obtained. A selection of XRD results representing the progress of racemization is shown in Figure 9. These results must represent the gradual changes that occur in the liquid state on annealing as a function of temperature and time, but in order to assess the extent of racemization, it is first necessary to crystallize the liquids. For those samples that crystallized as a consequence of the mechanical grinding of quenched amorphous samples, the results were fully consistent and showed no evidence of any additional racemization during crystallization. It is possible that mechanical grinding had a small effect in promoting racemization, as observed in other systems, but present results showed that liquid-state annealing was the main factor that controlled the extent of racemization. It is also possible that the samples contained some amorphous phase that was not detected by XRD. IR Analysis. A second technique that was used, indirectly, to study the progress of racemization was IR spectroscopy of the crystallized products. A selection of results is shown in Figure 10
from which it is clear that peaks characteristic of R (or γ) L-P and R (or γ) DL-P are obtained in certain regions of the spectra. The doublet structure observed in the IR spectra of R L-P crystals at 3399 and 3335 cm1 due to the ν(NH) stretch is in good agreement with our earlier Raman studies.28 These two bands are attributed to different intermolecular NH 3 3 3 O interactions of the NH groups with the acid CdO groups on neighboring chains in the L-P crystal structure. In comparison, a single stronger ν(NH) stretching band of R DL-P is observed at much lower energy (3298 cm1, Δ = 37 cm1) because of the involvement of strong hydrogen bonds. The ν(CdO) carboxylate and amide vibrations at 1709 and 1641 cm1 in the R L-P34 are shifted to lower frequencies (1703, 1615 cm1) in the R DL-P structure due to their structural differences.29,35,36 The technique appeared not, however, to be sensitive to differences between R and γ polymorphs (with very small shifts, e1 to 5 cm1, of the characteristic bands). The main conclusion from the IR results is qualitatively similar to the XRD results showing gradual racemization with time in the liquid state. DSC Analysis. A third technique that was sensitive, indirectly, to the degree of racemization was DSC. In the early stages of racemization where it was possible to crystallize samples either on cooling or subsequent heating in the DSC, it was noted that the melting temperature of the γ polymorph gradually decreased with annealing time in the liquid state, as shown in Figures 6 and 11. In the region of formation of homogeneous γ polymorph, with a range of compositions extending from pure L toward DL, the associated phase diagram must be different from that shown in Figure 5 since the samples do not belong to a two phase mixture with an equilibrium eutectic temperature. Instead, solidus and liquidus curves are likely to vary smoothly with composition, giving depressed metastable eutectics, as indicated schematically in Figure 12. HPLC Analysis. A fourth technique that should give direct information on enantiomeric composition is HPLC. Here, preliminary HPLC measurements were carried out to confirm directly whether liquid state racemization of D- and L-P occurred. Results 3371
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Table 3. Enantiomeric Compositions of Samples Crystallised from Undercooled Melt, Determined by HPLC, in Comparison with the Starting Compositions starting
expected enantiomeric
sample
enantiomeric
material
composition
treatment
ratio L/D
L-P
99.1: 0.9 e2.0: g98.0a
D-P DL-P
b
50.3: 49.7
180 °C, 40 min 98.1: 1.9 200 °C, 2 h
41.4: 58.6
200 °C, 2 h
49.7: 50.3
a
Figure 11. DSC (using Perkin-Elmer DSC 7) curves on three continuous heatcool cycles of D-P between 40 and 175 °C at rate of 10 °C/min and holding at 175 °C for 1 min, showing gradually decreased melting temperature of the γ polymorph.
Data provided by Aldrich. b Sample for HPLC provided by Daicel Chiral Technologies (China) Co. Ltd.; this may have slightly different composition from the DL-P (from Aldrich, purity g99.0%) used for this study. HPLC analysis was performed by Daicel Chiral Technologies (China) Co. Ltd., Contact address: Part C, FL5, No. 16, XiYa Road, No 69, WaiGaoQiao Freetradezone, Shanghai, 200131, China.
The rate of racemization of molten L-P (and D-P) was found to be very temperature-dependent. A series of isothermal experiments was carried out in which L-P and D-P samples were heated in an oven for various times, quenched, crushed and analyzed by XRD and IR, with the results shown in Figure 13. The general transformation sequence γL-P f γL-P þ γDL-P f γDL-P was observed at six temperatures and the rate was very temperature-dependent. The kinetic data for racemization of L-P and D-P are superposable in Figure 13.
Figure 12. Schematic metastable phase diagram, dashed, showing limited ranges of γ L solid solutions, γ DL solid solutions, and γ D solid solutions with depressed eutectic temperatures. The equilibrium phase diagram is shown as thin continuous lines.
are summarized in Table 3. A small amount of racemization of L-P occurred after 40 min at 180 °C whereas a large amount of racemization of D-P occurred after 2 h at 200 °C. Measurements on a sample of γ DL-P showed almost no change in the racemic composition DL-P on melting at 200 °C for 2 h. These results therefore confirm the essential conclusion that spontaneous liquid state racemization of L- and D-P occur in the liquid state and at a rate that is very temperature-dependent. We do not know the compositional extent of the γ L-P solid solutions, which are presumed to form by the incorporation of D molecules into the L crystal lattice. Likewise, we do not know if there is a similar range of γ DL-P solid solutions or whether the γ DL-P polymorph represents some kind of molecular disorder in the DL crystal lattice while retaining the overall D/L ratio of unity. Nevertheless, with these limitations, we can sketch schematically a metastable phase diagram between L-P and DL-P, resulting from liquid-state racemization and crystallization of metastable phases, Figure 12. This shows schematic limited ranges of γ L-P and γ D-P solid solutions and a possible γ DL-P solid solution. These are separated by two-phase regions with conventional eutectic melting but at a reduced temperature compared to that of the equilibrium eutectic.
4. DISCUSSION The phase diagram showing melting of L-P, DL-P, and D-P mixtures has been determined experimentally and calculated using thermodynamic data of the enantiomers and the racemic compound. Since the formation of racemic DL-P was already wellknown, having a different crystal structure from that of L-P,29,35,36 it is not surprising that it exists on the phase diagram as a congruently melting phase. Excellent agreement is obtained between experimental and calculated phase diagrams. The polymorphism of D-P has been confirmed and is very similar to that of L-P in which the various transition temperatures are the same to within (2 °C. DL-P shows no evidence of any solid state phase transition between 180 and the melting temperature, 185 °C. There is evidence for new metastable polymorphs of L-P, D-P, and DL-P, which form as the first product of crystallization of liquid. We label these polymorphs γ to retain the sequence R0 , R, β, γ, which indicates the increasing temperatures of stability/formation of these various polymorphs. We label the single polymorph of DL-P as R and the metastable polymorph formed on crystallizing liquids as γ, for consistency. We do not know the crystal structures of the γ polymorphs but suspect that they are derived from those of L (D) and DL by having some of the molecules in the crystal packing arrangements in either a different orientation or in the different enantiomeric form. The observation of gradual, liquid-state racemization with time would suggest that γ L (γ D) does indeed contain some molecules in the opposite enantiomeric configuration. The rate of crystallization of liquid P samples is variable and appears to depend on the holding time in the liquid state, at least for short times, as shown in Figure 6. We attribute the slowing down of crystallization rate with holding time to the increased difficulty of nucleating phases of different composition to the 3372
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Figure 13. Compositional change by heating L-P and D-P at different temperatures and times; samples were cooled, crushed and analyzed by XRD and IR. Closed symbols used L-P; open symbols used D-P.
mother liquor. Thus, after short holding times, Figure 6, the liquid composition and that of the crystallizing γ D polymorph are essentially the same and long-range molecular diffusion is not necessary in order to crystallize the γ D phase. At longer holding times, however, especially when mixtures of γ D (or L) and γ DL phases are obtained, Figure 13, then, long-range counter-diffusion of L- and D- molecules within an initially homogeneous melt, is necessary for the two γ phases, of very different enantiomeric composition, to subsequently nucleate and crystallize. The frequent occurrence of amorphous phases and the generally slow crystallization rates may, therefore, have two origins: the difficulty of nucleating phases of different composition to that of the mother liquor, and the difficulty of nucleation because of the need to break strong hydrogen bonds between molecules, even within the liquid state. The most unexpected result is the racemization of L-P (D-P), which occurs in the liquid state without the need for solvent or catalyst. It appears to be a spontaneous, homogeneous process. The racemization can be followed by quenching samples after various time in the molten state to halt racemization and then analyzing, at leisure, and at room temperature, the products of crystallization. The rate of racemization is very temperature-dependent and, for instance, was incomplete after 24.5 h at 160 °C but was complete after only 1.5 h at 210 °C (according to the XRD and IR results, although some amorphous material of unknown chirality may have remained). The molecular structure of P is shown in Figure 1 with the chiral center marked by an *. Racemization requires temporary removal of the hydrogen at the chiral center giving a planar intermediate and therefore, an equal probability of D and L configurations resulting from subsequent reattachment of the hydrogen, thus converting the enantiomer into a racemic mixture, Scheme 1. The racemization rate of amino acids in aqueous solution is known to be affected strongly by the electronegativity and the size or nature of the side chains: lactams of amino acids (e.g., P) were found to racemize at a considerably slower rate than amino acids (e.g., Glu) due to the removal of the positive charge from the amino nitrogen.16 We are not able to comment on the impact of the nitrogen on homogeneous racemization: in fact,
Scheme 1. Possible Mechanism of P Racemization through Formation of a Planar Intermediate. Arrows Indicate the Direction of Electron Transfer
was readily formed by cyclization of L-Glu on heating;23 no racemization of L-Glu was detected under those experimental conditions and prolonged heating of L-Glu eventually caused the formation of DL-P rather than DL-Glu. The observation of liquid state racemization of L- and D-P has obvious implications for retaining these materials, especially at high temperatures, in an enantiomerically pure state. It also has implications for the determination of molecular purity by conventional melting point determination since, as shown, racemization leads to the onset of melting at a lower temperature, corresponding to the eutectic in the relevant phase diagram. We believe that the possibility of spontaneous, liquid-state racemization of pure amino acids has not been recognized previously. It remains to be seen whether this is a phenomenon of widespread occurrence; for instance, is it only a property of systems that form racemic crystals, such as DL-P, or is it also shown by conglomerate systems of simple eutectic character. The occurrence of homogeneous, spontaneous, liquid-state racemization also provides an additional variable that should be considered in studies of enantiomeric transformation and separation which could be of relevance to pharmaceuticals and other materials with chirality-dependent properties.
L-P
’ ASSOCIATED CONTENT
bS
Supporting Information. Tamman plot of P by DSC to estimate eutectic composition and compositional limit of DL solid solution (Figure S1) and powder XRD patterns of L-P polymorphs
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’ AUTHOR INFORMATION Corresponding Author
*E-mail: a.r.west@sheffield.ac.uk (A.R.W).
[email protected] (H.W.).
’ ACKNOWLEDGMENT This work was carried out with the support of the Diamond Light Source. Thanks to DLS scientists Drs. Julia Parker and Chiu Tang for carrying out XRD measurements at DLS. We thank Rob Hanson for help with low temperature DSC experiments and Drs Simon Jones and Xiaoyong Li for helpful discussion.
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