Stabilization of Calcium Oxalate Metastable Phases by Oligo(l

ARTICLE pubs.acs.org/crystal

Stabilization of Calcium Oxalate Metastable Phases by Oligo(L-glutamic acid): Effect of Peptide Chain Length Viktor Fischer, Katharina Landfester, and Rafael Mu~noz-Espí* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

bS Supporting Information ABSTRACT: The understanding of the crystal growth of calcium oxalate and its inhibition is of great importance not only in medicine and biomineralization, but also in industry, as undesired deposits of this mineral are often formed. In this work, oligo(L-glutamic acid)s of different and well-defined chain lengths were synthesized by solidphase synthesis to serve as a model system for acid-rich biogenic macromolecules during the precipitation of calcium oxalate. The kinetic parameters of the precipitation process in the presence of the model peptides and the characteristics of the products were investigated. The peptide additives inhibit the nucleation and crystal growth of calcium oxalate and control the morphology and the phase, being calcium oxalate dihydrate the promoted phase with long-chain peptides. The results showed that the chain length, rather than simply the amount of carboxylic groups, plays a deciding role in the morphology, phase, and stability of the resulting crystals. We showed how the presence of oligo(L-glutamic acid)s is able to stabilize metastable calcium oxalate dihydrate (COD) and calcium oxalate trihydrate (COT). Although COD and COT convert commonly under thermodynamic conditions to the thermodynamically stable monohydrate phase (COM), in the presence of the homopeptidic additives, the formed metastable phases remained stabilized over very long periods of time. To prove the incorporation of the additives in the crystals, carboxyl-functionalized latex nanoparticles—which are easy to observe by electron microscopy—were used as models of carboxylic macromolecular additives. The formation of calcium oxalate in the presence of the latex particles showed that the additives become incorporated into the crystal, which can explain the stabilization of the metastable phases.

’ INTRODUCTION The inhibition of the formation of calcium oxalate has remained over the years a topic of major interest in medicine, materials science, and industry. The ubiquity of this apparently simple biomineral in nature and industrial processes explains the high research effort devoted to the understanding of its crystallization. Calcium oxalate is found in various shapes, phases, and properties in plants, constituting an essential component of their skeleton, and in pathological precipitates in the renal tract of humans and animals.1,2 Many studies have tried to understand how plants can control the formation of different calcium oxalate forms and how diseases like kidney stones could be avoided by suppression of the mineral precipitation. Moreover, in industrial processes, calcium oxalate appears as an undesired deposition product in pumps, boilers, heat exchangers, and sewage pipes, causing many efficiency problems and energy losses.35 In aqueous media, calcium oxalate crystallizes in three different hydrate modifications: the thermodynamically stable monoclinic monohydrate (CaC2O4 3 H2O, COM) or whewellite, the metastable tetragonal dihydrate (CaC2O4 3 2H2O, COD) or weddellite, and the metastable triclinic trihydrate (CaC2O4 3 3H2O, COT) or caoxite. COM and COD are present in plant r 2011 American Chemical Society

organelles like leaves, roots, blossoms, haulms, barks, and seeds, serving as a calcium source for the cytosol of the plant cell and also improving the mechanical stability.68 COM, often accompanied by small ammounts of COD, is the main component of kidney stones, consequence of a deficit of osteopontin and uromodulin (Tamm-Horsfall protein),1,911 whereas COT results of a bacterial indisposition of the renal tract.1214 In nature, the formation of inorganic crystals takes place under presence of biogenic macromolecules, which are able to control the crystallization.1518 Calcium carbonate, present in corals or shells of marine invertebrates, calcium phosphate, main constituent of bones and teeth, and calcium oxalate are the best examples of this phenomenon. Biominerals use biomacromolecules as matrices, nucleation seeds, and growth inhibitors (or promoters), providing an excellent control over shape, size, habit, and phase of the produced system. In the case of calcium oxalate, crystal chamber membranes serve in plants either directly as a nucleation promoter or may anchor or enclose other molecules Received: January 14, 2011 Revised: February 14, 2011 Published: March 17, 2011 1880

dx.doi.org/10.1021/cg200058d | Cryst. Growth Des. 2011, 11, 1880–1890

Crystal Growth & Design (or complexes) that promote the nucleation of the inorganic phase.7 The human body uses citrate,19 amino acids,20 acid-rich urine-proteins,21,22 the Tamm-Horsfall protein,1 inulin,3 and osteopontin911 to inhibit the crystallization of calcium oxalate. Some research groups have focused their investigations on the specific inhibition of osteopontin—an aspartic and glutamic acid-rich protein—and oligopeptide sequences of this protein on the mineralization of calcium oxalate, showing that the inhibition occurs even if only sequences of the protein are used and that the aspect ratio of the formed crystal is affected.10,11,2325 Furthermore, the influence of glutamic and aspartic acids,20,26,27 poly(glutamic acid),21,2831 poly(aspartic acid)21,2831 carboxylate-modified biopolymers,3 and model polyelectrolytic block and graft-copolymers3234 on the crystallization of calcium oxalates has also been studied. Most of the work dealing with peptides has been done with commercial materials of relatively high polydispersity and molar mass, which makes the systematization of the results difficult. However, the developments in solid-phase peptide synthesis enables now the preparation of sequences of defined composition and length. Therefore, in recent years, inspired by nature, the effect of well-defined amino acid sequences in crystallization of different minerals is attracting much attention. Although calcium carbonate is by far the most popular material,3538 silver nanoparticles39,40 and metal oxides, such as ZnO,4143 have also been investigated. In spite of the extensive literature, no systematic investigations have been done so far on how the chain length and the number of complexing groups affect the crystallization of biominerals. Taking the calcium oxalate as representative biomineral, in the present work we show the correlation of the chain length and the number of carboxylic groups with the inhibition of nucleation and crystal growth and the long-time stability of the metastable calcium oxalate phases formed in the presence of oligo(L-glutamic acid)s. Three homooligopeptides with a chain length of five (Glu5), ten (Glu10), and twenty units (Glu20) were synthesized by solid-phase synthesis and used at different concentrations as additives during the crystallization of calcium oxalate. We present evidence that calcium-ion complexation and adsorption of the oligomers to the crystal surface have an effect in different stages of the crystallization process, and we try to understand how crystal formation take place and how metastable phases are stabilized in the presence of biogenic additives.

’ EXPERIMENTAL SECTION Materials. For the peptide synthesis, a Wang resin (0.7 mol equiv, preload with Fmoc-Glu(OtBu)OH, Novabiochem, San Diego, CA), Fmoc- and tert-butyl-protected L-glutamic acid (Fmoc-Glu(OtBu) OH, Novabiochem), O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate (HBTU, Novabiochem), 1-hydroxybenzotriazole hydrate (HOBt, Acros Organics), piperidine (Alfa Aesar, p.a. 99%), N,N-dimethylformamide (DMF, Sigma Aldrich, p.a. 99.8%), N,Ndiisopropylethylamine (DIEPA, Sigma Aldrich, g 99%), N-methyl-2pyrrolidone (NMP, Sigma Aldrich p.a. 99%), trifluoroacetic acid (TFA, Sigma Aldrich, g99.5%), and triisopropylsilane (TIS, Sigma Aldrich, 99%) were acquired from commercial sources and used without further purification. For the crystallization experiments, a CaCl2 3 2H2O standard solution (0.1 M, Fluka), a potassium chloride solution (3.0 M, Merck, puriss. p.a. g99.5%), sodium oxalate (Sigma Aldrich, p.a. 99.5%), potassium hydroxide (Merck KGaA, p.a.), and L-glutamic acid (Sigma Aldrich,

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Scheme 1

99%) were obtained commercially and used as received. The synthesized oligo(L-glutamic acid)s were purified before use, as described below. Milli-Q water was used for all experiments and solutions. For the latex particle synthesis, 11-aminoundecanoic acid (Sigma Aldrich, 99%), methacryloyl chloride (Fluka, purum, dist., g97.0% GC), 2,20 -azobis(2-methylbutyronitrile) (Sigma Aldrich, purum g98.0%), and hexadecane (Sigma Aldrich, g99.0%) were used as received. Styrene (Sigma Aldrich, g99.0%) was distilled before use.

Microwave-Controlled Solid-Phase Peptide Synthesis and Peptide Purification. The syntheses of the pentamer (Glu5), decamer (Glu10), and eicosamer (Glu20) of the L-glutamic acid were performed on a CEM Liberty peptide synthesizer, using DMF/NMP as a solvent and following standard CEM Liberty coupling protocols. The loading of the preloaded Wang resin was 0.7% and a 1 mmol scale was applied. The amino acid (2 M in DMF) coupling was facilitated using HBTU/HOBt (0.5 M in DMF) and DIPEA (2 M in NMP). The release of the different oligomers was accomplished by a 24-h treatment with the cleavage mixture (95% TFA, 2.5% TIS, 2.5% H2O). The oligopeptides were isolated by diethyl ether precipitation and centrifugation. For purification, the solid was washed three times with diethyl ether by dispersion of the solid and centrifugation. The chemical integrity of the oligomers was characterized by means of MALDITOF (in DMF) and capillary electrophoresis (CE): Glu5 (M = 663.59 g mol1): MALDI-TOF m/z [Mþ]: 740 [H, þ2K], 724 [H, þNa, þK], 702 [þK], 686 [þNa], 664 [þH]; CE (electrophoretic mobility): 50.2 cm2 V1 min1. Yield: 557 mg (0.84 mmol; 84%). Purity (determined by CE): 91%. Glu10 (M = 1300.16 g mol1): MALDI-TOF m/z [Mþ]: 1385 [H, þ2K], 1370 [H, þK, þNa], 1354 [-H, þ2K], 1332 [þNa]. CE (electrophoretic mobility): 48.1 cm2 V1 min1. Yield: 1.05 g (0.80 mmol; 80%). Purity (determined by CE): 73%. Glu20 (M = 2600.29 g mol1): MALDI-TOF m/z [Mþ]: 2763 [5H, þ 2K, þ4Na], 2749 [5H, þK, þ5Na], 2677 [H, þ2K], 2638 [þK], 2622 [þNa], 2600 [þH]. CE (electrophoretic mobility): 46.7 cm2 V1 min1. Yield: 1.90 g (0.73 mmol; 73%). Purity (determined by CE): 70%.

Synthesis, Purification, and Characterization of CarboxylFunctionalized Particles. In the first place, the surfmer (N-methacryloyl-11-aminoundecanoic acid, Scheme 1) was synthesized. For that aim, 11-aminoundecanoic acid (4.16 g, 20.7 mmol) and sodium hydroxide (1.74 g, 43.5 mmol, 2.1 equiv) were dissolved in 70 mL of water and cooled to 0 °C. Subsequently, acrylic acid (3 mL, 3.24 g, 31.0 mmol, 1.5 equiv) was added dropwise under stirring to the reaction mixture during 30 min. The reaction mixture was allowed to warm to 25 °C and stirred for another 12 h. The mixture was acidified with HCl (pH 2) and the precipitate was filtered, washed three times with HCl and water, and dried under vacuum (3.02 g, 11.2 mmol, 54% yield). 1 H NMR (300 MHz, DMF δ/ppm): 12.22 (s, 1H), 7.89 (s, 1H), 5.74 (s, 1H), 5.33 (s, 1H), 3.22 (dd, J = 13.2, 6.8 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.93 (s, 3H), 1.64 1.45 (m, 4H), 1.30 (s, 12H). The latex particles were prepared by miniemulsion polymerization with styrene and a small amount of the surfmer (N-methacryloyl-11-aminoundecanoic acid) serving as comonomer and surfactant. The disperse phase was prepared by mixing styrene (6 g) with hexadecane (250 mg) and 2,20 azobis(2-methylbutyronitrile) (120 mg). The continuous phase was prepared by dissolving N-methacryloyl-11-aminoundecanoic acid (480 mg) 1881

dx.doi.org/10.1021/cg200058d |Cryst. Growth Des. 2011, 11, 1880–1890

Crystal Growth & Design and NaOH (1.8 mL, 1 M) in water (22.2 mL). Both phases were mixed and stirred for 45 min. The miniemulsion was achieved by ultrasonification for 3 min (Branson Digital Sonifier 450-D; 90% intensity, pulse 10.0 s, pause 2.0 s), while cooling in an icewater bath to avoid polymerization during homogenization due to heating. The reaction occurred under constant stirring at 72 °C in a closed flask and was stopped after 12 h. The resulting dispersion was filtered for purification. The particle size (mean diameter = 77 nm, σ = 11%) of the prepared particles was determined by using a Nicomp particle sizer (model 380, PSS, Santa Barbara, CA) at a fixed scattering angle of 90°. The solid content (equal to 20.6%) was determined by freeze-drying a portion of the dispersion under vacuum for 24 h. The surface-charge density (1.04 charged groups 3 nm2) was estimated by titration with poly(diallyl dimethylammonium chloride) (M€utek Analytik, M = 40 000100 000 g mol1) as previously reported.44 Crystallization Experiments. To prepare 250 mL of a 0.1 M stock solution of the oligo(L-glutamic acid)s, an amount of a potassium hydroxide solution (0.1 M) equal to the number of carboxylic groups (Glu: 5.0 mL, Glu5: 15.0 mL, Glu10: 27.5 mL, Glu20: 52.5 mL) was added to 0.25 mmol of the oligo(L-glutamic acid)s (Glu: 36.8 mg, 0.50 mmol COOH; Glu5: 166 mg, 1.5 mmol COOH; Glu10: 327 mg, 2.75 mmol COOH; Glu20: 650 mg, 5.25 mmol COOH) and the appropriate amount of water. The crystallization experiments of calcium oxalate were conducted at 25 ( 0.1 °C under constant stirring (300 rpm) in a jacketed reactor tempered with a thermostat (Lauda Ecoline RE240). A calcium-containing aqueous solution was prepared as follows: CaCl2 3 2H2O (0.1 M, 0,5 mL) and KCl (3.0 M, 0.5 mL, added to keep the ionic strength constant) were mixed with the pertinent amount of water (48.5 mL for the case of the blank in the absence of any additive). Afterward, the pH was adjusted to 8.5 by the addition of a KOH solution (0.1 M, 18 μL). In the experiments carried out in the presence of oligo(L-glutamic acid)s, a proper volume of the stock solutions was added to obtain overall peptide concentration of 0.2, 0.1, 0.01, and 0.001 mmol L1, adjusting the amount of water to keep a constant volume of 49.5 mL. The mixture was degassed by bubbling argon through and tempered for at least 10 min. The precipitation reaction was induced by adding sodium oxalate (0.1 M, 0.5 mL). At the starting point, the concentrations of Ca2þ and C2O42 ions were both of 1 mM. The decrease of calcium-ion concentration was monitored by an ionometer (MettlerToledo SevenMulti S47) equipped with a calciumselective electrode (MettlerToledo, DC240) and connected to a computer. After a reaction time of 1 h, the solution was filtered (Pall Life Science Supor-100, membrane filter, pore size 0.1 μm). The precipitates were washed three times with water (Milli-Q), dried under vacuum at 25 °C and collected for investigation. Light transmission measurements were conducted in the dark with a self-made setup comprised of a HeNe red laser source (JDSU, model 1145P, 633 nm, 25 mW), a thermostatted reaction chamber under constant magnetic stirring (300 rpm), a filter (Schott) that reduces the intensity of the transmitted light by a factor of 100, and a photodiode detector (photosensitive area =10 10 mm). The detected current is amplified by a transimpedance amplifier, coupled with a multimeter (Keithley 2010) connected to computer for data acquisition. The crystallization in the presence of the latex nanoparticles was performed in a similar way to the crystallization experiments with the oligomers. Instead of oligomer, 20 μL of the particle dispersion containing 4.12 μg (ca. 82 μg L1) of the functionalized particles was added to the crystallization solution. Evaluation of Data of Crystallization Kinetics. All measurements were performed three times for each concentration of additive to confirm repeatability and the average curve was considered in the data evaluation. The curves were normalized to the initial value of one to compare the features of the desupersaturation curves.

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Crystal growth rates correlate to desupersaturation rates, which were obtained from the fast desupersaturation period of the experimental calcium concentration curves. The fast crystal growth period was determined from the numeric differentiation of the desupersaturation curve. The time interval of the resulting differential peak—whose maximum represents the inflection point—corresponds to the fast growth period. Because the experiments were done in the absence of any seed and with additive, and the analysis of real data becomes very complex in terms of any known model, an empirical fitting using the socalled “derivative method”45 was used for the evaluation. Accordingly, the experimental concentration values corresponding to the fast growing period were fitted to a fourth-order polynomial in time cðtÞ ¼ A0 þ A1 t þ A2 t 2 þ A3 t 3 þ A4 t 4

ð1Þ

The desupersaturation rate, r, is then given by the derivation of this expression r ¼

dcðtÞ ¼ A1 þ 2A2 t þ 3A3 t2 þ 4A4 t 3 dt

ð2Þ

The inflection point of the desupersaturation curve (i.e., the point at which the change in the Ca2þ concentration is maximum) was calculated by equating the second derivative of eq 1 to zero d2 cðtÞ ¼ 2A2 þ 6A3 t þ 12A4 t 2 ¼ 0 dt 2

ð3Þ

The obtained values of t and the fitting parameters Ai were substituted in eq 2 to determine rmax (rate at the inflection point). In the case of the light transmission measurements, experimental data of intensity, I, versus time were fitted to the empiric equation IðtÞ ¼ t0 þ ðtend t0 Þ

Bn

tn þ tn

ð4Þ

in which the parameters t0 and tend correspond to the initial and final values, respectively, of the intensity within the fast growing interval (determined, analogously to the concentration desupersaturation curves, from the limits of the peak found in the first numerical derivative of the experimental curve) and B and n are fitting constants. The change in intensity of transmitted light per time unit, which correlates to the desupersaturation rate, is given by r ¼

dIðtÞ nBn t ðn 1Þ ¼ ðtend t0 Þ dt ðBn þ t n Þ2

ð5Þ

The rate in the inflection point, rmax, was determined substituting in eq 5 the value of the inflection time obtained from the abscissa intercept of the second derivative of eq 4. Induction periods and relative equilibrium Ca2þ concentrations were directly read from the experimental curves. Crystal Characterization. The resulting calcium oxalate precipitates were characterized by scanning electron microscopy (SEM) on an LEO Gemini 1530 (Zeiss) field-emission microscope operated with an extractor voltage of 5.4 kV. Powder X-ray diffraction (XRD) patterns were recorded on a Phillips PW 1820 diffractometer with CuKR radiation at 30 kV (5 s, Δθ = 0.02°). Thermogravimetric analysis (TGA) was carried out with a thermobalance Mettler Toledo ThermoSTAR TGA/ SDTA 851 under a nitrogen atmosphere and with a heating rate of 10 °C 3 min1 from room temperature to 900 °C.

’ RESULTS AND DISCUSSION Calcium oxalate was precipitated at a constant temperature of 25 °C from an aqueous solution in the presence of different concentrations of L-glutamic acid (Glu) and three oligo(L-glutamic acid)s with 5, 10, and 20 units (represented as Glu5, 1882


Crystal Growth & Design

Figure 1. Progress of the relative Ca2þ concentration (normalized to the initial value) measured by a Ca2þ-selective electrode during unseeded precipitation of calcium oxalate at 25 °C: (a) in the absence and in the presence of a constant concentration of 0.1 mmol L1 L-glutamic acid (Glu) and homopeptides with different chain length (Glu5, Glu10, and Glu20); (b) in the absence and in the presence of different concentration of Glu20.

Glu10, and Glu20, respectively). The pH was adjusted to 8.5 to ensure the deprotonation of the carboxylic groups and the additive concentration was varied from 0.001 to 0.2 mmol L1. This section has been divided in two parts: a first part about the effect of concentration and length of the oligo(gutamic acid)s on the crystallization kinetics, investigated by conductivity and light transmission measurements; and a second part discussing how the peptides influence not only the morphology of the resulting crystals, but very especially the crystal structure and the stability of metastable phases. 1. Crystallization Kinetics. The inhibition effect of the peptides was estimated by comparing the crystallization in their presence with a blank crystallization in the absence of any additive. The crystallization kinetics was studied by two different methods: (1) the measurement of the change in calcium concentration by means of a calcium-selective electrode, and (2) the measurement of the transmitted light (turbidimetry) during crystallization. Figure 1 shows the normalized calcium concentration (i.e., the concentration at each time, ct, divided by the initial concentration, c0) obtained from conductivity measurements of a Ca2þselective electrode in the absence and presence of the different

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Figure 2. Precipitation of calcium oxalate followed by measurement of the transmitted light: (a) in the absence and in the presence of a constant concentration (0.1 mmol L1) of monomeric Glu and peptides with different chain length (Glu5, Glu10, Glu20); (b) in the absence and in the presence of different concentrations of Glu20.

oligo(L-glutamic acid)s. These curves represent the desupersaturation of the system during calcium oxalate precipitation and show in all cases an induction period, tind, during which nuclei of critical size are formed. The induction period is followed by a fast decrease of the calcium concentration due to crystal growth. This fast growth interval has an inflection point and ends in a point after which only small concentration changes occur, ascribable to a slow crystal growth followed by Ostwald ripening. The cruves tend to an equilibrium concentration (ceq) corresponding to the solubility of the precipitated calcium oxalate. In some cases, this equilibrium concentration is reached beyond the time of indicated in Figure 1, limited to 1600 s for a better visualization of the initial evolution. Qualitatively, the main features of the desupersaturation curves are the clear increase of the induction period and the decrease of the supersaturation with increasing peptidic chain length and concentration. Moreover, an increase in the solubility of the precipitate is also observed. Light transmission measurements were monitored by a simple setup that registers the transmitted light of a laser source going through the reactor vessel during the precipitation. The transmission curves, of which a representation is contained in Figure 2, show an analogue progression to the measurements with the Ca2þ-selective electrode. Light transmission is an accurate method to determine the start—in means of induction period—and the end of the precipitation, since the formation of nuclei is well observed by the laser beam and the results are less sensitive to 1883



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absence (rblank) and in the presence (rmax) of an additive, an inhibition efficiency can be calculated according to R ¼

Figure 3. Induction period for the precipitation of calcium oxalate in the presence of different concentrations of monomeric L-glutamic acid and oligo(L-glutamic acid)s of different chain lengths. (Solid lines are merely drawn for facilitating the reading.)

local concentration fluctuations than the electrode measurements. Thus, the exact values of the induction period were obtained from measurements of transmitted light. The evolution of the induction periods with the concentration for the monomer and the oligo(L-glutamic acid)s is presented in Figure 3. The monomer (Glu) and the pentamer (Glu5) have only a moderate influence on tind, whereas a concentration of 0.1 mmol L1 of the long-chain oligomers Glu10 or Glu20 is able to increase it up to a factor of 2.6 and 4.6, respectively, with respect to the precipitation in the absence of peptides. The highest concentration (0.2 mmol L1) of the longest chain peptide (Glu20) has a dramatic effect on the induction period, which increased by a factor of 11. This indicates that a certain chain length of the oligomer is needed to significantly influence the nucleation rate, inversely proportional to the induction period (J µ 1/tind). The experimental data of the fast crystal growth period could not be linearized either by the classical method of Nancollas and Gardner46 nor by more complex models as those reported by Sikiric and F€uredi-Milhofer47 or Njegic-Dzakula et al.48 As our experiments were carried out without seeding in the presence of peptides and determining the number of nuclei becomes experimentally very complex, desupersaturation rates were calculated from the derivatives of an empirical polynomial fitting, which is a method reported in literature for analyzing real data of nonseeded systems.45 In a similar way, the experimental transmission data were fitted to the empirical expression given by eq 4, and the growth rates were obtained from equating the second derivative of this expression to zero. Although the analysis based on derivatives might be sensitive to measurement errors, this treatment of data has the advantage that it is neither influenced by all equilibria of Ca2þ nor by the mechanism of crystal growth; it simply reflects an experimental rate obtained from an experimental curve. For the determination of the rates from both calcium concentration and light transmission measurements, the fast growing range was taken into account. The calculated growth rates, rmax, correspond to the slope of the curve at the inflection point and, therefore, to the maximum value of the rate. From the rates in the

rblank rmax rblank

ð6Þ

Table 1 contains the calculated values of the growth rates, rmax, and inhibition efficiency, R, for the measurements of the calcium concentration by the ion-selective electrode, and for the light transmission measurements. Equilibrium concentrations (corresponding to the solubility of the precipitate) and the identified crystal phases (discussed in the next subsection) are also included in this table. The equilibrium concentrations were read from the Ca2þ concentration curves once a plateau (slope close to zero) had been reached. Comparing the rates obtained for the measurements of transmitted light with those of the Ca2þselective electrode, the former are slightly higher than the latter, which can be understood from the dependency of the transmitted and scattered light on the size, the amount of scattering particles, the refraction index and the form factor of the precipitating crystal phase. Thus, changes of phase, size, and form, as well as formation of new crystals (due to primary or secondary nucleation induced, for instance, by stirring) may cause a faster intensity decrease in comparison to the Ca2þ concentration registered by the electrode. The inhibition efficiency values for Glu and Glu5 measured with the electrode show an anomalous behavior, with no apparent trend between the inhibition of crystal growth and the increasing concentration. A similar behavior for shortchain low molecular weight additives, which was proven to be reproducible, was also observed by Komunjer et al.,26 who could not find a plausible explanation. We speculate that this odd trend could be related with the experimental response of the electrode to the complex equilibria of formation/dissolution of different species, especially taking into account that values obtained from light transmission measurements for Glu and Glu5 show that the crystal growth rate is almost not influenced by those additives. In a different manner, an increasing concentration of the oligomers Glu10 and Glu20 increases drastically the inhibition efficiency. Both, calcium concentration and light transmission measurements, show a comparable tendency. The increase of the induction period—which correlates to a decreased nucleation rate—and the crystal growth inhibition can be explained either by the complexation of calcium ions by the carboxylic groups of the oligo(L-glutamic acid)s or by the adsorption of the oligomer chains on the growing crystal surfaces; a combination of both effects is possible and expectable. Note that the concentration of carboxylic group cannot simply explain the differences in the effects of oligomers with different chain length. Although a concentration of 0.1 mmol L1 of Glu20 corresponds to the same amount of γ-carboxylic groups as a concentration of 0.2 mmol L1 of Glu10, the difference in crystal growth inhibition is of about 20%. This indicates that the chain length is the controlling parameter and the adsorption of oligomers is the predominant process. The evolution of the inhibition efficiency R with the oligomer concentration in the presence of Glu10 and Glu20, plotted in Figure 4, seems to confirm the adsorption process, as we will discuss in the following. If we assume that the crystal growth inhibition efficiency is proportional to the fractional coverage of surfaces, the experimental 1884



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Table 1. Relative Growth Rates (rmax) and Crystallization Inhibition Efficiency (R) Estimated from Ion-Selective Electrode and Light Transmission Measurements, Induction Periods (tind), Relative Equilibrium Concentration (ceq), and Crystal Phases for Different Concentrations of Additives Ca2þ-selective electrode 1

cadditive(mmol L )

rmax ( 10

blank Glu

Glu5

Glu10

Glu20

a

3 1

s )

light transmission

R

rmax ( 102 s1)

6.78

R

2.53

tind (s)

ceq

phasea

25

0.12

COT

0.001

7.11

0.05

2.45

0.03

40

0.15

COT

0.01

4.40

0.35

2.42

0.04

35

0.16

COT

0.1

6.19

0.09

2.47

0.02

30

0.18

COT

0.2 0.001

5.48 5.65

0.19 0.17

2.46 2.51

0.03 0.01

45 40

0.17 0.15

COT

0.01

6.51

0.04

2.53

0.00

40

0.16

COT

0.1

5.29

0.22

2.56

0.01

35

0.18

COT

0.2

6.27

0.08

2.17

0.14

45

0.15

0.001

5.23

0.23

2.12

0.16

40

0.17

COT

0.01

4.03

0.41

1.93

0.24

45

0.19

COD/COT

0.1

3.48

0.49

1.64

0.35

65

0.24

COD/COT

0.2 0.001

3.46 4.83

0.49 0.29

1.37 2.20

0.46 0.13

80 35

0.24 0.19

COT

0.01

4.25

0.37

1.99

0.21

40

0.32

COD

0.1

2.23

0.67

0.77

0.69

115

0.32

COD

0.2

1.19

0.82

0.36

0.86

275

0.36

As identified by X-ray diffraction: COD (calcium oxalate dihydrate), COT (calcium oxalate trihydrate).

Figure 4. Evolution of the inhibition efficiency R for the crystallization of calcium oxalate in the presence of oligo(glumamic acid)s with 10 and 20 monomer units: (a) calculated from growth rated determined by measurement of a calcium-selective electrode; (b) calculated from growth rates determined by measurement of the transmitted light. The discontinuous lines correspond to theoretical Freundlich-like adsorption expressions.

data of the inhibition efficiency R in Figure 4 can be fitted to a Freundlich-like isotherm of the form R ¼ Aðcpept Þ1=n

ð7Þ

being cpept the concentration of peptidic additive and A and n fitting parameters. From the measurements by the Ca2þ-selective electrode, the Freundlich exponents n for Glu10 and Glu20 are 12.6 and 3.9, respectively, and the values for the constant A are 0.6 and 1.2, respectively. The results indicate that Glu20 reaches a higher surface coverage than Glu10, which implies that the energy of

adsorption to free sites is higher for Glu10 than for Glu20. It has been reported that poly(L-glutamic acid) (M = 20007000 g mol1) presents a random-coil structure at pH 8.49 We can accordingly assume that the oligomer with more carboxylic groups in average pointing to the crystal surface has a higher inhibiting effect. It should be also kept in mind, as indicated in the next subsection, that a mixture of calcium oxalate dihydrate (COD) and calcium oxalate trihydrate (COT) were formed even at high concentrations of Glu10 and both phases have different surface energies. 2. Influence of Oligopeptides on Morphology, Phase, and Phase Stability. Investigations by X-ray diffraction (XRD) 1885



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Figure 5. X-ray diffraction patterns of calcium oxalate precipitated in the absence and in the presence of L-glutamic acid and oligo(L-glutamic acid)s with different chain length at a constant concentration of 0.1 mmol L1. Vertical lines show the position and relative intensity of known crystal phases: triclinic calcium oxalate trihydrate (in blue, JCPDS card No. 20232); tetragonal calcium oxalate dihydrate (in red, JCPDS card No. 17541).

showed that, using different concentrations and chain lengths of oligo(L-glutamic acid), calcium oxalate crystallizes under the same reaction conditions in two different phases: the triclinic COT (JCPDS card No. 20232) and the tetragonal COD (JCPDS card No. 17541). The last column of Table 1 reports the phases identified during the crystallization in the presence of Glu and the different peptides. As a representative example, powder XRD patterns of samples precipitated at a constant concentration of 0.1 mmol L1 of Glu, Glu5, Glu10, and Glu20 are presented in Figure 5 and compared with the patterns of the blank sample, precipitated in the absence of any additive. At the given reaction conditions, COT was the kinetically stable phase obtained without additives and in the presence of Glu and Glu5, even at the highest concentration. At least a chain of ten monomer units (Glu10) at a concentration of 0.01 mmol L1 is needed to stabilize the dihydrate and enforce the simultaneous crystallization of COT and COD. It is remarkable that in the presence of the largest peptide, Glu20, only COD was formed at concentrations g0.01 mmol L1. This leads to the conclusion that a certain concentration and chain length of the peptide is required to stabilize the COD nuclei and, consequently, to favor the growth of this phase. Under the argon atmosphere used in the experiments, the variation of pH during the precipitation was always of less than 0.15 units in the absence of any additive and remains almost constant—presumably because of a very mild buffer effect of the peptide—at the highest concentration of the long chain peptides (see Figure S1 in the Supporting Information). The investigation of the crystal morphology by SEM evidences that the morphology is dependent on the chain length and concentration of the added peptide. As shown in Figure 6, in the absence of additive, truncated prismatic platelets of triclinic COT are obtained. Similar morphologies are observed for all

Figure 6. SEM micrographs of calcium oxalate formed in the absence and presence of L-glutamic acid and the different oligo(L-glutamic acid)s (left column: blank sample and samples prepared in the presence of 0.1 mmol L1 of Glu, Glu5, and Glu10; right column: samples obtained at increasing concentration of Glu20). The red circles identify typical morphologies found in each sample.

concentrations of the monomeric Glu and for the lowest concentration (0.001 mmol L1) of Glu5, Glu10, and Glu20. The presence of Glu5 at a concentration of at least 0.01 mmol L1 leads to the formation of a rhombic COT morphology. The tetragonal dihydrate phase identified by XRD at concentrations equal or higher than 0.01 mmol L1 of Glu10 is confirmed in SEM micrographs through the presence of characteristic tetragonal bipyramidal crystals. Rhombic structures of COT could also be sporadically observed in the presence of Glu10, as expected from the diffraction results. The increase in the concentration of Glu20 (cf. the panels on the right in Figure 6) induced the formation of elongated COD crystals, showing dominant {100} faces. For the sake of clarity, a 1886



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Figure 7. Schematic representation of the morphology evolution of the calcium oxalate crystals prepared in the presence of oligo(L-glutamic acid)s.

schematic sketch of the morphological evolution is depicted in Figure 7. The elongation of the COD crystals can be explained through a specific adsorption of the peptides to the {100} faces, promoting the growth in the [001] direction and resulting in smaller {101} faces. This is perfectly consistent with the results from the kinetic investigation (cf. Figure 4). SEM micrographs also show that crystals formed at high concentration of peptide are in average smaller than those formed at lower concentrations. Another important observation is the occurrence of crystal interpenetration at high concentration of long-chain oligo(L-glutamic acid) (see, as an example, the inset in Figure 6 for 0.1 mmol L1 Glu20), which could be an indirect indication of a so-called aggregation mechanism for the crystal growth in the presence of the peptides.50,51 In order to investigate the thermodynamic stability of the formed metastable phases, the crystals were aged at room temperature for a period of 12 months. The aged samples were analyzed with XRD and TGA and compared with the original ones. Figure 8 shows the XRD patterns of the same samples presented in Figure 5, but after the aging process. Thermograms of as-prepared crystals and after aging are presented in Figure 9 for two representative cases: the blank sample without additive and the sample prepared in the presence of 0.1 mmol L1 of the longest chain peptide, Glu20. XRD patterns and the TGA curve show that the blank sample precipitated without additives was completely converted to the thermodynamically most stable phase, calcium oxalate monohydrate (COM), whereas samples with additives could be stabilized at different degrees in the metastable phases COD or COT. As seen in Figure 9a, TGA curves of COT presents two decomposition steps under 200 °C, the first one corresponding to the conversion to COM, and the second one to the conversion of COM to the anhydrous phase. Accordingly, only the second of these steps is identifiable in samples with only COM. The analysis of the total amount of water lost allows one to determine stoichiometrically the amount of water molecules contained in the products precipitated in the absence and presence of the peptides. Table 2 compiles the values of hydration stoichiometry

Figure 8. X-ray diffraction patterns of calcium oxalate precipitated in the absence and in the presence of L-glutamic acid and oligo(L-glutamic acid)s (0.1 mmol L1) after aging for 12 months. Vertical lines show the position and relative intensity of known crystal phases: monoclinic calcium oxalate monohydrate (in green, JCPDS card No. 20231); tetragonal calcium oxalate dihydrate (in red, JCPDS card No. 17541), triclinic calcium oxalate trihydrate (in blue, JCPDS card No. 20232);.

(n in CaC2O4 3 n H2O) calculated from TGA results and the phases identified by XRD in the original samples and after aging. A loss of 1.8 crystal water molecules (from 2.7 to 0.9) in the blank sample after aging is coherent with the phase transition from COT to COM. When monomeric Glu is used as an additive (0.1 mmol L1), approximately 50 mol % of COT was converted to COM. At the same concentration of Glu5, the transition of 1887



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Figure 9. Thermogravimetric analysis (TGA) curves and corresponding derivative curves of samples as precipitated (black lines) and after aging for 12 months (blue lines): (a) blank sample prepared in the absence of any additive; (b) sample prepared in the presence of 0.1 mmol L1 of Glu20.

Table 2. Hydration Stoichiometries Calculated from Weight Loss in TGA Thermograms and Crystal Phases for As-Prepared and Aged Calcium Oxalate Samples, Precipitated in the Absence and Presence of Oligo(L-glutamic acid)s (0.1 mmol L1) as-prepared samples weight lossa/%

hydration stoichiometryb

blank

27.8

2.7

Glu

29.0

2.9

Glu5

29.0

Glu10 Glu20

aged samples (12 months) phasesc

weight lossa/%

hydration stoichiometryb

phasesc

COT

11.7

0.9

COT

17.3

1.5

COMþCOT

2.9

COT

22.6

2.1

COMþCOT

27.0

2.6

CODþCOT

24.1

2.3

CODþCOT

22.0

2.0

COD

20.9

1.9

COD

COM

Total weight loss under 200 °C corresponding to hydration water. b Value of n in CaC2O4 3 nH2O, calculated from the weight loss the TGA curve. c As identified by X-ray diffraction: COM (calcium oxalate monohydrate), COD (calcium oxalate dihydrate), and COT (calcium oxalate trihydrate). a

Figure 10. SEM micrographs of calcium oxalate crystals formed in the presence of carboxylic-functionalized latex particles. The micrographs on the right show the incorporation of the particles in the crystal.

COT to COM decreases to approximately 30 mol %. Glu10 and Glu20 were able to stabilize almost completely the crystal phases of calcium oxalate as precipitated. With increasing chain length, the stability of the metastable phases increases dramatically, which could be explained by an incorporation of the peptides in the crystal structure that stabilizes the phase initially formed. To prove this claim, calcium oxalate was precipitated in an analogue way in the presence of carboxyl-functionalized polystyrene particles (82 μg L1), which can be used as a model for macromolecular carboxylic

additives and have the advantage of being easily observable by electron microscopy. The particles, as described in the Experimental Section, were prepared with an N-methacryloyl-11-aminoundecanoic acid surfmer to avoid influences of surfactant on the crystallization process. X-ray diffraction patterns of the samples (see Figure S2, Supporting Information) showed that at this very low latex concentration the phase obtained was COT, as in the absence of any additive. The most significant information of this experiment was, however, not the crystal phase, but the observation of the heavy incorporation of the polymer particles 1888


Crystal Growth & Design into the crystal, shown in the SEM micrographs of Figure 10. During the crystallization, adsorbed additive particles become incorporated into the growing crystal. This is similar to results previously found for zinc oxide and calcium carbonate.44,52,53 We consider that the incorporation of the model latex particles are an indirect proof of a similar incorporation of the peptides, which can explain the stabilization of the metastable phases obtained. The Ca2þ ions complexed by polyelectrolytes may serve as nucleation centers for building calcium oxalate crystals. The structure of the Ca2þ/polyelectrolyte complex can lead to different crystal structures by reducing the free energy of nuclei formation. The additives adsorb either specifically (as it seems to be the case for Glu10 and Glu20, in which an adsorption to the (100) face can be assumed) or unspecifically to the growing crystal surfaces, stabilizing the nuclei after formation.

’ CONCLUSIONS We have shown that the peptidic chain length is the controlling parameter for the crystal formation and growth mechanism of calcium oxalate precipitated from aqueous media in the presence acid-rich peptides, using homopeptides of L-glutamic acid as a model. Comparing the crystal growth rates of different chain lengths with same carboxylic group concentration, it was found that the concentration of carboxylic groups is less important for the crystal growth inhibition than the chain length. An inhibition of the nuclei formation leading to an increase in the induction period, a decrease in the crystal growth rate, and a higher solubility of the precipitate are observed with increasing chain length and concentration. The evolution of the kinetic parameters (inhibition efficiency calculated from growth rates) with the additive concentration can be well fitted to a Freundlichlike expression, which seems to indicate that adsorption of the peptide to the growing crystal faces takes place, being especially significant for molecules with longer chain. We also found that an increase in the chain length favors the formation of tetragonal calcium oxalate dihydrate (COD) over triclinic calcium oxalate trihydrate (COT), the latter being the kinetically stable phase under our experimental conditions. Calcium ions precomplexed by the peptides may serve as nucleation centers reducing the free energy of nuclei formation, and the structure of the formed calcium-ion complex may be determining for the structure of the resulting crystal. The elongation of the (100) face in COD crystals, observed in samples prepared with an increasing chain length, indicates a predominant specific adsorption of the oligopeptide on this face and a promotion of the growth in the [001] direction, which is consistent with the kinetics results. Time stability studies showed that oligo(L-glutamic acid)s are able to stabilize the metastable phases of calcium oxalate, COD and COT, avoiding the conversion to the thermodynamically stable phase, monoclinic calcium oxalate monohydrate (COM). The stability of the formed phase increased dramatically with increasing the chain length of the peptides, with almost no conversion of the formed COD metastable phase for the case of the largest chain. Crystallizations in the presence of carboxyl-functionalized particles, serving as observable additives, showed that the additives are heavily incorporated in the formed crystals. Thus, we conclude that the incorporation of the peptides and their interaction with the crystals is responsible for the long-time stability of the metastable phases.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Evolution of the pH during the precipitation of calcium oxalate (Figure S1) and XRD patterns of the sample prepared in the presence of carboxyl-functionalized latex particles (Figure S2) (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel.: þ49-6131-379410, Fax: þ49-6131-379100.

’ ACKNOWLEDGMENT The authors thank Dr. Patrice Castignolles for the help with the capillary electrophoresis measurements and their interpretation, and Matthias Maier and Julien Andrieu for their assistance with the peptide synthesizer. Alfons Becker is gratefully acknowledged for the technical support in the building of the light transmission setup. ’ REFERENCES (1) Shirane, Y.; Kurokawa, Y.; Miyashita, S.; Komatsu, H.; Kagawa, S. Urol. Res. 1999, 27, 426–431. (2) Wesson, J. A.; Ward, M. D. Elements 2007, 3, 415–421. € (3) Akın, B.; Oner, M.; Bayram, Y.; Demadis, K. D. Cryst. Growth Des. 2008, 8, 1997–2005. (4) Perera, C. O.; Hallett, I. C.; Nguyen, T. T.; Charles, J. C. J. Food Sci. 1990, 55, 1066–1069. (5) Horenstein, B. K.; Hernandez, G. L.; Rasberry, G.; Crosse, J. Wat. Sci. Technol. 1990, 22, 12–183. (6) Bouropoulos, N.; Weiner, S.; Addadi, L. Chem.—Eur. J. 2001, 7, 1881–1888. (7) Webb, M. A. Plant Cell 1999, 11, 751–761. (8) Franceschi, V. R.; Nakata, P. A. Annu. Rev. Plant Biol. 2005, 56, 41–71. (9) Taller, A.; Grohe, B.; Rogers, K. A.; Goldberg, H. A.; Hunter, G. K. Biophys. J. 2007, 93, 1768–1777. (10) Grohe, B.; O’Young, J.; Ionescu, D. A.; Lajoie, G.; Rogers, K. A.; Karttunen, M.; Goldberg, H. A.; Hunter, G. K. J. Am. Chem. Soc. 2007, 129, 14946–14951. (11) Wang, L. J.; Guan, X. Y.; Tang, R. K.; Hoyer, J. R.; Wierzbicki, A.; De Yoreo, J. J.; Nancollas, G. H. J. Phys. Chem. B 2008, 112 9151–9157. (12) Heijnen, W.; Jellinghaus, W.; Klee, W. E. Urol. Res. 1985, 13, 281–283. (13) Opalko, F. J.; Adair, J. H.; Khan, S. R. J. Cryst. Growth 1997, 181, 410–417. (14) Cody, A. M.; Cody, R. D. J. Cryst. Growth 1994, 135, 235–245. (15) B€auerlein, E. Handbook of Biomineralization; Wiley-VCH: Weinheim, Germany, 2007. (16) Mann, S., Biomineralization. Oxford University Press: Oxford, 2001. (17) Sangwal, K., Additives and Crystallization Processes; John Wiley: West Sussex, U.K., 2007. (18) Schweizer, S.; Taubert, A. Macromol. Biosci. 2007, 7, 1085–1099. (19) Kofina, A. N.; Demadis, K. D.; Koutsoukos, P. G. Cryst. Growth Des. 2007, 7, 2705–2712. (20) Brecevic, L.; Kralj, D. J. Cryst. Growth 1986, 79, 178–184. (21) Garti, N.; Tibika, F.; Sarig, S.; Perlberg, S. Biochem. Biophys. Res. Commun. 1980, 97, 1154–1162. (22) Fellstr€om, B.; Danielson, B. G.; Ljunghall, S.; Wikstr€om, B. Clin. Chim. Acta 1986, 158, 229–235. 1889



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Stabilization of Calcium Oxalate Metastable Phases by Oligo(l

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