- Email: [email protected]
Part I. Enzymatic synthesis of lactate and glycolate esters of fatty alcohols
Enzyme and Microbial Technology 25 (1999) 745–752
Part I. Enzymatic synthesis of lactate and glycolate esters of fatty alcohols Carlos Torres, Cristina Otero* Departamento de Biocata´lisis, Instituto de Cata´lisis, CSIC. Cantoblanco (28049). Madrid, Spain Received 30 September 1998; received in revised form 27 July 1999; accepted 10 August 1999
Abstract Optimum conditions were determined for the esterification reactions of lactic and glycolic acids with fatty alcohols (C8 –C16) in the presence of a lipase from Candida antarctica. This synthetic method gives nearly complete conversion to the desired ester in a relatively short time with high volumetric productivity. In acetonitrile the maximum yields of dodecyl lactate (95% in 48 h) and glycolate (87% in 24 h) were obtained in the presence of a desiccant and 0.28% (w/w) added water, respectively. The procedure permits one to increase substrate concentrations without significant adverse effects on the yield of lactate ester for lactic acid concentrations of 0.25 to 1 M. Similar yields of lactate ester (94 –96%) were obtained for alcohol chain lengths from C8 to C16. Although esterification of lactic acid with fatty alcohols is not favored in apolar solvents (e.g. n-hexane), esterification of glycolic acid in n-hexane produces high yields of the glycolate ester (96% in 4 h). In the solvent-free system, esterification of lactic acid with a fatty alcohol requires the presence of desiccant from the beginning of the process (yield of 70% in 48 h). For the reactions of glycolic acid, a strategy in which a desiccant is added after 24 h of reaction gives the maximum yield of the glycolate ester (91%) in a shorter time (48 h of total reaction time). Alternatively, transesterification between the alcohol of interest and ethyl lactate increases the maximum yield of dodecyl lactate (87% in 24 h). © 1999 Elsevier Science Inc. All rights reserved. Keywords: Candida antarctica lipase; Enzymatic synthesis; Esterification; Glycolic acid; ␣-hydroxy acids; Lactic acid; Lipase; Nonconventional media
1. Introduction The use of topical formulations containing hygroscopic compounds is of great interest since these compounds attract and retain moisture, a benefit in the treatment of particular skin diseases. Glycerol, lactic, and glycolic acids have been used in the formulation of both cosmetic and pharmaceutical products for topical application because of their humectant properties. However, ␣-hydroxy acids have limited applicability because of irritant effects attributed to their acidities [1–3]. Because the esters of ␣-hydroxy acids have reduced acidity, use of these compounds could circumvent most of the problems associated with use of these acids. These esters have already been used as humectants in several cosmetic and pharmaceutical formulations [2]. Moreover, the esters of glycolic acid have the added benefit of exhibiting antimicrobial activity [3]. * Corresponding author. Tel.: ⫹34-91-5854805; fax: ⫹34-915854760. E-mail address: [email protected] (C. Otero)
A significant problem in esterification of hydroxy carboxylic acids is that these compounds may act as both acyl donor and nucleophile in competitive reactions. To increase the selectivity of these reactions with respect to the desired esterification reaction of the ␣-hydroxy acid with an alcohol, one could either protect the hydroxyl group of the ␣-hydroxy acid, and/or use an enzyme that is selective for the desired reaction of the acid group with an alcohol thereby minimising reactions with the ␣-hydroxy group in which the lactic acid functions as a nucleophile. In addition, one can improve the conversion of the alcohol by employing a molar excess of lactic acid to circumvent problems associated with the presence of oligomers in commercially available lactic acid. Lipases have been used successfully as catalysts for esterification of molecules of variable structure that contain at least one hydroxy or acid group [4]. The mild reaction conditions and high selectivities characteristic of these enzymatic processes facilitate preparation of very pure products [5,6]. Moreover, the lipase-catalyzed reactions can be regioselective when polyfunctional compounds are used [6].
0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 1 1 7 - 9
746
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Lipase-catalysed polycondensation reactions of ␣-hydroxy acids have been observed in alcohol free systems [8]. However, in the presence of alcohols, the lipase-catalysed reactions of ␣-hydroxy fatty acids yield hydroxy esters as the major products [9]. Recently, lipase-catalysed esterification reactions of lactic acid in n-hexane have been reported [10]. The procedure permits preparation of high yields (89%) of the butyl ester. These yields decrease for alcohols with longer chain lengths (28% and 11% for octyl and decyl lactates, respectively) [10]. In this work, we have optimized a procedure for the enzymatic formation of ester linkages between lactic (or glycolic) acid and fatty alcohols of different chain lengths (C8 –C16). Optimisation of the yields of these reactions has permitted identification of those factors that have the most significant effects on the yield of the desired ester. The aim of this work was to overcome some very important limitations on earlier biocatalytic processes in order to obtain high productivity/selectivity towards the desired product, decrease the reaction time, and reduce potential toxicity effects associated with the use of organic solvents as the reaction medium. Our goal was to obtain nearly quantitative esterification of lactic and glycolic acids with long chain alcohols in a variety of solvents and in solvent free systems.
2. Materials and methods
mixture. In all cases, the solvents were dried with molecular sieves before use. The molar yields of the products were calculated with respect to the less concentrated reactant. 2.3. Purification of the esters All the esters were subsequently prepared in larger amounts (up to 2 mmol) and then purified. These pure materials were used in experiments to characterize their structures and as standards for the high-performance liquid chromatography (HPLC) analyses. The enzyme and the molecular sieves were removed from the reaction mixtures via filtration through a 0.1 mm sieve, and the solvent was then evaporated using a rotovap system. Further purification was accomplished by liquid chromatography. The filtered and evaporated reaction mixtures were dissolved in n-hexane. The lactic acid was extracted from these solutions by washing the mixture 3⫻ with the same volume of a 0.1 M NaCl solution. The remaining mixture was introduced into a silica gel column equilibrated with n-hexane. The chromatographic process consisted of successive elutions with 100 ml of hexane, and mixtures of hexane/chloroform in which the percentage of chloroform was successively increased. The desired ester was eluted with a solution of hexane/chloroform 60/40 (v/v). Since the last eluted fractions were contaminated with dodecanol, they were discarded.
2.1. Materials 2.4. Analysis of the reaction mixtures Novozym 435 (Lipase B from Candida antarctica, a non specific lipase immobilized on a macroporous acrylic resin Accurel EP-100) and Lypozyme (lipase from Mucor miehei immobilized on Duolite) were kindly provided by Novo Nordisk A/S (Bagsvaerd, Denmark). Lipase from C. rugosa, octanol, cetyl alcohol, and molecular sieves were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lipase from Pseudomonas sp. was obtained from Amano Chemicals (Nagoya, Japan). Lactic acid, glycolic acid, and dodecanol were purchased from Merck (Darmstadt, Germany). Acetone, acetonitrile and methanol were supplied by Scharlau (Barcelona, Spain). All chemicals were analytical grade. 2.2. Enzymatic reactions Reactions were performed by mixing the indicated amount of the corresponding ␣-hydroxy acid and the alcohol in a stopped glass bottle. After addition of the indicated amount of the enzyme and 2 ml of the organic solvent, the reaction mixture was maintained at constant temperature in an orbital shaker (Stuart Scientific, Surrey, England) for a known time at a specified temperature. When it is so indicated, some reaction mixtures were prepared as above without addition of the organic solvent. Where noted, molecular sieves were added to sequester excess water in the reaction
The course of the enzymatic reactions was monitored via analysis of the reaction mixtures by HPLC. At the indicated reaction time, the reaction was stopped by addition of 2 ml of dimethylformamide (DMF). The enzyme was eliminated by filtration and centrifugation. The volume of the final transparent solution was increased to 5 ml by addition of DMF. This solution was analyzed in an HPLC apparatus consisting of an L-7100 isocratic pump, a Lichrospher 100 RP-18 column, an RI-71 refractive index detector, and a D-7500 integrator. The injection volume was 20 l and the column temperature was 25°C. All components of the HPLC system were obtained from Merck– Hitachi Ltd. (Germany and Japan). Calibration analyses were performed using esters obtained and purified in our laboratory. The HPLC analyses indicated that the purities of these standards exceeded 99%. The eluant used for quantification of the dodecyl lactate and dodecanol was acetonitrile/methanol/water 50/35/15 (v/v). The retention times for dodecyl lactate and dodecanol were 5.8 and 6.9 min, respectively, at a flow rate of 1 ml/min. Results are expressed as the mean value of at least two independent experimental measurements. In all cases the experimental errors were below 4%.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
2.5. Mass spectrometry The mass spectra were recorded in the range 50 to 900 by using type VG Autospec MICROMASS (Manchester, UK) and via direct introduction into the solids probe with a temperature gradient of 50 to 300°C. Ionization voltage: 70 eV; ion source temperature 200°C. The molecular masses determined for dodecyl lactate, octyl lactate, cetyl lactate, and dodecyl glycolate were 258.3, 202.2, 314.3, and 244.3, respectively. Experiments were carried out in duplicate. 2.6. NMR analyses 1
H NMR spectra were recorded for solutions of the esters in methanol-2H4 by using a Varian XL-300 spectrometer operating at 30°C and 300 Mhz. The 1H NMR spectra of our lactic and glycolic esters were similar. As an example, consider the 1H NMR spectrum of dodecyl lactate: 4.24 (dd, 1H, J ⫽ 6.9, HO-CH-COO), 4.16 (m, 2H, CH2-CH 2OOC), 1.65 (m, 2H, CH 2 -CH2-OOC), 1.43 (d, 3H, J ⫽ 6.9, CH3-CH-OH), 1.25 (m, 18H, CH2-(CH2)9-CH3), 0.86 (t, 3H, CH3-(CH2)9-, J ⫽ 6.9).
747
Table 1 Enzymatic synthesis of dodecyl lactate catalysed by different biocatalysts Enzyme (mg)
Ester yield (%)
Hydrolytic activity (uc/mg biocat)
CRLa (25) PSLb (25) Lipozyme IM (50) Novozyme 435 (50)
1 1 2 44
3.6 ⫻ 10⫺3 1.0 ⫻ 10⫺3 24 ⫻ 10⫺5 5.7 ⫻ 10⫺6
Reaction conditions: 50 mg (0.55 mmol) lactic acid, 100 mg (0.55 mmol) dodecanol, 2 ml acetone, 24 h and 50°C. a Candida rugosa lipase b Pseudomonas sp. lipase c Units of enzymatic activity as mequivalents of acid liberated per minute at pH ⫽ 7.0 and 30°C.
molecular masses of the monomer, dimer, and trimer, respectively. Results are expressed as molar percentages of total monomeric lactic acid. All spectra were recorded in duplicate and the experimental errors were less than 4%.
3. Results
2.7. Karl Fischer measurements 3.1. Selection of the best biocatalyst Water contents were determined by the Karl Fischer method using a KF DL18 Mettler Toledo apparatus (Barcelona, Spain). The titrations of the reaction systems were carried out by adding an aliquot of the liquid reaction mixture to the titrator at the indicated reaction time. Results are expressed as the mean value of at least three independent experimental measurements. In all cases the experimental errors were below 5%. 2.8. Determination of the enzymatic activity The hydrolytic activity was measured by using a pH-stat DL 21 Mettler Toledo apparatus (Barcelona, Spain). An emulsion (40 ml) containing olive oil (200 l), buffer Tris/ HCl, 1 mM, acetonitrile (3%), and gum arabic [1.5% (v/v)] was sonicated for 30 min. After pretitration, a known weight of enzyme was added. The enzymatic activity was determined as the mequivalents of acid liberated per minute at pH 7.0 and 30°C (1 unit ⫽ 1 mequiv. acid/min). 2.9. Degree of polymerisation of lactic acid Commercial grade lactic acid (10 mg) dissolved in 1.5 ml acetonitrile were analyzed (1 l sample volume) by gas chromatography in a Carlo Erba MFC 500 apparatus with a splitless injector fitted with a OV1 100% methyl silicon column, a mass spectrometer detector (Ionization voltage: 70 eV, detector temperature: 250°C, temperature program: 45°C during 5 min increasing 8°C/min up to 300°C), and He gas. The lactic acid chromatogram contained three peaks at 15.17, 20.20, and 25.03 min. These peaks correspond to the
The reaction between lactic acid and dodecanol was carried out by using several lipases from different sources (Table 1). The initial molar ratio of lactic acid and dodecanol was 1 : 1 to facilitate identification of the lipase with the best selectivity for use of the ␣-hydroxy acid as an acyl donor. Both native and immobilized enzymes were employed. To compensate for the weight of the support, the quantities of the immobilized lipases (Novozym 435 and Lypozyme) were twice as great as those for the native (soluble) enzymes. The activities of these biocatalysts for the hydrolysis of olive oil were also evaluated (Table 1). No correlation was observed between the hydrolytic and synthetic activities. The highest conversion to the ester was obtained with Novozym 435 (44% dodecyl lactate in 24 h), which was selected for use in subsequent experiments. 3.2. Influence of organic solvents Of the various solvents examined for the reaction between lactic acid and dodecanol, the best yields were obtained using acetonitrile (see Table 2). The amount of water present in commercial-grade lactic acid was much greater than that associated with the other components of the reaction mixture. Analysis by gas chromatography indicated that the polymerised fraction of the commercial lactic acid was 32 ⫾ 2%. Hence, when one employs equimolar proportions of reagents (acid and alcohol) in acetonitrile, the yield of the ester was close to the maximum that could be obtained via direct esterification of the monomeric fraction of the lactic
748
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Table 2 Effect of solvent on ester yield in the enzymatic synthesis of dodecyl lactate Medium
Log P
Yield (%)
Dioxane Acetonitrile Acetone n-Hexane No solvent
⫺1.1 ⫺0.33 ⫺0.24 3.5 —
51 54 42 9.0 19
Conditions: lactic acid (100 mg, 1.07 mmol), dodecanol (200 mg, 1.1 mmol), Novozyme 435 (50 mg), and 2 ml of organic solvent, 200 rev./min, 24 h and 50°C. Solvent polarity in terms of log P, where P is the distribution coefficient of the solvent employed between isoctanol and water. Water contents (as determined by the Karl Fischer method) were 0.013%, 0.015%, 0.019%, 0.003%, 13%, 0.11%, 1.07% (w/w), for dioxane, acetonitrile, acetone, n-hexane, lactic acid, alcohol, and enzyme, respectively.
acid (68%). Thus, acetonitrile was selected for use in subsequent experiments. 3.3. Influence of the molar ratio of reactants In this series of experiments, the amounts of acetonitrile, Novozym 435, and one of the substrates were held constant whereas the quantity of the other substrate was decreased. In the presence of excess dodecanol (Table 3), the highest yield was obtained at a molar ratio of lactic acid to dodecanol of 1 : 10. In this case, the water content of the reaction mixture decreased significantly as the lactic acid content decreased, thereby increasing the molar ratio of dodecanol to water. This condition should have promoted a shift of the reaction equilibrium towards formation of the ester. In the case of reaction in the presence of excess lactic acid, the experiments were carried out such that the water content originating from the commercial ␣-hydroxy acid was held constant. The optimum yield of the ester corresponded to a 5 : 1 molar ratio of lactic acid to dodecanol. High yields of ester (⬎87%) can be obtained under optimal conditions regardless of which substrate is present
Table 3 Effect of the molar ratio of reactants in the enzymatic synthesis of dodecyl lactate in acetonitrile Molar ratio Lactic/dodecanol
Yield (%)
1 : 10 1:5 1:4 1:2 1:1 2:1 4:1 5:1 10 : 1
90 81 79 72 53 77 85 87 84
Conditions: A. 205 mg (1.1 mmol) dodecanol. B. 100 mg (1.1 mmol) lactic acid.; 2 ml acetonitrile, 50 mg Novozym 435, 24 h and 50°C.
Fig. 1. Enzymatic synthesis of dodecyl lactate in acetonitrile in the presence and absence of molecular sieves. Conditions: 100 mg (1.1 mmol) lactic acid, 40 mg (0.21 mmol) dodecanol, 50 mg of Novozyme 435, 2 ml of acetonitrile, and 200 mg of molecular sieves at 30°C.
in excess. However, because the reaction mixtures containing an excess of the ␣-hydroxy acid provide sufficient monomeric lactic acid to achieve 100% conversion of the alcohol to the ester and because of the difficulties of purifying the ester when excess alcohol is present, a 5 : 1 molar ratio of lactic acid to alcohol was used in subsequent experiments. 3.4. Influence of molecular sieves Because hydrolysis is merely the reverse of esterification, the degree of hydration of the medium plays an important role in determining the maximum extent of esterification [7]. Thus, the effect of use of a desiccant on the reaction yield was also studied. The incubation temperature was 30°C because a previous study of the effect of the reaction temperature (data not shown) indicated that yields close to 80% could be obtained in 24 h at this moderate temperature. The presence of the molecular sieves decreased the initial reaction rate, but increased the conversion ultimately achieved (Fig. 1). The decrease in the initial rate is attributed to the affinity of the molecular sieves for polar substances. Hence, this desiccant could adsorb some of the lactic acid, thereby diminishing the effective concentration of this substrate in the reaction medium. Because the molecular sieves also absorb the water formed by the reaction, the presence of this desiccant shifts the equilibrium of the reaction toward synthesis of the ester. When the desiccant was present, the final yield of the ester increased from 87% to 95%. The water content of the reaction medium in both the presence and the absence of molecular sieves was investigated. The water generated during the reaction [0.1% (w/ w)] was negligible in comparison with the water entering the medium with the commercial lactic acid. Hence, this water should not have a significant effect on the conversion.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
749
the water in excess of that necessary to bring about complete esterification. However, the presence of a higher concentration of molecular sieves makes it difficult to achieve effective contact between the different components of the heterogeneous reaction mixture, because of difficulties in maintaining uniform suspension of the solids (immobilized biocatalyst, molecular sieves). 3.6. Influence of the chain length of the alcohol
Fig. 2. Effect of the concentrations of reactions on the enzymatic synthesis of dodecyl lactate in acetonitrile. Conditions: lactic acid/dodecanol molar ratio (5 : 1), the enzyme/alcohol ratio [5 : 4 (w/w)], and the amount of desiccant (200 mg) were held constant; 48 h and 30°C.
However, addition of the molecular sieves decreased the effective water content of the reaction medium from 1.3% to 0.4% (w/w). This effect is responsible for the higher conversions to the ester observed in the presence of the molecular sieves. 3.5. Effect of substrate concentration The use of maximum concentrations of substrates in the solvent is a challenge to be met in industrial implementation of any reaction. With the goal of increasing the concentration of reactants in acetonitrile without causing adverse effects on either the reaction rate or the yield, we probed the effect of using large amounts of reactants at 30°C (Fig. 2). The effects of adding different quantities of substrates to a fixed volume of solvent are depicted. Similar conversions were obtained in 48 h for concentrations of dodecanol between 0.05 and 0.2 M. The lower conversions obtained below 0.05 M dodecanol can be a consequence of the decrease in the reaction rate associated with the lower concentration of the substrate. When dodecanol was below 0.2 M, the quantity of molecular sieves employed (200 mg) and their capacity for water [20% (w/ w)] were sufficient to sequester both the water present in the original reagents (primarily that associated with the commercial lactic acid) and that produced by reaction. For these conditions one can maintain the degree of hydration of the medium necessary to sustain the activity of the enzyme for esterification of the ␣-hydroxy acid. The solutions containing reactant concentrations above 0.2 M were more viscous and lower reaction rates occur at these concentrations. In these cases, the amount of desiccant employed (200 mg) was slightly smaller than that required for sequestration of
Enzymatic esterification of lactic acid with alcohols of different chain lengths (C8 and C16) was carried out at 30°C in media which were 0.1 M in the alcohol, 0.5 M in lactic acid, and contained 50 mg of Novozym 435, 200 mg molecular sieves and 2 ml acetonitrile. In all cases, the yields of the esters obtained in 48 h were similar (94 –96%). This fact demonstrates the general applicability of this methodology for preparing different lactic esters of fatty alcohols under the same reaction conditions. 3.7. Preparation of glycolic ester with dodecanol The synthesis of dodecyl glycolate was investigated using the same molar ratio of reagents (5 : 1 of acid/alcohol), an identical charge of the biocatalyst and a temperature that is optimal for preparation of dodecyl lactate in acetonitrile. Surprisingly, the reaction in acetonitrile produced the lowest yield of the ester (Fig. 3A). In an effort to obtain a better understanding of the reasons for the low conversion in acetonitrile, this reaction was repeated in the presence of different amounts of water (Fig. 3B). In the presence of 0.28% (w/w) added water, the yield of ester increased to a value comparable to those obtained in the other solvents investigated (Fig. 3A). The lower water content of the glycolic acid [0.11% (w/w)] relative to that of lactic acid [13% (w/w)] is the source of the necessity for adding water to increase the reaction rate when the solvent is acetonitrile. Further optimization studies were carried out in hexane, because of its relatively low toxicity and the ease of subsequent purification of the ester (low solubility of glycolic acid in this medium). The reaction conditions employed were identical to those for the series of experiments intended to elucidate the effect of the solvent. As was the case for synthesis of dodecyl lactate, the molecular sieves increased the yield of dodecyl glycolate. Esterification of glycolic acid gave a yield of 96% ester in 4 h. 3.8. Reactions in the absence of any solvent To minimize the toxicity of products formed via the enzymatic reaction, it would be desirable to eliminate the use of a solvent. Consequently, esterifications of lactic and glycolic acids with alcohols were carried out in absence of an organic solvent. Initially the reaction was studied at the optimal conditions for reaction in the presence of solvent. However,
750
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Fig. 3. Enzymatic synthesis of dodecyl glycolate. Effect of the (A) organic solvent and (B) water content on the reaction in acetonitrile. Conditions: 100 mg (1.3 mmol) of glycolic acid, 50 mg (0.27 mmols) of dodecanol, 50 mg of Novozym 435, and 2 ml of solvent with a 24-h reaction time at 30°C.
because of the low miscibility of the reagents, the rates of the synthesis of the lactic and glycolic esters were slow, and the yields of the corresponding esters were inferior to those obtained in the presence of solvent. In an effort to improve the conversions, the temperature was increased to 60°C. Esterification of lactic acid in a solvent free system should not be favored because of the relatively high water content of this substrate. Hence, different strategies were attempted to increase conversion to the ester. Although use of excess lactic acid would facilitate the purification process, the yield obtained with a 5 : 1 ratio of acid to alcohol was compared to that for a 1 : 5 ratio (excess dodecanol). Use of excess alcohol permits one to reduce the water content introduced with the commercial lactic acid, thereby increasing the yield of the ester (see Fig. 4A). In addition, we utilized an alternative strategy based on a transesterification reaction of the ethyl ester of lactic acid that has much
lower water content [0.09% (w/w)] than the commercially available lactic acid. The results are also shown in Fig. 4A. The highest yield (87%) after 24 h of reaction was that obtained via the transesterification reaction. At 60°C, both the ester yield and the rate of reaction of glycolic acid were greater than those obtained at 30°C. In 3 days, the biocatalyst produced a yield of 86% of the glycolate ester (Fig. 4B). As was the case for reactions in organic solvents, use of a desiccant decreased the reaction rate. Because the presence of molecular sieves should permit one to increase the ultimate extent of conversion, a strategy was developed to decrease the required reaction time (see Fig. 4B). The method basically consists of carrying out the reaction in stages. The first stage is a rapid reaction carried out in the absence of molecular sieves. The second stage is intended to reach equilibrium in a medium containing molecular sieves so as to reduce its degree of hydration. The
Fig. 4. Enzymatic synthesis of (A) dodecyl lactate and (B) dodecyl glycolate in absence of organic solvent. Conditions. (A): 50 mg of Novozym 435 and 200 mg of molecular sieves at 60°C; (■), 100 mg of lactic acid and 40 mg dodecanol; (Œ), 20 mg of lactic acid, and 205 mg of dodecanol; (), 125 mg of ethyl lactate and 40 mg of dodecanol. (B): 100 mg of glycolic acid, 50 mg of dodecanol, 50 mg of Novozym 435, and 200 mg of molecular sieves at 60°C.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
final yield of the ester increases from 86% to 91% when molecular sieves are added 24 h after starting the reaction. Furthermore, this approach reduces the total reaction time to 48 h.
4. Discussion Of the different lipases tested in the present study, Novozyme 435 is the one that best uses ␣-hydroxy acids as acyl donors. This lipase has previously been employed in different esterification reactions with a variety of acids and alcohols. For these reactions Novozym 435 has both high activity and thermostability [5,12]. In previous studies, the required reaction times were much longer (several weeks) than those for the optimal conditions employed in the present study (4 – 48 h). In our work, Novozym 435 did not exhibit significant deactivation at either 30°C or at 60°C, in presence and absence of solvent, respectively. From et al. [10] have described the enzymatic synthesis of lactic acid esters in n-hexane. This work demonstrated that apolar solvents are not appropriate for biotransformation reactions involving long chain alcohols. The effective solvation of these alcohols by apolar solvents reduces their activity coefficients and decreases the reaction rates [13]. The study herein reported shows that the degree of hydration of the reaction medium is largely determined by the high water content [13% (w/w)] present in the lactic acid purchased from the vendor. This water content could produce saturation of the more hydrophobic solvents, causing a shift in the reaction equilibrium to favour the hydrolysis reaction. In addition, the higher solubility of lactic acid in polar solvents might contribute to the much higher yield of ester observed in acetonitrile relative to n-hexane. In the present work, we have developed a method for the nearly quantitative synthesis of lactic or glycolic esters of fatty alcohols. High yields of lactate can be prepared in acetonitrile via the indicated protocol (which includes addition of a desiccant). For the reactions of glycolic acid, the water content must be increased in acetonitrile because of the relatively low degree of hydration of commercial sources of this substrate. Although n-hexane does not facilitate esterification of lactic acid with C8 to C16 alcohols, nearly complete conversion of glycolic acid to the ester could be achieved in this solvent. The relatively low water content of commercially available glycolic acid permits the preparation of glycolate esters in n-hexane in a very short time (4 h). The synthetic procedure reported here employs low temperature reaction conditions and is not limited by the chain length of the alcohol in the range C8 to C16. This method can be used in the presence of an excess of either substrate (acid or alcohol). It permits the use of relatively high concentrations of substrates while maintaining good yields and selectivity. Use of highly concentrated reaction mixtures reduces the time necessary to achieve a specified conver-
751
sion, the size of the reactor required and the cost of the process (less organic solvent must be removed during purification of the reaction mixture). In less toxic solvent-free systems, it is possible to obtain yields similar to those obtained in the presence of organic solvents by increasing the temperature to 60°C. The increased temperature enhances both the reaction rate and the yield of the ester, results that can be attributed to the kinetic effect of temperature and the higher solubility of the ␣-hydroxy acid in the alcohol. The reduced rate of glycolic acid esterification in presence of desiccant may be a consequence of interactions (adsorption) between the acid and the drying agent and/or reduction of the water content below that necessary for the progress of the reaction [11,15]. To overcome this problem, we have developed a strategy consisting of first carrying out the reaction in the absence of molecular sieves and after 24 h of reaction adding this desiccant to the reaction mixture. This approach reduces the time necessary to accomplish the initial stage of the reaction, while maintaining a capability for shifting the equilibrium position of the reaction towards the esterification during the final stage. Thus, the ester yields were increased to 90% and the required time was reduced by 24 h. Comparison of the three different strategies studied for the solvent-free esterification of lactic acid (addition of desiccant, use of excess alcohol, and transesterification), indicates that the transesterification reaction with ethyl lactate gives the best result (87% dodecyl lactate in 24 h) and better permits one to overcome the problem of the high water content of commercial grade lactic acid. By comparison of the reaction times required for the reactions of lactic acid (Fig. 4A) with those for glycolic acid (Fig. 4B), one can readily see that both substrates have similar reactivities. We hypothesise that the ability of the enzyme to bind these substrates is very comparable because of similarities in both molecular size and structure, as well as in the values of the acidity constants (1.37 ⫻ 10⫺4 and 1.5 ⫻ 10⫺4 for lactic and glycolic acid, respectively [16]). The preference of C. antarctica lipase for the different enantiomers of lactic acid has been previously studied [10]. It has been demonstrated that this lipase catalyses the esterification of both enantiomers of lactic acid with almost equal rates. To obtain better enantioselectivity, it would be preferable to esterify the hydroxyl group present in lactic acid. When the chiral center is located on the alcohol side, earlier investigations have reported good stereoselectivity [17]. In conclusion, the degree of hydration of the commercial ␣-hydroxy acids determines: 1) whether additional water or a desiccant must be employed; and 2) which reagent should be used in excess. These factors, in turn, govern which of two alternative strategies should be used: direct esterification or transesterification of the acid.
752
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Acknowledgments This work has been financed by the Spanish CICYT (BIO96-0837), and a predoctoral fellowship for C.T. We thank Germaine de Capuccini S.A. for their personal and technical support, and Professors Charles G. Hill and Manuel Bernabe´ for comments on the manuscript.
[8]
[9] [10] [11]
References [1] Greaves W. Topical ␣-hydroxy acid derivative for relieving dry itching skin. Cosmet Toilet 1990;105:61– 4. [2] Datta R, Tsai S-P, Bonsignore P, Moon S-H, Frank JR. Technological and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol Rev 1995;16:221–31. [3] Bailey AV. Antimicrobial properties of some erucic acid-glycolic acid derivatives. J Am Oil Chem Soc 1989;66:932– 4. [4] Kirchner G, Scollarm MP, Klibanov AM. Resolution of racemic mixtures via lipase catalysis in organic solvents. J Am Chem Soc 1985;107:7072– 6. [5] Arcos JA, Bernabe´ M, Otero C. Quantitative enzymatic production of 6-O-acylglucose esters. Biotechnol Bioeng 1998;57:505–9. [6] Arcos JA, Bernabe´ M, Otero C. Quantitative enzymatic production of 1,6-diacyl fructofuranoses. Enz Microb Tech 1998;22:27–35. [7] Lopez R, Montero E, Sa´nchez F, Can˜ada J, Fernandez–Mayoralas A. Regioselective acetylations of alkyl -D-xylopyranosides by use of
[12]
[13]
[14]
[15]
[16]
[17]
lipase PS in organic solvents and application to the chemoenzymatic synthesis of oligosaccharides. J Org Chem 1994;59:7027–32. Ohya Y, Sugitou T, Ouchi T. Polycondensation of ␣-hydroxy acids by enzymes or PEG-modified enzymes in organic media. Pure Appl Chem 1995;A32:179 –90. Hayes DG. The catalytic activity of lipases toward hydroxy fatty acids-A review. J Am Oil Chem Soc 1996;73:543–9. From M, Adlercreutz P, Mattiasson B. Lipase catalyzed esterification of lactic acid. Biotechnol Lett 1997;19:315–7. Zaks A, Klibanov AM. The effect of water on enzyme action in organic media. J Biol Chem 1987;263:8017–21. Fartasch M, Teal J, Menon GK. Mode of action of glycolic acid on human stratum corneum-ultrastructural and functional evaluation of the epidermal barrier. Arch Dermatol Res 1997;289:404 –9. Kirk O, Bjo´rkling F, Godtfredsen SE, Larsen TO. Fatty acid specificity in lipase-catalyzed synthesis of glucoside esters. Biocatalysis 1992;6:127–34. Janssen AEM, Halling PJ. Specificities of enzymes “corrected for solvation” depend on the choice of the standard state. J Am Chem Soc 1994;116:9827–30. Omar IC, Nishio N, Nagai S. The role of water on the equilibrium of esterification by immobilized lipase packed-bed column reactor. Biotechnol Lett 1988;10:799 – 804. Van Ness JH. Hydroxy Carboxylic acids. In: Grayson M, Eckroth D, editors. Kirk Othmer Encyclopedia of Chemical Technology, 3rd Ed. NY: John Wiley & Sons, 1981. p. 80 –103. Sinisterra JV, Llama EF, Campo CD, Cabezas MJ, Moreno JM, Arroyo MJ. Stereoselectivity of chemically modified ␣-chymotrypsin and immobilized lipases. J Chem Soc Perkin Trans 1994;2:1333– 6.
Part I. Enzymatic synthesis of lactate and glycolate esters of fatty alcohols Carlos Torres, Cristina Otero* Departamento de Biocata´lisis, Instituto de Cata´lisis, CSIC. Cantoblanco (28049). Madrid, Spain Received 30 September 1998; received in revised form 27 July 1999; accepted 10 August 1999
Abstract Optimum conditions were determined for the esterification reactions of lactic and glycolic acids with fatty alcohols (C8 –C16) in the presence of a lipase from Candida antarctica. This synthetic method gives nearly complete conversion to the desired ester in a relatively short time with high volumetric productivity. In acetonitrile the maximum yields of dodecyl lactate (95% in 48 h) and glycolate (87% in 24 h) were obtained in the presence of a desiccant and 0.28% (w/w) added water, respectively. The procedure permits one to increase substrate concentrations without significant adverse effects on the yield of lactate ester for lactic acid concentrations of 0.25 to 1 M. Similar yields of lactate ester (94 –96%) were obtained for alcohol chain lengths from C8 to C16. Although esterification of lactic acid with fatty alcohols is not favored in apolar solvents (e.g. n-hexane), esterification of glycolic acid in n-hexane produces high yields of the glycolate ester (96% in 4 h). In the solvent-free system, esterification of lactic acid with a fatty alcohol requires the presence of desiccant from the beginning of the process (yield of 70% in 48 h). For the reactions of glycolic acid, a strategy in which a desiccant is added after 24 h of reaction gives the maximum yield of the glycolate ester (91%) in a shorter time (48 h of total reaction time). Alternatively, transesterification between the alcohol of interest and ethyl lactate increases the maximum yield of dodecyl lactate (87% in 24 h). © 1999 Elsevier Science Inc. All rights reserved. Keywords: Candida antarctica lipase; Enzymatic synthesis; Esterification; Glycolic acid; ␣-hydroxy acids; Lactic acid; Lipase; Nonconventional media
1. Introduction The use of topical formulations containing hygroscopic compounds is of great interest since these compounds attract and retain moisture, a benefit in the treatment of particular skin diseases. Glycerol, lactic, and glycolic acids have been used in the formulation of both cosmetic and pharmaceutical products for topical application because of their humectant properties. However, ␣-hydroxy acids have limited applicability because of irritant effects attributed to their acidities [1–3]. Because the esters of ␣-hydroxy acids have reduced acidity, use of these compounds could circumvent most of the problems associated with use of these acids. These esters have already been used as humectants in several cosmetic and pharmaceutical formulations [2]. Moreover, the esters of glycolic acid have the added benefit of exhibiting antimicrobial activity [3]. * Corresponding author. Tel.: ⫹34-91-5854805; fax: ⫹34-915854760. E-mail address: [email protected] (C. Otero)
A significant problem in esterification of hydroxy carboxylic acids is that these compounds may act as both acyl donor and nucleophile in competitive reactions. To increase the selectivity of these reactions with respect to the desired esterification reaction of the ␣-hydroxy acid with an alcohol, one could either protect the hydroxyl group of the ␣-hydroxy acid, and/or use an enzyme that is selective for the desired reaction of the acid group with an alcohol thereby minimising reactions with the ␣-hydroxy group in which the lactic acid functions as a nucleophile. In addition, one can improve the conversion of the alcohol by employing a molar excess of lactic acid to circumvent problems associated with the presence of oligomers in commercially available lactic acid. Lipases have been used successfully as catalysts for esterification of molecules of variable structure that contain at least one hydroxy or acid group [4]. The mild reaction conditions and high selectivities characteristic of these enzymatic processes facilitate preparation of very pure products [5,6]. Moreover, the lipase-catalyzed reactions can be regioselective when polyfunctional compounds are used [6].
0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 1 1 7 - 9
746
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Lipase-catalysed polycondensation reactions of ␣-hydroxy acids have been observed in alcohol free systems [8]. However, in the presence of alcohols, the lipase-catalysed reactions of ␣-hydroxy fatty acids yield hydroxy esters as the major products [9]. Recently, lipase-catalysed esterification reactions of lactic acid in n-hexane have been reported [10]. The procedure permits preparation of high yields (89%) of the butyl ester. These yields decrease for alcohols with longer chain lengths (28% and 11% for octyl and decyl lactates, respectively) [10]. In this work, we have optimized a procedure for the enzymatic formation of ester linkages between lactic (or glycolic) acid and fatty alcohols of different chain lengths (C8 –C16). Optimisation of the yields of these reactions has permitted identification of those factors that have the most significant effects on the yield of the desired ester. The aim of this work was to overcome some very important limitations on earlier biocatalytic processes in order to obtain high productivity/selectivity towards the desired product, decrease the reaction time, and reduce potential toxicity effects associated with the use of organic solvents as the reaction medium. Our goal was to obtain nearly quantitative esterification of lactic and glycolic acids with long chain alcohols in a variety of solvents and in solvent free systems.
2. Materials and methods
mixture. In all cases, the solvents were dried with molecular sieves before use. The molar yields of the products were calculated with respect to the less concentrated reactant. 2.3. Purification of the esters All the esters were subsequently prepared in larger amounts (up to 2 mmol) and then purified. These pure materials were used in experiments to characterize their structures and as standards for the high-performance liquid chromatography (HPLC) analyses. The enzyme and the molecular sieves were removed from the reaction mixtures via filtration through a 0.1 mm sieve, and the solvent was then evaporated using a rotovap system. Further purification was accomplished by liquid chromatography. The filtered and evaporated reaction mixtures were dissolved in n-hexane. The lactic acid was extracted from these solutions by washing the mixture 3⫻ with the same volume of a 0.1 M NaCl solution. The remaining mixture was introduced into a silica gel column equilibrated with n-hexane. The chromatographic process consisted of successive elutions with 100 ml of hexane, and mixtures of hexane/chloroform in which the percentage of chloroform was successively increased. The desired ester was eluted with a solution of hexane/chloroform 60/40 (v/v). Since the last eluted fractions were contaminated with dodecanol, they were discarded.
2.1. Materials 2.4. Analysis of the reaction mixtures Novozym 435 (Lipase B from Candida antarctica, a non specific lipase immobilized on a macroporous acrylic resin Accurel EP-100) and Lypozyme (lipase from Mucor miehei immobilized on Duolite) were kindly provided by Novo Nordisk A/S (Bagsvaerd, Denmark). Lipase from C. rugosa, octanol, cetyl alcohol, and molecular sieves were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lipase from Pseudomonas sp. was obtained from Amano Chemicals (Nagoya, Japan). Lactic acid, glycolic acid, and dodecanol were purchased from Merck (Darmstadt, Germany). Acetone, acetonitrile and methanol were supplied by Scharlau (Barcelona, Spain). All chemicals were analytical grade. 2.2. Enzymatic reactions Reactions were performed by mixing the indicated amount of the corresponding ␣-hydroxy acid and the alcohol in a stopped glass bottle. After addition of the indicated amount of the enzyme and 2 ml of the organic solvent, the reaction mixture was maintained at constant temperature in an orbital shaker (Stuart Scientific, Surrey, England) for a known time at a specified temperature. When it is so indicated, some reaction mixtures were prepared as above without addition of the organic solvent. Where noted, molecular sieves were added to sequester excess water in the reaction
The course of the enzymatic reactions was monitored via analysis of the reaction mixtures by HPLC. At the indicated reaction time, the reaction was stopped by addition of 2 ml of dimethylformamide (DMF). The enzyme was eliminated by filtration and centrifugation. The volume of the final transparent solution was increased to 5 ml by addition of DMF. This solution was analyzed in an HPLC apparatus consisting of an L-7100 isocratic pump, a Lichrospher 100 RP-18 column, an RI-71 refractive index detector, and a D-7500 integrator. The injection volume was 20 l and the column temperature was 25°C. All components of the HPLC system were obtained from Merck– Hitachi Ltd. (Germany and Japan). Calibration analyses were performed using esters obtained and purified in our laboratory. The HPLC analyses indicated that the purities of these standards exceeded 99%. The eluant used for quantification of the dodecyl lactate and dodecanol was acetonitrile/methanol/water 50/35/15 (v/v). The retention times for dodecyl lactate and dodecanol were 5.8 and 6.9 min, respectively, at a flow rate of 1 ml/min. Results are expressed as the mean value of at least two independent experimental measurements. In all cases the experimental errors were below 4%.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
2.5. Mass spectrometry The mass spectra were recorded in the range 50 to 900 by using type VG Autospec MICROMASS (Manchester, UK) and via direct introduction into the solids probe with a temperature gradient of 50 to 300°C. Ionization voltage: 70 eV; ion source temperature 200°C. The molecular masses determined for dodecyl lactate, octyl lactate, cetyl lactate, and dodecyl glycolate were 258.3, 202.2, 314.3, and 244.3, respectively. Experiments were carried out in duplicate. 2.6. NMR analyses 1
H NMR spectra were recorded for solutions of the esters in methanol-2H4 by using a Varian XL-300 spectrometer operating at 30°C and 300 Mhz. The 1H NMR spectra of our lactic and glycolic esters were similar. As an example, consider the 1H NMR spectrum of dodecyl lactate: 4.24 (dd, 1H, J ⫽ 6.9, HO-CH-COO), 4.16 (m, 2H, CH2-CH 2OOC), 1.65 (m, 2H, CH 2 -CH2-OOC), 1.43 (d, 3H, J ⫽ 6.9, CH3-CH-OH), 1.25 (m, 18H, CH2-(CH2)9-CH3), 0.86 (t, 3H, CH3-(CH2)9-, J ⫽ 6.9).
747
Table 1 Enzymatic synthesis of dodecyl lactate catalysed by different biocatalysts Enzyme (mg)
Ester yield (%)
Hydrolytic activity (uc/mg biocat)
CRLa (25) PSLb (25) Lipozyme IM (50) Novozyme 435 (50)
1 1 2 44
3.6 ⫻ 10⫺3 1.0 ⫻ 10⫺3 24 ⫻ 10⫺5 5.7 ⫻ 10⫺6
Reaction conditions: 50 mg (0.55 mmol) lactic acid, 100 mg (0.55 mmol) dodecanol, 2 ml acetone, 24 h and 50°C. a Candida rugosa lipase b Pseudomonas sp. lipase c Units of enzymatic activity as mequivalents of acid liberated per minute at pH ⫽ 7.0 and 30°C.
molecular masses of the monomer, dimer, and trimer, respectively. Results are expressed as molar percentages of total monomeric lactic acid. All spectra were recorded in duplicate and the experimental errors were less than 4%.
3. Results
2.7. Karl Fischer measurements 3.1. Selection of the best biocatalyst Water contents were determined by the Karl Fischer method using a KF DL18 Mettler Toledo apparatus (Barcelona, Spain). The titrations of the reaction systems were carried out by adding an aliquot of the liquid reaction mixture to the titrator at the indicated reaction time. Results are expressed as the mean value of at least three independent experimental measurements. In all cases the experimental errors were below 5%. 2.8. Determination of the enzymatic activity The hydrolytic activity was measured by using a pH-stat DL 21 Mettler Toledo apparatus (Barcelona, Spain). An emulsion (40 ml) containing olive oil (200 l), buffer Tris/ HCl, 1 mM, acetonitrile (3%), and gum arabic [1.5% (v/v)] was sonicated for 30 min. After pretitration, a known weight of enzyme was added. The enzymatic activity was determined as the mequivalents of acid liberated per minute at pH 7.0 and 30°C (1 unit ⫽ 1 mequiv. acid/min). 2.9. Degree of polymerisation of lactic acid Commercial grade lactic acid (10 mg) dissolved in 1.5 ml acetonitrile were analyzed (1 l sample volume) by gas chromatography in a Carlo Erba MFC 500 apparatus with a splitless injector fitted with a OV1 100% methyl silicon column, a mass spectrometer detector (Ionization voltage: 70 eV, detector temperature: 250°C, temperature program: 45°C during 5 min increasing 8°C/min up to 300°C), and He gas. The lactic acid chromatogram contained three peaks at 15.17, 20.20, and 25.03 min. These peaks correspond to the
The reaction between lactic acid and dodecanol was carried out by using several lipases from different sources (Table 1). The initial molar ratio of lactic acid and dodecanol was 1 : 1 to facilitate identification of the lipase with the best selectivity for use of the ␣-hydroxy acid as an acyl donor. Both native and immobilized enzymes were employed. To compensate for the weight of the support, the quantities of the immobilized lipases (Novozym 435 and Lypozyme) were twice as great as those for the native (soluble) enzymes. The activities of these biocatalysts for the hydrolysis of olive oil were also evaluated (Table 1). No correlation was observed between the hydrolytic and synthetic activities. The highest conversion to the ester was obtained with Novozym 435 (44% dodecyl lactate in 24 h), which was selected for use in subsequent experiments. 3.2. Influence of organic solvents Of the various solvents examined for the reaction between lactic acid and dodecanol, the best yields were obtained using acetonitrile (see Table 2). The amount of water present in commercial-grade lactic acid was much greater than that associated with the other components of the reaction mixture. Analysis by gas chromatography indicated that the polymerised fraction of the commercial lactic acid was 32 ⫾ 2%. Hence, when one employs equimolar proportions of reagents (acid and alcohol) in acetonitrile, the yield of the ester was close to the maximum that could be obtained via direct esterification of the monomeric fraction of the lactic
748
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Table 2 Effect of solvent on ester yield in the enzymatic synthesis of dodecyl lactate Medium
Log P
Yield (%)
Dioxane Acetonitrile Acetone n-Hexane No solvent
⫺1.1 ⫺0.33 ⫺0.24 3.5 —
51 54 42 9.0 19
Conditions: lactic acid (100 mg, 1.07 mmol), dodecanol (200 mg, 1.1 mmol), Novozyme 435 (50 mg), and 2 ml of organic solvent, 200 rev./min, 24 h and 50°C. Solvent polarity in terms of log P, where P is the distribution coefficient of the solvent employed between isoctanol and water. Water contents (as determined by the Karl Fischer method) were 0.013%, 0.015%, 0.019%, 0.003%, 13%, 0.11%, 1.07% (w/w), for dioxane, acetonitrile, acetone, n-hexane, lactic acid, alcohol, and enzyme, respectively.
acid (68%). Thus, acetonitrile was selected for use in subsequent experiments. 3.3. Influence of the molar ratio of reactants In this series of experiments, the amounts of acetonitrile, Novozym 435, and one of the substrates were held constant whereas the quantity of the other substrate was decreased. In the presence of excess dodecanol (Table 3), the highest yield was obtained at a molar ratio of lactic acid to dodecanol of 1 : 10. In this case, the water content of the reaction mixture decreased significantly as the lactic acid content decreased, thereby increasing the molar ratio of dodecanol to water. This condition should have promoted a shift of the reaction equilibrium towards formation of the ester. In the case of reaction in the presence of excess lactic acid, the experiments were carried out such that the water content originating from the commercial ␣-hydroxy acid was held constant. The optimum yield of the ester corresponded to a 5 : 1 molar ratio of lactic acid to dodecanol. High yields of ester (⬎87%) can be obtained under optimal conditions regardless of which substrate is present
Table 3 Effect of the molar ratio of reactants in the enzymatic synthesis of dodecyl lactate in acetonitrile Molar ratio Lactic/dodecanol
Yield (%)
1 : 10 1:5 1:4 1:2 1:1 2:1 4:1 5:1 10 : 1
90 81 79 72 53 77 85 87 84
Conditions: A. 205 mg (1.1 mmol) dodecanol. B. 100 mg (1.1 mmol) lactic acid.; 2 ml acetonitrile, 50 mg Novozym 435, 24 h and 50°C.
Fig. 1. Enzymatic synthesis of dodecyl lactate in acetonitrile in the presence and absence of molecular sieves. Conditions: 100 mg (1.1 mmol) lactic acid, 40 mg (0.21 mmol) dodecanol, 50 mg of Novozyme 435, 2 ml of acetonitrile, and 200 mg of molecular sieves at 30°C.
in excess. However, because the reaction mixtures containing an excess of the ␣-hydroxy acid provide sufficient monomeric lactic acid to achieve 100% conversion of the alcohol to the ester and because of the difficulties of purifying the ester when excess alcohol is present, a 5 : 1 molar ratio of lactic acid to alcohol was used in subsequent experiments. 3.4. Influence of molecular sieves Because hydrolysis is merely the reverse of esterification, the degree of hydration of the medium plays an important role in determining the maximum extent of esterification [7]. Thus, the effect of use of a desiccant on the reaction yield was also studied. The incubation temperature was 30°C because a previous study of the effect of the reaction temperature (data not shown) indicated that yields close to 80% could be obtained in 24 h at this moderate temperature. The presence of the molecular sieves decreased the initial reaction rate, but increased the conversion ultimately achieved (Fig. 1). The decrease in the initial rate is attributed to the affinity of the molecular sieves for polar substances. Hence, this desiccant could adsorb some of the lactic acid, thereby diminishing the effective concentration of this substrate in the reaction medium. Because the molecular sieves also absorb the water formed by the reaction, the presence of this desiccant shifts the equilibrium of the reaction toward synthesis of the ester. When the desiccant was present, the final yield of the ester increased from 87% to 95%. The water content of the reaction medium in both the presence and the absence of molecular sieves was investigated. The water generated during the reaction [0.1% (w/ w)] was negligible in comparison with the water entering the medium with the commercial lactic acid. Hence, this water should not have a significant effect on the conversion.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
749
the water in excess of that necessary to bring about complete esterification. However, the presence of a higher concentration of molecular sieves makes it difficult to achieve effective contact between the different components of the heterogeneous reaction mixture, because of difficulties in maintaining uniform suspension of the solids (immobilized biocatalyst, molecular sieves). 3.6. Influence of the chain length of the alcohol
Fig. 2. Effect of the concentrations of reactions on the enzymatic synthesis of dodecyl lactate in acetonitrile. Conditions: lactic acid/dodecanol molar ratio (5 : 1), the enzyme/alcohol ratio [5 : 4 (w/w)], and the amount of desiccant (200 mg) were held constant; 48 h and 30°C.
However, addition of the molecular sieves decreased the effective water content of the reaction medium from 1.3% to 0.4% (w/w). This effect is responsible for the higher conversions to the ester observed in the presence of the molecular sieves. 3.5. Effect of substrate concentration The use of maximum concentrations of substrates in the solvent is a challenge to be met in industrial implementation of any reaction. With the goal of increasing the concentration of reactants in acetonitrile without causing adverse effects on either the reaction rate or the yield, we probed the effect of using large amounts of reactants at 30°C (Fig. 2). The effects of adding different quantities of substrates to a fixed volume of solvent are depicted. Similar conversions were obtained in 48 h for concentrations of dodecanol between 0.05 and 0.2 M. The lower conversions obtained below 0.05 M dodecanol can be a consequence of the decrease in the reaction rate associated with the lower concentration of the substrate. When dodecanol was below 0.2 M, the quantity of molecular sieves employed (200 mg) and their capacity for water [20% (w/ w)] were sufficient to sequester both the water present in the original reagents (primarily that associated with the commercial lactic acid) and that produced by reaction. For these conditions one can maintain the degree of hydration of the medium necessary to sustain the activity of the enzyme for esterification of the ␣-hydroxy acid. The solutions containing reactant concentrations above 0.2 M were more viscous and lower reaction rates occur at these concentrations. In these cases, the amount of desiccant employed (200 mg) was slightly smaller than that required for sequestration of
Enzymatic esterification of lactic acid with alcohols of different chain lengths (C8 and C16) was carried out at 30°C in media which were 0.1 M in the alcohol, 0.5 M in lactic acid, and contained 50 mg of Novozym 435, 200 mg molecular sieves and 2 ml acetonitrile. In all cases, the yields of the esters obtained in 48 h were similar (94 –96%). This fact demonstrates the general applicability of this methodology for preparing different lactic esters of fatty alcohols under the same reaction conditions. 3.7. Preparation of glycolic ester with dodecanol The synthesis of dodecyl glycolate was investigated using the same molar ratio of reagents (5 : 1 of acid/alcohol), an identical charge of the biocatalyst and a temperature that is optimal for preparation of dodecyl lactate in acetonitrile. Surprisingly, the reaction in acetonitrile produced the lowest yield of the ester (Fig. 3A). In an effort to obtain a better understanding of the reasons for the low conversion in acetonitrile, this reaction was repeated in the presence of different amounts of water (Fig. 3B). In the presence of 0.28% (w/w) added water, the yield of ester increased to a value comparable to those obtained in the other solvents investigated (Fig. 3A). The lower water content of the glycolic acid [0.11% (w/w)] relative to that of lactic acid [13% (w/w)] is the source of the necessity for adding water to increase the reaction rate when the solvent is acetonitrile. Further optimization studies were carried out in hexane, because of its relatively low toxicity and the ease of subsequent purification of the ester (low solubility of glycolic acid in this medium). The reaction conditions employed were identical to those for the series of experiments intended to elucidate the effect of the solvent. As was the case for synthesis of dodecyl lactate, the molecular sieves increased the yield of dodecyl glycolate. Esterification of glycolic acid gave a yield of 96% ester in 4 h. 3.8. Reactions in the absence of any solvent To minimize the toxicity of products formed via the enzymatic reaction, it would be desirable to eliminate the use of a solvent. Consequently, esterifications of lactic and glycolic acids with alcohols were carried out in absence of an organic solvent. Initially the reaction was studied at the optimal conditions for reaction in the presence of solvent. However,
750
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Fig. 3. Enzymatic synthesis of dodecyl glycolate. Effect of the (A) organic solvent and (B) water content on the reaction in acetonitrile. Conditions: 100 mg (1.3 mmol) of glycolic acid, 50 mg (0.27 mmols) of dodecanol, 50 mg of Novozym 435, and 2 ml of solvent with a 24-h reaction time at 30°C.
because of the low miscibility of the reagents, the rates of the synthesis of the lactic and glycolic esters were slow, and the yields of the corresponding esters were inferior to those obtained in the presence of solvent. In an effort to improve the conversions, the temperature was increased to 60°C. Esterification of lactic acid in a solvent free system should not be favored because of the relatively high water content of this substrate. Hence, different strategies were attempted to increase conversion to the ester. Although use of excess lactic acid would facilitate the purification process, the yield obtained with a 5 : 1 ratio of acid to alcohol was compared to that for a 1 : 5 ratio (excess dodecanol). Use of excess alcohol permits one to reduce the water content introduced with the commercial lactic acid, thereby increasing the yield of the ester (see Fig. 4A). In addition, we utilized an alternative strategy based on a transesterification reaction of the ethyl ester of lactic acid that has much
lower water content [0.09% (w/w)] than the commercially available lactic acid. The results are also shown in Fig. 4A. The highest yield (87%) after 24 h of reaction was that obtained via the transesterification reaction. At 60°C, both the ester yield and the rate of reaction of glycolic acid were greater than those obtained at 30°C. In 3 days, the biocatalyst produced a yield of 86% of the glycolate ester (Fig. 4B). As was the case for reactions in organic solvents, use of a desiccant decreased the reaction rate. Because the presence of molecular sieves should permit one to increase the ultimate extent of conversion, a strategy was developed to decrease the required reaction time (see Fig. 4B). The method basically consists of carrying out the reaction in stages. The first stage is a rapid reaction carried out in the absence of molecular sieves. The second stage is intended to reach equilibrium in a medium containing molecular sieves so as to reduce its degree of hydration. The
Fig. 4. Enzymatic synthesis of (A) dodecyl lactate and (B) dodecyl glycolate in absence of organic solvent. Conditions. (A): 50 mg of Novozym 435 and 200 mg of molecular sieves at 60°C; (■), 100 mg of lactic acid and 40 mg dodecanol; (Œ), 20 mg of lactic acid, and 205 mg of dodecanol; (), 125 mg of ethyl lactate and 40 mg of dodecanol. (B): 100 mg of glycolic acid, 50 mg of dodecanol, 50 mg of Novozym 435, and 200 mg of molecular sieves at 60°C.
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
final yield of the ester increases from 86% to 91% when molecular sieves are added 24 h after starting the reaction. Furthermore, this approach reduces the total reaction time to 48 h.
4. Discussion Of the different lipases tested in the present study, Novozyme 435 is the one that best uses ␣-hydroxy acids as acyl donors. This lipase has previously been employed in different esterification reactions with a variety of acids and alcohols. For these reactions Novozym 435 has both high activity and thermostability [5,12]. In previous studies, the required reaction times were much longer (several weeks) than those for the optimal conditions employed in the present study (4 – 48 h). In our work, Novozym 435 did not exhibit significant deactivation at either 30°C or at 60°C, in presence and absence of solvent, respectively. From et al. [10] have described the enzymatic synthesis of lactic acid esters in n-hexane. This work demonstrated that apolar solvents are not appropriate for biotransformation reactions involving long chain alcohols. The effective solvation of these alcohols by apolar solvents reduces their activity coefficients and decreases the reaction rates [13]. The study herein reported shows that the degree of hydration of the reaction medium is largely determined by the high water content [13% (w/w)] present in the lactic acid purchased from the vendor. This water content could produce saturation of the more hydrophobic solvents, causing a shift in the reaction equilibrium to favour the hydrolysis reaction. In addition, the higher solubility of lactic acid in polar solvents might contribute to the much higher yield of ester observed in acetonitrile relative to n-hexane. In the present work, we have developed a method for the nearly quantitative synthesis of lactic or glycolic esters of fatty alcohols. High yields of lactate can be prepared in acetonitrile via the indicated protocol (which includes addition of a desiccant). For the reactions of glycolic acid, the water content must be increased in acetonitrile because of the relatively low degree of hydration of commercial sources of this substrate. Although n-hexane does not facilitate esterification of lactic acid with C8 to C16 alcohols, nearly complete conversion of glycolic acid to the ester could be achieved in this solvent. The relatively low water content of commercially available glycolic acid permits the preparation of glycolate esters in n-hexane in a very short time (4 h). The synthetic procedure reported here employs low temperature reaction conditions and is not limited by the chain length of the alcohol in the range C8 to C16. This method can be used in the presence of an excess of either substrate (acid or alcohol). It permits the use of relatively high concentrations of substrates while maintaining good yields and selectivity. Use of highly concentrated reaction mixtures reduces the time necessary to achieve a specified conver-
751
sion, the size of the reactor required and the cost of the process (less organic solvent must be removed during purification of the reaction mixture). In less toxic solvent-free systems, it is possible to obtain yields similar to those obtained in the presence of organic solvents by increasing the temperature to 60°C. The increased temperature enhances both the reaction rate and the yield of the ester, results that can be attributed to the kinetic effect of temperature and the higher solubility of the ␣-hydroxy acid in the alcohol. The reduced rate of glycolic acid esterification in presence of desiccant may be a consequence of interactions (adsorption) between the acid and the drying agent and/or reduction of the water content below that necessary for the progress of the reaction [11,15]. To overcome this problem, we have developed a strategy consisting of first carrying out the reaction in the absence of molecular sieves and after 24 h of reaction adding this desiccant to the reaction mixture. This approach reduces the time necessary to accomplish the initial stage of the reaction, while maintaining a capability for shifting the equilibrium position of the reaction towards the esterification during the final stage. Thus, the ester yields were increased to 90% and the required time was reduced by 24 h. Comparison of the three different strategies studied for the solvent-free esterification of lactic acid (addition of desiccant, use of excess alcohol, and transesterification), indicates that the transesterification reaction with ethyl lactate gives the best result (87% dodecyl lactate in 24 h) and better permits one to overcome the problem of the high water content of commercial grade lactic acid. By comparison of the reaction times required for the reactions of lactic acid (Fig. 4A) with those for glycolic acid (Fig. 4B), one can readily see that both substrates have similar reactivities. We hypothesise that the ability of the enzyme to bind these substrates is very comparable because of similarities in both molecular size and structure, as well as in the values of the acidity constants (1.37 ⫻ 10⫺4 and 1.5 ⫻ 10⫺4 for lactic and glycolic acid, respectively [16]). The preference of C. antarctica lipase for the different enantiomers of lactic acid has been previously studied [10]. It has been demonstrated that this lipase catalyses the esterification of both enantiomers of lactic acid with almost equal rates. To obtain better enantioselectivity, it would be preferable to esterify the hydroxyl group present in lactic acid. When the chiral center is located on the alcohol side, earlier investigations have reported good stereoselectivity [17]. In conclusion, the degree of hydration of the commercial ␣-hydroxy acids determines: 1) whether additional water or a desiccant must be employed; and 2) which reagent should be used in excess. These factors, in turn, govern which of two alternative strategies should be used: direct esterification or transesterification of the acid.
752
C. Torres, C. Otero / Enzyme and Microbial Technology 25 (1999) 745–752
Acknowledgments This work has been financed by the Spanish CICYT (BIO96-0837), and a predoctoral fellowship for C.T. We thank Germaine de Capuccini S.A. for their personal and technical support, and Professors Charles G. Hill and Manuel Bernabe´ for comments on the manuscript.
[8]
[9] [10] [11]
References [1] Greaves W. Topical ␣-hydroxy acid derivative for relieving dry itching skin. Cosmet Toilet 1990;105:61– 4. [2] Datta R, Tsai S-P, Bonsignore P, Moon S-H, Frank JR. Technological and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol Rev 1995;16:221–31. [3] Bailey AV. Antimicrobial properties of some erucic acid-glycolic acid derivatives. J Am Oil Chem Soc 1989;66:932– 4. [4] Kirchner G, Scollarm MP, Klibanov AM. Resolution of racemic mixtures via lipase catalysis in organic solvents. J Am Chem Soc 1985;107:7072– 6. [5] Arcos JA, Bernabe´ M, Otero C. Quantitative enzymatic production of 6-O-acylglucose esters. Biotechnol Bioeng 1998;57:505–9. [6] Arcos JA, Bernabe´ M, Otero C. Quantitative enzymatic production of 1,6-diacyl fructofuranoses. Enz Microb Tech 1998;22:27–35. [7] Lopez R, Montero E, Sa´nchez F, Can˜ada J, Fernandez–Mayoralas A. Regioselective acetylations of alkyl -D-xylopyranosides by use of
[12]
[13]
[14]
[15]
[16]
[17]
lipase PS in organic solvents and application to the chemoenzymatic synthesis of oligosaccharides. J Org Chem 1994;59:7027–32. Ohya Y, Sugitou T, Ouchi T. Polycondensation of ␣-hydroxy acids by enzymes or PEG-modified enzymes in organic media. Pure Appl Chem 1995;A32:179 –90. Hayes DG. The catalytic activity of lipases toward hydroxy fatty acids-A review. J Am Oil Chem Soc 1996;73:543–9. From M, Adlercreutz P, Mattiasson B. Lipase catalyzed esterification of lactic acid. Biotechnol Lett 1997;19:315–7. Zaks A, Klibanov AM. The effect of water on enzyme action in organic media. J Biol Chem 1987;263:8017–21. Fartasch M, Teal J, Menon GK. Mode of action of glycolic acid on human stratum corneum-ultrastructural and functional evaluation of the epidermal barrier. Arch Dermatol Res 1997;289:404 –9. Kirk O, Bjo´rkling F, Godtfredsen SE, Larsen TO. Fatty acid specificity in lipase-catalyzed synthesis of glucoside esters. Biocatalysis 1992;6:127–34. Janssen AEM, Halling PJ. Specificities of enzymes “corrected for solvation” depend on the choice of the standard state. J Am Chem Soc 1994;116:9827–30. Omar IC, Nishio N, Nagai S. The role of water on the equilibrium of esterification by immobilized lipase packed-bed column reactor. Biotechnol Lett 1988;10:799 – 804. Van Ness JH. Hydroxy Carboxylic acids. In: Grayson M, Eckroth D, editors. Kirk Othmer Encyclopedia of Chemical Technology, 3rd Ed. NY: John Wiley & Sons, 1981. p. 80 –103. Sinisterra JV, Llama EF, Campo CD, Cabezas MJ, Moreno JM, Arroyo MJ. Stereoselectivity of chemically modified ␣-chymotrypsin and immobilized lipases. J Chem Soc Perkin Trans 1994;2:1333– 6.