2Depto. de Sistemas Biológicos, Universidad Autónoma Metropolitana, Col. Villa Quietud, Coyoacán, C.P. 04960. México, D.F., México. 3Instituto de Quımica ...
Biotechnology Letters 22: 575–578, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Aminolysis of 2-hydroxy esters catalyzed by Candida antarctica lipase Gerardo Valerio-Alfaro1,∗ , Hugo S. Garc´ıa1 , H´ector Luna2 & Raymundo Cruz Almanza3 1 UNIDA,
Instituto Tecnol´ogico de Veracruz, 91897 Veracruz, Ver., M´exico de Sistemas Biol´ogicos, Universidad Aut´onoma Metropolitana, Col. Villa Quietud, Coyoac´an, C.P. 04960 M´exico, D.F., M´exico 3 Instituto de Qu´ımica, Universidad Nacional Aut´ onoma de M´exico, Circuito Exterior, Ciudad Universitaria, Coyoac´an, C.P. 04510 M´exico, D.F. M´exico ∗ Author for correspondence (Fax: +29 34 57 01; E-mail: geval@ itver.edu.mx) 2 Depto.
Received 4 October 1999; Revisions requested 3 November 1999/8 December 1999; Revisions received 30 November 1999/9 February 2000; Accepted 10 February 2000
Key words: aminolysis, Candida antarctica, 2-hydroxy amides, lipase
Abstract Candida antarctica lipase catalyzed the aminolysis of 2-hydroxy esters with amines in organic solvents to yield the corresponding 2-hydroxy amides. The reactions proceeded at 28–30 ◦ C in dioxane for 6 h with 3 mM substrates with yields ranging between 45% (w/w) (for branched substrates) to 88% (w/w) (for linear substrates). Although the reaction was not enantioselective, because of its simplicity it represents an alternative method for the synthesis of functionalised amides.
Introduction Optically active 2-hydroxy acids and their esters and amides derivatives are versatile and important intermediates used as chiral auxiliary building blocks, bioactive compounds, and can easily be converted into chiral halo-esters, glycols, epoxides, and amino acids (Coppola 1997). Lipases (EC 3.1.1.3) can catalyze many synthetic organic reactions and are suitable for the kinetic resolution of racemic alcohols, carboxylic acids and esters, due to their enantioselectivity in both aqueous and organic media (Haraldsson 1992). In organic solvents, the enzymatic resolution of bifunctional 2-hydroxy acid by esterification (Parida & Dordick 1991), and by transesterification of their esters, is possible (Kanerva & Sundholm 1993). Furthermore, chemical enantioselective synthesis of alkylamides involves drastic reaction conditions, which are not compatible with most functional groups. Enzymes can catalyze the in situ resolution of either esters (Vörde et al. 1996, Hacking et al. 1998), or amines (Kitaguchi et al. 1989, Reetz & Dreisbach 1994, Alfonso et al. 1999), in organic solvents with a high degree of enantioselectivity.
The lipase from Candida antarctica (CAL) has a high catalytic efficiency in the resolution of chiral esters, amines, and diamines through aminolysis and ammonolysis, including regio-, chemo-, and geometric selectivity (Quirós et al. 1995). The enzymecatalyzed synthesis of 2-hydroxy amides from 2hydroxy esters, to the best of our knowledge, has not been reported with this enzyme. In the present work, we studied the use of CAL as a catalyst for the aminolysis in organic solvent. Dioxane was selected as the solvent because it has been widely used in this kind of bioreactions with CAL.
Materials and methods Candida antarctica lipase (CAL-B, Novozym SP-435, 7000 propyl laureate units (µmol of ester produced per minute) g−1 of immobilized enzyme [PLU] was purchased from Novo Nordisk. All solvents, substrates and other chemicals were obtained from Aldrich. Dioxane was dried by refluxing and distilling from sodium, and kept over molecular sieves. All prod-
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Scheme 1.
Scheme 2. Table 1. Candida antarctica lipase-catalyzed aminolysis of 2-hydroxy esters (I), with n-butylamine. Entry
1 2 3 4 5 6b 7
α-Hydroxy ester sustrate (I)a
Conversionc (% w/w)
a: R 1 = n-bu, R 2 = Me, R 3 = H b: R 1 = Et, R 2 = R3 = Me c: R 1 = Et, R 2 = n-Pr, R 3 = H d: R 1 = Et, R 2 = n-Bu, R 3 = H e: R 1 = Et, R 2 = n-Hex, R 3 =H f: R 1 = Me, R 2 = Phe, R 3 = H g: R 1 = Et, R 2 = i-Propil, R3 = H
81 87 88 78 69 45 81
a Mean values from experiments performed by duplicate for 6 h in dioxane at 28–30 ◦ C. b Reaction time: 20 h. c Monitored by GC-MS.
Table 2. Candida antarctica lipase-catalyzed aminolysis of lactic acid esters (III) with with n-butylamine in dioxane. Entry
R2 (2-hydroxy ester absolute configuration)
Conversion (% w/w)
8 9 10 11 12 13
g: (R) methyl h: (S) ethyl i: (R,S) ethyl j: (R,S) sec-butyl k: (S)-n-butyl l: (S) benzyl
88 87 86 87 70 80
Results and discussion 1 H-NMR
ucts were characterized by IR and spectra. Reactions were monitored by TLC and GC. In a representative procedure for the preparation of N-(alkyl)-2-hydroxy amides, a solution of racemic nbutyl lactate (3.0 mmol) in 3 ml dioxane, was mixed with 100 mg CAL, and then a solution of the amine (3.15 mmol) in 3 ml dioxane was added at 28–30 ◦ C. The mixture was stirred and the progress of the reaction was monitored by GC and TLC analysis. After 6 h the reaction mixture was filtered and washed with dichloromethane. The filtrate was concentrated and the residue purified by column chromatography to give the 2-hydroxy amide (the unreacted 2-hydroxy ester was eventually isolated to evaluate enantioselectivity).
In general, the aminolysis of the 2-hydroxy esters (I) tested (Scheme 1), with n-butylamine proceeded with similar rate and good yields, (Table 1). CAL is able to catalyze the reaction of a wide range of substrates including linear and branched substrates (C3 – C8 ), and also methyl mandelate and methyl malate (an 2-hydroxy diester). The reaction time was stablished at 6 h as standard because at this time the best conversion/reaction time ratio was obtained. Long chain substrates as ethyl 2-hydroxy caproate (entry 4), methyl-2-hydroxy octanoate (entry 5), and methyl mandelate (entry 6) gave slower reactions (probably due to steric hindrance). Comparing linear and branched substrates (entry 1 vs. entry 2, and entry 3 vs. entry 7), similar conversion rates were observed.
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Scheme 3. Table 3. Effect of the amine in the Candida antarctica lipase-catalyzed aminolysis of 2-hydroxy esters (V) in dioxane.
Entry
Amine (% conversion) sec-Butyl n-Pentyl
α-Methyl benzyl
3-Aminopropanol
14 Ethyl lactate 15 n-Butyl lactate 16 Ethyl-2-hydroxy caproate
a: 55 a: 58 a: 50
c: 60 c: 65 c: n.r.a
d: 55 d: 60 d: 45
b: 81 b: 85 b: 64
a No reaction was detected.
The aminolysis of methyl malate was regioselective and proceeded with good conversion (86%), toward the ester group at position 1. However, enantioselectivity was not observed; even when the reactions were stopped at ca. 50% conversion, the 2-hydroxy amide (II) obtained, as shown by the analysis of the corresponding Mosher’s derivatives, was virtually racemic. These results are in sharp contrast with the high enantioselectivity reported for 3-hydroxy esters (Sánchez et al. 1999). Apparently, the proximity of the hydroxyl group to the reactive site has an important effect on the stereoselectivity of the aminolysis. To prove the effect of the alcoxyl group during the aminolysis, several lactic acid esters (III) were reacted with n-butyl amine as nucleophile (Scheme 2). The biocatalyzed reaction was fast in all cases (conversions over 80% in 6 h), regardless the alcoxyl group (R 2 : OMe, -OEt, -O-i-butyl, -O-benzyl), and the 2-hydroxy ester absolute configuration (Table 2). In order to prove the enzyme selectivity with respect to the nucleophile, several amines were tested (Scheme 3, Table 3). Based on these results ethyl (entry 14), n-butyl (entry 15) lactates, and ethyl 2hydroxy caproate (entry 16) (esters (V)) were selected. CAL accepts a variety of amines as substrates, however, with α-methyl benzylamine (entries 14c–16c) and sec-butylamine (entries 14a–16a), the reactions were slower than with the other amines (entries 1– 6 and 14b–16b), probably due to steric hindrance.
The biocatalytic process with 3-amino-1-propanol was chemoselective toward the amino group, but it reacted slower than with the other amines, maybe because of a decrease in nucleophilicity of the amino group in this compound. Finally, we tested the catalytic activity of other hydrolases for the aminolysis of the aforementioned 2hydroxy esters (I); among the commercially available enzymes, lipases from Pseudomonas cepacia, Candida cylindraceae, and Rhizomocur miehei, and the protease Subtilisin Carlsberg, only CAL gave better results for conversion of 2-hydroxy esters to the amide (II). In conclusion, this report describes a simple biocatalytic method to prepare 2-hydroxy amides from their esters under mild conditions. Although the lipase from Candida antarctica was able to accept a wide range of 2-hydroxy esters and amines as substrates, this biotransformation did not show enantioselectivity, probably because of the electron withdrawing effect of the hydroxyl group in the 2-hydroxy esters.
Acknowledgements We thank the financial support of CONACYT (Grant No. 0494P-N9506) and COSNET (Grants No. 607.96P and 856.98-P). We also thank the valuable discussion and suggestions of Dr Vicente Gotor.
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