The synthesis of n-butanol and cinnamic alcohol esters of glucuronic acid and the esterification of ascorbic acid. (vitamin C) with phenylbutyric acid was ...
Biotechnology Letters, Vol 20, No 11, November 1998, pp. 1091–1094
Lipase-catalyzed esterification of unusual substrates: Synthesis of glucuronic acid and ascorbic acid (vitamin C) esters Ralf T. Otto1,2, Uwe T. Bornscheuer1, Holger Scheib1, Jürgen Pleiss1, Christoph Syldatk2 and Rolf D. Schmid1* Institute of Technical Biochemistry1 and Institute of Biochemical Engineering2, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany The synthesis of n-butanol and cinnamic alcohol esters of glucuronic acid and the esterification of ascorbic acid (vitamin C) with phenylbutyric acid was performed with lipase from Candida antarctica B (CAL-B, SP435) in a mainly solid-phase system. Products were obtained in 15 to 22 % yield. Computer modelling based on the structure of CAL-B was used to elucidate the access of glucuronic acid to the catalytic site of the lipase. A fixation of glucuronic acid via H-bonds to Q157, D134 and H224 during the transition state was observed. Keywords: Candida antarctica, lipase, glucuronic acid, vitamin C
Introduction In vivo, glucuronic acid esters are found in urine where they are involved in the conversion of arylalkanoic acids. Partly, they are used to target antitumor drugs like Paclitaxel (De Bont et al., 1997). Moreover, esters based on glucuronic acid should certainly have quite similar properties like esters from glucose. Aliphatic glucose esters are well known as surfactants and emulsifiers which are widely used in pharmaceutical, cosmetic, petroleum and food industries and their properties are comparable to chemically prepared surfactants. However, they can also be produced from renewable-resources and are advantageous due to their biodegradibility (Fiechter, 1992). Also, attention is paid to arylaliphatic sugar esters concerning both their medical and pharmaceutical potential. Glucose esters of cinnamic acid and hydroxylated derivatives like caffeic acid (Kimura et al., 1987; Ata et al., 1996) as well as plant extracts containing these compounds (Abdallah et al., 1994; Nicoletti et al., 1990; Slagowska et al., 1987) were investigated for applications such as tumor treatment and as antioxidative agents. Traditionally, they are isolated as bitter glucosides from plants like certain Prunus sp. and Rheum. sp. encountered with low yields and difficult product isolation (Ushiyama et al., 1989). Ascorbic acid (vitamin C, 2-oxo-L-gulonic acid-g-lactone) is one of the most important antioxidants. Esterified ascorbic acids, like 6-O-palmitoyl-L-ascorbic acid, are used in e.g. creams and baby milk (Humeau et al., 1995). © 1998 Chapman & Hall
However, chemical modifications of vitamin C are limited due to its distinct instability. To overcome this problem, an enzymatic synthesis under mild reaction conditions can be performed. As published earlier (Cao et al., 1997; Ducret et al., 1995; Adelhorst et al., 1990), high yields and regioselectivities were obtained in the lipase-catalyzed syntheses of sugar esters based on aliphatic long-chain fatty acids. Additionally, lipases react also with bulky aromatic carboxylic acids in the synthesis of glucose as well as b-aryl- and alkylglycoside esters (Otto et al., 1998a; Otto et al., 1998b). To date, only the lipase-catalyzed synthesis of sugar esters was reported with the sugar serving as the alcohol component which was esterified with carboxylic acids. In the present study, we show for the first time, the esterification of sugar acids with alcohols yielding sugar esters. Also, a mild procedure to synthesize esters of vitamin C is presented. Materials and methods Enzyme and chemicals Immobilised Candida antarctica B lipase preparation (SP435) was a gift from Boehringer Mannheim, Penzberg, Germany. All chemicals were purchased from Fluka, except for b-D(1)-glucose (Sigma). Analyses Thin layer chromatography (TLC) of reaction products was performed in chloroform:methanol:water (65:15:2 by vol.) Biotechnology Letters ⋅ Vol 20 ⋅ No 11 ⋅ 1998
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R.T. Otto et al. and spots were visualized under UV-light and after dipping in acetic acid:conc. sulfuric acid:anisaldehyde (100:2:1 by vol.) reagent followed by heating to 150°C for 5 min. 13CNMR spectra were recorded on a Bruker AM 400 (100.6 Mhz). General procedure for enzymatic reactions and product isolation The reaction mixture consisted of equimolar amounts of substrates (glucuronic acid and alcohols as well as ascorbic acid) and t-butanol, 2 eq. (w/w of substrates) as adjuvant, and 0.5 eq. (w/w of substrates) of activated molecular sieve 3Å, 10 mesh) for the adsorption of water generated during esterification. The reaction mixture was incubated in a round bottom flask, placed in an oil bath which was thermostated to 60 °C and stirred by a magnetic bar (250 rpm). The reaction was initiated adding lipase [1.25 to 1.5 eq. (w/w of substrates)]. Samples were directly taken from the reaction mixture and analysed using TLC. Finally, the glucose esters were extracted with 25 ml dichloromethane by stirring at room temperature for 15 min. Organic solvent was removed in vacuo and the crude product was purified using silica gel chromatography (ethyl acetate: methanol, 10:1 v/v). 6-Butyl-D-glucuronide 5 mmol of glucuronic acid 1 and 5 mmol of n-butanol 2 were esterified and product 4 was isolated as described above: yield: 22% (270 mg), colorless oil, Rf: 0.49 (chloroform: methanol:water, 65:15:2 by vol.); 13C-NMR (CD3OD): d (ppm) 5 14.0 (C-4), 20.1 (C-3), 31.7 (C-2), 65.9 (C-1), 72.7 (a-C-5'), 73.2 (b-C-4'), 73.4, 73.6, (a-C-2',C-3'), 74.5 (b-C-2'), 75.9 (a-C-4'), 77.2, 77.4 (b-C-3', C-5'), 94.5, 98.7 (a/b-C-1'), 170.9, 172.1 (a/b-C5O). Anal. calcd. for C10H18O7 (250.24): C, 47.99 ; H, 7.25. Found: C, 47.39 ; H, 7.37. 6-Cinnamyl-D-glucuronide 5 mmol of glucuronic acid 1 and 5 mmol of cinnamic alcohol 3 were esterified and product 5 was isolated as described above: yield: 15% (230 mg), colorless oil, Rf: 0.60 (chloroform:methanol:water, 65:15:2 by vol.); 13CNMR (CD3OD): d (ppm) 5 65.4 (C-1), 72.9 (a-C-5'), 73.2 (b-C-4'), 73.4, 73.5, (a-C-2',C-3'), 74.4 (b-C-2'), 75.9 (a-C-4'), 77.1, 77.4 (b-C-3', C-5'), 94.5, 98.8 (a/bC-1'), 127.4 (C-2), 129.1, 129.5 (Ar, C-3) 135.3 (C-4), 170.5, 171.9 (a/b-C5O). Anal. calcd. for C15H18O7 (310.30): C, 58.06 ; H, 5.85. Found: C, 56.06 ; H, 5.95. 6-O-Phenylbutyroyl-L-ascorbic acid 5 mmol of ascorbic acid and 5 mmol of phenylbutyric acid were esterified and product 6 was isolated as described above: yield 210 mg (15%), brownish oil, Rf 0.35 (chloroform:
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methanol:water, 65:15:2 by vol.); 13C-NMR ([D6]DMSO): d (ppm) 5 28.3 (C-3), 34.7 (C-2), 36.6 (C-4), 66.5 (C-6'), 68.7 (C-5'), 77.9 (C-4'), 120.5 (C-2), 127.6 (C-8), 130.1 (C-6, C-7, C-9, C-10), 141.33 (C-5), 155.2 (C-3’), 173.9 (C-1'), 175.5 (C5O). Anal. calcd. for C16H18O7 (322.32): C, 59.63 ; H, 5.63. Found: C, 58.50 ; H, 5.82. Modelling The protein structure of CAL-B (pdb entry 1lbs) was obtained from the Protein Data Bank (PDB, Bernstein et al., 1977) based on the crystallisation data published by Uppenberg et al. (1994). Structural models of the substrates were created using SYBYL 6.3 (Tripos Inc. St. Louis, MO). The substrates were docked into the binding site according to results from Grid Software (Goodford, 1995; dry-, methyl-, and water-probe to indicate binding regions for hydrophobic, small and non-polar as well as hydrophilic groups, respectively) and further guided by the position of the covalent phosphonate inhibitor of 1lbs. The substrates were docked into the binding site mimicking the first tetrahedral intermediate which is supposed to be the rate-limiting step in hydrolysis reactions of organic esters. The lipase-substrate complexes were energy minimised using the Powell method. Results and discussion Synthesis of glucuronic acid esters The esterification of glucuronic acid with n-butanol or cinnamic alcohol with lipase from Candida antarctica B (immobilized preparation SP 435) was performed in a mainly solid-phase system in the presence of t-butanol (Scheme 1) at moderate yields. During the course of the reaction, after approximately 30 hours, the mixtures started to partly solidify and the esters precipitated or crystallized from the liquid phase. A sufficient yield (22%, 270mg) was achieved with an equimolar reaction mixture of n-butanol and glucuronic acid. Using the more bulky cinnamic alcohol, the reaction still occured and the sugar ester was obtained in 15% (230mg) yield. Products were easily recovered at high purity by means of silica gel chromatography. An excess of alcohol did not improve the yields. Comparing the esterification of glucuronic acid with alcohol to the usually performed synthesis of sugar esters from sugar alcohols (e.g. glucose) and fatty acids, our method gives access to a great variety of new sugar ester compounds. A wide range of aliphatic as well as arylaliphatic and aromatic alcohols might be used and new properties (e.g. surfactants) can be expected from the resulting products. Two aspects exist why esterification reactions using glucuronic acid differ from reactions including fatty acids which represent the natural substrate of lipases: (1) Sugar
Lipase-catalyzed synthesis of arylaliphatic glycolipids
Scheme 1 alcohol 3.
Candida antarctica lipase B catalyzed esterification of glucuronic acid 1 with n-butanol 2 or cinnamic
Figure 1 The binding site of Candida antarctica lipase B with modelled cinnamyl glucuronic acid ester. Residues from the “bottom” of the binding site are coloured in black, from the “top” in white. Residues forming the alcohol binding pocket are grey. Only the residue side chains are displayed. Hydrogen atoms are omitted.
acids are hydrophilic, whereas fatty acids are hydrophobic. (2) Sugar acids represent bulky acyl donors and, therefore, differ considerably from the smaller and more flexible aliphatic fatty acid chains. In order to understand both access and fixation of glucuronic acid in the catalytic site of Candida antarctica lipase, computer modelling based on the published structure (1lbs) of CAL-B was performed. The substrate binding pocket located on top of the central bsheet was accessible by a bean-like ellipse of 9.5 3 4.5Å. The binding pocket for the scissile acyl group, the hydrophobic crevice, described by Pleiss et al. (1998) is built from the hydrophobic A141, L144, V149, V154, and I285 which residues are located at the “top” of the binding site (Figure 1: white residues). The “bottom” of the substrate binding site in CAL-B is mostly hydrophilic including
residues S105, D134, T138, Q157, D187 and H224 (Figure 1: black residues). Width and hydrophobicity of the hydrophobic crevice gives glucuronic acid easy access to the bottom of the binding site, especially the catalytic triad. The position of the substrate is fixed by H-bonds from the glucuronic acid hydroxy groups to D134, Q157 and the catalytic H224. However, residues W104, L278, A282 and I285 form the alcohol binding pocket (Figure 1: grey residues). The alcohol binding site is wide enough to even catalyse the reaction of bulky cinnamyl alcohol with glucuronic acid. Esterification of vitamin C Vitamin C esters are commonly used in e.g. cremes or baby milk and as stabilizers of the oxidation-prone vitamin C. Biotechnology Letters ⋅ Vol 20 ⋅ No 11 ⋅ 1998
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Scheme 2
Candida antarctica lipase B catalyzed esterification of vitamin C with phenyl butyric acid (15 %, 210 mg).
Taking advantage of the mild reaction conditions in combination with a lipase-catalyzed synthesis and the ability to esterify a hydrophilic with a hydrophobic compound in a solid-phase system, it was possible to synthesize the phenyl butyric acid ester of vitamin C (Scheme 2). To date, only the transesterification reaction between palmitic acid methyl ester and L-ascorbic acid was reported (Humeau et al., 1995). Although, no activated substrates were used, the vitamin C ester was produced in moderate yields (15%, 210 mg). 13CNMR spectra confirmed that undesired side-reaction of the labile vitamin C did not occur and that acylation took place with excellent regioselectivity towards the primary hydroxy function. Acknowledgments Financial support to R. T. Otto by the Fonds der Chemischen Industrie (Frankfurt, Germany) and the gift of CAL-B by Boehringer Mannheim (Penzberg, Germany) are gratefully acknowledged. References Abdallah, OM, Kamel, MS and Mohamed, M.H. (1994). Phytochemistry 37:1689–1692 Adelhorst, K, Björkling, F, Godtfredsen, SE and Kirk, O (1990). Synthesis, 112–115 Ata, N, Oku, T, Hattori, M, Fujii, H, Nakajima, M and Saiki, I (1996). Oncol Res 8:503–511 Berger, S, Otten, MG and Steinbach, K (1992). Liebigs Ann Chem, 1045–1048 Bernstein, FC, Koetzle, TF, Williams, GJ, Meyer, EE, Brice, MD,
Rodgers, JR, Kennard, O, Shimanouchi, T and Tasumi, M (1977). J Mol Biol 112:535–542 Cao, L, Fischer, A, Bornscheuer, UT and Schmid, RD (1997). Biocatal Biotransform 14:269–283 De Bont, DB, Leenders, RG, Haisma, HJ, Van der MeulenMuileman I and Scheeren, HW (1997). Bioorg Med Chem 5:405–414 Ducret, A, Giroux, A, Trani, M and Lortie, R (1996). Biotechnol Bioeng 48:214–221. Fiechter, A (1992). Trends Biotechnol 10:208–217 Goodford, PJ (1985). J Med Chem 28:849–857 Humeau, C, Girardin, M, Coulon, D and Miclo A (1995). Biotechnol Lett 17:1091–1094 Kimura, Y, Okuda, H, Nishibe, S and Arichi, S (1987). Planta Med 53:148–153 Nicoletti, M, Galeffi, C, Messana, I, Marini-Bettolo, GB, Garbarino, JA and Gambaro, V (1988). Phytochemistry 22:639–641 Nihro, Y, Sogawa, S, Izumi, A, Sasamori, A and Sudo, T (1992). J Med Chem 35:1618–1623 Nilsson, KG (1987). Carbohydr Res 167:95–103 Otto, RT, Bornscheuer, UT, Syldatk, C and Schmid, RD (1998a). Biotechnol Lett 20:437–440 Otto, RT, Bornscheuer, UT, Syldatk, C and Schmid, RD (1998b). J Biotechnol 64:231–237 Pleiss, J, Fischer, M, Schmid, RD (1998). Chem Phys Lipids, in press Slagowska, A, Zgorniak-Nowosielska, I and Grzybek, J (1987). Pol J Pharmacol Pharm 39:55–61 Uppenberg, J, Ohrner, N, Norin, M, Hult, K, Kluywegt, GJ, Patkar, S, Waagen, V, Anthonsen, T and Jones, TA (1995). Biochemistry 34:16838–16851 Ushiyama, M, Kumagai, S and Furuya, T (1989). Phytochemistry 28:3335–3339
Received: 26 August Revisions requested: 15 September Revisions received: 24 September Accepted: 24 September
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