Study of Vitamin Ester Synthesis by Lipase-Catalyzed Transesterification in Organic Media Thierry Maugard,* Joana Tudella, and Marie Dominique Legoy Laboratoire de Ge ´nie Prote ´ique et Cellulaire, UPRES 2001, Universite ´ de La Rochelle, Avenue Michel Cre ´peau, 17042 La Rochelle Cedex 1, France Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results. 1. Introduction Vitamin A (retinol), vitamin C (ascorbic acid), and derivatives are valuable agents in the treatment of photoaging, skin cancer, and numerous skin disorders. These vitamins are related, in part, through their ability to act as antioxidants (1). Antioxidants stabilize reactive, potentially harmful, free radicals generated after UV exposure and during normal and pathological metabolic processes. If not quenched, these free radicals contribute to photoaging and photocarcinogenesis by producing DNA mutations (2). It is well-known that therapeutic doses of topically applied vitamins frequently cause skin irrita- tions, which interfere with treatment. To solve these problems, several authors have conjugated vitamins with carboxylic acids (3). Carboxylic acids are also useful in the treatment of aging skin, especially R- and -hydroxy acids. The most commonly used are glycolic acid, L-lactic acid, and sali- cylic acid, which seem to exert slight but significant effects in reducing skin discolorations and roughness when applied in a cream. These acids are known to improve skin function and appearance by accelerating desquamation and promoting stratum corneum renewal (4). Unfortunately, these acids penetrate too quickly into the deep epiderm and are irritating to the skin, particu- larly at concentrations of 5% or higher and for individuals with sensitive skin. These problems limit the availability of useful therapies for skin aging (5). Vitamins conjugated to carboxylic acids are believed to hydrolyze in vivo and yield an active vitamin and active hydroxy acid agent (3). Both compounds provide antiaging effects when made available to the skin. Esters of vitamin and hydroxy acid are unusually effective as skin conditioners, with significant reductions in the irritation problems characteristic of vitamins and hy- droxy acids in nonesterified form. Hydrolytic enzymes such as lipases have become increasingly popular in recent years for chemical syn- thesis. This is especially so in the pharmaceutical and cosmetic industries where regulatory pressure has en- couraged the development and marketing of natural compounds. This article reports the acylation of vitamins A and C from hydroxy acids catalyzed by the immobilized lipase from Candida antarctica (Novozym) in organic media. A convenient process that provides high reaction yields for the production of retinyl L-lactate and ascorbyl L-lactate is described. 2. Materials and Methods 2.1. Biological and Chemical Materials. Novozym SP 435 (lipase from Candida antarctica type B, im- mobilized on an acrylic resin) and Lipozyme (lipase from Rhizomucor miehei immobilized on an anionic macroporous resin, Duolite 568N) were from Novo Industries (Den- mark). Porcine pancreatic lipase, Candida cylindracea lipase, and Rhizopus arrhizus lipase were from Sigma Chemical Co. (U.S.). The solvents, all analytical grade, retinol, ascorbic acid, L-methyl lactate, and L-lactic acid were obtained from Sigma. 2.2. General Procedure for the Enzymatic Reac- tion. Reactions were conducted in screw-cap glass vials. In standard conditions, 0.5 mmol of vitamin was reacted with 0.5 mmol of L-methyl lactate, 0.25 g of 4 Å molecular sieves, and 100 mg of lipase in 5 mL of solvent. The reaction mixture was stirred under positive argon pres- sure at 55 °C and protected from light. These conditions were used except when otherwise stated in the text. 2.3. HPLC Analysis. Quantitative analyses of reac- tants and products were conducted using an HPLC system from Hewlett-Packard (processor, pump, UV * Corresponding author. E-mail: tmaugard@univ-lr.fr. 358 Biotechnol. Prog. 2000, 16, 358-362 10.1021/bp000034a CCC: $19.00 © 2000 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/02/2000