1014
P.G. Quintana, A. Baldessari / Steroids 74 (2009) 1007–1014
used as nucleophile and solvent, is economic and has low toxicicty,
and the lipase is recyclable and biodegradable.
[6] Ponec M, Kempenaar JA. Biphasic entry of glucocorticoids into cultured
human skin keratinocytes and fibroblasts. Arch Dermatol Res 1983;275:334–
44.
[7] Caron D, Queille-Roussel C, Shah VP, Schaefer H. Correlation between the
drug penetration and the balanching effect of topically applied hydrocortisone
creams in human beings. J Am Acad Dermatol 1990;23:458–62.
[8] Kasuya Y, Ishimaru H, Shibasaki H, Furuta T. Stable isotope methodology for
kinetic sutdies of interconversion of cortisol and cortisone in a human subject.
Steroids 1998;63:122–9.
[9] Brazzini B, Pimpinelli N. New and established topical corticosteroids in der-
matology: clinical pharmacology and therapeutic use. Am J Clin Dermatol
2002;3:47–58.
4. Discussion
In our previous works we found that enzymes proved to be
useful catalysts in steroid transformations. In this report, we use
a lipase as biocatalyst in esterification and transesterification reac-
tions of hydrocortisone with variable chain length saturated and
them novel compounds. All the products were identified by spec-
troscopic methods, 1H and 13C NMR spectra are reported. Signal
assignments were made by comparison of the data with previous
reports on hydrocortisone [26,27,34].
[10] (a) Bommarius AS, Riebel BR. Biocatalysis, fundamentals and applications.
Weinheim: Wiley–VCH; 2004;
(b) Buchholz K, Kasche V, Bornscheuer UT. Biocatalysis and enzyme technol-
ogy. Weinheim: Wiley–VCH; 2005;
(c) Saha BC, Demirjian DC. Applied biocatalysis in specialty chemicals and phar-
maceuticals. Washington, DC: ACS; 2000.
[11] Carrea G, Riva S, editors. Organic synthesis with enzymes in non-aqueous
media. Weinheim: Wiley–VCH; 2008.
In order to get the best reaction conditions we studied the influ-
ence of various reaction parameters and procedures, affording the
best results with C. antarctica lipase in the one-pot transesterifica-
tion.
[12] Gotor V, Alfonso I, García-Urdiales E, editors. Asymmetric organic synthesis
with enzymes. Weinheim: Wiley–VCH; 2007.
[13] Ferrero M, Gotor V. In: Patel RM, editor. Stereoselective biocatalysis. New York:
Marcel Dekker; 2000. p. 579–631.
[14] Cruz Silva MM, Riva S, Sá e Melo ML. Regioselective enzymatic acylation of
vicinal diols of steroids. Tetrahedron 2005;61:3065–73.
[15] Cruz Silva MM, Sá e Melo MM, Parolin M, Tessaro D, Riva S, Danieli B. Highly
selective lipase-mediated discrimination of diastereomeric 5,6-epoxysteroids.
Tetrahedron Asymm 2004;15:1173–9.
[16] Bertinotti A, Carrea G, Ottolina G, Riva S. Regioselective esterification
of polyhydroxylated steroids by Candida antarctica lipase B. Tetrahedron
1994;50:13165–72.
[17] Baldessari A, Maier MS, Gros EG. Enzymatic deacteylation of steroids bearing
labile functions. Tetrahedron Lett 1995;36:4349–52.
[18] Baldessari A, Bruttomeso AC, Gros EG. Lipase-catalysed regioselective deacety-
lation of androstane derivatives. Helv Chim Acta 1996;79:999–1004.
[19] Brutomesso AC, Baldessari A. Lipase-catalysed deacetylation of androstane and
pregnane derivatives: influence of ring D substitution. J Mol Catal B: Enzym
2004;29:149–53.
[20] Brutomesso AC, Tiscornia A, Baldessari A. Lipase-catalyzed preparation
of biologically active esters of dehydroepiandrosterone. Biocatal Biotransf
2004;22:215–20.
It is important to emphasize the completely regioselective
behavior shown by the C. antarctica lipase. Only the 21 hydroxyl
group actsas substrateof theenzymaticreactionand 21acyl deriva-
tives are exclusively obtained. It was also observed that the yield
depends on the chain length and unsaturation grade of the acylating
agent but not on the stereochemistry of the double bond.
Finally, this work also describes the application of the
enzymatic approach to the preparation of 3,11,17␣-Triacetoxy-
21-hydroxypregna-3,5-dien-20-one, novel compound obtained by
enzymatic alcoholysis of a tetra-acyl derivative of hydrocortisone.
Based on current results and the general behavior displayed by CAL
B, the enzyme acts only at carbon 21 of hydrocortisone both in
acylation and alcoholysis reactions.
[21] Rustoy EM, Ruiz Arias IE, Baldessari A. Regioselective enzymatic synthesis of
estradiol 17-fatty acid esters. ARKIVOC 2005;xii:175–88.
[22] Monsalve LN, Machado Rada MY, Ghini AA, Baldessari A. An efficient enzy-
matyic preparation of 20-pregnanes succinates: chemoenzymatic synthesis
of 20-hemisuccinyloxy-5␣ H-pregnan-3-one. Tetrahedron 2008;64:1721–
30.
[23] Faramarzi MA, Yazdi MT, Sahfiee A, Zarrini G. Microbial transformation
of hydrocortisone by Acremonium strictum PTCC 5282. Steroids 2002;67:
869–72.
[24] Baldessari A, Mangone CP. One-pot biocatalyzed preparation of substi-
tuted amides as intermediates of pharmaceuticals. J Mol Catal B: Enzym
2001;11:335–541.
This biotechnological procedure shows several advantages such
as regioselectivity and low environmental impact. Through the
regioselective behavior of the enzyme it is possible to obtain the
desired product free of secondary by-products. Biocatalytic reac-
tions offer a way for achieving green chemistry goals. The lipase is
biodegradable and consequently more friendly to the environment
than chemical catalysts. On the other hand, ethyl carboxylates and
carboxylic acid are less toxic than most acylating agents commonly
used in traditional synthetic procedures. Moreover, as the enzyme
is insoluble in the reaction medium, it is easily removed by filtra-
tion at the end of the process and can be re-used. In the acetylation
reaction of hydrocortisone, CAL B keeps 78% of its activity after ten
reaction cycles.
[25] Marwah P, Marwah A, Lardy HA. Microwave induced selective enolization of
steroidal ketones and efficient acetylation of sterols in semisolid state. Tetra-
hedron 2003;59:2273–87.
[26] Antonucci R, Bernstein S, Heller M, Lenhard R, Littell R, Williams JH. Steroidal
cyclic ketals. III. Hydrocortisone and related steroids. J Org Chem 1953;18:
70–2.
[27] Kirk DN, Toms HC, Douglas C, White KA. A survey of the high-field 1H NMR
spectra of the steroid hormones, their hydroxylated derivatives and related
compounds. J Chem Soc Perkin Trans II 1990;9:1567–94.
Acknowledgements
[28] Hickey JP, Butler IS, Pouskouleli G. Carbon NMR spectra of some representative
hormonal steroids. J Magn Reson 1969;38:501–6.
We thank UBA (project X010) and ANPCyT (project PICT 2005-
32735) for partial financial support. We are grateful to UMYMFOR
(CONICET-FCEN) for the analytical and spectroscopic determina-
tion.
[29] Priego J, Ortíz-Nava C, Carrillo-Morales M, López-Munguía A, Escalante J,
Castillo E. Solvent engineering: an effective tool to direct chemoselectivity in a
lipase-catalyzed Michael addition. Tetrahedron 2009;65:536–9.
[30] Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in
organic solvents. Biotechnol Bioeng 1987;30:81–7.
[31] Baldessari A, Mangone CP. Enzyme-catalyzed preparation of novel fatty
acid derivatives of pyridoxine with surfactant activity. Biocatal Biotransf
2002;20:275–9.
[32] Monsalve LN, Rosselli S, Bruno M, Baldessari A. Lipase-catalysed prepara-
tion of acyl derivatives of the germacranolide cnicin. J Mol Catal B: Enzym
2009;57:40–7.
[33] Procopiou PA, Baugh SPD, Flack SS, Inglis GGA. An extremely powerful acy-
lation reaction of alcohols with acid anhydrides catalyzed by trimethylsilyl
trifluoromethanesulfonate. J Org Chem 1998;63:2342–7.
References
[1] Munk A. Glucorticoid biology—a historical perspective. In: Goulding NJ, Flower
RJ, editors. Glucocorticoids (milestones in drug therapy). Basel: Birkhauser Ver-
lag; 2001. p. 17–34.
[2] Brunton LL, Lazo JS, Parker KL. Goodman and Gilman’s the pharmacological
basis of therapeutics. 11th ed. New York: The Mc-Graw Hill Companies, Inc;
2006.
[3] Hughes J, Rustin M. Corticosteroids Clin Dermatol 1997;15:715–21.
[4] Wiedersberg S, Leopold CS, Guy RH. Bioavailability and bioequivalence of top-
ical glucocorticoids. Eur J Pharm Biopharm 2008;68:453–66.
[5] Ponec M, Kempenaar JA, De Kloet ER. Corticoids and cultured hyman epider-
mal keratinocytes: specific intracellular binding and clinical efficacy. J Invest
Dermatol 1981;76:211–4.
[34] Furuta T, Eguchi N, Yokokawa A, Shibasaki H, Kasuya Y. Synthesis of multi-
labeled cortisols and cortisones with 2H and 13C for study of cortisol
metabolism in humans. Steroids 2000;65:180–9.