steroids 71 ( 2 0 0 6 ) 429–434 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini Andrea Romano a , Diego Romano a , Enzio Ragg b , Francesca Costantino c , Roberto Lenna c , Raffaella Gandolfi d , Francesco Molinari a,* a Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universit` a degli Studi di Milano, via Celoria 2, 20133 Milano, Italy b Dipartimento di Scienze Molecolari Agroalimentari, Universit ` a degli Studi di Milano, via Celoria 2, 20133 Milano, Italy c Industriale Chimica, via Grieg 13, 21047 Saronno (VA), Italy d Istituto di Chimica Organica “Alessandro Marchesini”, Universit` a degli Studi di Milano, via Venezian 21, 20133 Milano, Italy article info Article history: Received 4 April 2005 Received in revised form 28 December 2005 Accepted 11 January 2006 Published on line 31 March 2006 Keywords: Hydroxylation Botryodiplodia malorum Colletotrichum lini Dehydroepiandrosterone (DHEA) 15,16-Methylene-3--hydroxy-5- androsten-17-one abstract An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15,16-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7- and 15-hydroxylation of 1 and 7-hydroxylation of 2, while B. malorum gave 7-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glu- cose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7,15-dihydroxy-1 production were improved obtaining 5.8 g l -1 (recovered product) from 7.0 g l -1 of substrate. The structures of the hydroxylated products were assigned by a combi- nation of two-dimensional NMR proton–proton and proton–carbon correlation techniques. © 2006 Elsevier Inc. All rights reserved. 1. Introduction Microbial modification of steroids is a long-established method for the preparation of a number of hydroxysteroids [1–3], but large scale transformations are sometimes limited by low yields due to low solubility of the substrates and by inhibition effects [4]. Microbial hydroxylations are among the most studied and useful transformations since they can be achieved under mild conditions with high chemo-, regio- and stereoselectivity [5]. Hydroxylation at positions 7 and 15 with controlled stereoselectivity allows for the ∗ Corresponding author. Tel.: +39 0250316695; fax: +39 0250316694. E-mail address: francesco.molinari@unimi.it (F. Molinari). production of important pharmaceutical intermediates [2]. Hydroxylation at positions 7 and 15 of dehydroepiandros- terone (3-hydroxy-5-androsten-17-one, DHEA, 1) has been reported by using Colletotrichum lini allowing for the prepa- ration of 3,7,15-trihydroxy-5-androsten-17-one, while a process for preparing 3,7-dihydroxy- 5 -steroids is based on the hydroxylation of the position 7 of 15,6- methylene-3--hydroxy-5-androsten-17-one (2) catalyzed by Botryodiplodia malorum [7]. The products of these bio- transformations (3,7,15-trihydroxy-5-androsten-17-one and 3,7-dihydroxy- 5 -steroids) are key-intermediates for 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.01.014