Bio-transformation of artemisinin using soil microbe: Direct C-acetoxylation of artemisinin at C-9 by Penicillium simplissimum Abhishek Goswami a , Partha P. Saikia a , Nabin C. Barua a, * , Manobjyoti Bordoloi a , Archana Yadav b , Tarun C. Bora b , Binod K. Gogoi b , Ajit Kumar Saxena c , Nithasa Suri c , Madhunika Sharma c a Natural Products Chemistry Division, North-East Institute of Science & Technology (CSIR), Jorhat 785 006, Assam, India b Biotechnology Division, North-East Institute of Science & Technology (CSIR), Jorhat 785 006, Assam, India c Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180 001, India article info Article history: Received 12 June 2009 Revised 21 October 2009 Accepted 22 October 2009 Available online 29 October 2009 Keywords: Artemisinin Bio-transformation Penicillium simplissimum Anticancer activity Microbial strain abstract Potent antimalarial compound artemisinin, 1 was bio-transformed to C-9 acetoxy artemisinin, 2 using soil microbe Penicillium simplissimum along with C-9 hydroxy derivative 3. The products were character- ized using high field NMR and MS–MS data. The absolute stereochemistry of the newly generated chiral centers has been ascertained by COSY and 1D NOESY experiments. This is the first Letter of direct C-acet- oxylation of artemisinin using microbial strains. Ó 2009 Elsevier Ltd. All rights reserved. Artemisinin, 1 is a sesquiterpene lactone endoperoxide iso- lated 1 in 1971 from the Chinese medicinal plant Artemisia annua L. Due to their potent antimalarial activity, low toxicity, and fast action, artemisinin and a few of its analogue viz. artemether, artether, artesunate have gained importance over the years as a new generation antimalarial drugs, especially in the treatment of multi-drug-resistant malaria strains. 2 However, sensitive nature of the artemisinin molecule has restricted extensive chemical transformation on this naturally occurring endoperoxide for devel- oping more potent antimalarial derivatives. In this context, several authors have reported findings on bio-transformation of artemisi- nin which include introduction of a keto function in the C-9 posi- tion of artemisinin by Streptomyces griseus ATCC 13273, 3 conversion to 3a-hydroxy-deoxyartemisinin and deoxyartemisinin by Mucor polymorphosporus and Aspergillus niger, 4,5 conversion to 9b-hydroxy artemisinin and 3a-hydroxy artemisinin, 5–7 conver- sion to 10-hydroxy artemisinin and 9b-hydroxy-11a-artemisinin, 7 and conversion to 5b-hydroxy artemisinin. 8 However, direct C- acetoxylation with microorganism has not been previously known. In connection with our ongoing program on bio-transformation of phytochemicals for value addition using microbial strains 9 we screened several strains isolated from untapped mega biodiversity hot zone of Indo-Burma belt for biocatalytic activity using potent antimalarial compound artemisinin 1, with an aim to synthesize novel analogues for using as scaffold for synthetic manipulation. During this study, it has been observed that fungal strain Penicil- lium simplissimum 10 converted artemisinin into C-9 acetoxylated compound 2 (20.6% yield), mp 113 °C, [a] D 75 (c 0.5, CHCl 3 ) and C-9 hydroxylated compound 3 (31.3% yield), 11 mp 175 °C, [a] D +55 (c 0.25, CHCl 3 )(Scheme 1). Fungus strains were isolated from forest soil samples collected from Tripura and Kaziranga National park, Assam, India. The strains were isolated in selective isolation medium using Roseben- gal chloramphenicol agar 12 and maintained as pure culture after repeated sub-culturing in Potato Dextrose Agar (PDA) which is composed of (g/L): potato infusion (200.0), dextrose (20.0), agar (15.0). PDA was also used for routine maintenance of the fungus strains. All the strains were preserved in mineral oil as stock cultures at 4 °C with a periodic check for viability. A panel of 40 freshly isolated fungus strains was selected to screen for bio-transformation of artemisinin using environmen- tally benign technique. 13 Out of the fungus strains tested, only two were able to produce metabolites polar than artemisinin in the screening. However, P. simplissimum produced two major metabolites and preparative TLC (1:2 EA/hexane) enabled us to obtain 2 and 3 in 20.6% and 31.3%, respectively. The stereo chemical assignments for both the bio-transformed products have been determined using high field NMR. The COSY spectrum recorded at 300 MHz for compounds 2 and 3, revealed 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.10.097 * Corresponding author. Tel.: +91 376 2372948; fax: +91 376 2370011. E-mail address: ncbarua12@rediffmail.com (N.C. Barua). Bioorganic & Medicinal Chemistry Letters 20 (2010) 359–361 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl