Microbiological hydroxylation of androst-1,4-dien-3,17-dione byNeurospora crassa

Abstract

Nine hydroxy-derived androstadiene compounds were isolated from the fermentation broth of Neurospora crassa when incubated in the presence of androst-1,4-dien-3,17-dione (ADD; I) for 7 days. Hydroxylations at 6b, 7b, 11a, 14a-positions and 17-carbonyl reduction of the substrate were the characteristics observed in this biotransformation. Their structures were determined by spectroscopic methods as 17b-hydroxyandrost-1,4-dien-3-one (II), 14a-hydroxyandrost-1,4-dien-3,17-dione (III), 6b-hydroxyandrost-1,4-dien-3,17-dione (IV), 11a-hydroxyandrost-1,4-dien-3,17-dione (V), 6b,17b-dihydroxyandrost-1,4-dien-3-one (VI), 7b-hydroxyandrost-1,4-dien-3,17-dione (VII), 14a,17b-dihydroxyandrost-1,4-dien-3-one (VIII), 6b,14a-dihydroxyandrost-1,4-dien-3,17-dione (IX), and 11a,17b-dihydroxyandrost-1,4-dien-3-one (X). A new steroid substance, 6b,14a-dihydroxyandrost-1,4-dien-3,17-dione (IX), was also characterized during this study. The best fermentation condition was found to be 7-day incubation at 258C and pH values of 5.0 Á6.0 in the presence of 0.05 g 100 mL (1 of the substrate. At a concentration above 0.075 g 100 mL (1 , the biotransformation was completely inhibited.

Microbial conversion of androst‐1,4‐dien‐3,17‐dione by Mucor racemosus to hydroxysteroid‐1,4‐dien‐3‐one derivatives

“…Steroid products were characterized using spectral data ( 13 C NMR, 1 H NMR, FTIR, MS), melting points and optical rotations. 17 β ‐Hydroxyandrost‐1,4‐dien‐3‐one (II): crystallized from chloroform; yield 9.4%; mp 170–173 °C, [α] D + 30°(CHCl 3 );14, 15 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.76; Retention time: 27.17 min; IR ν max 3426, 1659, 1615 cm −1 ; MS (EI) m/z (%) 286 (M + , C 19 H 26 O 2 ; 25), 227 (8), 159 (8), 147 (15), 122 (100), 91 (18), 77 (15); 1 H NMR (CDCl 3 )δ 0.84 (3H, s, H‐18), 1.29 (3H, s, H‐19), 3.66 (1H, t, J = 8 Hz, H‐17), 6.06 (1H, s, H‐4), 6.21 (1H, d, J = 10 Hz, H‐2); 13 C NMR (CDCl 3 )δ 155.9 (C‐1), 127.7 (C‐2), 186.4 (C‐3), 123.8 (C‐4), 169.2 (C‐5), 81.4 (C‐17), 11.1 (C‐18), 18.7 (C‐19). 14 α ‐Hydroxyandrost‐1,4‐dien‐3,17‐dione (III): crystallized from ethanol; yield 33.2%; mp 285–288 °C, [α] D + 92° (EtOH);14, 15 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.71; Retention time: 20.8 min; IR ν max 3408, 1743, 1654, 1614 cm −1 ; MS (EI) m/z (%) 300 (M + , C 19 H 24 O 3 ; 6), 161 (15), 147 (28), 134 (63), 122 (100), 91 (79), 55 (50); 1 H NMR (CDCl 3 )δ 1.2 (3H, s, H‐18), 1.36 (3H, s, H‐19), 6.13 (1H, s, H‐4), 6.28 (1H, d, J = 10 Hz, H‐2), 7.12 (1H, d, J = 10 Hz, H‐1); 13 C NMR (CDCl 3 )δ 155.5 (C‐1), 127.6 (C‐2), 186.3 (C‐3), 124.1 (C‐4), 167.9 (C‐5), 80.5 (C‐14), 217.8 (C‐17), 17.9 (C‐18), 18.4 (C‐19). 15 α ‐Hydroxyandrost‐1,4‐dien‐3,17‐dione (IV): crystallized from chloroform; yield 5.5%; mp 245–247 °C, [α] D + 125°(CHCl 3 );14, 16 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.5; Retention time: 19.17 min; IR ν max 3411, 1736, 1657, 1605 cm −1 ; MS (EI) m/z (%) 300 (M + , C 19 H 24 O 3 ; 20), 272 (8), 167 (15), 154 (100), 86 (22), 70 (60); 1 H NMR (CDCl 3 )δ 0.91 (3H, s, H‐18), 1.22 (3H, s, H‐19), 4.38 (1H, m, H‐15), 6.09 (1H, s, H‐4), 6.20 (1H, d, J = 10 Hz, H‐2), 7.02 (1H, d, J = 10 Hz, H‐1); 13 C NMR (CDCl 3 )δ 155.6 (C‐1), 127.6 (C‐2), 186.4 (C‐3), 123.7 (C‐4), 168.9 (C‐5), 69.9 (C‐15), 216.0 (C‐17), 15.3 (C‐18), 18.7 (C‐19). 15 α ,17 β ‐Dihydroxyandrost‐1,4‐dien‐3‐one (V): crystallized from methanol; yield 25.7%; mp 126–129 °C, [α] D + 57° (MeOH);16 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.67; Retention time: 19.68 min; IR ν max 3432, 1659, 1615 cm −1 ; MS (EI) m/z (%) 302 (M + , C 19 H 26 O 3 ; 6), 287 (9), 276 (12), 213 (7), 212 (11), 188 (32),154 (16), 138 (9), 83 (20), 49 (100); 1 H NMR (CDCl 3 )δ 0.84 (3H, s, H‐18), 1.27 (3H, s, H‐19), 3.91 (1H, t, J = 9.6 Hz, H‐17), 4.13 (1H, sx, J = 8.8 Hz, and J = 3.6 Hz, Hβ‐15), 6.09 (1H, s, H‐4), 6.25 (1H, d, J = 9.4, H‐2), 7.07 (1H, d, J = 9.4 Hz, H‐1); 13 C NMR (CDCl 3 )δ 155.6 (C‐1), 127.5 (C‐2), 186.3 (C‐3), 123.7 (C‐4), 168.9 (C‐5), 72.3 (C‐15), 78.5 (C‐17), 12.6 (C‐18), 18.7 (C‐19). 14 α ,17 β ‐Dihydroxyandrost‐1,4‐dien‐3‐one (VI): crystallized from ethanol; yield 3.9%; mp 222–225 °C, [α] D + 70° (EtOH);15, 17 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.6; Retention time: 18.62 min; IR ν max 3417, 1650, 1609 cm −1 ; MS (EI) m/z (%) 302 (M + , C 19 H 26 O 3 ; 13), 283 (30), 266 (15), 188 (5), 173 (12), 150 (58), 121 (100), 91 (64), 55 (43); 1 H NMR (CDCl 3 )δ 0.94 (3H, s, H‐18), 1.27 (3H, s, H‐19), 4.30 (1H, t, J = 8 Hz, H‐17), 6.06 (1H, s, H‐4), 6.22 (1H, d, J = 10 Hz, H‐2), 7.05 (1H, d, J = 10 Hz, H‐1); 13 C NMR (CDCl 3 )δ 155.2 (C‐1), 125.4 (C‐2), 184.8 (C‐3), 121.7 (C‐4), 168.3 (C‐5), 80.3 (C‐14), 75.9 (C‐17), 13.6 (C‐18), 16.9 (C‐19). 6 β ,17 β ‐Dihydroxyandrost‐1,4‐dien‐3‐one (VII): crystallized from chloroform; yield 3%; mp 189–194 °C, [α] D + 59°(CHCl 3 );…”

“…6 β ,17 β ‐Dihydroxyandrost‐1,4‐dien‐3‐one (VII): crystallized from chloroform; yield 3%; mp 189–194 °C, [α] D + 59°(CHCl 3 );15, 18 R f (acetone/chloroform/ethyl acetate, 3.5:5.5:1, v/v/v): 0.55; Retention time: 18.12 min; IR ν max 3414, 1658, 1616 cm −1 ; MS (EI) m/z (%) 302 (M + , C 19 H 26 O 3 ; 6), 284 (28), 266 (10), 225 (5), 188 (15), 151 (22), 121 (100), 91 (100), 55 (51); 1 H NMR (CDCl 3 )δ 0.83 (3H, s, H‐18), 1.45 (3H, s, H‐19), 3.66 (1H, t, J = 8.5 Hz, H‐17), 4.54 (1H, brs, H‐6), 6.17 (1H, s, H‐4), 6.21 (1H, d, J = 10 Hz, H‐2), 7.07 (1H, d, J = 10 Hz, H‐1); 13 C NMR (CDCl 3 )δ 157.4 (C‐1), 126.6 (C‐2), 186.8 (C‐3), 125.8 (C‐4), 166.1 (C‐5), 73.7 (C‐6), 81.5 (C‐17), 11.2 (C‐18), 20.4 (C‐19).…”

Fungal transformation of androsta-1,4-diene-3,17-dione by Aspergillus brasiliensis

“…Biotransformation of this substrate, has already been reported by some other fungi, such as Mucor racemosus, Acremonium strictum , Cephalosporium aphidicola and Neurospora crassa leading to the production of different compounds [ 11 - 14 ].…”