Notes Bull. Korean Chem. Soc. 2003, Vol. 24, No. 10 1541 Biotransformation of 6-Deoxotyphasterol in a Liverwort, Marchantia polymorpha Young-Soo Kim, Tae-Wuk Kim, and Seong-Ki Kim * Department of Life Science, Chung-Ang University, Seoul 156-756, Korea Received July 11, 2003 Key Words : Brassinosteroids, Biotransformation, 6-Deoxotyphasterol, Liverwort, Marchantia polymorpha A series of our researches revealed that a Bryophyte, Marchantia polymorpha, contained steroidal plant hormones, collectively named brassinosteroids (BRs), such as 6-deoxo- castasterone (7), castasterone (8) and brassinolide (9), 1,2 and potent biosynthetic precursors of the BRs, campesterol (1) and campestanol (2). 3 Together with demonstrating the presence of a biosynthetic sequence, 7 → 8 → 9, the result strongly suggested that a biosynthetic pathway from 1 to 9 via 2, 7 and 8, so called the C6-oxidation pathway found in higher plants, 4-8 is operative to produce BRs in the lower plants (Fig. 1). 9 Nevertheless, biosynthetic intermediates between 2 and 7 in the C6-oxidation pathway have not been identified from the lower plant, so that the precise pathway to synthesize 7 from 2 in M. polymorpha still remains to be elucidated. This prompted us to investigate in vivo and in vitro conversion(s) of [26, 28- 2 H6]- and [ 2 H0]-6-deoxotypha- sterol (6), a potent biosynthetic precursor of 7, in cultured cells of M. polymorpha in this study, 10,11 which gives information on biosynthetic processes for A-ring hydroxyl- ation to produce the BRs identified from the lower plant. First, [ 2 H6]-6 was fed to the media of suspension cultured cells of M. polymorpha to investigate in vivo conversion of 6 in the lower plant. After incubation for 7 days, the cells were harvested and extracted with 80% methanol followed by chloroform. The extract was reduced to aqueous phase, combined with the media, and purified by column chromato- graphies by the guidance of the rice lamina inclination assay. 12 The final purification for metabolites of [ 2 H6]-6 was carried out by a reversed phase HPLC. The obtained HPLC fractions were analyzed by a Preparative TLC, giving rise to BR-like purple-bluish spots in the HPLC fraction 42, 43, 50- 52, 55 and 56. Among them, the fraction 55 and 56 was correspondent to the retention time of [ 2 H6]-6 added as a substrate, proposing that the active compounds in the fraction 42, 43 and 50-52 were metabolites of [ 2 H6]-6 in the Marchantia cells. The fractions were thus derivatized to be a methaneboronate (MB) or MB-trimethylsilylic ether (TMSi), and then analyzed by GC-MS/Selected Ion Monitoring (SIM). As summarized in Table 1, a bis-MB (BMB) of the active compound in the fraction 42 and 43 showed characteristic ions for [ 2 H6]-7 BMB at m/z 504 (M + , 39%), 489 (13), 273 (100) and 161 (34) at the same GC retention time as that of authentic [ 2 H6]-7 BMB. A MB of one of the active compounds in the fraction 50-52 gave a mass spectrum at m/z 462 (M + , 47%), 447 (7), 385 (5), 301 (8), 246 (12), 231 (100) and 161 (45) whose mass spectrum and GC retention time were identical to those of authentic [ 2 H6]-6-deoxo-3-dehydrotea- sterone ([ 2 H6]-5) MB. In addition, a MB-TMSi ether of other active compound in the fraction appeared the same mass spectrum at m/z 536 (M + , 45%), 521 (42), 479 (19), 446 (20), 431 (29), 305 (28), 215 (100) and 161 (41) at the equal Figure 1. The late C6-oxidation pathway to produce BRs possibly involved in M. polymorpha. Asterisks indicate steroids identified from M. polymorpha. Dashed arrows indicate steps which are not verified yet. 1, campesterol; 2, campestanol; 3, 6-deoxocathasterone; 4, 6- deoxoteasterone; 5, 6-deoxo-3-dehydroteasterone; 6, 6-deoxotyphasterol; 7, 6-deoxocastasterone; 8, castasterone; 9, brassinolide; a, 2 22R- hydroxylase; b, 3 23R-hydroxylase; c, 4 dehydrogenase; d, 5 reductase; e, 6 2a-hydroxylase; f, 7 oxidase; g, 8 oxidase.