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Proc. Indian Ac ad. Sci. (Chem . Sci.), Vol. 106, No. 5, October 1994, pp. 1203-1212.9 Printed in India.

Preparat ive ly useful transform at ions o f stero ids and m orph ine a lka lo idsb y M ucor piriformisK M M A D Y A S T H ADepartment of Organic C hemistry, Indian Institute of Science, Bangalore 560012, IndiaAbstract. A versatile fungus isolated in our laboratory and identified as Mucor piriforraishas b een shown to effect novel and preparatively useful transformations in steroids andmorphine alkaloids. The o rganism v ery effectivelycarries out hy droxylation of various C19and C~1 steroids at 7 and 14-positions. Although the organism is capable of catalysing hydro-xylation at 6fl and liar-positions, these are n ot the major activities. The 14~-hydroxylaseappears to h ave a broad substrate specificity. How ever, steroids with a bulky substitution atC-17 a-position or without the 4-en-3-one group are not accepted as substrates by the14~t-hydroxylase system. Studies have dem onstrated how various C19 and C2~ steroidscan be m odified to yield new structures w hich are either difficult to prepare by traditionalmethods or hitherto unknow n. The organism also very efficiently and selectively carries ou tthe N-dealkylation o f tbebaine and its N-variants. Interestingly, the nor-com pound formeddoes no t get further metabolized. Since thebaine is very often used as a starting material tosynthesize various m orphine agon ists as well as antagonists, and one o f the steps involvedin their preparation is the N -deaikylation reaction, the m icrobial process could certainlyoffer an alternative approach.K e yw or ~ S t e r o i ds ;morphine alkaloids; M ucor piri formis;hydroxylation; N-dcalkylation;transformation.

1 . In tr od u c t i on

I n r e c e n t y e ar s , t h e m o s t s i g n i f ic a n t d e v e l o p m e n t t h a t h a s t a k e n p l a c e i n t h e fi el d o fs y n t h e t i c c h e m i s t r y h a s b e e n t h e a p p l i c a t i o n o f b i o l o g i c a l s y s t e m s t o c h e m i c a lr e a c ti o n s . O n e o f t h e b i o l o g i c a l s y s t e m s w h i c h a p p e a r s t o h a v e t h e g r e a t e s t p o t e n t i a li n sy n t h e ti c o r g a n i c c h e m i s t r y i s m i c r o o r g a n i s m s o r e n z y m e s i s o l a te d f r o m t h e m .M o d i f i c a t io n o f n a t u r a l p r o d u c t s u s i n g m i c r o o r g a n i s m s h a s b e e n a v e r y u s e fu l m e t h o di n s y n t h e t ic o r g a n i c c h e m i s t ry . R e a c t i o n s c a t a l y z e d b y m i c r o b e s o f t e n o f fe r s i g n if ic a n ta d v a n t a g e s i n c l u d i n g t h o s e o f e f fi c ie n c y , r e g i o s p e c i f i c it y , s te r e o s e l e c t iv i t y , e t c . T h ec o n d i t i o n s u n d e r w h i c h m i c r o b i a l r e a c ti o n s t a k e p l a ce a re m i ld a n d h e n c e c o m p o u n d ss e n s i t i v e t o h e a t , a c i d s a n d b a s e s c a n b e e a s i l y s u b j e c t e d t o s u c h t r a n s f o r m a t i o n s .M i c r o b e s a n d m i c r o b i a l e n z y m e s a r e b ei n g u s e d a s r e ag e n t s i n v a r io u s o r g a n i cs y n th e s is ( R o s a z z a 1 9 8 2; Y a m a d a a n d S h i m i z u 1 98 8 ; C r o u t a n d C h r i s t e n 1 9 89 ; D a v i e se t a l 1 98 9). T h e s c o p e o f m i c r o b i a l r e a c t i o n s is w i d e n e d c o n s i d e r a b l y d u e t o t h ef i n d i n g th a t t h e s e r e a c t i o n s c a n a l s o b e c a r r ie d o u t i n o r g a n i c s o l v e n t s o r i n e m u l s i o n so f w a t e r a n d i m m i s c i b le o r g a n i c s o l v e n t s. T o d a y , c h e m i s t s a r e e x p l o r i n g t h e p o s s i b i l it yo f u s i n g m i c r o b e s o r e n z y m e s i s o la t e d f r o m t h e m i n t h e s y n t h e s is o f d i ff e re n t c h i ra ls y n t h o n s a n d p h y s i o l o g i c a l l y a s we ll a s c o m m e r c i a l l y i m p o r t a n t c o m p o u n d s .

T h e f ie ld o f m i c r o b i a l t r a n s f o r m a t i o n o f s t er o id s g o t a t r e m e n d o u s b o o s t a f t er t h es u c c es s fu l l l ~ - h y d r o x y l a t i o n o f p r o g e s t e r o n e b y t h e f u n g u s , R h i z o p u s a r r h i z u s

1 2 0 3

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1204 K M M adyas t ha(Pe t e r son an d M ur ra y 1952). Trans fo rma t i on o f st e ro i ds by va r i ous fung i have ga i nedi ndus tr i al i m por t a nce s i nce these me t h ods can be used i n t he syn t hesi s o f s t e ro i da lhorm one s and th e i r ana logs . Hy dro xy la t ion a t speci fic pos i t ions of a s te roid moleculecan b e ef f ic iently car r ied o ut us ing micro organism s. In fac t , s ignif icant amo un t o fwork has a l r eady been ca r r i ed ou t on t he mi c rob i a l s t e ro i d t r ans fo rma t i ons andexce ll ent r ev iews have ap pea red i n t he l i t e r a t u r e on t h is t op i c (C ha rney an d Herzog1967; I i zuka and Nai to 1981; Mahato et al 1989).In our e f for t s to f ind sui table microorganisms which can ef fec t novel and usefult ransformat ions in some of the representa t ive s te roids , we i sola ted a fungal s t ra inident i f ied as M uco r pir iformis which has been show n to ef fec t novel a r id prepara t ive lyuse fu l t r ans fo rma t i ons o f some o f t he s t e ro i ds and mo rph i ne a l ka lo i ds. T he p r e sen tpape r i s conf i ned t o va r i ous t r ans fo rma t i ons m ed i a t ed by t h is ve r sa t il e mi c roorgan i smand the scope of i t s synthe t ic appl icabi l i ty . However , comple te potent ia l of M u c o rpiriformis as a tool in organ ic synthes i s i s ye t to be es tabli shed.

2. Tran sform ations of C21 and C i9 steroids by M ucor p iri formisFung i be l ong i ng t o t he genus M u c o r have been r epor t ed t o ma i n l y e ff ect hydrox y l a t i onat 14~t, l l0t, 7ct an d 6fl-posi tions of variou s ste roids (T am m et al 1963; Vezina an dSi ngh 1975 ; Ho l l and and R i eml and 1985 ; M ady as t ha an d Sr i va t san 1987; Kr i shn anet al 1991). How ever , mu ch i n fo rma t i on has been cove red by pa t en t s (M ur ray an dPe t e r son 1957 ; Do dso n a nd T we i t t 1960 ; C h a rney an d H erzog 1967). M uco r piri formisi so la ted in our l a bo ra to ry has bee n show n to b e versa ti le in e f fec ting t ransform at ionsin both C21 and C19 s teroids . As far as we know only two repor t s have appeared inthe l i t e ra ture on the t rans form at ions of s te roids ; v iz . 21-hydrox y-4-pregnene-3 ,20-d i one (M ur ray and Pe t e r son 1957) and 17c t-21-d i hydroxy-4 -p regnene -3 , 20-d ione(Eroshin 1962) by Mucor piriformis. The se repor t s indica te the abi l i ty of th i s organismto in t rodu ce hyd roxy l funct ion a t C-9 , C-11 an d C-6 pos i t ions . H owev er , M u c o rpiriformis i so la t ed in ou r l abora t o ry i s kn ow n t o hyd roxy l a t e mos t l y a t 7 and 14posi t ions in various C19 and C21 steroids.2.1 Transformations o f progesterone (1)M ucor piri formis has been show n t o t r ans fo rm p roges t e rone (1) p r edom i nan t l y in t o140t -hydroxyproges terone 2) which fur the r ge t s h yd rox yla te d a t 6fl , 7~t or 7fl pos i tions ,t he r eby y i e l d ing t he co r r e spond i ng d i hydrox yprog es t e rones ( fi gure 1 ) (M adya s t haand Sr iva t san 1987) . Th e org anism a l so pro duc es 5f l , 140t -dihydroxypregnane-3 ,20-d i one (_6) a s a m i nor me t abo l i te . T i m e co ur se expe r i men t s have c l ea rl y dem ons t r a t edt ha t 14~-hydroxy l a t ion is t he f i rs t s tep i nvo l ved i n t he t r ans fo rm a t i on sequ ence( figure 1). In fac t in 12 h , near ly 75% of prog es teron e (1) ge t s m etabol i sed m ost ly to14~t -hydroxyproges terone(2) whereas prolon ging the inc ub at ion to 48 h resul t s in thefo rma t i on o f d i hydroxy proges t e rone s f igu re 1, com pou nds 3 ,4 and_5). Trans fo rma t i onof p rog este ron e (_1) into co m po un ds _5 an d _6 (figure 1) by M ucor a l s has no t beenrepo r ted ear l ie r . Ear l ie r s tudies have indica ted tha t M u c o r species have r ig id s te reo-se lec tivi ty in the i r ab i l ity to h yd rox yla te var ious C19 and C21 s teroids (Vezina andSingh 1975) . However , i t i s in teres t ing to note tha t M. piriformis i so la ted in ourlabo ra tory readi ly hyd roxyla tes 14~t -hydroxyproges terone (_2) a t both 7~t an d7fl-positions.

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T r a n s fo r ma t io n s o f s t e r o id s a n d a l k a lo id s b y M p i r if o r mi s 1205H3C~.C,J-O

H3C~ C~0 0~ _ H3C~ C~ 0

M3C C 0 0OH ~ ~ OH

0H3C~c~0 ~ ~ H3C~.. C~0

0 0~'7~'~ ' ' / ' '0H5 4Figure 1 . T r a n s f o r m a t i o n s o f p r o g e s t e r o n e ( _1 ) b y M. pir i formis .

2.2 Tran s form at ions o f 17c t-hydroxypro#es terone (Z )M u c o r p i r i f o rmi s has no t been u sed to s tudy th e t ransfo rma t ions in 17~t-hydroxypro-ges t e rone (7 ) , 16 -dehydroproges t e rone (12) a nd p r egnen o l one (17) . Hence t heses tud i e s have been ca r r ied ou t t o e s tab l ish w he t he r o r no t a h ydrox y l g roup a t t he17~t-pos it ion or a C-16(17) doub le b on d in a p roge s teron e ske le ton wo uld ef fec t themo de o f t r ans fo rm a t i on by t h is o rgan i sm. I t i s a lso o f i n te r e s t to f ind ou t t he func t i ona ls ignif icance of 4-en-3-one gro up in a C21 s teroid m olecule and for th is rea sonpregneno l one (17) wh i ch i s devo i d o f t h is g ro up has been chosen a s t he subs t r a te .The o rgan i sm t r ans fo rms (Mady as t ha and Joseph 1994) 17~-hydroxy proges t e rone(7) into 170t-200t-dihydroxypregn-4-en-3-one (8), 7~,17~t-dihydroxypregn-4-ene-3,20-dione (9_),6fl,17~t,20~-trihydroxypregn-4-en-3-one(l_Q)an d 11~ 17~20~t-trihydroxypregn-4-en-3-one (11) ( figure 2) . I t i s in teres ting to no te th a t the presen ce of a hyd roxy l a t17~t-posi t ion ster ical ly hinders hydroxylat ion at 14~t-posi t ion. This is actual ly inaccorda nce w i th t he ea r l ie r r epor t t ha t an a l ky l subs t it u t ion a t t h e 17~-posi ti on ina s t e ro i d mol ecu l e p r even t s hydroxy l a t i on a t t he 14~-pos it ion b y M u c o r g r is c o -c y a n u s(Singh e t a l 1967) . Th e pro xim i ty of the bulk y 17ct- subst ituent pro bab ly h indershy dro xy lat io n at 14or-posit ion an d direc ts it to 7~t-posit ion. H ow ev er, s teroids withhyd roxy l gro up a t 17fl -posi tion as in t es tos tero ne (21) is readi ly t ransform ed to i t s14~t-hydroxy derivat ive (Krishnan e t a l 1991) . Ear l ie r , i t has bee n dem on st ra te d tha tincub at ion of 170t -hydroxyproges terone (7) wi th a Baci l lus species resul ts in thefo rma t i on o f a s i de -cha i n c l eaved p roduc t (M aha t o and B ane r j ee 1986). I t appea r st h a t M. p i r i f o r mi s i s not capable of c leaving the C17 s ide-chain in a C2x s teroid .Surp ri s ing l y , t he o rgan i sm showed i ts un i que ab i l it y t o r educe t he C -20 ke t o g roup ,a r eac t i on neve r been r epo r t ed ea r l ie r in o rgan i sms o f t he o rde r Mu c o r a le s . H o w e v e r ,t he r educ t i on o f C -20 ke t o g rou p h as no t been obse rved i n t he ca se o f p roges t e rone(1), 16 -dehy droxy prog es terone (12) and preg nen olon e (17) (f igures 1 , 3 and 4). Time

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1206 K M M a d y a s t h aCH3 CH

8

r

~ .HC -- OH HC--OH

0 0OH - - 1 1

Figure2. Transformationsof 17at-hydroxyprogesterone (7) b y M. piriformis.

CN3 CNC =O C =O

O 01.~3 1 4\ /-

r

~H3 CHC ' O C = O

. o 6 . o . .. .- - . 1 5

Figure 3. Transfo rmation s of 16-dehydroprogesterone (12) b y M. piriformis.

~ H 3C ~ O

~ o ~C = O C = O

H O ~ ~ , O H 1 8 H O , , I . _ ~H O ~ - 19

Figure 4. Transform ations of pregnenolone (17) by M. piriformis.

c o u r s e st u d i e s h a v e i n d i c a te d ( M a d y a s t h a a n d J o s e p h 1 99 4 ) t h a t 1 7 ~ , 2 0 ~ - d i h y d r o -xypreg n-4-en-3-on e (8 , f igure 2) i s the in it i a l h io t ra nsfo rm at ion pro du c t fo rm ed whicht h e n g e t s h y d r o x y l a t e d e i t h e r a t t h e 6 /3 - o r 11 ~ - p o s i ti o n ( M a d y a s t h a a n d J o s e p h 1 99 4 ).

2.3 T r a n s f o r m a t i o n s o f 1 6 - d e h y d r o p r o g e s t e r o n e ( 1 2 )V a r i o u s f u n g a l s p e c ie s h a v e b e e n r e p o r t e d t o t r a n s f o r m 1 6 - d e h y d r o p r o g e s t e r o n e(12) in to d i f fe ren t me tabol i tes involv ing hyd roxy la t ion a t l l0 t- and 21-pos i t ions ,

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T r a n s fo r ma t io n s o f s t e r o id s a n d a lk a lo id s b y M p i r i f o r m i s 1207dehyd rogena t ion a t C-1 pos i t ion and r educ t ion o f t he C-16 (17) dou b le bon d (Ki t aand Shul l 19 59; Las kin 1963; Vezina e t a l 1963) . In co nt ras t to these obse rvat ions ,M . piriformis very efficiently carries ou t (Ma dy asth a an d Jo seph 1994) 14a-hyd roxylat ionof 16-dehydroproges terone (12 , f igure 3) . Time-course s tudies have indica ted tha tthe organism in i tia tes the hydro xyla t ion a t the 140t-pos it ion to y ie ld 14~ -hydroxypregna-4 ,16-diene-3 ,20-dione (13) which fur ther ge ts hydroxyla ted a t the 7or-pos i t ion (14 ,f igure 3). In fac t , a t the end of 48 h , near ly 80 ~ of the subs t ra te (12) ge ts transfo rm edto 7~t ,14~-dihydroxypregna-4-16:diene-3 ,20-dione (14 , f igure 3) and th is metabol i tege ts accumula ted in the fermenta t ion medium in s igni f icant leve ls . The reduct ion ofthe 4 -en -3 -one g rou p r e su lt ing in t he fo rma t ion o f me tabo l i t e s 15 and 16 ( fi gu re 3 )is no t a m a jo r ac t i v it y o f t h is o rgan i sm s ince bo th t hese me tabo l i t e s a r e fo rme d inve ry low l evel s even a t t he end o f 48 h (M ady as tha and Joseph 1994). How ever , i t isin teres t ing to note th a t the reduct ion of the 3-k eto grou p i s no t s te reose lec t ive s inceme tabol i tes w i th bo th 3~t- and 3f l -hydroxyl funct ions h ave been i so la ted ( f igure 3). I ti s gra t ify ing to , note tha t the m etabo l i tes 1 3 , 14 , 15 an d 16, ( figure 3), a l l der ivedf rom 16-dehydroproges t e rone (12 ) , appea r t o be h i the r to unknown .

2.4 Trans format ions o f pregnenolone (17)To f ind ou t whe the r t he o rgan i sm has t he ab i l it y t o i somer ize 5 -en -3 -o l to 4 -en -3 -onesys tem in C x9 and C 21 s te ro ids , represen ta t ive s te ro ids be longin g to th is ca teg ory v iz .p regneno lone (17 ) and dehydroep iandros t e rone (26 ) have been used a s subs t r a t e s .These subs t ra tes have a lso provided an opp or tu ni ty to f ind out the effec t of a 5-en-3f l-o lsys tem on the m ode o f t r ans fo rma t ion by M. piri formis. Severa l repor ts have appearedin li te ra ture regarding the microbia l conve rs ion of pregnen olone (17) in to pro ges terone(! ) as a resul t of the i some r iza t ion of the 3f l -hydroxy -5-ene to the 4-en-3-one s ys tem(Pe r lman 1952 ; Ca pek e t a l 1957). I n add i t i on to conv e r s ion o f p r egneno lone (17 ) top roges t e rone (1 ), t he re a r e r epor t s on the fu r the r co nve r s ion o f p roges t e rone (1) t ohyd roxy la ted p roge s teron es w i th hy drox yl fun ct ion a t 7f l , 1 le t , 12fl , or 15or-pos it ions( T an a n d S m i t h 1 9 6 8 ; N a m b o o r i e t a l 1980).

The orga nism t ransform s pregn eno lone (17) to 3f l, 7c t -d ihydroxyp regn-5-en-20-one(18) and 3fl,7ct, 1 ~-tr ihydro xyp regn-5 -en-2 0-on e (19, f igure 4) (M ad ya sth a an d Jo seph ,unpub l i shed obse rva t ion ) . 7~ t -Hydroxy la ti on seems to be one o f t he cha rac t e r is t icfea tures of M. piri formis. Hydroxy la t i on a t t he 7o r -pos i t i on may no t be due to t hereac t iv i ty of the a l ly lic pos i t ion . The organ ism doe s not h ave the abi l i ty to c leavethe C1T s ide-chain in pregn enolo ne (17 , f igure 4) . M os t o f the ear l ie r rep or ts w herepregn enolo ne (17) has been used as the su bs t ra te , pro ges te rone (1_) has been sh ow nto be form ed as a resul t o f the i som er iza t ion of the 5-en-3fl -o l sys tem to the 4-en-3-onesys tem (Pe r lman 1952 ; Ta n an d S mi th 1968 ; Ho l l and and T ay lo r 1979). M . p i r i fo r mi sappears to lack the i somerase and in th is respect d i f fers f rom the o ther organismstes ted on var ious C2x s tero ids .Cur so ry exam ina t ion o f t he me tabo l i t e s fo rme d f rom d i f fe r en t C2 : s t e ro ids t e s t edreveals tha t M. p i r i f o r mi s very efficiently c arries o ut th e 14~t-hydroxylation of C2xs t ero ids . How ever , t he re appea r s t o be a r ig id s t ruc tu ra l r equ i r emen t fo r t he o rgan i smto accept subs t ra tes for 14ct -hydroxyla t ion reac t ion . The organism readi ly conver tss t e ro ids wi th a 4 -en -3 -one g roup and wi tho u t b u lky su bs t i t u t i on a t t he 170 t-posi tionto the i r respect ive 14~t-hydroxy der iva t ives . The organ ism is a l so k no w n for i ts ab i l i tyto car ry out 7c t -hydroxyla t ion .

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1208 K M M adyas t ha2.5 Transformations o f androstenedione (20)And ros t encd i one (20) appea r s t o be a goo d subs t r a te f o r M. piriformis. At a subs t ra tecon cen t ra t ion o f 0-5 g per l it re , v i r tua l ly a l l the subs t ra te ad ded ge t s t ransfo rme d in tome t abo l i te s by t he end o f 24 h . The o rgan i sm t r ans fo rms andro s t ened i on e (20) i n t otes tos terone (21), 14~t -hydroxy andros tenedione 22), 7c t -hyd roxya ndros ten edionc 23),14ct -hydroxytes tos terone (24) an d 70t,14~, 17f l - t r ihydroxyand ros t -4-en-3-one (25)(f igure 5) (Krishnan et al 1991 ; Madyas t ha and Joseph , unpub l i shed obse rva t i on ) .T i me-cour se expe r imen t s ca r r ied ou t w i t h an dros t ened i on e (20) have c l ea r ly i nd i ca tedt ha t d u r i ng t he ea r l y s tages o f incub a t i on (24 h ), hydroxy l a t i on t akes p l ace bo t h a tthe 7~t- an d 14~t-pos it ions. Howev er , the m ajo r m etabol i t e form ed a t the en d of 24 hhas bee n show n to be 14ct -hydroxytes tos terone (24, figure 5, 45~ ) . T his m etabo l i t e(24) cou ld hav e be en form ed f rom 22 ( f igure 5) in the p resence o f a 17-keto oxido-reductase or f rom tes tos terone (21) by 14~t-hydroxyla tion . Th e m ajor m etabo l i t efo rmed a t t he en d o f 24h v i z. 14c t -hydroxy t cs t ost e rone (24) ge t s f u r t he r hyd roxy l a t edat the 7~t -posi tion resul t ing in the form at ion of a t r ihyd rox y co m po un d (25, f igure 5 ,M ady as t ha a nd Joseph , unpub l i shed obse rva ti on ). The leve l o f t h is t r i hydro xycom pou nd i nc r eases a t t he end o f 48 h w i t h con com i t an t dec r ease i n t he l evel o f 24(f igure 5) , suggest ing a product precursor relat ionship.The fo rma t i on o f t e s tos t e rone (21) and 14~-hydroxy t e s t os t e rone (24) from andro -s tenedione (20) has bee n repo r ted ear l i e r us ing Phyc om yces blakesleeanus (Smith et al1989) . A l t hough t he r educ t i on o f t he 17 -ke t o g roup has been r epor t ed i n s eve ra l

/ ~o

2 3 24

25F i g u r e 5 . T r a n s f o r m a t i o n s o f a n d r o s t e n e d i o n e (2 _.0 )b y M . p ir i f o r m i s .

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T r a n s fo r ma t io n s o f s t e r o id s a n d a l k a lo id s b y M p i r if o r mi s 1209microbia l sys tems, i t has nev er been show n to be present in M. pir i formis . Similar ly,the abi l ity of microb es to ca r ry ou t 140t-hydroxyla tion of and ros te ned ione (20) hasbeen demonst ra ted ear l i e r (Singh e t a l 1967; Crab b e t a l 1980) . However , such anac t iv i ty has neve r been shown i n Mucor p ir i formis . The o rgan i sm a l so accep t s te s t-os t e rone (21) r ead il y a s subs t r a te a nd t he m ode o f tr ans fo rm a t i on i s ve ry s imi l a r t ot ha t obse rved w i t h and ros t ened i one (20). The o rgan i sm r ead i ly ca r ri e s ou t hydro -xyla t ion at bo th 7~t- an d 14~t-posit ions. I t is interest ing to no te th at testo stero neprop i ona t e i s ve ry poor l y accep t ed a s subs t r a te (M adya s t ha and Joseph , unpu b l i shedobservat ion) .2.6 T r a n s fo r ma t io n s o f d e h y d r o e p ia n d ro s t e ro n e ( 26 )Incubat ion of deh ydroe piand ros teron e (26) wi th M. pir i formis yie lds mo st ly me tabol i t esform ed as a resul t of hydro xyla t ion a t the 7~t-pos it ion as wel l as red uct ion of the17-keto gro up ( f igure 6) (M ady as tha a nd Joseph, u npu bl i shed resul ts ). Th e orga nismtransforms deh yd roep iand ros te ron e (26) in to 3f l ,17f l -d ihydroxyand ros t -5-ene (27) ,3f l -hydroxyandros t -5-ene-7 ,17-dione(28) , 3f l ,17f l -d ihydrox yandro s t -5-en-7-one (29),3f l ,70t-dihydrox y-andro st-5-en-17-on e (20) an d 3fl ,7~t ,17fl- tr ihydroxya ndrost-5-ene(31). I t is in teres ting to n ote tha t the orga nism fai ls to c ar ry o ut 14ct -hydroxyla tionof dehy droep i andros t e ro ne (26). S imi l a r obse rva ti on h as a l so bee n m ade i n t he ca seof pregneno lone (17) . Both these co m po un ds (17, 26) con ta in 5-en-3f l-ol sys tem int he ir s t ruc t u r e and i t appea r s t ha t one o f t he s t r uc t u r a l r equ i r emen t s needed fo r t he14~-hydroxylase to a ccept a s te roid m olecule as a sub s t ra te i s the presence of a

OH

27

. o ~29

fo

OH 0

3~OH

Figure 6. Transfo rmations f dehydroep iandrosterone2_6)by M. piriformis.

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1210 K M M a d y a s t h a4-en -3 -one g rou p in i t s mo lecu le . Th i s i s suppor t ed by the f ac t t ha t M . p i r i f o r m i s h asthe ab i li t y t o ca r ry ou t hydro xy la t i on o f p roges t e rone (1) , 16 -dehydroprog es t e rone(12) , and ros te ned ion e (20) and tes tos te rone (21) a t 14or-pos it ion . The o the r in teres t ingaspect i s tha t the org anism is no t ca pab le of isomer iz ing 5-en-3f l-o l to 4-en-3-one , ar eac t ion m any o rgan i sms a r e cap ab le o f pe r fo rming . In t he ca se o f dehy droep iandro -s t e rone (26 ) wh ich i s devo id o f 4 -en -3 -one g roup , t he o rgan i sm p re fe rs t o ca r ry ou thydro xy la t i on a t 7~ t-posi ti on . Th i s 7g -hyd roxy la t i on m ay n o t be due to t he r eac t iv i tyof the a l ly lic pos i t ion , but ra th er d ue to the geom etr ica l na tu re of the ac t ive si te .Ea r l ie r it ha s been sho wn tha t a R h i z o p u s spec i e s t r ans fo rms dehydroep iandros t e rone(26) to 3f l , 7g-d ihyd roxy andro s t -5-en -17-o ne (30) , 3f l -7f l -d ihydroxyan dros t -5-en-17-one and 3 f l -hydroxyandros t -5 -ene -7 ,17 -d ione (28 ) (Dodson e t a l 1959) . Thu s 7 -hydro xy la t i on o f dehy droep iandro s t e rone (26 ) appea r s t o be s t e reospeci fi c . A b s i d i ar egn ier i , be long ing to t he o rde r M u c o r a l e s h a s b e e n s h o w n t o t r a n s f o r m d e h y d r o -ep i andro s t e rone (26 ) i n to i t s 7g -hyd roxy la t ed de r iva t ive (Bel l e t a11975 ) . The r educ t ionof t he 17 -ke to g rou p o f deh ydroe p iand ros t e ron e (26) t o 17 f l -hydroxy l has no t beenrepor t ed by fung i o f t he o rde r M u c o r a l e s .

Stud ie s ca r r i ed ou t wi th M . p i r i f o r m i s dem ons t r a t ed t he ve r sa t il i ty o f t hi s o rgan i smin in t rod ucing hy dro xyl gro ups a t the 7~t- an d 14~t-pos it ions in var io us C19a n d C21s tero ids . Tran sform at ion of 17~-hyd roxyproges terone (7) and 16-deh ydrop roges terone(12) by th is organ ism has resul ted in the form at ion of m etab ol i tes (11 , 13 , 14 , 15and 16 ) wh ich a r e h i t he r to unkn ow n . In f ac t, M . p i r i fo r m i s can be use d as an eff ic ientr e ag e n t t o p r e p a r e s o m e o f t h e se n o v e l c o m p o u n d s .

3 . 1 4 a t - H y d r o x y l a t i o n b y c e l l -f r e e e x t r a c t o f M ueor p iri formisM u c o r p i r i f o r m i s i so l a t ed i n ou r l abo ra to ry has t he un ique ab i l i t y t o hydroxy la t eva r ious C19 and C21 st e ro ids (M ad yas th a a nd S r iva tsan 1987 ; Kr i shnan e t a l 1991;M ady as tha and Josep h 1994). Ce l l- fr ee ex t r ac t p r epa red f rom induced vege t a ti ve ce llcu l tu re s -o f M . p i r i f o r m i s fo l l owing the p rocedure deve loped ea r l i e r ( J ayan th i e t a l1 98 2; M a d y a s t h a e t a l 1 98 4; M a d y a s t h a a n d J o s e p h 1 9 9 3) h a s b e e n s h o w n t o c o n t a i n

Table 1. Su bst rate specificity of the microsom al 14~z-hydroxylasr from M ucorpiriformis.14a-hydroxylated pro du ct formedSubstrate (% conversion)

Progesterone 69Testosterone 65Androstenedione 3016-Dvhydroprogesterone 811,2-Dehydrotestosterone 4517a-HydroxyprogesteroneEpitestosterone17~t-Ethynyl-19-nortestosterone3fl-Hydroxyandrost-5-ene-7,17-dioneThe ab ov e experiments were c arried out using acetone-washed microsomes asdescribed earlier (Madyastha and Jos eph 1993).

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Tran s fo rma t ions o f s t e ro id s and a l ka lo id s by M p i r i fo rmi s 1211high 14ct-hydroxylase act ivi ty. M os t o f the 140t ,hydroxylase act ivi ty is asso ciated w iththe m icrosom es (105,000 g sedimen t ) prepare d f rom the ac t ive ce l l- f ree ext rac t . BothN A D P H a n d 0 2 a r e n e c es s a ry f o r t h e h y d r o x y l as e a c ti v it y ( M a d y a s t h a a n d J o s e p h1993). M icrosom es readi ly conv er t var ious C19 and C21 s teroids to the i r respect ive14~t-hydroxy com pou nds ( tab le 1 ) (M ady as t ha and Joseph 1993) . M i c rosomesprepa red f rom the un indu ced ce ll s a re dev oid of 14~ -hydroxylase ac t iv ity .

4 . T r a n s f o r m a t i o n o f t h e b a i n e a n d i t s N - v a r i a n t sThe w ork p r e sen t ed so f a r c l ea rl y dem ons t r a t e s t he un i que ab i li ty o f M. p i r i f o rmi st o ca r ry ou t p r epa ra t i ve l y use ful st e ro id t r ans fo rma t i ons . To our g r ea t su rp r i s e wehave now dem ons t r a t ed t ha t t h i s o rgan i sm ca n be used a s an e f fi c ien t r eagen t f o reffec ting N -dea lkyla t ion of thebaine (32), an i soquinol ine a lka loid , and i ts N-v ar iantswhere t he a l ky l g roup on n i t r ogen is va r i ed f rom m e t hy l t o e t hy l , n -p ropy l , i sop ropy l,n-butyl and cyc lopro pylm ethyl ( figure 7a) . Th e o rganism essent ia lly car ries ou t N -dealky la t ion result ing in the forma t ion of nor co m po un d (33) wi th h igh yie lds ( ,,, 80)(M ady as tha a nd Re ddy 1994). I t i s in teres t ing to no te tha t the s ize of the a lkyl groupon ni t rogen does n ot have an y s ignif icant e ffec t on th e N-de alky la t ion reac t ion. Themost s igni f icant par t of th i s microbia l sys tem i s tha t the nor thebaine (33) formeddoes no t ge t f u r t he r me t abo l ized . The o rgan i sm a l so ca r r ie s ou t N-de me t hy l a t i on o fthe Die l s -A lder ad du ct o f thebain e (32) in h igh yie lds ( figure 7b) (M ady as th a an dRed dy 1994).Th ebaine (32) i s extens ive ly used as a s ta r t ing m ater ia l to synthes ize var iousmorph i ne agon i s t s and an t agon i s t s . One o f t he s t eps i nvo l ved i n t he i r p r epa ra t i on

( a ) R Y i e l d ('/,)H 3 C O ~ , ~ H 3 C O ' ~ CH 81N R H CHzCH2CH3 86

H3CO" "32 H3C 33 CH (CH 3) 2 88- - - - CHzCH .,CH*H3- - - 89R : CH 3 CH2---< 92(b)

It3CO', H 3 C O " ~

O ~ ' ~ .R 0 . ~ ' " R

R Y i e ld ( % )CH3 83OCH3 62

Figur e 7. (a) N-dealkylation of thcbaine (32) and its N-variants by M. piriformis. (b)N-demethylationof the D iels-Alderadduct by M. piriformis.

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1212 K M Madyasthai s the N-de alky lation reaction which is norm al ly carried ou t chemically. Since thechemical method involves harsh react ion condi t ions as wel l as hazardous and tox icreagents, microbial method could offer al ternative approach. Our studies havedemonstrated the sui tabi l i ty of M . p i r i fo r mis as an efficient reagent to carry outN-dea lkylat ion of thebaine (32) and i t s N-variants.

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