3-I4-I2-(cyclpprppylmcthoxv)cthyllphcnpxyI-l-chlprPEifli?an-2-pne__(3Q).-
Ten yeast s tra in s w ere screened. The yeasts w ere sto red at 4 ° C on YM agar slo pes. YM b ro th w as o b tain ed from D ifco lab o ra to ries, D e tro it, M ichigan.U.S.A. "O gihara 1" was d o n ate d by P rofesso r Y. O gihara, Departm ent o f P h arm aco gno sy , F acu lty o f P h arm aceu tical S c ie n c e s, C ity U n iversity, N agoya, Japan, and is O rie n ta l B aker's Y east (O riental Y east Co. Ltd. Jap an ). "OY" y easts w e re iso lated from O riental Y east and w ere donated by Enzym atics, U .K . BLY is B u rto n s larger yeast. N C Y C y e a s ts were obtained from the N atio n al C o lle ctio n o f Y east C u ltures, N orw ich, U.K.
All operatio ns w ere d on e u n d e r sterile co n d itio n s. A 2 5 m l conical flask containing Y M m ed ia (1 0 m l) was innoculated w ith a lo o p fu l o f cu ltu re, and shaken at 30 ° C for 24 h o u rs . Then a one in ten d ilu tion w as perform ed to fresh YM m ed ia (50m l). A f te r 24 hours
3 - [ 4 - [ 2 - ( c y c lo p r o p y lm e th o x y ) e t h y l ] p h e n o x y ] - l - c h l o r o p r o p a n -2-o n e ( 3 0 ) (50m g, lg/L ; d isso lv e d in ethanol (0.5m l) 1% v /v ) w as added. The reaction s were m onitored by U.C., (to lu en e : diethyl ether; 10 : 1). R f (30) = 0.51. Rf (31) = 0.27, R f (2 9 ) = 0 .2 2 . The reactions w hich produced th e most am ount o f alc o h o l p ro d u c t 3 -[ 4 -[2 - (c y c lo p r o p y lm e lh o x y ) e t h y l ] p h e n o x y ] - l - c h lo ro p ro p an -2 -o l (2 9 ) w e re w orked -u p. T he s u s p e n s io n w as c e n trifu g e d (5,0 00 r.p.m . / 10 m ins). T h e supernatant was rem oved, and the pellet w as resuspended in w a te r (5 0 m l) and ce n trifu g ed (5 ,0 0 0 r.p .m . / lOmins). The co m b ined s u p e rn a ta n ts w e re ex tra cted w ith e th e r (2x 10 0m l), dried
(M g S (>4), filtere d and ev a p o rate d under reduced p ressure. FC (silica 16g.
light petroleum e th e r (b.p. 4 0 -6 0 °C ) : diethyl ether. 9 : 1, to 4 : 1, to 1 : 1), afforded p ro d u c t (2 9 ) as a n o il. The % ee w as dete rm in e d by syn th esising the (R )-O -a cety lm an d elic e s te r (32) as d esc rib ed abov e (m aking sure th at the reaction s w e re co m p le te by t.I.c. b efore th e re a c tio n s w ere w orked-up ). The results are given T ab le 2.5, Section 2.7. T h e fo llow in g y easts w ere not
worked-up. S a c c h a r o m y c ts cerevisia e NCYC 1765, OY P2, OY R l, OY R2, BLY
B , because th e reduction o f (30) was very slow. The m ajo r product in th ese c a s e s w as 4 -[ 2 -(c y c lo p ro p y lm e th o x y )e th y l]p h e n o l (3 1 ), w h ic h was
identified by t.l.c. and 1H n.m.r.
O th er m ic ro b io lo g ic a llv m ed iate d re d u c tio n o f
3 - L 4 : J 2 - ( c v c l o D r o D v l m e t h o x Y l c t h v l lp h e n o x v 1 - l - c h lo r o p r o p a n - 2 - o n e (3 0 L - T hese r e d u c tio n experim ents w ere p erfo rm ed by E. T idsw ell (D ep artm e n t o f M icro b io lo g y and Botany, U n iversity C o lle g e o f W ales, A berystw yth). T o a g ro w in g c u l t u r e o f m icroo rg anism was a d d e d
3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ] - l - c h l o r o p r o p a n - 2 - o n e ( 3 0 ) (lm g /m l, 0 .0 5 0 g ). The redu ction was m o n ito re d by t.l.c., (to lu ene : d ieth y l ether; 10 : I ) . R f (30) = 0.51, R f (31) = 0.27, R f (29) = 0.22. A fter work-up th e d e s ire d p r o d u c t
3 -[ 4 - [ 2 - ( c y c lo p r o p y lm e ty l ) e t h y l) p h e n o x y ] -1 - c h lo ro p r o p a n -2 - o l (2 9 ) a n d th e p h en o l b re ak d o w n p ro d u c t (31 ) w ere se p a ra te d by flash
c h ro m a to g ra p h y . FC (6g silic a , (lig h t p e tro le u m e th e r (b.p. 40 -60 ° C ) : diethyl e th e r; 4 : 1, to 2 : 1). T he alcohol (2 9 ) was converted into the (R )- O -a c e ty lm a n d e lic ester (3 2 ), as d e s c rib e d above (m aking sure th at th e reactio n s w e r e com plete by t.l.c . b efore th e reaction s w ere w o rk ed -u p). T he full r e s u lt s arc given b elow :
m i c r o o r g a n i s m C a r b o n g r o w t h s o u r c e (3 1 ) / y % ( 2 9 ) / y % ( 2 9 ) / c e % ( 2 9 ) R /S C lo s tr id iu m p a steu ra n iu m 6013 G lu c o s e n .d . 9 6 7 R C . ty r o b u t y r i c u m LAI (Aux P) G lu c o s e n .d . 3 2 7 R C .ty r o b u ty r ic u m L A I (A ux P) C r o t o n a t e 70 8 4 4 R m utant o f A u x P (TM V) g l u c o s e 47 3 39 R m utant o f A u x P (TM V) c r o t o n a t e 4 6 0 L a c t o b a c i l l u s b rev is f r u c t o s e 16 32 28 S ( i . ) - 3 - i 4 - [ 2 - ( C v c l o p r o p v l m e t h o x v l e t h v l l p h e n o x v l - l - c h l o r o - 2 - a c e to x v p ro p a n e (3 3 ) .- To an ice cold so lu tio n o f
( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ]-1 - c h l o r o p r o p a n - 2 - o l (29 ) (3 .0 9 g , llm m o l) in d ry d ich lo ro m eth a n e (20m l) and dry p y rid ine (10 m l), w a s ad d ed acetic an h y d rid e (10 m l, lOSm m ol). T he re actio n m ix tu re
was allow ed to slow ly c o m e to room tem perature. T .I.c., (lig h t petroleum ether (b.p. 40-60 °C ) : d ieth y l ether, 1 : 1), R f (29) = 0.23, R f (3 3 ) = 0.53. After 14 h o urs the re actio n m ix tu r e w as d ilu te d w ith diethy l e th e r (1 0 0 m l),
washed with 1M HC1 (5 0 m l), saturated NaHC0 3 solution (2 x 5 0 m l) (VIGOROUS
!), saturated sodium c h lo rid e (50 m l), dried (M g S0 4), filte re d and evaporated
u n der reduced p re ssu re, to y ie ld a colourless liqu id (33) (3 .0 8 g , 87% yield), hom ogeneous by t.l.c. and *H n.m .r.; v (C D C I3) 3 000 (w), 2 9 3 0 (m ), 2 850 (s), 2 700 (w ), 1 735 (s). 1 605 (m ), 1 580 (m), 1 505 (s), 1 365 (m ). 1 225 (s), 1170 (m), 1 085 (s), and 1 0 4 0 c m' 1 (s); 6h (220 M Hz; solvent C D C I3; standard M e4S i) 0.2 0 (2H , m , c y c lo p ro p a n e m ethylene protons), 0.5 5 (2 H , m, cyc lo p ro p an e m e th y le n e p r o to n s ), 1.06 (1 H , m . cy c lo p ro p y l m eth in e proton), 2.13 (3H, s. C ( 0 ) C / / j ) . 2.87 (2H. d. J 9 Hz, AtC H2 ). 3 .3 0 (2H . d. J 7 Hz. O C / /2-cyclopropyl p ro to n s), 3 .63 (2H, t, J 9Hz, ArCH2CH 2 ), 3 .8 4 (2H , m, A rO C //2), 4.17 (2H, m , C H2C l). 5.35 (1H, m . C tfO A c), 6.89 (2H . d . y0rtho 10 Hz. A r-/ / ) , and 7.20 (2H. d , / 0rtho 10 Hz. Ar-H); 8C (100 MHz; solv en t C DCI3) 2.78 (C -12), 10.46 ( C - l l ) . 20.65 (C -2 ’), 35.32 (C -8), 45.34 (C -l), 66 .0 2 (C -10), 71.04
(C -3), 71.52 (C -2). 75.38 (C -9 ), 114.46 (C -5), 129.75 (C-6). 131.9 6 (C -7). 156.59
(C -4), and 169.81 (C -l'); m /z (E l) 328 (M + . 1%). 326 (M +. 3 ). 137 (36), 135
(100), 107 (10). 91 (6), an d 7 7 (5) HRM S, found (m /z): 32 8.12 58 ,
Ci7H 23 0 437CI. requires: 3 2 8 .1 2 5 6 : Found (m /z): 326.1281, C1 5H2i0335C] re q u ires: 326.1 2 85 .
Initial enzvm c s creen in g e x p e rim e n t, for th e ability o f an en z y m e to re so lv e racem ic 3 - f 4 - f 2 - ( c v c l o D r o p v lm e t h o x v ) e t h v l lp h e n o x Y 1-1 - c h lo r o - 2 - a c e to x y p ro n a n e (3 3 1 .- T e n enzym es w ere screened for th e ir ab ility to hy d ro ly se (33). A s u s p e n s io n o f
( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ] - l - c h l o r o - 2 - ac etoxypropane (3 3 ) (2 0 m g ), phosphate b u ffer, pH 7, lOOmM (1m l) and enzym e (c a . lOm g) w a s v ig o ro u s ly stirred at room te m p e ra tu re . The
reactions w e re m onitored by t.l.c ., ( lig h t petroleum e th e r (b.p. 4 0 -6 0 ° C ) : diethyl ether; 1 : 1), R f (33) = 0.53, R f (29) = 0.23. R eaction s that appeared to term inate at 50% co m p letio n w ere d ilu te d with w a te r (5 m l) and e x tra cted with d ieth y l eth e r (3 x 10 m l), d ried (M g S O ij) filtere d an d ev apo rated u nd er reduced pressure. FC (silic a 14g, lig h t petroleum e th e r (b .p. 40-60 ° C ) : diethyl ether; 4 ; 1) to give startin g m ateria l (33 ) and p rod uc t alcohol (2 9). The alc o hol (29) w as co n v e rted in to its corresp o n d in g
(R )-O -a c e ty lm a n d e lic e s te r (3 2). T h e re sid u a l startin g m aterial (3 3 ) was hydrolysed chem ical. A typical p ro c ed u re is as follow s. A cold (-11 ° C ) s u s p e n s io n o f 3 - [ 4 - [ 2 - ( c y c lo p r o p y lm e th o x y ) e t h y l ] p h e n o x y ] - l - c h l o r o - 2 - ac eto xyp ropa n e (33 ) (0.1 l l g , 0 .3 4 m m o l) and an h y d ro u s p o tassium ca rbo n ate (0 .0 9 4g , 0 .6 8m m ol) in a n h y d ro u s m ethan o l (4m l) w as stirred fo r 30 m inutes. T .l.c., (lig h t petroleum e th e r (b.p . 40-60 ° C ) : diethyl ether; 1 : 1), Rf (33) = 0.53, R f (2 9 ) = 0.23. T h e reaction m ixture w as diluted with w ater
(20m l) and extracted w ith dieth yl e th e r (2 x2 5m l), d ried (M gS0 4), filtered
and ev a p o rate d u n d er reduced p re s s u re to yield a co lo u rle ss liq u id (29) (0.092g, 96% yield), hom ogeneous by t.l.c . and *H n.m .r. The alcohol (29) was th en conv erted in to its co rre s p o n d in g ( R )- 0 -a c e ty l m an d elic e s te r (3 2 ) (m aking sure that th e reaction w as co m p le te by t.l.c.). T h e resu lts are as fo llo w s :
Enzym e, source, co d e and s u p p lie r T im e
/ d a y s S M (3 3 ) % ee,A C P ( 2 9 ) % ee,AC c E e n z y m e q u a n t i t y
lipase, C a n d id a c y lin d r a c e a , S igm a n.d. n .d . n .d . n .d . 11 m g
lipase A, A s p e r g illu s n ig e r , A m ano 4 73,S 2 9 .R 0 .7 2 4 1 2m g
lipase P, P seu d o m o n a s s p ., A m ano n.d. n .d . n .d . n .d . 1 4 m g
lipase F -A P15, R h izo p u s sp., A m ano 4 10,R 28.S 0 .2 6 2 lO m g
lipase M , M ucor s p ., Am ano 4 25 ,R 54,S 0 .3 2 4 1 1 nig
lipase N, R hizo pu s s p ., Amano 4 18.R 27.S 0 .4 0 2 15 m g
P o rcin e p an c rea tic lip a s e . S igm a 36.R 54.S 0 .4 0 5 l l m g
lipase G e o tr ic h iu m c a n d id u m , (B) 4 n.d. n .d . n .d . n .d . 1 Omg
lipase P s e u d o m o n a s flu o r e s c e n s , (B; 4 >97 ,R 57.S 0 .6 3 14 1 Omg
PL E , B o eh rin g e r M an n h eim 2.R 5.S 0 .2 9 1 lOpI
b lan k ( c o n tr o l)
S cale -u p o f the lip ase ex P seudom onas flu o re s c e n t c a ta ly s e d resolution o f ra cem ic 3 - i 4 - 1 2 - ( c v c l o o r o p v l m e t h o x v ) e t h v l l p h e n o x v l - 1 - c h l o r o - 2 - a c e to x v p ro p a n e (3 3 ) .- A suspension o f
( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ] - l - c h l o r o - 2 - ac eto xy pro p a ne (3 3 ) (l.OOg), phosphate bu ffer, pH 7 , lO O m M (100m l) and lip ase from P s e u d o m o n a s flu o r e s c e n s (B iocatalysts L td .) (0 .4 6 9 g ) was v ig o ro u sly s tirre d at room tem perature fo r 3days. T h e u s u a l determ inatio n o f th e E v alu e as described above gave (R )-(33) (0 .1 9 3 g , 19% yield. 90% ee)
and (S )-(29) (0.598g, 68% yield. 36%ee), c = 0.714. E = 6.
( i )- 3 -l 4 - [ 2 - ( C v c l o D r Q P v l m e t h Q x v ) e t h v ll p h c n o x v l - 1 - c h i p r o - 2- h u ta n o v lo x v p ro p a n e (3 4 1 .- To an ice cold solution, u n d e r an argon a tm o sp h ere, o f ( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ] - l - c h lo ro p ro p a n -2 -o l (2 9 ) (0 .4 1 5 g , 1.46m m ol) in d ry d ic h lo ro m e th a n e (10 m l) and d ry p y rid in e (2 m l), w as added 4 -d im e th y la m in o p y rid in e (cat.) and d is tille d b u tano ic an hydride (0.36m l, 2.2m m ol). The re a c tio n was allow ed to slo w ly com e to room tem p eratu re, t.l.c., (lig h t p etro le u m e th e r (b.p. 40-60 ° C ) : diethyl ether, 1 : 1), R f (29) = 0.23, R f (34) = 0.84, a f te r 15 hours the re a c tio n m ix tu re w as d ilu te d with with d ic h lo ro m e th a n e (20m l), washed w ith 1M HC1 (1 0 m l), saturated sodium hydrogen c a rb o n a te (20m l), w ater
(1 0 m l), d ried (M gS0 4), filtered and then evap orated u n d e r reduced
p re ssu re to yield (3 4 ) as a co lourless oil (0.465g, 90% y ie ld ) , hom ogeneous by t.i.e. and >H n.m .r.; v (C DC I3) 2 960 (w), 2 930 (m). 2 8 6 0 (m ), 1 730 (s), 1 650 (s), 1 580 (w). 1 505 (s). 1 235 (s). 1 170 (s). and 1 0 85 c m1 (s); 8H (220 M H z; so lvent C D C I3; standard Me4Si) 0.18 (2H , m . c y c lo p ro p a n e m ethylene
p ro to n s ), 0.53 (2H , m . cyclopropane m ethylene p ro ton s), 0 .9 5 (5H , m. C H 3
and cyclopropyl m ethine proton), 1.67 (2H, ni, C (0)C H2C / /2C H3), 2.37 (2H, t, J
10 H z. C (0 yCH2). 2.87 (2H. d, J 9 Hz. A1C//2 ). 3.30 (2H. d , / 7 H z. OC H 2-
cyclopropyl protons), 3.63 (2H, t, / 9H z, ArCH2C / /2). 3.85 (2H, m, A1O C / /2). 4 .37 (2H . m , C H2C l), 5.38 (1H . m . CH ). 6.91 (2H. d. J onho 10 Hz, A r-H ), and 7.23
(2H , d, 7 ortho 10 H z, Ar-H); 8c (100 MHz; solvent CDCI3) 2.77 (C-12), 10.45 (C-
11), 13.37 (C -4'), 18.24 (C-3’), 35.32 (C -8), 35.92 (C -21), 42.47 (C -l), 66.1 2 (C- 10). 70.76 (C -3). 71.53 (C -2), 75.39 (C -9), 114.48 (C -5), 129.73 (C-6), 131.94 (C- 7). 156.61 (C -4). and 172.59 (C -l'); rn/z (E l) 354 (M +. 1%). 354 (M + . 3). 163 (1 00 ). 107 (6). 91 (6). 77 (4). 55 (7 2). and 43 (38); HRM S, found (m /z): 356 .1 5 6 9 , C i9H2 7 0 4;l 7Cl requires: 356.1568; Found (m /z): 354.1598, C l9H 2 7 0 4^ C l requires: 354.1598. E n zy m a tic h y d ro ly s is o f the bu ty rate
(♦ ) - 3 - f 4 - [ 2 - ( c v c l o p r o p v l m e t h o x y ) e t h y l lo h e n o x y l - l - c h l o r o - 2 -
( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l ] p h e n o x y ] - l- c h l o r o - 2 -
b u tan o y lo x y p ro p a n e (3 4 ) ( c a 0 .0 6 g ), phosphate buffer, pH 7, lOOmM (3m l) and enz y m e (ca. 0 .0 3 g ) w as v ig o rously stirred at room tem perature f o r 14 h o u rs. E valu e d eterm in ed as usual.
lip a s e from e n z y m e / m g s m ( 3 3 ) / m g r e s i d u a l ( 3 3 ) % y,% ec,A C p r o d u c t ( 2 9 ) % y,% ee,A C c E P s e u d o m o n a s fl u o r e s c e n s 31 65 4 3 ,9 9 ,R 5 9 ,7 5 ,S 0 .5 7 35 L ip a s e M (A m ano) 37 62 65.15.R 20.82.S 0 .1 5 10 L ip a s e N (A m an o) 32 60 70.10.R 3 4 ,7 7 ,S 0 .1 1 10
S cale -u p o f the lip ase ex P seu d o m o n a s flu o re s c e n t catalysed re so lu tio n o f ra c e m ic 3-T4-Î 2 - ( c v c l o p r o p v l m e t h o x v l e t h v l l p h e n o x v 1-1 - c h lo r o - 2 - b u ta n o v lo x v n ro p a n c ( 3 4 ) .- A suspension o f
( ± ) - 3 - [ 4 - [ 2 - ( c y c l o p r o p y l m e t h o x y ) e t h y l J p h e n o x y J - l - c h l o r o - 2 - b u tan o y lo x y p ro p a n e (3 4) (0 .2 0 g ), ph o sp h ate b u ffer, pH 7, lOOmM (10 m l)
and lip ase ex P s e u d o m o n a s flu o r e s c e n s (B io catalysts, L td.) (0.10 g) w as v ig o ro u s ly stirred at room tem p eratu re fo r 14 hours. T he usual d ete rm in a tio n o f th e E valu e as d esc rib ed above gave (R )-(-)-(3 4 ) (0.089g,
44% yield. 95%ee l a ] D = -21.6 ° (c 0.89 CHCI3)) and (+ )-(S)-(29) (0.092g. 56%
yield . 82% ee, [oJD = +0-9 (c 0.76 CHCI3)). c * 0.537. E » 37. C o n v ersio n o f th e re sid u a l sta rtin g material (R ) - ( - ) - 3 - [ 4 - [ 2 -
( c v c l o p r o p y l m e t h o x Y ) e t h v H p h c n o x v l - l - c h l p r o - 2 - b u t a n p v l Q S Y p r p p a n e
I M ) __ (9 5% ce) to__ ( R H + ) - 3 -I 4 -[2 -(c y c lp p rp p v )ro g th P x v )c ih y i]p b g n fl.x Y l-l,2 - ep o x v p ro p a n e 1281. A nd s u b seq u e n t co n v e rsio n to (S )-(-)-3 -[4 -f2 - ( c v c l o p r o p v l m e t h o x v > e th v l l p h e n o x v l - 2 - h v d r o x v D r o p v la m in e ( 3 7 1 .- T o a s tir re d so lu tio n , u n d e r arg o n , o f (R )-(-)-3 -[4 -[2 -(c y c lo p ro p y lm e th o x y ) e th y l]p h e n o x y ] - l- c h lo r o - 2 - b u ta n o y lo x y p r o p a n e (3 4 ) (9 5 % e e ) (0 .0 6 3 g , 0 .1 8 m m o l) and fresh ly m ade p otassium re rf-b u to x id e (0 .0 2 4 g , 0.21m m o l) in dry re rf-b u ta n o l (2 m l) was heated at 60 ° C for tw o hours, t.l.c ., (light p etroleu m ether (b .p. 40-6 0 ° C ) : diethyl ether; 1 : 1), R f (34) = 0.58, R f (28).=