2.5 HYPRQLYTIC APPROACH
2.8.2 SUBSTRATE MODIFICATION
F rom the en zy m e screen in g experim ents the lip ase from P s e u d o m o n a s flu o r e s c e n s had been selected. T w o m odifications to the su b strate w ere m ade both designed to in crease the length o f the acyl group (Schem e 2.27).
( 2 9 ) R = CH3 (33), 87% yield. R = C3H7, (34), 90% yield.
C o n d itio n s: R = C5H7, (35), 85% yield.
R = CH3, (33), acetic anhydride, pyridine, DMAP, CH2C12. R = C3H 7, (34), butyric anhydride, pyridine, DMAP, C H2C12. 108
R = C5H7, (35), hexanoic acid, DCC, DM AP, CH2C12. S ch em e 2.27
Both (34) and (35) w ere good substrates fo r the lipase from P seu d o m o n a s flu o re sc en s. T hey w ere both hydrolysed ap p ro x im ately three tim es faste r than th e co rresp o n d in g acetate (33). The E value also in creased considerably
(Table 2.7). A maximum v alu e, fo r this set o f ex p erim en ts, was achieved fo r the butyrate (34), E valu e = 35
Table 2.7 E ffect of su strate m o dification on both th e rate of hydrolysis and ihS-gnamiPSglgçtivHy.
o
o
0 X D Ar' — - V . B io c ata ly st^ H20 A r ' sm ca. 60m g • O ^ - (R ) Cl OH Ar (S ) Cl sm # R T i m e / h r s E 3 3 c h3 7 2 1 4 3 4 c h3c h 2c h 2 1 4 3 5 3 5 CH3(CH2)3CH2 1 4 2 7 6 2The e n a n tio m eric ex cesses and the su b seq u en t E values were d eterm in ed in the usual m anner (see S ectio n 2.8.1, Schem e 2.25). T h e butyrate ex p erim en t was rep ea ted on a slightly larger scale (X 2) to give (sm ) (34) = 44% yield, 95% ee; (p) (29) = 56% y ield , 82%ee; c = 0 .5 3 7 , E = 37. The (R)-(-)-residual starting m aterial (3 4 ) was converted in two step s in to the known ( S ) - ( - ) -a m in o a lc o h o l104 (37) in 58% yield, see Section 2.10.
Tw o f u r th e r experim ents w ere co n d u cted u sin g the butyrate (34), e m p lo y in g the lipases from M ucor species and R h iz o p u s ja v a n ic u s . T hese two lipases had p reviously been found to be the b est en zy m es for th e resolution o f th e butyrate (2 6 ) (see Section 2 .5 , Table 2.3). It w as found th a t both reactions proceeded w ith an E value o f 10. H ow ever, the conversion was quite low afte r 14 hours i.e. lipase from R h izo p u s ja v a n ic u s gave (sm) (3 4 ) = 15%ee, (p) (2 9 ) = 82%ee; c = 0 .1 5 , E = 10. The lipase fro m M u c o r species produced (sm) (34) = 10% ee, (p) (29) = 77%ee; c = 0.11, E = 10.
So w ith co n d itio n s m axim ised in the h y d ro ly tic direction we turned o u r attention to u sin g the lipase fro m P seu d o m o n a s fl u o r e s c e n s in the rev erse, esterificatio n , direction.
2 .9 LIP A SE CATALYSED ESTERIFICATION
The es te rific a tio n was attem p ted using tw o d iffe re n t system s. O ne e m p lo y s potentially rev ersib le co n d itio n s, w hilst the other uses irre v e rs ib le co n d itio n s.
2 .9 .1 POTENTIALLY REVERSIBLE CONDITIONS22
In these ex p erim en ts the acyl donor w as the free acid. The reaction solvent em p lo y ed was iso o ctan e and the enzym e, as usual, was used d ire c tly from the bo ttle (Table 2.8):
T ab le 2,8 P o te n tia lly reversible lip a se cataly sed esterificatio n o f ch lo ro h v d rin (291.
+ RCOOH B io c a ta ly s t
(29) ca . 50mg B io cataly st:
lipase from P s e u d o m o n a s flu o re sc e n s , iso o c ta n e , 55 °C. R E c c h3 c h3c h 2c h 2 CH3(CH2)4CH2 no reaction 1 0 4 7 4 0 . 1 2 0 .0 5
A lthough the E v a lu e is very high, th e rate o f conversion is very slow , giving o nly 12% conversion afte r 24 hours. In another experim ent b u ta n o ic anhydride w as used as the ac y l donor. H ow ever, n o n -en zy m atic esterificatio n occurred (as observed in a control ex p e rim en t in which no enzym e was p resen t).
The "sense" o f th e stereochem istry w as preserved i.e . the (S) en an tio m er w as th e faste r reacting en a n tio m er in th e
hydrolysis reaction, sim ilarly th e (S) en an tio m er w as esterified faster than the (R) en a n tio m er (Schem e 2 .2 8 ).109
O F A S T F A S T (S ) Cl OH SLOW SLOW S ch em e 2.28
B ecause of the low rate o f reactio n , the conditions o f the este rifica tio n were ch a n g ed .
2 .9 .2 IRREVERSIBLE CONDITIONS 14
T h e acyl donors in this ca se w ere eith er isopropenyl acetate7 8
o r vinyl acetate.7 9 T hese h av e a m ajor ad v an tag e o v er using the free acid as the acyl d o n o r, in th at co ncom itant w ith este r form ation an enol is fo rm ed w hich rapidly tau to m erises to the p aren t aldehyde or keto n e. T h e position o f enol to the aldehyde o r ketone equilibrium lies firm ly on the side o f the aldehyde or ketone. A ccordingly, the o v e ra ll este rifica tio n reactio n is essen tially irreversible. T h re e ex p erim en ts w ere set up. In one, vinyl acetate was used b o th as acyl donor and as th e organic solvent. In th e o th er tw o e x p e rim en ts, iso p ro p en y l a c etate was
used as the acyl donor. The organic s o lv en t was either TH F or C H2CI2. These solvents were chosen m erely on the grounds that the substrate (29) was soluble in b o th.7 9 The lipase catalysed esterification was extrem ely clean in all th ree cases. The rate o f conversion w as greater when vinyl a c e ta te was used, and slowest when CH2CI2 w as used as solvent. The experim ents using vinyl acetate and THF were w o rk ed -u p . The w ork-up w as trivial. It involved sim ply filtering o ff th e enzyme, and evaporation o f the solvent, follow ed by flash chrom atographic separation o f product (3 3 ) and starting m aterial (29). The E value was determ ined as usual. The re su lts are given below (Table 2.9):
Table 2.9 L ipase catalysed irre v ersib le resolution of
ch lo ro h v d rin (29). q oA c h3o „ i ♦ , A R+ A r ' O - ' S Cl “ “ ~ Cl „ ? H o At'0~ V r A ( OH OAc (2 9 ) (p), (S)-(33) Cl (sm ), (R )-(2 9 ) B io cataly st:
lipase from P seu d o m o n a s flu o re sc e n s , stir at room tem perature.
S o lv en t R T i m e / h r s c E THF c h3 9 6 0 . 2 2 1 3 0 CH2CI2 c h3 slow n.d. n .d. V inyl H 4 8 0 .3 3 1 5 9 a c e ta te 6 6
The re a c tio n using vinyl acetate w as scaled-up to l g (20X). S ince th is reaction was the most en an tio selectiv e s o far ac h ie v e d , and the rate o f conversion, although lo w e r than in the h y d ro ly tic reaction, was acceptable, especially g iv e n the d ram atic increase in the E value (Schem e 2.29).
u n
. - » - S *
i
a
" OAc Ar ( l g ) ' V N j Cl ( S )-(3 3 ) 47% yield, >98%ee. Ul 1 r ' ° ^ S Cl ( R ) - ( 2 9 ) 4 7 % yield, 8 8% ee. B io c a ta ly s t:lip ase fro m P seu d o m o n a s flu o r e s c e n s 65 h o u r s , room tem perature
c = 0.47 E = 502
Schem e 2.29
The ap p a re n t E value was 502. H ow ever, care sh o u ld be taken w hen in terp retatin g E values g reater than 100, s in c e very ac cu rate determ ination o f the %ee is required f o r a subsequent ac cu rate calculation o f the extent o f conversion c.7 0 -81 What can be s ta te d positively is that with E values g reater th a n 100 the re a c tio n is essentially com pletely e n a n tio selectiv e. T h e above re a c tio n gave optically pure (S )-(3 3 )-p ro d u ct. T h e residual sta rtin g m aterial (29) (8 8% ee) w as re-in tro d u ced to fresh en zy m e. The contam inating 6% o f (S)-(29) w as su ccessfu lly " sip h o n ed off". The conventional w ork-up gave o p tic a lly pure (R )-(2 9 ) (Schem e 2.30).
Cl Cl (R )-(2 9 ), 8 8% ee. (R )-(2 9 ),87% yield, 42% overall yield, >98%ee B io c a ta ly st:
lipase from P seu d o m o n a s flu o re sc e n s 3 d ay s, ro o m tem perature.
Schem e 2.30
A ccordingly, w ith this result, an enzym atic approach to b o th enantiom ers o f o p tically pure (5-blocker precu rso r (% ee > 98) had been a c h ie v e d , with an overall yield o f 89% . To co m p lete the p ro ject s u c c e s s fu lly it w as necessary to determ ine th e a b s o lu te s te r e o c h e m is tr y .
2 .1 0 DETERMINATION OF THE ABSOLUTE STEREOCHEMISTRY
The op tically p u r e (S)-(+)-(33), obtained by the lipase fro m P s e u d o m o n a s flu o r e s c e n s - c a ta ly s e d en a n tio s e le c tiv e este rifica tio n , w a s converted in to the (S )-(-)-e p o x id e-(2 8 ) in 95% yield by tre a tm e n t w ith potassium fe /7 - b u to x id e ( S c h e m e 2 .3 1 ) .
,o
o
O 'A o, - x y
( S H + H 3 3 ) ( S ) - ( - ) -( 2 8 ) C o n d itio n s: p o ta s s iu m /e r t- b u to x id e , /e rf-b u tan o l, 60 °C, 3 0 m in s. Schem e 2.31W hen sodium m ethoxide h a d been used fo r the above conversion a significant am o u n t o f methyl ether (36) had been produced, eith er by n u cleo p h ilic ring opening o f epoxide (28) or d irect Sn2 displacem ent o f the chloro group in the starting m a terial (33):
T h e epoxide (S )-(-)-(2 8 ) w a s then converted into the known (3-amino alcohol (R )-(+ )-(3 7) 104 in 57% yield. The product was iso lated by flash ch ro m ato g rap h y (Schem e 2.32).
O H