A p p l i c a t i o n s of E n z y m e s to t h e P r e p a r a t i o n of
O p t i c a l l y A c t i v e C o m p o u n d s .
B y
S u s a n M . O pel t
S u b m i t t e d for t h e d e g r e e of D o c t o r o f P h i l o s o p h y
D e p a r t m e n t o f C h e m i s t r y
U n i v e r s i t y o f W a r w i c k
T H E B R IT ISH L IBR AR Y
d o c u m e n ts u p p l yc e n t r eB R IT IS H T H E SES
N O T I C E
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T H E B R I T I S H L IB R A R Y D O C U M E N T SUPPLY CENTRE
L ist o f tables. x
A c k n o w le d g e m e n ts . xii
D e c la ra tio n . xiii
S u m m a r y . x iv
A b b r e v i a t i o n s xv
C h ap ter 1: A spects o f S electiv ity in L ipase-C atalysed
B io tr a n s f o r m a tio n s . 1
1.1 In tro d u c tio n . 1
1.1.1 D istribution o f lipases. 2
1.1.2 A ssay o f lipase activ ity . 3
1.1.3 M echanism o f lip ase action. 4
1.1.4 The use o f biotransform ations in organic
s y n th e s is . 7
1.1.5 L ip ases and stereo selectiv ity . 8
1.1.6 Types o f sele ctiv ity in lip ase-catalysed
r e a c tio n s . 1 3
1.1.7 Aims o f this review . 1 5
1.2 Pig P ancreatic Lipase. 16
1.2.1 PPL -catalysed reactio n s of prim ary alcohols. 17
1.2.2 Reactions o f secondary alcohols using PPL. 2 5
1.2.3 R eg io selectiv ity in PPL -catalysed reactions. 3 1
1.2.4 The use o f PPL fo r resolution o f chiral acids. 3 4
1.2.5 R eactions of lactones. 3 7
1.2.6 C onclusions on th e use o f PPL in organic
s y n th e s is . 3 9
1.3 The Lipase from C a n d id a cylindracea. 4 3
1.3.1 R esolutions o f secondary alcohols. 4 4
CONTENTS
1.3.2 E sterolytic reactio n s w ith ch iral acids. 5 4
1.3.3 R esolution o f prim ary alcohols. 6 0
1.3.4 R eg io selectiv e re a c tio n s o f carbohydrate
s u b s tr a te s . 6 2
1.3.5 C oncluding co m m ents. 6 4
1.4 Lipase P from P seu d o m o n a s flu o r e s c e n s . 6 5
1.4.1 L ipase P -cataly sed este ro ly tic reactio n s
of secondary alcohols. 6 5
1.4.2 Esterolytic reactions o f lipase P on
prim ary alco h o ls. 7 4
1.4.3 Resolution o f chiral acids. 7 6
1.4.4 C onclusions. 7 7
1.5 O ther M icrobial Lipases. 7 9
1.5.1 Lipase SAM II from P seu d o m o n a s sp. 7 9
1.5.2 The lipase from M u co r m ieh ei. 8 2
1.5.3 The lipase from A s p e r g illu s niger. 8 6
1.5.4 the lipase from P s e u d o m o n a s fr a g i. 8 8
1.5.5 The lipase from P s e u d o m o n a s a eru g in o sa . 9 0
1.5 .6 .W h eatg erm lip ase. 9 1
1.5.7 The lipase from C h r o m o b a c te riu m visc o su m . 9 2
1.6 Typical E xperim ental P ro ced u res For
B io tra n s fo rm a tio n s . 9 6
1.6.1 H ydrolysis w ith PPL. 9 6
1.6.2 H ydrolysis w ith PPL. 9 6
1.6.3 H ydrolysis with CCL. 9 7
1.6.4 E sterification w ith PPL. 9 8
1.6.5 Esterification w ith C C L. 9 8
1.6.6 G eneral p ro ced u re fo r th e lipase-catalysed
1.7 C oncluding C om m ents. 101
C hapter 2: R esolution o f a B eta-B lo ck er P recu rso r. 1 0 9
2 .1 I n tr o d u c tio n . 1 0 9
2 .2 Synthesis o f th e R acem ic a - C h lo r o h y d r in . 1 1 1
2 .3 A M ethod o f C hiral A nalysis. 1 1 3
2 .4 E nzym atic H y d ro ly ses o f d ie ster ( 1 1 9 ) . 1 1 4
2 .5 T ran sesterificatio n o f the a - C h l o r o h y d r in . 1 1 8
2 .6 Sum m ary and C o n clu sio n s. 1 1 9
C hapter 3: R esolution of M ethyl
4 - ( p - c h lo r o p h e n y lth i o ) b u ta n o a te . 1 2 0
3 .1 I n tr o d u c tio n . 1 2 0
3 .2 Synthesis o f the R acem ic Esters. 121
3 .3 H y d ro ly sis o f M eth y l 3 aceto x y 4
-( p - c h l o r o p h e n y lth io ) b u ta n o a te . 1 2 2
3 .4 H y d ro ly sis o f M e th y l3 b u ta n o y lo x y 4
-(p - c h lo ro p h e n y lth io ) b u ta n o a te . 1 2 4
3 .5 T ra n se s te rific a tio n R eactio n s. 1 3 0
3 .6 C o n clu sio n s. 131
C hapter 4: R eso lu tio n o f 3 M e th y l4 o x o 4
-(4 -a m in o b e n z y l)b u ta n o ic acid . 1 3 2
4 .1 I n tr o d u c tio n . 1 3 2
4 .2 H ydrolysis w ith PLE. 1 3 8
4 .3 In te resterificatio n w ith P L E . 1 4 2
4 .4 H ydrolysis o f th e C orresponding A m ide
Ester w ith PLE. 1 4 3
4 .6 In te resterificatio n w ith L ip ases. 14S
4 .7 R eduction w ith B akers' Y east. 1 45
4 .8 D evelopm ent o f a R eliable M ethod o f Chiral
A n a ly s is . 1 4 7
4 .9 R eduction w ith C andida g u illie rm o n d ii. 1 4 7
4 .1 0 R eduction w ith o th er M icro o rg an ism s. 1 4 8
4 .1 1 Investigation o f the L ability o f the Product
under the F erm en tatio n C o n d itio n s. 1 5 0
4 .1 2 Sum m ary and C o n clu sio n s. 151
C hapter 5: Resolution o f K etones via H ydrolysis o f a
C orresponding O xim e Ester. 1 53
5.1 In tro d u ctio n . 1 53
5.2 P reparation and H ydrolysis o f A cetone
O xime A cetate. 1 5 4
5.3 Preparation and H ydrolysis of
2-M ethyl cyclohexanone O xim e A cetate. 1 55
5.4 P rep aratio n o f 2 ,6 -D im e th y lc y clo h ex a n o n e
O xime A cetate. 1 6 0
5.5 Enzym atic H ydrolysis o f the O xim e A cetate. 1 7 0
5.6 P reparation and Enzym atic H ydrolysis
o f N orcam phor O xim e A cetate. 1 7 2
5.7 P reparation and H ydrolysis o f N o rcam phor O xim e
B utanoate and H exanoate. 1 7 9
5.8 C onclusions. 1 8 0
C hapter 6: R esolution o f C hiral K etones by Enzym atic
H ydrolysis o f an Enol A cetate. 1 8 2
6 .2 R esolution o f 2 -M eth y lcy clo h e x an o n e. 1 8 4
6.2 .1 E xperim ents w ith the N o n -ch iral
Enol A cetate. 1 8 4
6 .2 .2 Experim ents w ith the C h iral
Enol A cetate. 1 8 6
6 .3 R esolution o f 2 ,6 -D im e th y lc y clo h ex a n o n e. 1 8 9
6 .4 R esolution o f N o rcam p h o r. 1 9 2
6.4.1 Synthesis o f the Enol A cetate. 1 9 2
6 .4 .2 E n z y m e-C ataly sed R e so lu tio n s. 1 9 4
6 .5 Sum m ary and C o n clu sio n s. 1 9 7
C h ap ter 7: E xperim ental D etails. 1 9 9
7.1 In tro d u ctio n . 1 9 9
7.1 Experim ental to C hapter 2. 2 0 0
7.3 Experim ental to C hapter 3. 2 0 8
7.4 Experim ental to C hapter 4. 2 1 6
7.5 Experim ental to C hapter 5. 2 2 3
7.6 Experim ental to C hapter 6. 2 3 5
LIST OF FIGURES.
Fig. 1 V ariation o f ec for d ifferent E values. A: ec o f the
rem ainin g su bstrate, B: ee o f th e pro d uct. 10
F ig. 2 C o m p u te r-g e n era te d cu rv es from e q u a tio n s 9 and 10
sh o w in g p ercen ta g e e n a n tio m e ric e x c e s s fo r (A ) p ro d u c t,
and (B ) rem ainin g s u b s tra te as a fu n c tio n o f p ercen tage
co n version at d ifferent v alues o f E and K. 13
F ig. 3 Som e prim ary alco ho ls re solved v ia P PL -catalyscd
r e a c t i o n s . 18
Fig. 4 C om po u nd s h y d rolysed n o n s c lc c tiv c ly by PPL. 20
Fig. 5 F u rth er p rim ary a lc o h o ls re so lv e d u sin g PPL. 20
Fig. 6 P rim ary alc o h o ls re so lv e d usin g P PL . 21
Fig. 7 A lcohols resolved usin g PPL and lip ase P. 22
Fig. 8 S eco n d ary alc o h o ls re s o lv e d usin g P P L -cata ly se d re a c tio n s . 25
Fig. 9 H y drolysis o f esters o f bicy c lic alc o h o ls with PPL. 30
Fig. 10 P ro d u cts o f P P L -catalyscd h y d ro ly s e s o f fully c stcrificd
s u g a r s . 31
Fig. 11 H exosidcs acylatcd by PPL. 33
Fig. 12 C hiral ac id s resolved by P P L -m c d iatcd reaction s. 34
Fig. 13 Esters o f d iacid s hy droly sed by PPL. 36
F ig. 14 C is and tr a n s dioxolanc co m p o u n d s used as su b strates for
e n z y m e -c a ta ly s e d h y d r o ly s e s . 36
Fig. 15 P P L -cata ly se d fo rm a tio n an d h y d ro ly s is o f
l a c to n e s . 37
F ig. 16 S u b strates used in d ete rm in a tio n o f the active site m odel
o f PPL. 40
Fig. 18 C y clic seco n d ary alcohols re so lv e d via
C C L -cataly sed reactions. 4 4
Fig. 19 C om po u nd s with a n o rb orn ane ty p e s tru c tu re
h y d ro ly sed w ith high sele c tiv ity . 4 8
Fig. 20 E lu cid atio n o f the b ridg ehe ad re q u ire m e n ts for
a selec tiv e reaction. 4 9
Fig. 21 G en eral su b strate m odel fo r th e h y d ro ly s is o f
n o rb o rn an e-ty p e esters by C C L . 4 9
Fig. 22 S econdary alcohols resolved by C CL. 53
Fig. 23 A survey o f C CL-catalysed re actio ns o f ac id s. 5 5
Fig. 24 P rim ary alco ho ls resolved v ia C C L -c a ta ly se d re actio n s. 6 0
Fig. 25 C C L -cataly sed reactions on sug ars. 6 2
Fig. 26 C om pounds deprotected using P P L and C CL. 6 3
Fig. 27 S econ dary alcohols resolved w ith lip ase P. 6 5
Fig. 28 P ro d u cts o f esterificatio n re a c tio n s o f s e c o n d ary
alco h o ls using lipase P. 6 9
Fig. 29 C y clic secondary alcohols re so lv e d w ith lip ase P. 71
Fig. 30 P ro d u cts o f lipaseP -catalysed h y d ro ly s is o f
c y c lo p e n ta n e d ie s te r s . 71
Fig. 31 B icyclic esters hydrolysed by lip ase P. 7 2
Fig. 32 A ctive site model for lipase P. 73
Fig. 33 E x o -s u b s tra te s hydrolysed w ith h ig h s e le c tiv ity
using lip ase P. 73
Fig. 34 N ew m an pro jectio ns o f e n d o and e x o s u b s tra te s . 7 4
Fig. 35 P rim ary alco h ols resolved by lip ase P -c a ta ly se d re actio n s. 75
Fig. 36 C hiral acids resolved with lip a s e P. 7 6
Fig. 37 C om pounds resolved using lip ase SAM II. 7 9
Fig. 39 R elativ e ra te s o f e s te rific a tio n o f o cta n ol w ith
v ario u s a c id s.(a ) study o f th e effec t o f b ra n ch in g
in th e ac id c h a in .(b ) com parison o f the ra te s o f reaction
o f c y c lo h e x y l and p h en y l-su b stitu te d ac id s. 83
Fig. 40 P ro du cts o f M M L -cataly sed e s te r h y d ro ly sis. 85
Fig. 41 P ro d u cts o f h y d ro lytic re actio n s ca taly sed by the
lip ase from A niger. 87
Fig. 42 P roducts o f reactions ca talysed by lipase from P. a eru g in o sa . 91
Fig. 43 R c g io s e le c tiv e re actio n s c a taly sed by lip ase
from C. visco su m . 93
Fig. 4 4 M inim al s te ro id s tru c tu re . 94
Fig. 45 A lco h o ls re so lv e d by P P L -cata ly sc d h y d ro ly s is
o f an e s te r deriv ative. 102
Fig. 46 Esters re so lv ed by lipase P. 104
Fig. 47 G e n e ra lis e d su b strate e s te rs for h y d ro ly s is . 107
Fig. 48 S tru c tu re s o f ea rly b e ta -b lo c k e rs. 109
Fig. 4 9 G e n era l b e ta -b lo c k e r s y n th e s is . 110
Fig. 50 The b e ta -b lo c k e r p rc cu so r to be resolved. 1 11
Fig. 51 M osher's e s te r o f the a - c h l o r o h y d r i n . 114
Fig. 52 P ro d u cts re co v ered from the en z y m atic h y d ro ly sis. 115
Fig. 53 A u to titra to r traces for the h y d ro ly sis with and
w ith o u t s u rf a c ta n t. 126
Fig. 54 NM R sp e c tra for the o p tically ac tiv e alcohol
(a) w ith o u t sh ift re ag en t, (b) w ith sh ift re agen t,
(c) w ith s h ift reagent and added racem ic alcoh ol. 128
Fig. 55 E x am ples o f P L E -cataly sed re actio n s. 133
Fig. 56 The co m p o un d to be resolved. 134
Fig. 57 The v a s o d ila to r (1 2 2) and the targ et dru g m olecule (1 2 3) . 134
by b ak e rs' y e a s t. 135
F ig. 59 C o m p o u nd s o b tain ed by reduction o f a d ouble
b o n d u sin g b a k e rs ' y e a s t. 136
Fig. 60 E x am p les o f o p tic a lly p ure co m po un ds ob tain ed
by re d u ctio n w ith C . klu y v e ri. 137
Fig. 61 C o n fo rm a tio n o f 2 -m e th y lc y c lo h e x a n o n e o x im e . 156
Fig. 62 M in im ised co n fo rm atio n s (PC M O D EL ) for
2 .6 - d im e th y lc y c lo h e x a n o n e o x im e: (a ) c is d ia x ia l,
(b) c i s d ieq u a to ria l (c ) t r a n s . 163
F ig. 63 Part o f the 1H N M R spectrum o f
2 .6 - d im e th y lc y c lo h e x a n o n e d e c o u p le d at
C-2 and C -6 in turn. 166
Fig. 64 T h e e n a n tio m e rs o f 2 ,6 -d im c th y lc y c lo h e x a n o n c o x im e . 168
LIST O F TABLES.
T able 1 P P L -cata ly se d e ste rific a tio n o f sec o n d ary alc o h o ls. 2 7
T ab le 2 Influence o f acid and a lc o h o l in re solu tion . 34
T able 3 Influence o f the acid m o iety in resolution o f a
se c o n d a ry a lc o h o l. 5 4
T able 4 Study o f th e effect o f a c y la tin g agent on the
e n a n tio s e le c tiv ity o f C C L -c a ta ly se d e s te rific a tio n s . 61
T able 5 In v estig atio n o f the e ffe c t o f variation o f the acid
com p o n en t in the h y d ro ly sis o f esters o f a chiral alco h ol. 67
T able 6 E s te rific a tio n o f v ario u s se c o n d a ry alc o h o ls
cataly sed by lipase P. 70
T able 7 T ra n se sté rific a tio n o f e s te r s o f secon dary alco h ols
cataly sed by lipase P. 70
T able 8 Num ber o f reactions o n e a c h su b strate type
cataly sed by each o f th e co m m o n enzym es. 106
T able 9 L ipases u sed for a s c re e n in g ex perim ent for
hydro lysis o f ester (1 2 6) . 144
T able 10 R esu lts o f the red u ctio n s perfo rm ed with bacteria
and b a k e rs ' yeast. 149
T able 11 C om parison o f predicted , 3 C NMR chem ical shifts
(S pred) f ° r m ajor and m in o r isom ers o f
2 -m eth y lcyc io hexan on e o x im e w ith tho se observ ed (S ob s)- 157
T able 12 Enzym es used in screen in g fo r h y d ro lysis of
2 -m e th y lc y c lo h e x a n o n e o x im e a c e ta te . 159
T ab le 13 P redicted values for the , 3 C NM R shifts o f
th e cis and tr a n s iso m e rs. 162
T ab ic 14 P redicted and ex p erim ental 13C NMR shifts for the
ring ca rb o n atom s o f 2 ,6 -d im cth y lcy c lo h e x an o n c
T able 15 S im u late d 1 H NM R d ata for the tw o c i s co n fo rm atio n s. 167
T able 16 F req u e n cies and r e la tiv e in te n s itie s o f th e ob serv ed peak s. 168
T able 17 E n zy m e s used in th e screen in g fo r h y d ro ly s is
o f th e ox im e a c etate. 170
T ab le 18 C o rre la tio n o f th e d a ta reported by H a w k e s with
ex p e rim en tally d e te rm in e d d a ta fo r th e m ajo r o x im e isom er. 174
T able 19 A ssign m en t o f th e NM R s ig n als o f norcam ph or. 174
Table 20 R e s u lts o f the s c r e e n in g ex p e rim en t f o r h y d ro ly sis
o f th e cnol ac etate. 188
T able 21 L ip ase s used in th e screen in g e x p e rim e n t for
c n a n tio s c lc c tiv e h y d ro ly s is o f th e cn o l a c etate o f
2 ,6 - d im e th y lc y c io h e x a n o n c . 191
T able 22 L ip ase s u sed fo r th e tr a n s e s té rific a tio n re actio n . 192
T able 23 R esu lts o f a s c re e n in g ex p e rim en t fo r the
tra n s e s té rific a tio n o f n o rc a m p h o r c n o l ac e ta te . 196
T ab le 24 R esu lts o f fu rth e r sc re e n in g w ork o n th e
ACKNOWI -F-DGEMENTS.
I wish to express my gratitude to Professor D.H.G . C ro u t for his
co n stan t advice and enco u rag em en t th ro u g h o u t th e co u rse of
th is work.
I am also indebted to Professor G. M orris at the U niversity of
A berystw yth, and to Dr. M .B. M itchell of S m ithK line B eecham.
Thanks are also due to the technical staff o f th e C hem istry
Departm ent for th e ir assistance in running N M R and M ass
s p e c tra .
Financial assistance from S.E.R .C . and Sm ithkline B eecham pic is
DECLARATION.
The w o r k described in this thesis is the original o f the author,
ex c ep t w h ere acknow ledgem ent has been made to resu lts and
ideas p rev io u sly published. It w as ca rried out in the
D e p a rtm e n t of C hem istry, U n iv ersity o f W arw ick betw een
O c to b e r 1986 and S eptem ber 1990 and has not been subm itted
SUMMARY.
The in tro d u ctio n to this thesis is in the form o f a review en titled 'A s p e c ts o f Selectivity in L ipase C atalysed
B io tran sfo rm atio n s'. Each o f the m ost w idely used lipases have been d iscu ssed . The reactions o f each lipase have been ex ten siv ely re v ie w e d with the aim o f estab lish in g w hether any trends h av e ap p e a re d in the ch ara cte ristic s o f com pounds accep ted as s u b stra te s.
The rem ain in g ch ap ters co v er five unrelated studies in areas of reso lu tio n s o f c h ira l com pounds using b io tran sfo rm atio n s. In C hapter 2 th e resolution o f a P -b lo ck e r p recu rso r was attem p ted v ia lip ase-cataly sed h y d ro ly sis. l-C h lo ro -2 - h y d ro x y -3 [4 (2 -a c e to x y e th y l)p h e n o x y ]p ro p a n e w as o b ta in ed in high e n a n tio m e ric excess from hydrolysis o f the co rresponding butyrate e s te r w ith lipases from M u c o r or R h iz o p u s sp. Yields were low h o w e v e r, owing to enzym e inhibition by the butyric acid b y p ro d u c t.
In C h ap ter 3 th e resolution o f m ethyl 3-hyd ro x y -4 -(p - ch lo ro p h en y lth io )-b u tan o a te w as carried out. H ydrolysis o f the corresponding b u tanoate ester w ith lipase P gave the R
enantiom er o f th e desired com pound in high en antiom eric excess. T ra n sesterific atio n o f the racem ic alcohol w ith vinyl acetate, ag ain cataly sed by lipase P, furnished the opposite en an tio m er in 62% ee.
C hapter 4 is co n cern ed with the resolution o f a chiral acid, nam ely 3 -m e th y l-4 -o x o -4 (4 -a m in o b e n z y l)b u ta n o ic acid . T his was attem p ted b y hydrolysis o f an ester using pig liver esterase and various lip a s e s and via m icrobial reduction o f the co rresp o n d in g un satu rated com pound. The reac tio n s w ere all found to b e n o n -stereo selectiv e.
C hapters 5 an d 6 discuss novel m ethods for the enzym atic resolution o f keto n es.
The e n a n tio s e le c tiv e enzym atic h ydrolysis o f oxim e esters is discussed in C h a p te r 5. The resulting optically enriched oxim es may read ily be cleaved to the ketones. This m ethod was unsuccessful in the resolution o f the 2-m ethyl- and 2,6- d im eth y lcy clo h ex an o n es. A low enantiom eric ex c ess was achieved in th e resolution o f norcam phor, and attem pts to im prove th is u sin g a purified enzym e and by variation o f the ester ch ain w e re unsuccessful. H ow ever, this rep resen ts the first ex am p le o f the indirect enzym atic resolution o f ketones. In C h ap ter 6 th e enantioselective hydrolysis o f enol acetates of three k eto n es w as attem pted. This m ethod of resolution was unsuccessful in the resolution o f 2-m ethyl and 2,6-
A B B R E V IA T IO N S .
'h NMR P roton N u clear M agnetic R esonance S p ectro sco p y .
13C NM R Carbon N u clear M agnetic R esonance Spectroscopy.
TLC T h in Layer C h ro m a to g rap h y .
HPLC H igh P ressu re L iq u id C h ro m ato g rap h y .
GLC G as L iquid C hro m ato g rap h y .
IR In fra red s p ectro sco p y .
s sin g le t,
d d o u b le t,
t tr ip le t,
q q u a r t e t,
m m u ltip le t.
b r b ro a d .
J coupling co n sta n t (H z),
p p m parts per m illio n .
TMS te tr a m e th y ls il a n e .
ee en a n tio m eric e x c e s s
a m easu red r o ta tio n .
[oc]d specific ro ta tio n m easured at 589 nm .
c co n c en tratio n (g /1 0 0 ml).
DMSO D im ethyl su lp h o x id e .
THF T e tr a h y d r o f u r a n .
TMEDA T e tr a m e th y le th y le n e d ia m in e .
CHAPTER 1
ASPECTS OF SELECTIVITY IN LIPASE CATALYSED
BIOTRANSFORMATIONS.
11 INTRODUCTION
Lipases (E .C .3.1.1.3.) are a m em ber o f th e family of
carboxylesterase hy d ro ly tic enzym es a n d in nature ca taly se the
hydrolysis of fatty acid esters o f g ly c ero l according to the
general eq uation below :
Scheme 1. H ydrolysis o f triglycerides.
Some lipases do catalyse further h y d ro ly sis of the d ig ly cerid e to
m onoglyceride or even glycerol, and c a ta ly sis may occur at a pH
other than that which generates the fa tty acid in its ionised
form. O ther esters are also susceptible to attack by lipases.
Lipases may be d istin g u ish ed from o th e r carb o x y lesterases by
the physical nature o f the substrate req u ired for cataly sis.
Because o f the fatty nature of th eir tr u e substrates, lipases will
act upon an em ulsion o f a substrate, a n d display very low
activity when the substrate is fully d isp ersed in w a te r.1 F o r
exam ple pure pancreatic lipase is u n ab le to catalyse the
hydrolysis o f triacetin at a low co n c en tratio n but a high activity
develops when the co n cen tratio n is in c reased such that
em ulsified particles begin to form .2
,o. R
h2o
+ RCOO
O lipase
This was attributed to the fact th at the methyl e s te r w as
com pletely dissolved w hile the octyl e s te r existed as an
e m u ls io n .2
1.1.1 D istribution o f lipases.
Lipases have been found in and isolated from an im al, plant and
m icrobial sources, and lipases from each class have b ee n used in
b io tr a n s f o r m a tio n s .
A nim al lipases.
Three types o f lipase have been defin ed in anim als: th e lipases
discharged into the digestive tract, tissu e lipases and m ilk
li p a s e s .1 Among digestive lipases, that o f the p an c re as has been
isolated and used for biotransform ations. D espite its low
abundance com pared to other pancreatic enzym es (2 .5 % o f the
total pancreatic protein in the p ig 1 ) th is enzyme is im portant in
the digestion of fats. Pig pancreas contains a high activ ity of
lipase com pared to that from other anim als (1 3 ,0 0 0 U/g
com pared to 4,300 U/g in the horse pancreas, 2 ,800 U /g in the
cow and 1,700 U /g in sheep).4 Pig pancreatic lip ase has been
p urified and utilised ex ten siv ely in b io tran sfo rm atio n s. The lipase from C andida cy lin d ra cea hydrolysed o cty l
2-chloropropionate but not the m ethyl e s te r o f the sam e acid.
Few studies have so far been m ade on plant lipases b u t high
M icro b ial lipases.
Lipases are found widely in bacteria, yeasts and fu n g i. Most of
these enzym es are exocellular (i.e. excreted in to th e culture
m ed iu m ) and conditions may be optim ised for lipase
p r o d u c tio n .53 In some cases, for exam ple with C a n d id a
p a r a lip o ly tic a and C andida cy lin d ra cea the b io sy n th e sis of
lipase can be induced by addition o f g lycerides, ch o lestero l or
su rfactan ts to the culture medium. In other ca ses, addition of
such inducers inhibits biosynthesis o f the lipases ( eg with
P seu d o m o n a s fr a g i and G eo trich u m c a n d id u m).* M any
m icro b ial lipases are now com m ercially a v a ilab le as pow ders
w hich can simply be used as a chem ical reag en t th u s widening
th eir appeal to the synthetic organic chem ist.
1.1.2 Assay vf lipase aciivii^
The lipase-catalysed hydrolytic reaction m ay be fo llo w ed eith er
through disappearance o f ester or form ation o f alco h o l or acid.
V arious insoluble substrates have been u tilised in lip ase assays,
in clu d in g Tween 80, p -n itro p h en y lac eta te and p
-nitrophenylbutyrate. The water soluble T w een 20 has also
been u sed.6
M ethods of choice for routine work include titratio n o f fatty
acids eith er by conventional m ethods or by use o f a recording
p H -S tat and the continuous titration o f acids released from an
olive oil emulsion stabilised by gum arabic. E m u lsio n s of
trib u ty rin have also been used w ith satisfacto ry resu lts. More The lip ase isolated from w heatgerm has been u tilised in some
recently h ig h sp eed stirring o f a tw o-phase system
(iso o ctan e/a q u eo u s buffer) h as been proposed. This appears to
be useful fo r assay o f solid lipid substrates as well as liquids
such as o liv e o il.6a
1.1.3 Mechanism <?f lipase aciioiL
The m ech an ism o f lipase hydrolysis has been considered to be
the same as fo r serine proteases such as a -c h y m o tr y p s in w h ich
hydrolyses p e p tid e bonds via an acyl enzym e in term ed iate.
Schem e 2 . E n z y m e m echanism involving an a cyl-enzym e
i n t e r m e d ia te .
w h e re P i is the liberated alcohol, P2 is the product a n d E -P2 is the acyl enzyme intermediate.
Inactivation o f a -c h y m o try p s in w ith
d iiso p ro p y lp h o sp h o flu o rid ate (sp ecific for serine) im p licated a
serine (re s id u e 195) in the m echanism . A ffinity labelling
studies fu rth e r showed that H istidine 57 was involved. X-Ray
studies sh o w e d that His 57 and Serine 195 were adjacent and
that the c a rb o x y l side chain o f A spartate 102 was also close
b y .7a,b F u r th e r X-ray and chem ical studies elucidated the
"charge relay netw ork" of the catalysis. The catalytic triad has
also been d em o n strated by NMR experim ents with a - l y t i c
protease ( a n o th e r serine protease) in organic so lv en t.8 His 57
ren d erin g that resid u e m o re nucleophilic and allow ing attack on
the carbonyl group o f th e peptide to be cleaved.
A tra n sien t tetrah ed ral in term ed iate form s as A sp 102 is
p recisely oriented to p a rtia lly neutralise the charge on the
im idazole ring. The sto red proton is donated to the N-
com ponent of the p ep tid e bond to form th e acyl enzym e
in te r m e d ia te .
In the deacylation step th e charge-relay system draw s a proton
aw ay from a w ater m o lecu le, generating a hydroxyl ion which
attacks the acyl en zy m e interm ediate in th e reverse o f the
acylation step. The c h a rg e relay system is shown below .9
Schem e 3. The charge re la y system o f a - c h y m o tr y p s in
There is now much e v id en ce to indicate that lipases operate by
a sim ilar m echanism . E a rly claims that pancreatic lipase was a
"su lp h y d ry l enzym e" w e re discredited, although th e enzym e
was shown to have tw o sulphur groups; one near the site As
Ser 195
O - H O '
responsible for attractin g the e n z y m e to the hydrophobic
interface and th e other near th e a c tiv e site .10
It was an early suggestion th at a s e rin e or threonine residue
might be the acylation s ite ,11 and la te r studies of the
inactivation o f pig pancreatic lip ase w ith bile salts proved that a
serine residue w as indeed in v o lv e d .12 H istidine has also been
im plicated in the m echanism o f p ig pancreatic lip a se .13,14
Kinetic studies on lipase reactio n s, an d trapping and isolation of
the acyl-enzym e interm ediate a lso su p p o rt this ac y latio n and
deacy latio n m e ch an ism .15,16
Recently X -ray cry stallo g rap h ic s tu d ie s o f hum an pancreatic
lipase and the lipase from M u co r m ie h e i have show n that both
of these enzym es have a catalytic tria d sim ilar to that o f a -
c h y m o tr y p s in .17,18 Human p a n c re a tic lipase has an A sp-H is-Ser
triad chem ically analogous to th a t o f the serine proteases.
Serine 152 is the active serine (as is the case for the porcine
enzyme) and the triad is covered b y a surface loop. The same
situation has been revealed for the fu n g al lipase from M u c o r
m ie h e i, with Ser 144, His 257 and A sp 203 form ing the triad
which is again buried under a lo o p fo ld ed over the surface. A
central 0 - pleated sheet in the s tru c tu re also bears a close
resem blance to the structure o f ca rb o x y p e p tid a se A, another
serine pro tease.
This evidence confirm s that the lip a s e s act via a m echanism
1.1.4 The use o f b io iransform aiions in o rg an ic synthesis.
S ev eral review s have ap p eared o ver recen t y e a rs which
d escrib e som e o f the ap p licatio n s o f b io tran sfo rm atio n s in
org an ic synthesis. Lipase reactio n s are in c lu d ed in such
l i te r a tu r e .19,20,21
A w ide range o f lipases is utilized in biotransform ations.
A m o n g st them pig pancreatic lipase has been m ost widely used.
As the natural substrates o f lipases are fatty acid esters of
g lycerol it follow s that lipases are m ost fre q u e n tly used to
reso lv e esters o f chiral alco h o ls by en a n tio selectiv e hydrolysis.
H ow ever exam ples are also know n o f highly selective
h y drolyses o f esters of ch iral acids. H y d ro ly tic reactions may
be perform ed in a buffer system or it may be necessary to add
a co so lv en t or to use a biphasic system to o v erco m e low
so lu b ility o f the substrate. A nother technique used to
o v erco m e solubility problem s is em u lsific atio n with polyvinyl
alco h o l, although the effects o f the su rfactan t m ay not alw ays
be beneficial as it may com pete for the in terfacial region and
cau se in h ib itio n .22
Lipases are also stable and active in a w ide ran g e of organic
so lv en ts and have been used fo r resolution pro ced u res via
e ste rifica tio n and tran se sterific atio n reactio n s in organic m edia.
S ev eral review s and articles have appeared in w hich enzyme
ap p licatio n s in organic m edia have been d ic u ss e d .23' 29 The
m ain problem s associated w ith reactions in o rg an ic solvents is
that they are much slow er than reactions in aqueous media and
that the reactions are rev ersib le. V arious app ro ach es have
A num ber o f m ethods have been d ev ised to enhance sele ctiv ity
in lipase-catalysed reactions and these have been rev iew e d
r e c e n tly .30
1.1.5 Lipases and stereoselectivity.
The enantioselectivity of lipase-catalysed reso lu tio n is d u e to
com petition betw een the tw o enantiom ers o f the su b strate . In
an ideal case o f enantiodiscrim ination th e (/?) or (S) e n a n tio m e r
o f the substrate will be accepted by the enzym e w hile the other
enantiom er binds very w eakly to the enzym e or not at all. In
this instance both isomers can be o b tain ed essen tially
enantiom erically pure. In some cases one isom er may be
bound very tightly and irreversibly to the enzym e, actin g as a
com petitive inhibitor. In this instance th e other isom er is
selectively but incom pletely converted to the pro d u ct as m ore
and more o f the enzyme is bound in the unproductive com plex.
G enerally how ever, both enantiom ers are co n v erted to p ro d u ct
but one reacts very much faster than the other. In such cases
in order to com pare the selectivities o f d ifferen t en zy m es and
substrates the enantiom er ratio E has been introduced. T h is is a
biochem ical constant for the en zy m e-cataly sed reactio n on a
particular substrate that is independent o f tim e and s u b strate
c o n c e n tra tio n .3 1
In hydrolysis the reaction is essentially irrev ersib le. The
substrate enantiom ers act as co m p etitiv e in h ib ito rs o f each
other. H ow ever, the concentration o f th e faster reacting
enantiom er (designated A) d ecreases m ore rapidly than th a t of
B, the slow er reacting enantiom er. This releases B from th e
hydrolysis o f B. Equation 1 shows the reactions o f each
e n a n tio m e r .
k, *2 .
k .. k-2 D l i * FA
k',
B ---‘ - Enz-B - k*2 ^ Enz-B*
— ► Enz + PB
k' i k'-2 Eq 1.
(PA and PB are products from enantiom ers A and B.)
W hen the enzym e system obeys M ichaelis M enten kinetics
( k - i> > k2 k .i > > k2 ) the enantiom er ratio or E value is
determ in ed from th e relative rate constants o f binding and
c a ta ly s is .
A 0 and B0 are the concentrations of A and B at zero time. It is
possible to calculate the ee o f the products PA and PB in terms
o f the "conversion ratio"
c (*+ B)
( A0+B0) Eq. 3
This gives the expression for the enantiom er ratio given below:
l n ( [ ' c ] [ ] e e s ] )
E = — ; --- f Eq. 4 , n ( [! c ] [ l + ee s ] )
w h ere c= co n v ersio n
ees= ee o f the rem aining substrate.
The conversion m ay be taken from GLC or H PL C analysis o f the
ee;
c = ---*— Eq. 5
e e s+ e e p
w here e e p = ee o f the product.
This eq u a tio n can be plotted graphically and Fig 1 show s plots
o f p e rc e n ta g e en a n tio m eric excess versus the percen t
co n v ersio n fo r various enantiom er ratio s, E. A show s the ee of
the rem ain in g su b strate and B show s the ee o f the product.
Figure 1 . Variation o f ee fo r different E values.
From Fig. 1A it can be seen that the ee o f the unhydrolysed
substrate increases as the reactio n proceeds, eventually
reaching a value o f 100%. H ow ever, the d egree o f conversion
required to attain an ee o f 100% is dependent on the
enantiom er ratio E. At high values o f E, an ee of 100% of the
rem aining substrate is attained at ju st over 50% co nversion, but
at low values o f E, high optical purity o f the rem aining substrate
is only attained at a much higher degree of conversion. In such
cases, yield is sacrificed in o rd er to attain high optical purity.
W ith regard to product, the rev erse situation holds. In all cases
(Fig. IB ), the ee falls sharply after 50% conversion. At high E
values, the product has high optical purity up to 50%
conversion, whereas at low E values, the optical purity is
defined at time zero and falls o ff rapidly, w ith a more rapid
decrease, the low er the E value. The significance of the curves
shown in Fig. 1 is that they allow one to predict the degree of
conversion required in a system o f known E value to achieve
the best com prom ise betw een d eg ree o f co n v ersio n (yield) and
optical purity o f eith er substrate or product.
As an indication, an enzym e w ith an E value > 100 w ould be
considered to be highly selective, but effectiv e resolutions can
be carried out in system s w here E has a value as low as 10.
However, in such cases yield w ill be sacrificed in order to
achieve high optical purity, as noted above.
This relationship does not apply in the case o f
biotransform ations in lo w -w ater system s w hen the reactio n s
are reversible. Equation 6 d escrib es the esterificatio n o f the
enantiom ers o f an alcohol in the presence o f an excess o f acyl
Enz + A
k-i
K2
: Enz + Pb
k 2 Eq 6
The actio n o f the enzym e is to sp eed up the attainm ent of
equilibrium ; it does not change the position o f equilibrium . The
eq u ilib riu m co n sta n t therefore d e p e n d s only on the initial and
final states and the eq u ilib riu m co n sta n ts for the enantiom ers
should b e id en tical.
k . i k - 2 A_ _B^
k i " k 2 Pa Pb
K= Eq 7
Thus eq u a tio n 7 show s that the p referre d ch irality for forw ard
and rev erse reactio n s is the sam e. If A is the enantiom er which
is este rified faster PA w ould be th e faster reacting enantiom er
if the reactio n w ere driven in rev erse by hydrolysis or by
tra n se sterific atio n ag ain st an a c h ira l alcohol.
An e x p ressio n in co rp o ratin g th e therm o d y n am ic param eter K is
required for calcu latio n of the en a n tio m er ratio E.
Eq 8 ■ n [ l - C + K ) (' ■ t ì
l n [ l - ( l +K ) (
'-£ 1
To c o rrelate the conversion w ith th e enantiom eric excesses o f
substrate and pro d u ct the fo llo w in g equations have been
d e r iv e d .
In^l - ( l+ K ) ( c + e e s { 1 c ) ) ]
ln [ l - ( l + K ) ( c - e e s { 1 - c } ) ] Eq 9
In 1
-(
l + K ) c ( l + e e p)J
l n [ l - ( 1+K) c( 1 - e e p ) ]
The graphs show n in Fig 2 have been rep roduced from the
work o f Sih et a l30 and show com puter cu rv es generated from
equations 9 and 10. This provides an ov erv iew o f the
in terrelatio n sh ip s betw een c, ees and eep for fixed values o f E
and K.
Figure 2. C om puter g en e ra ted curves fr o m eq u a tio n s 9 a nd 10
show ing p ercen ta g e en a n tio m eric excess f o r (A ) p ro d u ct a n d (B)
rem aining su b stra te a s a fu n c tio n o f the p ercen ta g e conversion
at different values o f K fo r an E value o f ¡00.
The values o f K were (a) 0. (b ) 0.1, (c) 0.5, (d) 1.0. (c) 5.0.
These plots show that a sm all increase in the value of K has a
pronounced effect on the en antiom eric p u rity o f product and
rem aining substrate even for a system w ith a high E value.
1.1.6 Types o f Selectivity in L ipase-C atalvsed R eactions.
Since lipases have m ainly been used in the kin etic resolution of
esters o f ch iral alcohols, a m ajor co n sid eratio n concerns
selectiv ity w ith respect to prim ary and seco n d ary alcohol sites.
[image:30.346.76.225.143.209.2]catalysed esterolytic reactions. A m ajor d istin ctio n therefore is
betw een those lipases th at ca taly se estero ly tic reactio n s of
prim ary alcohols and those th a t cataly se este ro ly tic reactions of
secondary alcohols. Such d istin ctio n s are rarely , if ever
absolute. However, the litera tu re contains c lear cases when
certain lipases have proved to be m ore effectiv e than others in
cataly sin g en an tio selectiv e reac tio n s o f prim ary alcohols, and
another group has been found, in p ractice, to be m ore effective
in cataly sin g en a n tio selectiv e reactio n s of seco n d ary alcohols.
Since the normal su b strates o f lipases are (p resu m ab ly )
glycerides, i.e. glycerol esters o f long-chain carb o x y lic acids it is
not surprising that en a n tio selectiv ity in hy d ro ly sis o f esters of
chiral alcohols often shows a dependence on chain length of the
carb o x y late com ponent. E n an tio selectiv ity can th erefo re be
optim ised by correct choice o f acyl com ponent.
A key factor in estero ly tic transform ations is th e absolute sense
o f chiral discrim ination disp lay ed by the enzym e. As noted
above. X-ray cry stallo g rap h ic stru ctu res o f lip ases have only
recently been published and even in these cases the full atom
coordinates have not been released . A ccordingly, for mapping
o f the active site, one is still dependent m ainly upon structure-
activity studies. H owever, su fficien t data have been
accum ulated for certain enzym es to perm it th e construction of
outline models o f the active sites.
S im ilar, but much less ex ten siv e d ata are av a ilab le w ith respect
to enantioselective reactions o f esters o f ch iral acids.
These are the principal types o f selectivity to w hich reference
1*1.7 Aims of this review.
H aving briefly introduced the field o f lipase catalysed
biotransform ations the aim o f this review is to d iscu ss the
reactio n s and resolutions cataly sed by various lipases. A
su rv ey of the ex ten siv e literatu re show s that lipases have
w id ely d ifferen t selectiv ities with resp ect to su b strate.
H ow ever, very few ground rules exist w hereby one m ig h t
p red ict the m ost appropriate lipase for use in a given
application. The objective o f this review is to draw to g e th er the
d iffu se inform ation in the literatu re and to attem pt to ex tract
fro m it such indications as there may be of aspects o f the
selectiv ity of the various enzym es. The literature in clu d ed was
o b tained from a search o f the C hem ical A bstracts D atabase, but
it is not intended to be a fully com prehensive survey. F o r
in stan ce most o f the patent literature has not been in clu d ed and
th e search specifically ex clu d ed the reactions o f oils and fats.
M any reactions have been perform ed with m ore than one lipase
1,2 PIG PANCREATIC LIPASE
Pig p an c re atic lipase is the only m am m alian lip ase in com m on
use in b io tran sfo rm atio n s. It has alread y been m entioned that
p an creatic lipase constitutes only a sm all p ro p o rtio n o f the total
p an creatic protein and this has led to co n sid e rab le d ifficu lty in
its p u rificatio n , mainly because o f the clo se asso ciatio n o f lipids
w ith the en zy m e. The m ost satisfa cto ry te ch n iq u es th erefo re
start fro m defatted pancreas pow der. T w o m o lecu lar form s
w ith lip ase activity can be separated, but the iso electric p o in ts,
am ino acid com positions and specific activ ities o f the tw o form s
d iffer little from each o th e r.32 The co m p o sitio n o f the rat
enzym e is also very sim ilar to that o f the p o rcin e en z y m e.1
The co m m o n ly used pig pancreatic lip ases are crude enzym e
p rep ara tio n s ( for exam ple "p an creatin ") co n tain in g as little as
17% p ro te in , not all of w hich is necessarily the lipase. Thus the
b io tran sfo rm atio n may be ca taly sed by an o th er en zy m e p re se n t
in the m ix tu re. A ttem pting to im p ro v e a b io tran sfo rm atio n
using a p u rified PPL may lead to the d esired activ ity declining
or d isap p e arin g altogether. In rep o rts o f P P L -cataly sed
h y d ro ly sis o f am ino acid e s te rs 33 and p ep tid e sy n th esis34
purified P P L did not catalyse the reactio n s at all and crude PPL
exhibited a low er activity than papain. T his, co u p led w ith the
low y ield s obtained, suggests that the reac tio n s are, in fact,
catalysed by the protease im purities in th e cru d e PPL. D espite
such lim itatio n s PPL has been utilised ex ten siv ely for
b io tran sfo rm atio n s. It has been d em o n strated th a t P PL when
at tem peratures o f up to 100°C .35 P PL has also been m odified
by alkylation to ren d er it more activ e.36 H ow ever, because
crude PPL has been used in so m any biotransform ations,
interpretation o f th e results is made d ifficu lt by the absence of
precise in fo rm atio n on the nature o f th e enzym e(s) actually
cataly sin g the o b serv ed reaction.
It has been rep o rted that pancreatin h y d ro ly ses the esters from
sn-1 and sn-3 p o sitio n s o f 3 -O -o ctad e cy l-l ,2 -d io ctad ecen o y l-
sn-glycerol at e q u a l rates,37 showing that it is selective for
esters o f prim ary alcohols. Thus it is not surprising to find that
PPL has been u sed extensively for reactio n s o f prim ary alcohols.
PPL has been u tilized in the selective acylation of a prim ary
alcohol function in the presence o f a secondary alcohol m oiety38
and has also b een show n to acylate an am ino function in
preference to a seco n d ary alcohol.39
1,2,1 P PL -caia iy s e d r e a c t i o n s o f p r i m a r y a l c o h o l s ,
In Fig. 3 are show n some primary alco h o ls resolved using PPL.
In each case (ex ce p t when specified), the structure shown
corresponds to th a t o f the en antiom er m ost rapidly released by
Figure 3. Prim ary a lc o h o ls resolved via P P L -ca ta lysed rea c tio n s .
RCOO OH
R-CgH,,
25%, 70%ee +30% MeOH, 48%, 84%ee
Ref (40)*
OCH2Ph
HO OAc
( R )
+15% THF, 40%, 80%ee Ref (41)
Ph
HO OAc
(fl)-(+) 83%ee
Ref (43)
HO OAc
(*)-<♦)
45%, 88%ee Ref(42)
AcO OH
(S) 63%, 96%ee
Ref (45)
NHZ
HO OCO(CH2)3C H 3
( R )
>97%ee Ref (46)
OCH2Ph
HO OAc
(-) 79%, 41%ee
Ref (49)
R
AcO OH
R=OCH2CH=CH 2 70%ee R=OCH2C6H5 70%ee
Ref (47)$
AcO OH
34%conversion 95%ee Ref (50)
NHZ
HO OAc
(A) 77%, 97%ee
Ref (48)*
*=product of an este rifica tio n reaction, not hydrolysis.
[image:35.344.56.301.27.346.2]It may read ily be seen th a t variation in the su b stitu e n t at the
2-position has a dram atic effect on the selectivity o f the
re a c tio n .
In one ex a m p le of a hydrolytic reac tio n ,45 variation o f the
solvent w as found to increase the enantiom eric ex c ess from
85% ee in w ater/TH F 8 5:15, to 9 3 % ee in w ater/ /e r f - b u t a n o l
90:10, to 96% ee in w ater/ diiso p ro p y l ether 85:15. A
diiso p ro p y l eth er : w ater solvent sy stem has also been used
e l s e w h e r e .42 In this exam ple the product m onoester was
reesterifie d and rehydrolysed to g iv e the op p o site en a n tio m er
o f the corresp o n d in g benzyl ester (Schem e 4).
Schem e 4. H ydrolysis o f the corresponding b en zyl ester42
tsnu
rS
BzO OAc
rS
(S)-(-) 8 8 % e e
H ydrolysis o f the follow ing com pounds resulted in racem ic
F ig u re 4. C om pounds hydrolysed non s e le c tiv e ly by PPL.
In F ig . 5 are show n more exam ples o f p rim ary alcohols w hich
h av e been resolved using P P L -cataly sed re a c tio n s.
F ig u re 5. Further prim a ry alco h o ls reso lved using PPL.
96%. > 99% ee R ef <5 2 >
R el (51)
Q c C °
8 7 % e eRef (53)
PrCOO Oh
5 5 % e e Ref (54)
C
OCOPr OH84%, 94% ee
80%. 9 6 % e e
\ J ^
OCOMeS ^ ^ O H
Ref (55)
O C O M e
O H
[image:37.343.64.315.15.395.2]The effect o f rin g size in th is structure type has been
s tu d ie d .51,52 PPL hydrolysed all esters o f cyclic com pounds
tested w ith alm o st co m p lete selectiv ity .
Fig. 6 show s further prim ary alcohols resolved via P PL -
cataly sed re a c tio n s .
Figure 6. P rim a ry alcohols resolved w ith PPL.
(CH2)nOH
r2«r1«CH3 h ig h e e ee dependent on th e acid
Ref (56) (S)
O
R1
(S)
Ret (58)*
8 9% ee
Ref (57)
CH2OH
C l ^ s'“c h2c i
(«)-(♦)
+recovered substrate in 90% ee @ 75% conv.
Ref (59)
= >
R1
C H 2OH
R=(CH2)9C H 3: 38% conv. >95% ee R=(CH2)4C H (C H 3)2: 25% conv. >95%ee
R ’ ,R2,R3= H or CH 3
60%. >90%ee
R ef (60)
O ,— o
....r 0K
rA ^ oh ...
(
(fl)-(-)
c h2o h
(-)
>80% ee 9 7 % e e
Ref (62) Ref(63)
Ref (61)
OCOMe (5)_ 38%
NHCOMe
>95% enantioselectivity
Ref (39)
[image:38.344.62.319.19.379.2]B ian ch i et al58 have described reso lu tio n of prim ary alcohols v ia
tra n se sterific atio n w ith ethyl acetate, m ethyl p ro p io n a te or
m eth y l acetate. Six exam ples were g iv en using lipase P (from
P se u d o m o n a s flu o r e s c e n s ) and PPL in its free or im m obilised
form (Fig. 7). The selectivity was th e same for the tw o
e n z y m es, the (S) e s te r being form ed p referen tially .
Im m obilisation o f PPL had no effect on the selectivity. In
c e rta in cases, nam ely resolution o f ( I ) and (2 ) w ith ethyl
a c etate as acyl donor, and (5 ) w ith m ethyl pro p io n ate as acyl
d o n o r a more selective reaction was achieved w ith PPL. The
re su lts indicated in Fig. 7 relate to residual u n esterified
s u b s t r a t e .
F ig u re 7. Alcohols resolved usina P P L a nd lipase P (results a iv en
fo r P P L ).5*
ethyl acetate*, 9 l% e e
or m ethyl propionate, 88%ee ethyl acetate, 80% ee
(D
(2)
methyl propionate, 66%ee
A nother s tu d y reported on th e e ffe c t o f electron
d o n atin g /w ith d raw in g effec ts o f th e alcohol su b strate in
tr a n s e s t e r if ic a ti o n .64
Scheme 5 . T ra n sesterifica tio n w ith va rio u s alco h o ls.
PPL
CH3C O2CH2CH3 ♦ XCH2CH2CH --- CH3COOCH2CH2X + EtOH
, 1. ? 3.
X - C I , Br. MeO, BuO. M e ^ , B2N. a, b a. b. a, b
Class 1 co v e rs electron w ith d raw in g , 2, electro n d o nating, 3
more s tro n g ly electron do n atin g su bstituents. M oving from a to
b in each class gives an increase in steric bulk and in
n u cleo p h ilicity . Reactions w ere perform ed w ith the acylating
agent as so lv en t. With PPL the less sterically hindered 'a'
alcohols sh o w e d m arked a c tiv ity , g iving 80-90% conversion
after 48h. 'b' Alcohols w ith b u lk ier substituents, in spite of
better n u cleo p h ilicity , ex h ib ited a very slow reactio n and gave
only 2 6 -3 8 % conversion in 48h. This suggests that steric factors
are m ore im portant than electro n ic factors in governing lipase
activity to w a rd s tra n se ste rific a tio n .
This was also suggested in the follow ing study w hich considered
variation o f alcohol and acyl d o n o r65 (S ch em e 6).
Scheme 6.
RCC>2CH=CH + R1OH --- ► RCO2R ' + CH3CHO
R. R 1 = various alkyl groups
R eactions w ere performed in reflu x in g THF. H ighest yields were
obtained w ith R, R 1 as straight chain alkyl, fo r exam ple with
In troduction o f R 1 as an arom atic m oiety, R ! = PhCH2 o r as a
m oiety c o n ta in in g a double bond (for exam ple R 1 =
C H2= C H - C H2-) also gave a high yield. Introducing eith er R or R 1
as iP r re d u c e d the yields and as te r t-B u elim in ated activ ity
altogether. R eactions were m uch slow er w ith R 1 as iP r or 2-
Pent sh o w in g possible en a n tio selectiv ity , although th is was not
c o n f ir m e d .
T ra n se s te rific a tio n has a lso been attem p ted w ith org an o m etallic
s u b s tr a te s 66 (Schem e 7).
Sch em e 7.
R ’ COOEt + R*OX , T-- R'C OOR2 + EtOX
R 2« hexyl or cyclohexyl
R 1 - acetyl, butyryl, capryloyl or lauryl. X — H, SnBua, SiMe3
Silyl eth ers w ere poor su b strates g iv in g slow er reac tio n s than
the co rre sp o n d in g alcohol. Stannyl eth ers in co n trast reacted
three fold fa s te r than the co rresponding alcohol at a 1M
c o n c en tratio n and the effect was larg er at higher concentrations.
S tannyl e th e r s o f prim ary alcohols pro v ed to be b etter
substrates th a n those of secondary alco h o ls and th o se of
tertiary a lc o h o ls did not reac t at all. U se o f activated esters led
to faster re a c tio n rates. Equilibrium co ncentrations w ere the
sam e irre sp e c tiv e of w hether alcohol o r stannyl eth e r were
1.2.2 Reactions o f secondary alcohQls using P P L ,
PPL has also been used in resolutions o f seco n d ary alcohols as
shown in Fig. 8.
Figure 8. S ec o n d a ry alcohols reso lve d by P P L -ca ta lysed
r ea ctio n s.
O
OTBS
OAc O H
R » long chain akyl or alkenyl. ee rem aining substrate >92% @ 50% conv
(1 R.AS)
80-90% , >90%ee
Ref (68)
(1S.4R)
25% , 100% ee
R = H, aryl, alkyl.
45%, 42-96% ee (1 ft,4 S )
Ref (67)* R e f(69) Ref (70)*
OH
OH
48%, 95% ee. + 45% diester.
Ref (71)*
H
OAc 31.6% , 100%ee
Ref (72)*
(1 ft,2ft,5ft)
E -7 0
Ref (73)
rem aining substrate 75%, 9 0% ee
Ref (74)*
(S) 25-28% . 6 0 -9 7 % e e
Ref (78)*
+ (ft) ester Ret (79)* (+) >96% ee
Ref (75, 76)
>95% ee
[image:42.344.53.309.29.408.2]O. OCOPr
Ref (80)'
(fl)-(-) 75% , 90% ee
Ref (81)*
(«) The acyl donor influences the rate and selectivity
Ref (82)*
(a ) : R -E t < 50% conv., 1 00% ee (b ) :R -P r 1 6 % conv., 1 00% ee
Ref (83)
Ph^ < A R
OH
‘ OH
,OH
(S) 46% conv., 95% ee
Ref (84)
*=products o f an este rifica tio n reaction
**= removal o f R co ntam inant from an optically enriched
s u b s t r a te
It is im m ediately e v id e n t that PPL w ill accept a w ide variety o f
structures and tre a t them all w ith high e n a n tio d isc rim in a tio n .
A detailed study h as been made on the effect o f tem perature,
chain length o f su b stra te and enzym e im m o b ilisatio n on ester
form ation and e n a n tio se le c tiv ity o f P P L -cataly sed este rifica tio n
of aliphatic (C 5 -C 1 0 ) 2-alkanols and p h en y lalk an o ls.* 5 The
various substrates w e re esterified w ith d o d ecan o ic acid in n-
heptane, stirred at d efin ed tem peratures for v a rio u s lengths o f
time. PPL w as fo u n d to act preferentially on th e R- e n a n tio m e r
in all cases.
Table 1. PPL catalysed este rific a tio n o f secondary a lcohols.85
OH
♦ r’c o o h
P P L
rv h e p ta n e
HO
R ^ ^ C H , (S)
alco h o l T(°C) conv.(% ) ale. ee(% ) e s t e r
ee(% ) E
2 -h e x a n o l 4 0 3 6 4 6 .1 8 8 .3 2 7
7 0 3 0 3 3 .8 9 5 .9 7 1
2 -o c ta n o l 4 0 3 9 6 0 .1 8 8 .0 7 1
7 0 3 1 4 1 .1 9 3 .1 2 7
2 -d e c a n o l 4 0 2 7 3 9 .0 9 4 .9 5 5
7 0 3 4 4 6 .5 9 3 .0 4 4
1 - p h e n y le th a n o l 4 0 3 5 5 4 .8 9 5 .4 7 1
1 p h e n y l 1
-7 0 3 3 5 2 .7 9 2 .7 4 2
p ro p a n o l 4 0 1 9 1 9 .9 9 0 .8 2 6
1 p h e n y l 2
-7 0 1 8 1 9 .6 8 6 .9 1 7
p ro p a n o l 4 0 1 5 1 2 .7 85.1 1 4
7 0 1 4 1 3 .6 8 3 .7 1 3
W hen reactio n s with 1-p h en y leth an o l and 1-p h e n y l-1-p ro p an o l
were perform ed at 4 0 °C w ith im m obilised enzym e, E values of
392 and 604 resp ectiv ely w ere obtained.
At 70°C the en a n tio selectiv ity was on average 5.2% higher w ith
2-alkanols than at 4 0 °C .
E nantioselectivity was in c re a se d by the introduction o f an
arom atic group adjacent to the chiral ce n tre and d ecreased by
increasing the distance b etw e en the two. Im m obilisation o f PPL
led to an im provem ent in en an tio selectiv ity . This w as believed
to be due to enhancem ent o f the catalytically active surface and
removal o f w ater from th e enzym e preparation. The
PPL "straight from the bottle" and P P L that had previously been
d eh y d rated under vacu u m 81 ( S c h e m e 8).
Schem e 8. Esterification o f racem ic su lca to l
O
+ R’^ ^ O R
(R)-(-)-sulcatol laurate, 90%ee
The enantioselectivity was u n affec ted by the leaving group (O R )
o f the ester, and increasing chain le n g th o f the acid also had
little effect. However, use o f th e a c tiv a te d ester trifluoroethyl
laurate gave a four fold increase in E value. A com bination of
enzym e dehydration and ester s e le c tio n gave a ten fold increase
in the enantiom er ratio and the r e s u lts obtained un d er
optim ised conditions are shown in S ch em e 8.
Sulcatol has also been resolved b y PPL -catalysed esterificatio n
w ith trichloroethyl butyrate in a n h y d ro u s eth er.80
The selectivity of PPL for seco n d ary alcohols was a lso exploited
in enrichm ent o f an optically e n r ic h e d product from another
r e a c tio n .79 The (S) enantiom er o f th e desired pro d u ct was
obtained in 90%ee by bakers' y e a st red u ctio n o f the
corresponding ketone. The m inor (R) isom er w as rem oved by
selective esterification using lipase P to gain an increase to
97% ee. A final esterification w ith P P L under anhydrous
It has also been dem onstrated that PPL is selectiv e for the (R)
isom er in the esterification o f 2 -o ctan o l.82
PPL has been used for an in teresterifica tio n reaction on a
m ixture o f cis and tr a n s isom ers o f esters o f monocyclic
disecondary diols as shown in Schem e 9.72
Schem e 9.
4 5 .8 %
* this yield corresponds to 53% from the c is isom er in the
s u b s t r a te .
This exam ple shows that PPL catalyses th e reaction of the c is
isom er much faster than that of the tr a n s diacetate. In this
reaction PPL gave the mono ester m ost selectiv ely com pared to
other enzym es; lipase P for example gave m ostly diols.
PPL has proved to be sensitive to ring size and steric bulk o f
substituents in the hydrolysis o f racem ic e s te rs of bicyclic
a lc o h o ls .73' 77 The hydrolysis of co m p o u n d s (6 -9 ) was stu d ied 75
Figure 9. H ydrolysis o f esters o f bicyclic a lc o h o ls w ith PPL.
R(
t-H OAc H H H
(a) R - COCH3
(b) R - CCXCHaJsCHg
The alcohol from ( 6 a ) was obtained in >94% ee. The alcohol from
hydrolysis o f (7) was obtained in low en a n tio m eric excess and
com pound (8 ) was not hydrolysed at all. In co n trast, hydrolysis
of r a c-(9 ) gave the dextrorotatory alcohol in > 9 6 % ee with PPL.
The alcohol shown in Scheme 10 w as obtained in >95% ee by
hydrolysis o f an activated ester and by use o f a tw o step
p ro c e d u re .77 The product was isolated at 30% co n version and
the enriched ester was then further h y d ro ly sed . The opposite
enantiom er of the alcohol product w as then o b ta in ed by
chem ical hydrolysis of the rem aining su b strate .
Schem e 10.
(+)-95% ee
[image:47.342.51.308.30.393.2]1.2.3 R egioselectivitv in PPL catalysed reactio n s.
There are several exam ples o f the use o f PPL for the
regioselective hydrolysis or tran se sterific atio n o f sugars and
their esters. The products of such reactions are show n in Fig. 10.
Figure 10. Products o f PPL ca ta lysed h yd ro ly sis o f fu lly
esterified sugars.
* products o f esterification o f n o n-esterified sugars.
PPL has been shown to hydrolyse selectively the ester from the
chem ically unreactive C-4 po sitio n o f 1,6 -a n h y d ro -2 ,3 ,4 -tri-0 -
acetyl-(3-D -glucopyranose to give the 2 ,3 -d ia ceta te (1 2 ) in 42%
yield after 24h. reaction tim e. In this ex a m p le, how ever the
reaction had to be perform ed at a non-optim um pH to avoid
com peting non-enzym atic hydrolysis o f the C -2 and C-3
a c e ta te s .
The butyryl groups o f ester (1 3 ) were bound m uch more tightly
and reactions could be perform ed nearer to th e pH optimum of
the enzyme w ithout fear o f acyl m ig ratio n .89
OH OMe
(1 0)'
OMe OH OH
(11)*
OH OAc
(1 2)
Ret (87) 42 % Yield
Ref (88)
OH
(13)
OH (14) R - CO Pr
Ret (89)
6 5 % yield Ref (90)
[image:48.345.80.224.73.206.2]catalysed hydrolysis of th e tri-butanoyl e s te r the C-2 acyloxy
group was hydrolysed p referen tially in a s o lv e n t system
com prising m ethanol:w ater 1:4 to give este r (1 3 ). Increasing
the m ethanol content to 50% led to alm ost ex clu siv e deacy latio n
at C-4.
In the chem ical hydrolysis of these co m p o u n d s the sele ctiv ity
decreased w ith increasing size o f the acyl g ro u p but the
opposite is true for enzym atic hydrolysis.
In the case o f the tributanoyl sugar ester su b strate in the
reaction leading to m onoester (1 4 )(F ig . 10) th e ester at C -2 was
cleaved first followed by that at C-4. U sing the lipase from
C a n d id a cylin d ra cea the 3,4-di-O -butanoyl d eriv ativ e w as
obtained in 90% yield after 29h. reaction tim e. With PPL the
reaction w as found to give the 3 -O -butanoyl d eriv ativ e ( 1 4 ) in
65% yield (+ the 4-O -acetyl derivative in 19% yield) after 52h.
Sim ilar resu lts were o b tained for the co rresp o n d in g triacety l
ester but w ith lower yields and selectivity.
PPL was also shown to be selective for the h ydrolysis o f the
prim ary este r function o f peracetylated p y ra n o s e s .9 1
The acylation o f D -(1 7 ),(1 8 ) and L -( 1 5 ) ,( 1 6 ) hexosides have
been c o m p ared 87 (see Fig. 11). The esterificatio n s were
perform ed in TH F w ith 2,2,2 -triflu o ro eth y l b u ty ra te and the