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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

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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 e

B 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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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-

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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 .

(18)

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

(19)

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

(20)

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

(21)

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

(22)

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 '

(23)

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

(24)

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

(25)

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

(26)

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

(27)

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.

(28)

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

(29)

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 ) ]

(30)

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]
(31)

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

(32)

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

(33)

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

(34)

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

(35)

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]
(36)

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

(37)

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 e

Ref (53)

PrCOO Oh

5 5 % e e Ref (54)

C

OCOPr OH

84%, 94% ee

80%. 9 6 % e e

\ J ^

OCOMe

S ^ ^ O H

Ref (55)

O C O M e

O H

[image:37.343.64.315.15.395.2]
(38)

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]
(39)

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

(40)

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

(41)

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

(42)

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]
(43)

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.

(44)

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

(45)

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

(46)

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

(47)

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]
(48)

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]
(49)

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

Figure

Figure 2. Computer generated curves from  equations 9 and 10
Figure 3. Primary alcohols resolved via PPL-catalysed reactions.
Figure 4. Compounds hydrolysed non selectively by PPL.
Fig. 6 shows further primary alcohols resolved via PPL-
+7

References

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