ESTERASES

Pig liver esterase (P.L.E.) [EC .3.1.1.1] consists of a series of isozym es w ith sim ilar sp ecificities.6 0 One of the best

applications of P.L.E. is the hydrolysis o f m e so com pounds. M e so com pounds are superb substrates from a practical point o f view in so m uch as the enzym e reaction can proceed in theory to q u an titativ e yield, w ith com plete stereo sp ecificity (1 0 0% yield,

1 0 0% ee) . 17- 18

a

C 0 2Rc o2r R = El

a:

c o2r

a

C 0 2H ^ ^ C 0 2R c o2r 50 : 50 Schem e 1.23

R ecently Jones has proposed a cubic space model to account for these p rev io u sly m y stify in g rev ersals o f stere o ch em istry.6 1

Electric eel acetylcholinesterase (E.E .A .) [E C .3.1.1.7] has also been used for the stereo sp ecific hydrolysis o f m e s o com p o u n d s. Here a rem ark ab le ch an g e in stereo sp ecificity w as o b served (Schem e 1.24).62-63

OAc OAc 94% yield, 9 9 % ee OAc OH 39% yield, 1 0 0% ee Schem e 1.24

This rev ersal has a parallel in a reaction described in Chapter five o f this thesis, nam ely the hydrolysis o f c i$ - 3 ,6 - d ia c e to x y cy clo h ex en e (2) w hich resulted in racem ic h y d ro x y acetate (3) (S chem e 1.25). OAc OAc OH OAc OH OAc ( 2 ) ( 3 ) 50 : 50 Schem e 1.25 1.3.2 L IP A S E S

Lipases (T riacy lg ly cero l lipase; T riacy lg ly cero l acy lhydrolase [E C .3.1.1.3]) are com m only used biocatalysts. In n atu re their function is to hydrolyse triacyl glycerides (4) to produce long

chain fatty acids (5 ) and the parent glycerol (6) (S c h e m e 1 .2 6 ).64 0 C (0 )R |— OH 0 C (0 )R + 3 H 20 --- — - L -O H + 3RCOOH 0 C (0 )R L -o h (4 ) (6) (5 ) Schem e 1.26

They act at o il/w ater interfaces and are therefore well suited to hydrolysing w ate r-in so lu b le su bstrates. B acteria l lipases are extracellular and are therefore easy to p u rify in bulk q u an tities. In 1985 they represented 3% o f the W orld enzym e m a rk et.6 5

They are used in th e food industry to m odify the properties of oils and fats.65

R ecently, two papers have appeared in N a tu r e , both d escribing the X -ray structure o f lipases.6 6 -67 Both lipases appear to have a catalytic triad akin to the charge relay system o f the serine proteases (Schem e 1.27).68 A sp His

J

V

O j H His Schem e 1.27 2 4

The active serine is buried inside the enzym e and is protected by a loop o f am ino acid residues. This loop has to m ove to allow access by the substrate to the active site.

As w ell as hydrolysing m eso com pounds lipases have been ex ten siv ely applied to resolve racem ic m ixtures into their an tip o d es by the stereo selectiv e h y d ro ly sis/e sterificatio n o f one enantiom er. In the follow ing cases the (R ) enantiom er is the faster reacting (Schem es 1.28 and 1.29).22

(A ) R esolution o f racem ic alcohol/achiral acid ester:

Schem e 1.28

(B ) R esolution o f racemic acid/achiral alcohol: O

(S) Schem e 1.29

The d egree o f en a n tio selectiv ity a p articu lar en z y m e expresses w ith respect to a g iv en substrate can be q u an tified , by determ in in g the E valu e as derived by S ih.6 9

For a given racem ic m ixture, let us consider este rs o f racemic alco h o ls. A ssum e th a t a particular enzym e h y d ro ly ses the (R) e n a n tio m er p re fe re n tia lly (S chem e 1.30).

( R ) + h2o FAST (P) + c h3c o2h = SLOW R ' ^ O A c + H20 --- (S) R = alkyl > CH3 S ch em e 1.30 T h e r e f o r e : ki k2 r ~ —1 E n z— R --- ► E + P k- i k ’i k’2 S M * Enz— S --- E + Q k’-i

S ince the reaction is carried out in w ater (55 m o lar) at low s u b strate c o n c en tratio n s, h y d ro ly sis is es s e n tia lly irrev ersib le.

R OH + CH3C 0 2H

(Q)

Under M ichaelis-M enten conditions (i.e. [S] -> 0, k .i » k2 , and k '.i » k'2 ), the relative rates of form ation of P/Q (the enantiom eric ratio or E value) is given by:

E =

W h e re :

[R] = concentration of rem aining (R ) substrate at tim e t. (R] 0 = Initial concentration of (R) at time = 0

H ow ever, if the form ation of the enzym e substrate com plex is rate lim iting (i.e. all active sites o f the enzym e are saturated ([S] >2Km):

i.e. k2 » kj and k'2 » k’j Then E = ( k ,/k ',)

The d ifferen ce in the free energy o f the tw o diastereom eric transition states is related to the E value:

AAG# = -RT In E

if, for example then

AAG# = 12 M m ol'* (3 k c a lm o f1)

The E value describes a given resolution for a given enzym e. The h igher th e E value the b etter th e stereoselectivity. Sih estab lish ed that the E value can be d eterm ined by m easuring tw o p aram eters % ee(P) and % ee(sm) at the sam e conversion (c), (p = product, sm = residual startin g m aterial).

The conversion (c) can be phy sically determ ined, by g.l.c., or h.p.l.c. for exam ple, or m ore read ily from th e eq u a tio n:6 9

C = e e (sm )

e e (sm) + e e (p)

The E value is given by:

ln( [ 1 -c] [ 1 -e e (sm)] ) E = --- ln ( [ l - c ] [ l+ e e (sm)])

This allow s the relationship betw een % ee(sm ) and c and between % ee(p) and c to be plo tted (F igure 1.1 and 1.2) . 2 2

Z E N fW T lO C IE FI lC EXCE SS Z EN fl N T IQ tlE RI C EX CE SS

FIGURE 1.1 P lot o f the relatio n sh ip betw een %ee (startin g

_ ,

22 m aterial) and % conversion for various E values.

FIGURE 1.2 Plot o f the relatio n sh ip betw een % ee IprpduCI) 22

and % conversion for various E values.

T h u s the E value is independent of degree o f conversion (for a g iv e n set o f conditions). One can sim ply determ ine from the two g ra p h s when to term inate the reaction to obtain the desired e n a n tio m e r w ith th e desired o p tical purity.

F ro m inspection o f the graphs several points can be made: ( 1 ) Even for a large E value the reaction m ust be term inated

at or before c=0.5 if optically pure product is desired. ( 2 ) For E > 100 the reaction is essentially com pletely

s te r e o s e le c tiv e .

( 3 ) W ith low E values (for exam ple E=5) optically pure starting m aterial can be obtained, but at the expense of chem ical y ield .

( 4 ) For practical purposes the E value should be > 20. ( 5 ) For E>100 an accurate determ ination of E is difficult to

a c h ie v e. 7 0 *8 1

F o r a given reso lu tio n the en antioselectivity can be im proved in s e v e r a l ways:

(1) M odification o f the substrate (e.g. Table 1.4) . 71

Table 1.4 S u b stra te m odification o f racem ic este r (7) leads to an in c re a s e in enantigseleciiY ity.

PPL 0 C ( 0 ) R H20, 6hrs (+ ,-)(7 ) O _{— OH} + 0 C (0 )R

PPL = porcine p an creatic lipase

R Conversion, c E c h3 0 .6 0 4 c2h 5 0 .6 0 11 c3h 7 0 .6 0 1 3 c4h 9 0 .6 0 16 C5H „ 0 .6 0 16

(2 ) Screening o f other biocatalysts to find an enzym e with a larger E v alu e.2 2

(3 ) R ecycling th e product.22 This has successfully been em ployed w ith E v alues as low as 10. The pro d u ct after initial exposure to th e en z y m e is isolated, esterified and re-exposed to the sam e en z y m e.

(4 ) The en z y m e can be redesigned by enzym e engineering to produce an e n z y m e with more d esirab le p ro p erties. For exam ple using s ite directed m u tag en sis the p ro tease subtilisin was converted in t o a more stable form by rep lacin g a readily oxidisable m e th io n in e resid u e. 7 2 -73 This technique holds the promise o f a lterin g the properties o f an enzym e at will e.g. pH profile, te m p e ra tu re stability, su b strate s p ecificity , etc. H ow ever

this represents a labour in ten siv e, highly sk illed task. In 1986 it was p rojected to cost U S $ 1 ,0 0 0 ,0 0 0 per en z y m e.7 2

(5 ) C atalytic antibodies ca n be raised to a m im ic o f the reactio n ’s putative tra n sitio n state (e.g. S ch em e 1.31).74 For the hydrolysis:

S ch em e 1.31

If a ju d icio u s choice o f sta b le T.S. analogue is selected then it is possible to achieve 1 0 3-1 0 5 rate acceleratio n s when com pared to the b ase-catalysed h y d ro ly sis reaction. T h is technique is still in its infancy but it o ffers th e rew ard o f "enzym e like catalysts w ith ta ilo red sp ecificities” .7 4

(6) Enzym e im m o b ilisatio n to allow ea sie r separation o f pro d u ct fro m en zy m e.5 5

( 7 ) U tilisin g the e n z y m e in the rev erse/esterificatio n direction. This can be ac h ie v e d by using biphasic system s, or even b etter, in very low w ate r systems. T h e technique is easily ap p licab le in a standard ch e m ica l lab o rato ry . It circum vents several o f the problem s asso cia ted w ith en zy m es in aqueous s o lu tio n s:2 *

(A ) S ubstrates are usually m ore so lu b le in o rganic solvents. (B ) Sim ilarly, substrates are o fte n m ore stable in organic

s o lv e n ts.

(C) Separation of insoluble e n z y m e and product is trivial. (D) The enzym es have the p o te n tia l to be recycled. (E) M any enzym es are, su rp risin g ly , more stable in organic

solvents, than in an a q u e o u s en v iro n m en t.6 5

(F ) The pH of the system no lo n g e r has to be regulated. (G) In several cases increased en a n tio selectiv ity is

o b s e r v e d.2 2

The principle o f m icroscopic re v e rs ib ility states th at the enzym e m echanism m ust be the same in th e forw ard directio n as in the reverse direction (for a given set o f conditions) . 75 So, if the enzym e is operating in the re v e rs e , esterificatio n , direction it can be deduced that the m echanism w ill be the exact reversal of the hydrolytic method. T herefore, it follow s that if one en an tio m er is hydrolysed faster th a n the other, then the sam e en antiom er will be esterified fa s te r, when the enzym e is operated in the reverse directio n . T h is argum ent holds for enzym es whose mechanism do n o t alter when the bulk m edia is changed from aqueous to an o rg an ic solvent, i.e. if the principle o f m icroscopic reversibility is o b serv ed (Schem e 1.32).

FAST

R * -O A c + HzO R*—OH + CH3COOH

(R) FAST R * -O A c (S) + h2o SLOW SLOW R * -O H S ch em e 1.32 CH3COOH

This allows access to eith er enantiom er by sim p ly altering the so lv en t and the nature o f the starting m aterial (S ch em e 1.33) . 7 6

/ ° y — r ^ o A c PPL h2o _{V ^ /v J - ^ o h} 7 8 % yield, 9 4 % e e \ _o ' — PPL CH30C(0)CH3 Y _ < / ^ U ^ - O A c 74 % yield,

PPL = porcine pancreatic lipase 6 8% ee

Schem e 1.33

E sterification reactions can be carried out as m e n tio n ed above in biphasic system s or using the free acid as acy l donor in an org an ic solvent such as isooctane. The problem s w ith such sy stem s is that as the reaction proceeds w ater is produced. The enzym e can now hydrolyse the ester and hen ce an equilibrium concentration o f ester w ill be achieved (S ch em e 1.34).

O

R - O H + R,OOOH | R - O ' ^ ' R , + H20

Schem e 1.34

F urtherm ore, as the reaction proceeds to e q u ilib riu m the enan tio selectiv ity can decrease, this can be sim p ly ex p lain ed . If fo r exam ple, the (R ) enantiom er is esterified p refere n tia lly then as the reaction proceeds the levels of the (R ) e s te r w ill increase.

H ow ever, as the reaction approaches equilibrium the (R ) este r will be hydrolysed preferentially. In the w orst case scen ario racem ic ester w ill achieved, if the reaction is left long en o u g h at e q u ilib r iu m. 7 7

The problem has been recently circum vented. Novel acy latin g agents have been intro d u ced, 80 w hich effectively mean th a t the este rifica tio n is operated under irrev ersib le c o n d itio n s. 7 8 -7 9 The acylating agents are enol acetates, the ester product being produced along w ith an enol w hich quickly tautom erises to an inactive aldehyde or ketone (Schem e 1.35).

R -O H + R

O

R j = H (vinyl acetate), R i = CH3 (isopropenyl acetate)

Schem e 1.35

Since no equilibrium is set up the faster reacting en a n tio m er is not rev ersib ly hyd ro ly sed , and the en an tio selectiv ity or enan tio sp ecificity w ill not d ecrease w ith conversion.

C H A P T E R TW O

C hem oenzvm atic sy n th esis of optically p ure (3-blocker.

In document The chemoenzymatic preparation of optically active molecules (Page 36-51)