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Lipase chemoselectivity – kinetics and applications
Cecilia Hedfors
Licentiate Thesis
School of Biotechnology Royal Institute of Technology
Stockholm 2009
© Cecilia Hedfors School of Biotechnology Royal Institute of Technology AlbaNova University Center 106 91 Stockholm Sweden ISBN 978-91-7415-275-3 TRITA-BIO Report 2009:7 ISSN 1654-2312 Printed in Stockholm, April 2009 UniversitetsService US AB Box 700 14 100 44 Stockholm Sweden
ABSTRACT
A chemoselective catalyst is preferred in a chemical reaction where protecting groups
otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei
lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a
transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl
acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour
of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times
higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase.
The KM towards the thiol was more than two orders of magnitude higher than the KM towards
the corresponding alcohol. This was the dominating contribution to the high chemoselectivity
displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as
catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction
without using protecting groups (paper II). Poly(ε-caprolactone) with a free thiol at one of
the ends was synthesized in an enzymatic ring-opening polymerization initiated with
mercaptoethanol or terminated with either 3-mercaptopropionic acid or γ-thiobutyrolactone.
SAMMANFATTNING
En kemoselektiv katalysator är att föredra i en kemisk reaktion där skyddsgrupper annars är
nödvändiga. I en transacyleringsreaktion med etyloktanoat som acyldonator och hexanol eller
hexantiol som acylacceptor var den kemoselektiva kvoten, definierad som (kcat/KM)OH /
(kcat/KM)SH, stor för både Candida antarctica lipas B och Rhizomucor miehei lipas (artikel I).
Den kemoselektiva kvoten för den ickekatalyserade reaktionen var 120, till fördel för
alkoholen. Jämfört med den ickekatalyserade reaktionen var kemoselektiviteten 730 gånger
större för Candida antarctica lipas B och tio gånger större för Rhizomucor miehei lipas. KM
för tiolen var mer än två tiopotenser större jämfört med KM för motsvarande alkohol. Detta
var det dominerande bidraget till den stora kemoselektiviteten som de båda lipaserna
uppvisade. I artikel II användes Candida antarctica lipas B som katalysator för enzymatisk
syntes av tiol-funktionaliserade polyestrar i en enkärlsreaktion utan användande av
skyddsgrupper. Poly(ε-kaprolakton) med en fri tiolgrupp i en av ändarna syntetiserades med
enzymatisk ringöppningspolymerisation initierad med merkaptoetanol alternativt terminerad
med 3-merkaptopropionsyra eller γ-tiobutyrolakton.
LIST OF PUBLICATIONS
This thesis is based on the following publications which are referred to by their Roman numerals:
I Lipase chemoselectivity between thiol and alcohol acyl acceptors in
a transacylation reaction. C. Hedfors, K. Hult and M. Martinelle. Manuscript II Thiol end-functionalization of poly(ε-caprolactone), catalyzed by
Candida antarctica lipase B. C. Hedfors, E. Östmark, E. Malmström, K. Hult and M. Martinelle. Macromolecules 2005, 38, 647-649.
TABLE OF CONTENTS
1. INTRODUCTION 1
2. ENZYME SELECTIVITY 3
2.1 Regioselectivity 32.2 Enantioselectivity 4 2.3 Chemoselectivity 5 2.4 The Michaelis-Menten equation 5
3. TRIACYLGLYCEROL LIPASES 7
3.1 Reaction mechanism 8 3.2 Candida antarctica lipase B 10 3.3 Rhizomucor miehei lipase 10 3.4 Active site titration of immobilized lipase 10
4. LIPASE CATALYZED CHEMOSELECTIVE TRANSACYLATION REACTIONS 13
4.1 Chemoselectivity 14
5. LIPASES IN POLYESTER SYNTHESIS 17
5.1 Polycondensation 17 5.2 Ring-opening polymerization 18 5.3 Selective lipase polymerization 20 5.4 Chemoselective thiol end-functionalization of polyesters 20
ACKNOWLEDGMENTS 23
REFERENCES 25
1. INTRODUCTION
The existence of life is fascinating. How was it created and why does is exist at all?
Independently of how life began, one of its keys is enzymes. Enzymes are proteins catalyzing
chemical reactions by lowering the energy barrier when a substrate is transformed to a
product, increasing the probability of a reaction. The rate enhancement of an enzyme
catalyzed reaction, compared to the uncatalyzed reaction, is often in the magnitude of 108
[Wolfeneden, 2006], but the range is wide. The enzyme with the highest rate enhancement
known is orotidine monophosphate decarboxylase (EC 4.1.1.23), which converts orotidine
monophosphate to uridine monophosphate. The rate enhancement of this enzyme is 1017, or
with other words, the half-time of the reaction is 18 milliseconds with enzyme and 78 million
years without [Miller et al, 2002]. Enzymes are also important in another perspective as they
often display substrate selectivity. This allows them to control reaction pathways, prohibiting
side-reactions that could cause unwanted effects in a living organism. This selectivity is a
powerful tool and we learn more and more how to use it in vitro. This thesis will consider two
lipases, Candida antarctica lipase B and Rhizomucor miehei lipase, both capable to
distinguish between an alcohol and a thiol in transacylation reactions, and also, the use of
Candida antarctica lipase B as catalyst in thiol end-functionalized polyester synthesis.
Ever since the discovery of enzymes and their ability to catalyze chemical reactions, scientists
have tried to take advantage of their properties. With the technical developments in the
biotechnology area, it is possible to produce enzymes on a large scale (fermenter size > 10
m3) to a relatively low cost; enzyme concentrates are sold from 5 €/L [Bornscheuer and
Buchholz, 2005]. This opens the door for more industrial processes to use enzyme catalysts
and benefit from their high substrate selectivity.
- 1 -
The production of enzymes reached a value of 2 billion € in year 2007 with Novozymes A/S,
which held a market share of 47 percent, as the dominating manufacturer [Novozymes report
2007]. Enzymes can be used as final products, for example in detergents or in pharmaceutical
and animal feed industries, or as a processing aid like in textile, leather and sugar industries.
Enzymes are also used in food and beverage production; alcohol, baking and dairy industries.
The two dominating market areas for enzymes, with respect to volume and value of the
enzyme, are food processing (40-45%) and detergent industry (35-40%) [Bornscheuer and
Buchholz, 2005]. The single largest enzymatic industrial process is the production of high
fructose corn syrup from corn starch. The starch is broken down to oligosaccharides with the
enzyme α-amylase (EC 3.2.1.1), and further to glucose by glucoamylase (EC 3.2.1.3). Xylose
isomerase (EC 5.3.1.5) converts the glucose to fructose, yielding the syrup used as a substitute
to sugar [Cheetham, 2000].
- 2 -
2. ENZYME SELECTIVITY
One of the main advantages of using enzymes as catalysts is that they often display substrate
selectivity, i.e. the ability to favor one of many possible substrates. A chemically catalyzed
multiple step process, possibly needing protecting groups, was with use of enzymes made in a
one-step reaction with good yield (paper II). There are different kinds of substrate selectivity,
enzymes can for example be regioselective, enantioselective or chemoselective.
2.1 Regioselectivity If the production of one structural isomer is favored above others, the reaction is said to be
regioselective [Smith and March, 2001]. Oligosaccharides are formed from monosaccharides
which are linked together by glycosidic bonds (figure 1).
O
O OOH
OH
O
OOH
OH
O
O
OH
OH
O
OOH
OH
O
O OOH
OH
α1,4-glycosidic bond
α1,6-glycosidic bond
OH
OH OH
OH
Figure 1. The structure of glycogen with the α1,4-glycosidic and α1,6-glycosidic bonds marked.
Depending on the linkage, different kinds of oligosaccharides are formed. In glycogen, the
bonds between the glucose molecules are mainly α1,4-glycosidic bonds, but one in twelve is
- 3 -
an α1,6-glycosidic bond, which makes the polymer branched. Glycogen synthase (EC
2.4.1.11) can only form the α1,4-glycosidic bonds to yield α-amylose and are thus
regioselective. The branching of α-amylose to form glycogen is accomplished by amylo-
(1,4→1,6)-transglycosylase (EC 2.4.1.18) [Voet and Voet, 1995].
2.2 Enantioselectivity Enantiomers are a pair of molecular entities which are non-superposable mirror images of
each other [Moss, 1996] and enantioselectivity is the ability to distinguish between the two
enantiomers. Aspartame is a sweetener 200 times as sweet as sucrose. In the production of
aspartame, thermolysin (EC 3.4.24.27) can be used for the peptide bond formation between
the two precursors; blocked L-aspartic acid and L-phenylalanine methyl ester (figure 2). The
enantioselectivity of the enzyme makes it possible to use the cheaper racemic DL-
phenylalanine methyl ester, instead of the pure L-form. The unreacted D-form can be
racemised and reused in the synthesis, leading to high atom efficiency. Thermolysin is also
regioselective and reacts only with the α-carboxylate of the aspartic acid and not with the side
chain β-carboxylate, which would give rise to a bitter tasting β-aspartame [Cheetham, 2000].
L-Asp-L-Phe-OMe
L-Z-Asp + DL-Phe-OMe
L-Z-Asp-L-Phe-OMe D-Phe-OMe
L-Z-Asp-L-Phe-OMe
D-Phe-OMe
thermolysinracemerisation
HCl
deblocking
(aspartame)
aspartame
H3NNH
O
O
O
O
O
Figure 2. Production scheme of aspartame. Thermolysin is used in the first step as an enantio- and regioselective catalyst. The produced di-peptide forms a complex together with the unwanted form of phenylalanine (D-Phe-OMe) and precipitates. The D-Phe-OMe can be racemised and used as starting material again. Z = benzyloxycarbonyl protecting group on the amine group of aspartate.
- 4 -
2.3 Chemoselectivity Chemoselectivity is the existence of a preferential reaction of a chemical reagent with one
functional group in the presence of other functional groups [Smith and March, 2001]. The
lipase of Burkholderia cepacia (EC 3.1.1.3) catalyzes acylation of 1-phenylethanol hundred
times faster than of the corresponding amine using methyl butyrate as acyl donor
[Cammenberg et al, 2006].
O
O
NH2
OH
O
O
NH
O
100:1
+
+
+
Bcl
Figure 3. In a transacylation reaction Burkholderia cepacia lipase (Bcl) prefer the alcohol over the amine as acyl acceptor by a factor of 100.
As we have shown, Candida antarctica lipase B catalyzed the acylation of alcohol 88 000
times faster than the corresponding thiol in a transacylation reaction (paper I).
2.4 The Michaelis-Menten equation Most enzymes follow the kinetics proposed by Michaelis and Menten in year 1913. Enzyme
(E) and substrate (S) first form an enzyme-substrate complex (ES), called the Michaelis-
Menten complex, which is a non-covalently bound intermediate. The complex can then either
dissociate or cross the reaction energy barrier to form enzyme and product (P), (equation 1).
The reaction rate (v) of product formation is dependent on the catalytic constants kcat and KM,
the substrate concentration [S] and the enzyme concentration [E], according to the Michaelis-
Menten equation (equation 2). The specificity constant, kcat/KM, is an apparent second-order
rate constant of the enzyme reaction reflecting the specificity and efficiency of the reaction
(equation 3). Enzyme selectivity is the ratio of the specificity constants of the two reactions.
- 5 -
E + S ES E + PEP (equation 1)
[ ] [ ][ ]S
SE
M
0
+=
Kkv cat (equation 2)
[ ] [ ]SEM
freecat
Kkv = (equation 3)
- 6 -
3. TRIACYLGLYCEROL LIPASES
Triacylglycerol lipases (EC 3.1.1.3), in this thesis called lipases, belong to the family of
hydrolases acting on ester bonds. Lipases hydrolyze triglycerides to glycerol and free fatty
acids. They are found in animals, plants, bacteria and fungi, where they play an important role
in the metabolism. Lipases have an α/β-hydrolase fold [Ollis et al, 1992], a conserved
catalytic triad (Ser, His, Asp/Glu) [Brady et al, 1990] and an oxyanion hole which stabilizes
the oxyanion in the transition state [Brzozowski et al, 1991]. Most of them share a consensus
sequence of Gly/Thr-X-Ser-X-Gly making up the nucleophilic elbow containing the catalytic
serine [Wong and Schotz, 2002].
The definition of lipases and how they differ to esterases, also acting on ester bonds, have
been discussed over the years. Many lipases have a lid, an amphiphilic loop covering the
active site, which undergoes a structural change upon binding to a lipid surface [Brzozowski
et al, 1991]. Most of the lipases having a lid show interfacial activation, i.e. the lipase activity
increases at the critical micellar concentration. This was for long the definition of a lipase, but
since not all lipases have a pronounced lid or show interfacial activation, the definition was
changed in 1997 to “a carboxyl-esterase that hydrolyse long-chain acyl glycerols” [Verger,
1997].
Lipases have found many industrial applications, as reviewed by Ghanem, 2007 and Houde et
al, 2004. Detergent formulas use proteases, cellulases and lipases with broad substrate
selectivity and good activity in basic environments and at high temperatures. The most
common lipase used in detergents is Thermomyces lanugiosus. It is produced by Novozymes
A/S with trade names like Lipolase® Ultra, Lipo Prime™ and Lipex®. Kinetic resolution is
an additional large area for lipases. For example is BASF producing more than 2 500 tons
annually of enantiopure secondary amines using lipase as catalyst. Lipases are also widely
- 7 -
used in oleochemical transformations, for example to make cocoa butter from cheaper
glycerides and to produce isopropyl myristate for use in cosmetics.
3.1 Reaction mechanism The reaction mechanism of lipases involves the three catalytical residues; serine, histidine and
aspartate/glutamate, which are arranged to lower the pKa of the serine hydroxyl group and
thus enable it to do a nucleophilic attack on the carbonyl carbon of the substrate (scheme 1,
E). The first substrate, an acyl donor, finds its way into the active site of the enzyme and a
Michaelis-Menten complex is formed. The histidine acts as a general base, activating the
serine hydroxyl group, which attacks the carbonyl carbon on the substrate, forming a
tetrahedral intermediate (scheme 1, TI 1). The oxyanion charge is stabilized by hydrogen
bonds to the residues of the oxyanion hole, while the positive charge of the histidine is
stabilized by aspartate/glutamate. The tetrahedral intermediate collapses and by expulsion of
the first leaving group, an alcohol, the acyl enzyme intermediate is formed (scheme 1, AE).
Asp / Glu
O
O
His
H-N N
Ser
H-O
oxyanion hole
Asp / Glu
O
O
His
H-N N-H
Ser
O
oxyanion hole
Asp / Glu
O
O
His
H-N N
Ser
O
oxyanion hole
Asp / Glu
O
O
His
H-N N-H
Ser
O
oxyanion hole
OR1 O
R2
R2
OR2
OOH
R1O R2
O
OHR1
H2O
HO R2
O
acylation
deacylation
Free enzyme (E) Thetrahedral intermediate (TI 1)
Acyl enzyme (AE)Thetrahedral intermediate (TI 2)
Scheme 1. The enzyme mechanism of lipases is exemplified by a hydrolysis reaction. The first substrate, an ester, is attacked by the serine in the enzyme active site creating an acyl enzyme and the first product, an alcohol. The second substrate, a water molecule, attacks the acyl enzyme and a new ester is formed and released from the enzyme.
- 8 -
The tetrahedral intermediates are flanked on both sides by transition states, where bonds are
broken and formed. The transition states are the highest energy barriers the reaction has to
overcome. The deacylation step consists of the release of the substrate in the acyl enzyme.
The second substrate, an acyl acceptor, attacks the carbonyl carbon in the acyl enzyme
forming the second tetrahedral intermediate (scheme 1, TI 2). A proton is transferred, now
from the second substrate via the histidine to the serine oxygen. The second product is
released from the enzyme, which is then ready for the next cycle.
Depending on the substrates, different outcomes can be achieved. An ester as acyl donor will
create an alcohol when forming the acyl enzyme. If the acyl acceptor is water, the product
will be an acid and the overall reaction is hydrolysis of the ester (scheme 1). If the lipase is
used in solvents, an alcohol can be used as acyl acceptor and the product will be a new ester.
In the same way a thiol or amine as acyl acceptor will produce a thioester or amide as product
(scheme 2).
R1O R2
O
R3O R2
O
R3S R2
O
R3NH
R2
O
Acyl enzyme
R1 OH
R3 OH
R3 SH
R3 NH2
Ser OH
Free enzyme
Ser O R2
O
Ser OH
Free enzyme
Ser OH
Free enzyme
Ser OH
Free enzyme
Thioester
Amide
Ester
Scheme 2. Lipases can form esters, thioesters or amides depending on the acyl acceptor.
- 9 -
3.2 Candida antarctica lipase B The lipase B from the yeast Candida antarctica consists of 317 amino acids and weights 33
kDa. The active site residues are serine 105, histidine 224 and aspartate 187. The oxyanion
hole consists of backbone and side group of threonine 40 and the backbone of glutamine 106
[Uppenberg et al, 1994].
Candida antarctica lipase B (CalB) often shows high enantioselectivity and is a popular
catalyst for resolution of alcohols, amines, acids and esters as reviewed by Ghanem, 2007 and
Gotor et al, 2006. Resolution of secondary alcohols and amines are made at industrial scale by
BASF [Schmid et al, 2001]. In polyesterification reactions, CalB shows high performance
and its use have extension to functionalization of polymers as reviewed by Varma et al, 2005.
A number of engineering approaches have been applied on CalB to change the substrate
specificity, alter the enantioselectivity or to make non-conventional reactions like aldol
reactions and Michael-type additions as reviewed by Hult and Berglund 2003 and 2007. CalB
is commercially available from Novozymes A/S and Boehringer Mannheim. In this thesis the
preparation Novozym 435, which is CalB immobilized on an acryl resin, was investigated.
3.3 Rhizomucor miehei lipase Rhizomucor miehei lipase (Rml) is a fungal lipase, has 269 amino acids and weights 29 kDa.
The catalytic residues are serine 144, histidine 257 and aspartate 203 [Brady et al, 1990]. The
backbones of serine 82 and leucine 145 constitute the oxyanion hole [Norin et al, 1994]. Rml
has a pronounced interfacial activation and the lid region is constituted of the amino acid
residues 85 to 91 [Brzozowski et al, 1991].
Rml is frequently used as an industrial catalyst, for example in fat modifications for
cosmetics, bio-diesel and food industry [Houde et al, 2004]. Resolution of chiral compounds
using Rml as catalyst has been reviewed by Alcántara et al, 1998. Lipozyme®, from
Novozymes A/S, is Rml immobilized on ion-exchange resin and was the preparation
investigated in this thesis.
3.4 Active site titration of immobilized lipase The reaction rate is dependent on the amount of active enzyme. To measure the amount active
lipase, the enzyme preparation can be titrated with an active site inhibitor. The inhibitor has a
leaving group whose concentration can be measured by UV-Vis or fluorescence spectroscopy
[Rotticci et al, 2000; Dijkstra et al, 2008].
- 10 -
In paper I, the amount of active enzyme of two immobilized enzyme preparations, Novozym
435 (CalB) and Lipozyme® (Rml), was measured by active site titration. The active site
inhibitor methyl 4-methylumbelliferyl hexylphosphonate (figure 4) was used, and the amount
of 4-methylumbelliferone released was measured using a fluorometer [Fujii et al, 2003;
Magnusson et al, 2005].
O OOPO
OC6H13
Figure 4. The active site inhibitor methyl 4-methylumbelliferyl hexylphosphonate.
The amount of active lipase was measured to 3.3 weight percent on Novozym 435 and 0.14
weight percent on Lipozyme® (table 1). The inhibition of immobilized enzyme was slow
compared to lipase in solution. After four days incubation with the inhibitor, a plateau value
was reached. This may be compared with inhibition of CalB solubilized in an aqueous buffer,
where a plateau value was reached after 30 minutes using the same inhibitor [Hedfors,
unpublished data].
Table 1. Active site titration using methyl 4-methylumbelliferyl hexylphosphonate.
Lipase nmol lipase / g carrier weight percent
Novozym 435 1000 3.3
Lipozyme® 35 0.14
- 11 -
- 12 -
4. LIPASE CATALYZED CHEMOSELECTIVE TRANSACYLATION REACTIONS
In paper I, the chemoselectivity between alcohol and thiol was investigated for the two
lipases CalB and Rml. The transacylation of ethyl octanoate with hexanol and hexanethiol as
acyl acceptors was used (scheme 3). The acyl donor concentration was kept constant to
achieve pseudo one-substrate kinetics. Apparent kinetic constants kcatapp, KM
app and
(kcat/KM)app were determined (table 2).
O R
O
XH
X R
O
OH
lipase
R = C7H15X = S or O
Scheme 3. Transacylation reactions with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptors.
Rml catalyzed the transacylation reaction with hexanol with a kcat ten times higher than CalB,
while the KM-values were almost the same (table 2). For the reaction with hexanethiol, neither
of the lipase preparations could be saturated with the thiol. The initial rates were directly
proportional to the hexanethiol concentration up to 1.8 M. Concentrations above 1.8 M were
not tested since that would have altered the reaction conditions severely. Only the specificity
ratio, (kcat/KM)app, from the Michaelis-Menten curve could be derived from these experiments.
Rml was 1 600 times more efficient in using hexanethiol as acyl acceptor than CalB (table 2).
In the hexanol reaction, Rml was 20 times more efficient.
- 13 -
Table 2. Apparent kinetic constants for the acyl transfer using ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor in cyclohexane.
Lipase (kcat/KM)app (s-1M-1) kcatapp (s-1)[a] KM
app (M)[a]
CalB
hexanol 710 14 ± 9 0.019 ± 0.002
hexanethiol 0.0081[b] - >1.8
Rml
hexanol 16000 130 ± 10 0.0084 ± 0.002
hexanethiol 13[b] - >1.8
[a] Non-linear regression of Michaelis-Menten equation.
[b] Calculated from rates as a function of substrate concentrations far below KM.
In the literature, only one KMapp-value for a thiol in a transacylation reaction with a lipase can
be found. In a reaction with oleic acid and butanethiol using Lipozyme as catalyst, the KMapp
of butanethiol is reported to 1.85 M [Caussette et al, 1997]. Reports of transacylation
reactions with thiol substrates, where the two lipases CalB and Rml are compared with respect
to their activities have been made by Weber et al, 1999. It is concluded that CalB and Rml
have the same enzyme activity when compared per gram immobilized enzyme after 24 h
reaction. Using the data from Weber et al. 1999, and combining it with the data from the
active site titration in table 1 (paper I), showed that Rml is 300 times faster than CalB in
transacylation with thiol. This is in line with our results (table 2), where the ratio between Rml
and CalB for the thiol transacylation reaction was 1 600. Iglesias et al. published a paper on
the chemoselectivity between thiols and alcohols for lipases in 1996. They found that only O-
acylation and no S-acylation occurs for mercaptoalcohols in a transacylation reaction using
the lipase from procine pancreatic, Candida cylindracea or Rhitzomucor miehei as catalysts.
The low reactivity of thiols for CalB has been noticed by Öhrner et al. in 1996, investigating
the acylation of secondary alcohols and their amine and thiol analogues.
4.1 Chemoselectivity There was a large difference in specificity constants (kcat/KM) between the two acyl acceptors,
hexanol and hexanethiol, for both CalB and Rml (table 2). The ratios of the specificity of the
two reactions, (kcat/KM)OH / (kcat/KM)SH, gave the chemoselectivity (table 3). Both lipases
- 14 -
showed a much higher selectivity for the hexanol as compared to the hexanethiol, and this
was the major contribution to the great chemoselectivity displayed by the lipases.
Table 3. Chemoselectivity between hexanol and hexanethiol in the acyl transfer reaction with ethyloctanoate as acyldonor.
Catalyst chemoselectivity ratios relative to uncatalyzed
(kcat/KM)OH / (kcat/KM) SH
CalB 88000 730
Rml 1200 10
(knon)OH / (knon)SH
Uncatalyzed[a] 120 1
[a] Background reactions with no enzyme. Vinyl octanoate was used as acyl donor since no reaction was detected within ten days using ethyl octanoate.
To put the chemoselectivity displayed by the two lipases into perspective, the
chemoselectivity of the uncatalyzed reaction was investigated. Using the same reaction
conditions as for the enzymes, no product was detected within ten days of reaction.
Consequently, the activated ester of vinyl octanoate was used. The transacyaltion reaction was
run in a competitive manner, and the ratio of the reaction constants (knon)OH / (knon)SH was
measured to 120. The enzymatic contribution was derived by dividing the chemoselectivity of
the lipases with the chemoselectivity of the uncatalyzed reaction. The enzymatic contribution
of the chemoselectivity was 730 for CalB and ten for Rml (table 3).
The conclusions that can be drawn from table 2 and 3, and from the data in the literature, are
that alcohols react more readily than thiols in transacylation reactions with lipases, and that
the thiol substrates posses high KM-values. Is the high KM the reason to the low kcat/KM for
thiols, as compared to the kcat/KM for the corresponding alcohols? The KM for hexanethiol is
more than two orders of magnitude higher than the KM for hexanol for both CalB and Rml. It
seems that the KM-effect is the dominating, or maybe the only reason to the high
chemoselectivity.
- 15 -
- 16 -
5. LIPASES IN POLYESTER SYNTHESIS
The production of polymers is a big and growing market. Synthetic polymers are found in
many products; plastics, fabrics and building materials just to mention a few. Today polymers
are made by chemical catalysis, but during the last ten years the interest in enzyme-catalyzed
polymerization has grown. The advantages of using enzymes as catalyst are that they often
display high selectivity, are non-toxic and operate under mild reaction conditions; i.e.
temperature and pH [Varma et al, 2005]. The group of enzymes receiving most attention in
polyester synthesis is lipases. Their hydrolytic activity can in a low water environment be
turned to, for example, ester synthesis and produce polyesters when suitable monomers are
provided. Lipases can make polyesters by either polycondensation or ring-opening
polymerization.
5.1 Polycondensation Polyesters can be synthesized from diacids (or diesters) and diols (AA, BB-type monomers)
or from hydroxy acids (AB-type monomers) by polycondensation.
HO OH HO OH
O O
HO O O O OH
O O O O
HOOH HO
OO
O O
O
m
+mn
m m
n n
p
mm
OHO
m
p
lipase
lipase
A)
B)
Scheme 4. Schematic mechanism of polycondensation reaction. A) Example of an AA, BB-type reaction with diacid and diol as monomers. B) Example of AB-type reaction with a hydroxy acid monomer.
- 17 -
To receive polyesters of high molecular mass, water produced during the reaction has to be
removed to shift the equilibrium from hydrolysis to elongation of the polyester. The
conventional way of receiving polyesters by polycondensation polymerization, is to use an
acidic catalyst at high temperature (> 200◦C) and under reduced pressure. At this high
temperature, side reactions such as dehydration of diols can occur [Varma et al, 2005]. By
using a lipase as catalyst, the temperature can be lowered, leading to less side reactions. The
two pioneering works on enzyme catalyzed polycondensation of diacids and diols by
Okumura et al. in 1984 and of hydroxyl acids by Ajima et al. in 1985 have been followed by
many more as reviewed by Varma et al. 2005.
5.2 Ring-opening polymerization Polyesters can be made by a ring-opening polymerization (ROP) reaction if the monomer
consists of a ring-closed ester, for example lactones or lactides (figure 5).
OO
O
O
1 2 3
O O O
O O
m n
Figure 5. Lactones (1), cyclic carbonates (2) and lactides (3) can be used as monomers in ring-opening polymerization.
R OH R O
OO
O
m
OH
m+lipase
R O
OO
O
m
OH
m n+1n+lipase
R O
O
OH
m
Initiation
Propagation
Scheme 5. Schematic mechanism of a ring-opening polymerization (ROP) reaction. The reaction is initiated by an alcohol or water and then the opened monomer can be used for further reaction.
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The advantage of using a ring-ester monomer is that no product is formed in the acylation
step. Thus, the atom efficiency is 100 percent and the equilibrium is shifted to elongation
since no by-product is formed which can hydrolyze/cleave the polyester. The ring-opening
polymerization requires an initiator to start the reaction. Thus, by defining the initiator to
monomer ratio the polymer length can be controlled.
Enzyme catalyzed ROP of lactones was first published independently by the groups of Knani
et al. and Uyama and Kobayashi, both in 1993. Organometallic initiators show high activity
towards small lactones (4-7 member rings) and the polymerizability decreases with the ring
size, since the driving force for ROP is the ring-strain [Duda et al, 2002]. Lipases, on the
other hand, show increasing polymerizability with increasing ring size [Duda et al, 2002; van
der Mee et al, 2006]. The reason for this was further investigated for CalB and it was shown
that when the lactone possessed a transoid conformation, which is only possible for larger
lactones, the reactivity in the enzyme increases [van Buijtenen et al, 2007]. With larger
lactones, the polymeric product becomes more like low density poly(ethylene) in its physical
properties, while the ester groups may still make it biodegradable [Focarete et al, 2001]
O
O
E-OH
O
OE-O
E-O
OOH
E-O
OOH
O
OO
nR
O
OOH
H n
n ≥ 1
O
OOH
Rn+1
R OH
A n = 0B n ≥ 1
A n = 0B n ≥ 1
A
B
Scheme 6. The mechanism of ring-opening polymerization; A initiation and B propagation. The first substrate, a ring-closed ester, is opened by the enzyme, creating the acyl enzyme. The second substrate, which is an alcohol or water in the initiation step (A), or the hydroxyl end of a polymer in the propagation step (B) attacks the acyl enzyme and the product is released.
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5.3 Selective lipase polymerization One of the major benefits of using lipases as catalysts in polymer synthesis is the possibility
of easily achieving selective polymerization. Enantiopure polyesters can be synthesized by
using ω-methylated lactones and CalB as catalyst [van Buijtenen et al, 2007]. It has also been
shown that CalB can selectively grow polymers from glycopyranosides [Córdova, 1998]. The
selectivity is towards the primary alcohol, being much more reactive than the secondary
alcohols in the glycopyranoside. CalB can grow polyesters selectively from dendrimeric
initiators [Córdova, 2001]. Also the use of a selective lipase in grafting from polymers has
been investigated [Duxbury, 2007]. In both these cases the selectivity is mainly due to the
relative steric hindrance of the hydroxyl groups in a partly grafted polymer/dendrimer
compared to the hydroxyl groups at the ends of the growing polyesters. It has to be
remembered that the substrate must be able to reach the active site of the enzyme for
polymerization or hydrolysis to occur.
5.4 Chemoselective thiol end-functionalization of polyesters Polyesters with free thiol ends are of interest due to their ability to cross-link with enes to
form networks [Dondoni, 2008]. The network makes the polymer more rigid and the
technique is used for example in production of polymeric films used in paints, lacquer and
glue. Thiol end-functionalized poly(ε-caprolactone) has been made chemically by Trollsås et
al, using Al(iOPr)3 as catalyst and α-(2,4-dinitrophenylthio)ethanol as combined initiator and
protection group [Trollsås et al, 1998, Carrot et al, 1999].
In paper II, we showed a novel route to thiol end-functionalized polyesters in a one-pot
synthesis using the chemoselective CalB as catalyst. As backbone in the polymer chain, ε-
caprolactone was used. The polymer was either initiated with mercaptoethanol or terminated
with γ-thiobutyrolactone or 3-mercaptopropionic acid (scheme 7). The enzyme used was the
commercially available Novozym 435, which is CalB immobilized on acrylic resin. The
degree of thiol functionalization was 70-90 percent. Since we utilized the high
chemoselectivity displayed by the lipase, no protection of the thiol group was needed.
For the initiation reaction with mercaptoethanol (A, scheme 7), 70% of the polymers were
initiated with the alcohol moiety of mercaptoethanol, giving the desired thiol end-
functionalized polymer. Of the remaining polymers, 20% were water-initiated and 10% were
initiated with the thiol moiety of mercaptoethanol. Using 6-mercapto-hexanol as initiator, the
degree of thiol functional polymers can be increased to 97% [Takwa et al, 2006].
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HSOH O
O
+ HSO
OO
OOH
n-1
O
O
HOO
O
OOH
n-1
+ S
O
HOO
SH
O
O
n
1 2 3
2 4
4
5 6
I)
II)
+ HOO SH
O
O
n4
7 8
HO SH
O
n
n
B)
A)
H2O +
Scheme 7. A) Mercaptoethanol (1) was used as initiatior in the polyesterification of ε-caprolactone (2) giving the thiol end-functionalized product (3). B) Poly(ε-caprolactone) was synthesized from ε-caprolactone (2) initiated with water. I) Poly(ε-caprolactone) (4) was end functionalized by γ-thiobutyrolactone (5) or as in II) with 3-mercaptopropionic acid (7).
Table 4. Analysis of the thiol functionalized polymer products.
Polymer product
I or T I:M T:M Time (h) solvent polymers with -SH (%)
Mn (Da)
Mw/Mn (Da)
Dp
3 1 1:30 24 bulk 70 2900 1.4 26
6 5 5:1 72 MTBE 90 2100 1.8 19
8 7 1:30 24 bulk 70 6700 2.7 59
The polymer product numbers correspond to the numbers in figure 7. I = initiator, T = terminator, M = monomer. The initiator to monomer, or terminator to monomer, ratio is specified. MTBE = methyl tertiary-butyl ether. Mn = number average molecular weight, Mw = weight average molecular weight, both measured by GPC (gel permeation chromatography). Dp = average polymer length.
- 21 -
For the two termination reactions, poly(ε-caprolactone) was first synthesized with water as
initiator. Termination with γ-thiobutyrolactone resulted in 90% thiol end-functionalized
polymers. The attractiveness of using γ-thiobutyrolactone, a ring-closed thioester, as
terminator is that no water by-product is formed, and thus the probability for hydrolysis of the
polymer is reduced. However, a large excess of terminator was needed since the thioester of
γ-thiobutyrolactone was not as good substrate for the enzyme as the esters in the poly-ε-
caprolactone. When 3-mercaptopropionic acid was used as terminator, 70% of the polymers
were thiol end-functionalized.
This new approach with thiol end-functionalization of polyesters using a chemoselective
lipase as catalyst has been followed up within the Biocatalysis group together with the
Coating technology group at KTH, by Takwa et al, giving macromonomers and materials
[Takwa et al, 2006 and 2008; Simpson 2008]. Kato et al. have made polyesters with free thiol
groups along the polyester chain by a polycondensation reaction of dimethyl 2-
mercaptosuccinate and hexane 1,6-diol using CalB as catalyst [Kato et al, 2009]. Also, Kerep
et al. reported that 2-mercaptoethanol initiated ring-opening polymerization of ε-caprolactone
with lipase and microwave irradiation gives a higher chemoselectivity than polymerization
with only lipase as catalyst [Kerep et al, 2007].
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ACKNOWLEDGMENTS Jag vill rikta ett stort TACK till följande personer: Prof. Karl Hult. För att jag fick möjlighet att göra mitt examensarbete i biokatalysgruppen och fascineras av enzymer, samt för att jag fick fortsätta som doktorand. Ditt intresse för biokemi smittar av sig och ditt sätt att ifrågasätta allt är utvecklande. Dr. Mats Martinelle. För bra handledning och lärorika diskussioner. Du tar dig alltid tid och gör allt lika noggrant. Prof. Eva Malmström och Emma Östmark. För bra samarbete och intressanta diskussioner om polymerkemi. Alla nuvarande och tidigare medlemmar i biokatalysgruppen. För alla trevliga stunder! För allt jag har lärt mig och all hjälp jag fått med både stort och smått. Nu kan jag sluta isolera mig framför datorn och återgå till labbet och vara mer social. Alla ni andra på plan 2. För trevliga samtal under lunch och fika. Speciellt tack till Lotta och Ela. Till mina vänner. För alla trevliga stunder, i lekparken, över en fika eller en trevlig middag. Till min stora släkt. Mamma och pappa, mormor och morfar, farmor och farfar, alla kusiner, morbröder, mostrar, farbröder och fastrar – ni betyder alla mycket för mig. Även alla släktingar på Davids sida vill jag tacka, speciellt Anna och Thomas. Till min lilla familj. David, Astrid och Irma, ni är så värdefulla för mig. Tack för allt stöd och all er omtanke.
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