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Enzymes as Catalysts in Synthesis of

Enantiomerically Pure Building Blocks—

Secondary Alcohols Bearing Two Vicinal Stereocenters

Rong Liu

Doctoral Thesis Stockholm, Sweden 2005

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan (KTH) i Stockholm framlägges till offentlig granskning för avläggande av

filosofie doktorexamen i organisk kemi. Fakultetsopponent: Professor Thorleif Anthonsen

Department of Chemistry Norwegian University of Science and Technology (NTNU)

Avhandlingen försvaras på engelska och finns även tillgänglig på KTHB websidan: http://www.lib.kth.se

© Rong Liu Dissertation for the degree of Doctor of Philosophy in Organic Chemistry, 2005. ISBN: 91-7178-129-3

Abstract Liu, R.; Enzymes as Catalys ts in Synthesis of Enant iomerical ly Pure Build ing Blocks: Secondary Alcolhols Bearing Two Vicinal S tereocenters. Royal inst i tu te o f Technology, SE-100 44 Stockholm, Sweden Enzymes as tools in organic synthesis have provided enormous advantages.

This thesis deals with the applications of enzymes in the kinetic resolutions of

racemic compounds. The stereochemistry of chiral compounds and the kinetics

of α/β hydrolase lipases are presented. From a practical point of view, the

handling of a large number of parameters that influences the kinetic

resolutions, especially enantioselectivity (E-value) are systematically

described. A variety of approaches employed for raising the yields to over 50%

are additionally discussed.

Methods for the preparation of synthetically useful chiral building blocks were

developed in this thesis. Thus, resolution of secondary alcohols bearing two

vicinal stereocentres are studied. These building blocks can serve as starting

materials for the synthesis of various enantiomerically pure compounds for

agrochemistry, pharmaceuticals, chemical industry, and particularly for the

total synthesis of pheromones.

Racemic 3-substitued 2-hydroxybutane derivatives were produced in fairly

high diastereomeric purities by a variety of chemical approaches, such as

epimerization, metal-catalysed asymmetric addition etc. Kinetic resolution of

these racemates was achieved by enzyme-catalysed reactions. Two lipases,

Candida antarctica lipase B and Pseudomonas cepacia lipase were found to be

useful in acylations as well as hydrolyses. In the biotransformations studied,

the presence and nature of the second vicinal stereocentre in the chiral

secondary alcohols investigated seemed to be important, e.g. in terms of the

efficiencies of sequential kinetic resolutions, and altering the selectivities as

well.

Keywords: enzyme, kinetic resolution, enantioselectivity, lipase, diastereoselectivity, epimerisation, metal-catalysed transformation, intramolecular alkylation.

iii

Abbreviations [α] Specific rotation aw Water activity Ac Acetyl Ar Aromate BD 2, 3 - butanediol Bn Benzyl c Conversion CALB Candida antarctica lipase B dr Diastereomeric ratio DAB 2, 3 - diacetoxybutane DMF Dimethylformamide DMSO Dimethylsulfoxide E Enantiomeric ratio ee Enantiomeric excess ES & EP Michaelis-Menten complex GC Gas chromatography kcat Turnover number KM Michaelis-Menten constant LC Liquid chromatography LDA Lithium diisopropylamide MS Mass spectrometry NMR Nuclear magnetic resonance PCL Pseudomonas cepacia lipase THF Tetrabuthydrofuran TS Transition state ∆G≠ Activation Gibbs free energy ∆H≠ Activation enthalpy ∆S≠ Activation entropy

List of articles This thesis is mainly based on the following papers quoted in the text by means of their Roman numerals: I

Chemoenzymatic preparation of (2S,3S)- and (2R,3R)-2,3-butanediols and their esters from mixtures of d,l- and meso-diols Liu, R.; Högberg, H.-E. Tetrahedron: Asymmetry 2001, 12(5), 771-778.

II

Lipase-catalysed kinetic resolution furnishes the four stereoisomers of 3-bromo-2-butanol or its acetate from d,l- and meso-2,3-butanediol Liu, R.; Berglund, P. and Högberg, H.-E Tetrahedron: Asymmetry 2005, 16(15), 2607-2611

III

Chemoenzymatic preparation of isomerically pure 3-bromo-2-butyl ethyl malonate ester Liu, R.; Berglund, P. and Högberg, H.-E. Manuscript

IV

Enantiopure building blocks for the synthesis of 3-methyl-2-alkanols. Diastereoselective methylmetal addition to a chiral 2-methylaldehyde followed by lipase catalysed esterification Larsson, M.; Andersson J.; Liu R.; Högberg H. -E. Tetrahedron: Asymmetry 2004, 15(18), 2907-2915.

V

Attempted intramolecular enolate alkylation of anti-3-bromo-2-butyl, ethyl malonate ester Liu, R. Manuscript

Published papers, were reprinted with the kind permission of Elsevier science Ltd, UK.

iv

v

Table of contents

1 Introduction .........................................................................................1 1-1 Chiral compounds and enantiomers .................................................1 1-2 Optical rotation and enantiomeric purity..........................................2 1-3 Preparation of optically active compounds ......................................5 1-4 Catalytic kinetic resolution...............................................................7

2 Enzymes as catalysts in kinetic resolutions........................................9 2-1 Kinetics of enzymatic catalysis ......................................................10 2-2 Enantioselectivity ...........................................................................11 2-3 Enhancement of enantioselectivity.................................................13

2-3-1 Handling of substrates and reagents............................................... 13 2-3-1-1 Screening and chemical modification of substrates .............................. 14 2-3-1-2 Substrate engineering ........................................................................... 14 2-3-1-3 Addition of a non-competitive inhibitor ................................................ 15 2-3-1-4 Altering reagents................................................................................... 15

2-3-2 Media engineering ........................................................................... 15 2-3-2-1 Organic solvents and solid enzyme....................................................... 16 2-3-2-2 Co-solvents............................................................................................ 17 2-3-2-3 Biphasic aqueous-organic solutions ..................................................... 17 2-3-2-4 Control of water activity (aw)............................................................... 17 2-3-2-5 Ionic liquids ........................................................................................... 18

2-3-3 Handling of enzymes ....................................................................... 18 2-3-3-1 Screening of enzymes ............................................................................ 18 2-3-3-2 Immobilization and purity of enzymes................................................... 18 2-3-3-3 Protein engineering .............................................................................. 19 2-3-3-4 Chemical modification .......................................................................... 21 2-3-3-5 Coenzymes ............................................................................................ 21 2-3-3-6 Metal ions in enzymes ........................................................................... 21

2-3-4 Optimization of the reaction condition............................................ 22 2-3-4-1 Temperature.......................................................................................... 22 2-3-4-2 pH ......................................................................................................... 22

2-4 Improvement of the yield in enzyme catalysed resolutions ..........23 2-4-1 Enantioconvergent synthesis ........................................................... 23 2-4-2 Dynamic kinetic resolution.............................................................. 24 2-4-3 The meso-trick and enantiotopos selectivity ................................... 25

vi

3 Lipases................................................................................................. 26 3-1 Candida antarctica lipase B form, CALB ...................................... 28 3-2 Pseudomonas cepacia lipases, PCL ............................................... 31

4 Chemoenzymatic preparations of secondary alcohols bearing two vicinal stereogenic centres...................................................................... 33

4-1 Building blocks for synthesis of chiral natural products ............... 33 4-2 2,3-Butanediol (BD), Paper I ........................................................ 36

4-2-1 Chirality of BD and the synthetic strategy ...................................... 36 4-2-2 Thermodynamically favoured epimerisation ................................... 37 4-2-3 Screening of lipases for the kinetic resolutions .............................. 39 4-2-4 Binary liquid-solid phase equilibrium – recrystallisation .............. 43

4-3 3-Bromo-2-butanol and its derivatives (Paper II and III) ............. 45 4-3-1 Objectives and the synthetic route ................................................... 45 4-3-2 The vicinal bistereogenic centres of the substrates........................ 47

4-4 Intramolecular enolate alkylation Exploratory experiments, Paper V.................................................................................................. 49 4-5 3-Methyl-4-(phenylsulfanyl)butan-2-ol, Paper IV ....................... 51

5 Conclusion .......................................................................................... 55 6 Acknowledgements ............................................................................ 56 7 References........................................................................................... 58

1 Introduction of stereochemistry

1 Introduction 1-1 Chiral compounds and enantiomers A chiral molecule is defined as one that, due to its structural asymmetry or dissymmetry, is not superimposable on its mirror image. Such non-identical pairs of molecular mirror images are called enantiomers and are related to each other in the same way as your left and right hand (Figure 1-1). Enantiomers have identical physical and chemical properties, but differ when they are placed in a chiral environment e.g. when interacting with other chiral compounds or when subjected to plane-polarised light (see below).

Figure 1-1: Chirality, origin of life

Nearly all biological polymers are asymmetric and /or dissymmetric, and usually occur in homochiral form, i.e. all its component monomers having the same handedness. For instance, except for glycine, all amino acids in proteins are ‘left-handed’ or in the L-form, while all ß-nucleotides, saccharides in DNA and RNA, are ‘right-handed’ or in the D-form. Many drugs in use can exist in stereoisomeric forms. The pharmacological effects of drugs are often based on their interactions with proteins, which are homochiral and function as signal carriers, enzymes or receptors. The metabolism of a pair of enantiomers of a drug may therefore be highly enantioselective, leading to drastically different pharmacological potencies and activities of the enantiomers [Williams and Lee, 1985; Caldwell, 1995; Drayer, 1986; Kroemer et al., 1996; Testa, 1986]. Usually, only one of the enantiomers of a drug displays the desired therapeutic activity, whereas the other can completely lack activity, or may have deleterious effects. Some examples of the variation of the biological activities of various enantiomeric pairs are shown in Figure 1-2.

1


O

Cl

NHCH3O

Cl

H3CHN

R-Ketaminepsychotic

S-Ketamineanaesthetic

CH2

C NH2

CO2H

HO

HO CH2

CH2N

CO2H

OH

OH

S-alpha-methyldopahypertensive

R-alpha-methyldopainactive

H2NOCH2C CO2H

H2N H

CH2CONH2HO2C

NH2H

S-Asparaginebitter

R-Asparaginesweet

R-Limoneneorange fragrance

S-Limonenelemon fragrance

mirror plane

Figure 1-2. Examples of enantiomers with different biological effects. 1-2 Optical rotation and enantiomeric purity Historically the concept of molecular chirality was actually based on the discoveries of polarization of light and that some crystals, which were mirror images of each other, were able to rotate plane polarized light with an equal angle but in opposite directions [Eliel and Wilen, 1994]. The two mirror image molecules are called optical isomer, namely enantiomers. The optical rotation can be determined by Biot's law (Equation 1-1), where [α], the specific rotation,

2


depends on the wavelength of the monochromatic light (nm) and the temperature. These are usually indicated as subscripts and superscripts respectively, e.g. [α]D

25

denotes the specific rotation for sodium-D line (589 nm) at 25 °C.

=[a]a

l * c Equation 1-1: Specific rotation. α is the observed rotation in degrees (°) either minus (–) (counterclockwise rotation) or plus (+) (clockwise rotation); c is the concentration of optically active compound (g/ml); l is the length of cell (dm). In an enantiomerically enriched mixture, the excess of one enantiomer over the other is called enantiomeric excess (ee) and this, as a percentage, is usually used to state the enantiomeric purity of a compound (Equation 1-2).

e.e. =moles of major enantiomer - moles of minor enantiomermoles of major enantiomer + moles of minor enantiomer

Equation 1-2: Enantiomeric excess (ee)

There are various techniques used for determination of enantiomeric purity. This includes non-separative and separative methods. Non-separative methods can also be applied to establish the absolute configuration of a chiral molecule, such as polarimetry, circular dichroism, nuclear magnetic resonance (NMR), isotopic dilution, X-ray crystallography and enzyme techniques [Allenmark 1988]. Polarimetry (or chiroptics) is an easy method commonly used in synthetic chemistry. The optical purity of a enantiomerically enriched substance is determined from its optical rotation [α]obs and the optical rotation of the pure enantiomer [α]max according to the Equation 1-3.

[ ][ ] 100(%)

max

×=αα obsee Equation 1-3

However, this method requires a relatively large amount of sample. Several factors can influence the measurements leading to erroneous ee-estimations, such as the presence of an impurity; the difficulty to control concentrations; and that solvent properties and other parameters may vary from time to time. In addition, for new compounds, the optical rotation of the pure enantiomer, [α]max, is

3


unknown. Thus optical rotation is not so useful for determination of enantiomeric purity. Because it gives the sign of the optical rotation, it is, however, a routine method for the determination of the absolute configuration of a chiral compound. Today the most common techniques used for determination of enantiomeric purity are separative methods, mainly chromatographic chiral separation techniques, in which gas chromatography (GC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are widely employed, thanks to their high efficiency and sensitivity. Enantioselective chromatographic separation techniques can be divided into indirect and direct methods. In the indirect method, enantiomers are derivatised with a large excess of an enantiomerically pure reagent to form a quantitative yield of two diastereoisomers. Thus the ee of the original enantiomers is equal to the de of the resulting diastereomers (de is the diastereomeric excess defined as the excess of one diastereomer over the other). The two resulting diastereomers possess dissimilar chemical and physical properties and can therefore be separated in achiral systems, for instance by NMR, where each of the diastereoisomers should give distinct chemical shifts. Normal capillary gas chromatography (GC) or high performance liquid chromatography (HPLC) can also be used to separate the diastereoisomers produced. The direct method requires a chiral separation medium. Thus, either the mobile phase is chiral (chiral solvent or chiral additives, e.g. metal complexation in chiral ligand-exchange chromatography, Davankov, 1977) or the stationary phase is chiral. The latter refers to a separation column with a chiral stationary phase, sometimes containing a special chiral selector, e.g. cyclodextrin or its derivatives, which can be dissolved in, or bound to a bulk liquid phase which coats or binds to a variety of carriers. Nowadays a great diversity of efficient enantioselective (chiral) columns are commercially available and have been widely used for assessing enantiomeric purity. In our investigations, GC and GC-MS equipped with a capillary column containing a chiral liquid phase have been employed for monitoring the efficiency of transformations, and analysis of enantiomeric purity. The enantiomeric excess, ee can then be calculated by Equation 1-4.

[ ] [ ][ ] [ ] 100(%) ×

+−=

SR

SR

AAAAee

Equation 1-4: R, S are a pair of enantiomers, [A] is the GC integration areas of corresponding enantiomer.

4


5

1-3 Preparation of optically active compounds Many bioactive natural products occur only in a single enantiomeric form and can often be obtained from its natural source by means of separation and purification techniques. This method has appreciatively brought about the plenty of "chiral pool". In synthetic chemistry there are two additional approaches can be employed for producing optically pure compounds. The first is stereoselective synthesis, the preferential formation of one stereoisomer over another in a chemical reaction. For instance (1) in Scheme 1-1, achiral reagents B2H6 or 9-BBN and MeMgBr, are able to stereoselectively attack the target molecules that possess of conformationally rigid structures. In these cases the stereoheterotopic faces of the double bonds lead to favour one diastereomer over the other [Smith, 1994; Carey and Sundberg, 1993; Xing et al., 1980]. Furthermore, asymmetric synthesis allow a non-chiral or a prochiral substrate transforming into a chiral non-racemic compound by addition of a chiral reagent, or by use of a chiral auxiliary ligand or by using a chiral catalyst. The latter two are more widely used in modern organic preparations. A chiral auxiliary ligand can be either used in an elaborate reagent, e.g. the R-BINOL derivative in (2) in Scheme 1-1 [Lin et al., 2001; Harada et al., 2004] or as a moiety covalently bounded to an achiral substrate. The major categories of chiral catalysts are organometallic complexes (3) [Anderson et al., 2004] and biocatalysts, i.e. enzymes (4) in Scheme 1-1 [Allain et al., 1993]. The second approach to enantiopure compounds is the separation of enantiomers, resolution. As mentioned in paragraph 1-1, enantiomers possess identical physical and chemical properties. Therefore it is difficult to separate them by common physical methods. In addition most conventional synthetic methods are usually not enantioselective. Many chiral compounds can therefore only obtained in form of the racemate, in which the (+)- and (–) enantiomers are present in a 1:1 ratio. Thus many methods for the resolution of racemic mixtures have been developed. One of them is kinetic resolution, in which the enantiomers of a racemic substrate react with different rates with a chiral, nonracemic reagent resulting in recovery of at least one of the enantiomers. A more detailed discussion of this method is presented in the following paragraphs.


OH

(1) With achiral reagents

(3) Metal transformation: organometallic chiral catalyst

O

OH

major minor

MeO

N

Ph

O

R

MeO

N

Ph

OH

R

NH2

R

N

Ot-Bu

SiMe3

FePdHI

FOP:

(2) Using chiral auxiliary ligands

Ar

O

Al(OiPr)3

OHOH

FOPAgOCOCF3

rt, CH2Cl2

63-96% e.e.

+ BH3SMe2 Ar

OH

+ CH3MgBr

up to 90% e.e.

(4) Biotransformation: biocatalyst

1) hydroboration

2) NaOH, H2O2

86% (with B2H6)100% (with 9-BBN)

2

OH

C4H9 chloroperoxidas

H2OH2O2

C4H9

O

96% e.e.

Scheme 1-1: Methods in stereoselctive synthesis.

6


7

1-4 Catalytic kinetic resolution Chemical reactions are initiated by collisions of molecules. Correct orientation of the colliding molecules will obviously be necessary. There are two crucial aspects that predominate the efficiency of a reaction between molecules. One is the electronic effect or chemical operator which is commonly referred to as reactivity or affinity, e.g. basicity / acidicity, neucleophilicity / electrophilicity, lipophilicity / hydrophilicity or polarity. The other is the steric effect that mainly determine the selectivity or specificity of reactions but can simultaneously affect the reactivity, for instance, steric hindrance can inhibit the attack of a reagent. A catalyst is defined as a substance which increases the velocity of a reaction without being changed or consumed itself in overall process. A catalyst interacts with either the substrate or reagent or both by utilising various types of binding effects prior to reaction, such as those mentioned just above . These serve to stabilise the activated complex at the transition state so that the transformation occurs with a lowered energy barrier (∆G≠) compared with that in the uncatalysed reaction. In other words, the reaction rate is increased by the catalyst. In addition, a chiral catalyst possesses a particular spatial structure, which leads to a selective or even specific recognition of one stereoisomer in the presence of the other(s). Such chiral discrimination depends on how well the chiral catalyst match sterically with one stereoisomer compared with the other(s), so that a more reactive activated complex can be formed with the former than the latter (Figure 1-3). In a kinetic resolution, the enantiomers in a racemic substrate (1:1-mixture of SR and SS) selectively recognise a chiral non-racemic reagent or catalyst and form a fitting and a misfitting diastereomeric substrate-catalyst-transition-state complex, which differ in transition-state energy (∆∆G≠, Figure 1-3) resulting in different reaction rates for the enantiomers. Thus the enantiomers can be resolved. Although metal catalysed transformations can be employed in kinetic resolutions of enantiomers, biocatalysis is nowadays developed fast and has turned out to be one of the most advantageous methods for this purpose. Thus biocatalysts are highly efficient resolving agents for many types of compounds, and further reveal other significant advantages such as mild and simple reaction conditions, good economy and low environmental impact.


SR + SS

PR + PS

∆∆G≠

GFreeEnergy

[SR-Cat]*

[SS-Cat]*

SR + SS PR + PS

ChiralCat

Reaction:

Reaction coordinate

rac-Substr Product mix

A misfit

A fit

Figure 1-3: Reaction energy profile for the kinetic resolution of enantiomers. SR and SS represent the substrate; while PR and PS are the products. The two peaks in the reaction

coordinate correspond to the transition states of the enantiomers.

8

2 Enzymes as catalysts in kinetic resolutions

2 Enzymes as catalysts in kinetic resolutions Enzymes are catalysts of chemical reactions in biological systems. They mainly consist of proteins which are built up in Nature from twenty different amino acids. The catalytic ability of enzymes depends upon their three-dimensional architectures, which are basically determined by the L-amino acid sequence. The three-dimensional structure of an enzyme, often reveals that it possesses an active site, the region where the reaction takes place. The active site is the core of the chemical operator in an enzyme, while the structural features of the surroundings of the active site, determines the steric selectivity. A rational explanation of the specific nature of enzyme action is the Three-Point Attachment rule suggested by A. G. Ogston (Figure 2-1) [Faber, 1995, loc. cit.]. In order for a substrate molecule to be held firmly in the three-dimensional space, there must be at least three different points of attachment of the substrate onto the active site of a catalyst. In Figure 2-1, case A displays that the structures are perfectly complementary, and the groups of substrate are correctly aligned and fit for binding to these sockets at active site of an enzyme. In case B, however, two of the groups will be misfit to their sockets, which hardly leads a stable binding.

C

M

L S

D

C

M

LS

DStereoisomers

Active site

L'

M'

S'L'

M'

S'

A B

Figure 2-1: Three-point attachment rule, the stereospecific nature of enzyme

9


2-1 Kinetics of enzymatic catalysis Many enzymatic reactions display a kinetic behaviour known as Michaelis-Menten kinetics (Equation 2-1).

[ ] [ ][S] K

k

M

cat

+⋅= SEν Equation 2-1

Enzyme catalysis requires that a substrate (S) initially binds non-covalently to the active site of the enzyme (E). The enzyme-substrate complex formed (ES), is called the Michaelis-Menten complex. A typical Michaelis-Menten curve is shown in Figure 2-2. As shown in this, KM, which is called the Michaelis-Menten constant, is defined as the substrate concentration at which the reaction rate, v, is half of its maximal value, i.e. v = 0.5⋅Vmax. The apparent first-order constant, kcat, also called turnover number, is the rate constant for conversion by the enzyme of the ES complex into product. The ratio kcat / KM is called the specificity constant k1 and represents the apparent second-order rate constant of the enzymatic reaction i.e. k1 = kcat / KM. The specificity constant is a very important one in the assessment of the overall efficiency of enzyme catalysis and is also used to determine the ability of an enzyme to discriminate between competing substrates.

νVmax

ν = (Vmax / KM)[S]Vmax

[S]

maxV21

KM

Figure 2-2: Michaelis-Menten kinetics, where reaction rate ν versus substrate concentration [S]

Enzymes integrate two independent processes, i.e. non-covalent binding and chemical catalysis, which are reflected in KM and kcat, respectively.

10


At a low concentration of substrate, where [S] << KM, the Michaelis-Menten equation describing the reaction rate is simplified to v = (kcat / KM)[E][S]. Michaelis-Menten equation is such a fundamental pattern in enzymatic kinetics. Many extensions and modifications of it are widely used, especially in steady state kinetic studies. For instance, the Lineweaver-Burk plot and the Eadie-Hofstee plot are linear diagrams derived from Michaelis-Menten equation. These diagrams are very useful for graphical data analysis that yields a variety of parameters and for the study of different types of inhibition [Fersht, 1999]. 2-2 Enantioselectivity The resolution of two enantiomers e.g. R and S in a racemate catalysed with an enzyme is an example of two competing reactions. As an integration of its unique spatial structure, an enzyme is chiral and will interact with the two enantiomers in different ways. If the reaction is irreversible, the ratio of the different reaction rates can be expressed as described in Equation 2-2. E is the enantiomeric ratio, that is the ratio between the specificity constants for the two competing enantiomers [Equation 2-3. Chen,1982].

[ ][ ] [ ] [ ]

[ ][ ][ ]SRE

SRv

SdRd ⋅=⋅==−

)K / (k)K / (k

S Mcat

R Mcat Equation 2-2

[ ] )K / (k

)K / (k

S Mcat

R Mcat=E Equation 2-3

According to the Arrhenius equation derived from transition state theory, the Gibbs free energy difference for a single enantiomer between the transition state and ground state is expressed as shown in Equations 2-4. Thus the free energy discrimination, (∆∆G≠ ), between the two diastereomeric enzyme-transition-state complexes in a kinetic resolution is described in Equation 2-5.

∆G≠ = −RT lnh

kT⋅ k1

= −RT ln

hkT

⋅kcat

KM

Equation 2-4

k = 81.3807 x 10-23 J/K (Boltzmann's constant) and h 6.62618 x 10-34 Js (Planck's constant)

11


∆∆G≠ = ∆G≠R - ∆G≠S = [ ]

)K / (k)K / (k

lnS Mcat

R McatRT− = - RT ln E

Equation 2-5 The E-value or enantiomeric ratio is thus a number, which describes the enantioselectivity of an enzyme and it is a kinetic constant that is independent of substrate concentration and conversion. By definition, an E-value for a reaction can be determined by evaluating the kinetic constants kcat and KM for both enantiomers. One deficit of this method is the difficulty of acquiring the appropriate enantiomerically pure substrates. When the appropriate Equations 2-6 and 2-7 are used, the E-value of a reaction can be calculated by determination of the conversion c and ee, either of the remaining substrate, ees, or of the product, eep [Rakels et al., 1993].

[ ][ ])ee (1 c - 1ln

)ee (1 c - 1ln

p

p

−+

=E or ( )[ ]( )[ ])ee (1 c - 1ln

)ee (1 c - 1ln

s

s

+−=E

Equation 2-6: by product Equation 2-7: by substrate

Because these equations are derived from irreversible kinetics, they can be used provided that the reaction is either irreversible or at least far from its equilibrium position. The conversion c can also be calculated from knowledge of ees and eep by using Equation (2-8).

c = S[ ]S[ ] 0

= ee see s + ee p Equation 2-8

Investigations led by Hult have revealed that the impact of errors in the determination of the conversion (c) on E is limited at low conversions (< 40%) [Hult et al., 1992]. An error in the conversion (c) has a large effect on the E-value at higher conversions, resulting in a faulty E-value. The dependence of the ee of the products and remaining substrates as a function of conversion and E-values is illustrated in Figure 2-3. In our practice, E-values were calculated from data obtained simultaneously for both ees and eep in one sample, using for instance GC analysis on a chiral column. When single point determinations are used, large errors in the E-value calculation usually result. Therefore, during the course of

12


the reaction multiple point analysis must be made so as to achieve an accurate determination of the E-value. This procedure allows determination of E by curve fitting [Kroutil et al., 1997; Rakels et al., 1994; Chen et al., 1987].

0

20

40

60

80

100

0 20 40 60 80 100

conversion %

e.e

%

E=100

E=20

E=5

Substrate Product

Figure 2-3: Enantiomeric excess (ee %) of substrate and product as a function of the conversion

(%) of substrate. The curves from upper to lower represent the E-values: 100, 20 and 5. 2-3 Enhancement of enantioselectivity For synthetic purposes, the highest possible enantioselectivity is desired in a kinetic resolution as it brings about the best optical purity (ee) of both the product and the remaining substrate. However, despite a low E-value in the reaction, a high ee of the remaining substrate (ees) can usually be achieved albeit in a low yield in irreversible reactions at high conversions. This relationship is evident from Figure 2-3. In order to achieve both high ee:s and yields in a kinetic resolution, improvement of E-value is an essential challenge when trying to develop an efficient enzymatic resolution. Enzyme-catalysed methods have been constantly developed and refined by both chemists and biologists during the passed two decades and many new practical means for the improvement of enantioselectivity and optimization of enzyme catalysis have been introduced [Kazlauskas et al., 2001; Berglund, 2001; Carceia et al., 2004].

2-3-1 Handling of substrates and reagents Numerous methods for the modification of substrates and/or reagents have been developed in order to enhance enzyme selectivity and/or catalytic activity. In the

13


following paragraphs a few of the strategies that organic chemists often use are illustrated.

2-3-1-1 Screening and chemical modification of substrates With the aim of improving a biotransformation, a screen of substrates involves the correct choice of substrate among a broad range of available ones to achieve better catalytic power and selectivity via better fit of the substrates to the given enzymes. Depending on the purpose of the transformation, the screening can be accompanied with design or modification of favourable substrates. The principle of the selection and modification is based on the properties of the desired substrates. The chemical properties that can be varied include nucleophilicity or electrophilicity as well as reactivity and size of leaving groups. More fundamental properties that can be changed and that affect the basic substrate structure include the size of substituent groups including e.g. straight or branch chain(s) as well as their location; change of other moieties including introduction of bi- or multi-chiral substrates etc [Nordin, et al. 2000; Nguyen, et al. 1999].

2-3-1-2 Substrate engineering In some cases, low molecular weight substrates, such as ketones, aldehydes, carboxylic acids and alcohols may fail to react in a desired biotransformation. The biocatalysed enantioselective 3-hydroxylation of cyclopentanone was not effective. Through chemical modification, however, the reaction worked. Thus a chiral auxiliary was attached to cylopentanone as a d/p-group, according to the "docking/protection" concept, to improve the stereoselectivity in the reaction with Beauveria bassiana ATCC 7159 (Scheme 2-1) [Raadt, 2001; Gubitz, 2003; Raadt, 2000; Riva, 2002].

O

NBz

O

NBz

OBnO

OBnO

HO

BzN

NBz

OHOii. B. bassiana ATCC 7159

84%

iii. BnBr/ NaH

iv. IR 120 (H , cat.)

Scheme 2-1: Substrate engineering: recycling of the chiral "docking/protection" group

i.

14


15

2-3-1-3 Addition of a non-competitive inhibitor In lipase-catalysed resolutions, selectivity can be enhanced by the addition of chiral bases, such as amines and alcohols. The additives act as non-competitive inhibitors, which act by inhibiting the transformation of one enantiomer more than the other. For instance, the addition of a chiral base of the morphinan-type (e.g. dextromethorphan) to the aqueous buffer used as solvent in the CRL catalysed resolution of 2-aryloxypropionates, led to approximately one order of magnitude raise of E-value [Guo and Sih, 1989].

2-3-1-4 Altering reagents Selection of reagent(s) involved in the reaction is another vital parameter to improve the efficiency of biotransformations. For instance, by changing the acyl donor in a lipase catalysed esterification of an alcohol from an alkyl ester to a vinyl one, significant improvement of E-value was usually obtained by selectivity improvement as well as by changing from a reversible to an irreversible esterification caused by the vinyl alcohol leaving group.

2-3-2 Media engineering Solvent properties have fundamental effects on the outcome of enzyme catalysed reactions, influencing their efficiency [in terms of turnover number, kcat, and selectivity (kcat/KM)]. It is thus expected that the enhancement of the selectivity towards enzymes can be controlled by varying solvents. Solvents possess a variety of properties, such as solubilising power, boiling point, dipole moment, polarity and polarizability, being protic or aprotic. They can be divided into three main categories: Protic solvents, which possess a proton-donating function such as –OH or –NH–, and thus have strong intermolecular forces such as hydrogen bonding and solvation effect. Water, alcohols, amines and carboxylic acids are typical examples. Dipolar aprotic solvents, which possess a large dipole moment and donor properties but lack acidic protons. Examples are dimethylsulphoxide (DMSO), acetonitrile (MeCN), dimethylformamide (DMF) and acetone. Non-polar aprotic solvents, which have only small dipole moments, and have merely weak intermolecular forces such as van der Waal- forces. This category includes hydrocarbons, halocarbons and ethers.


16

For a long time the general belief was that biocatalysis worked efficiently only in aqueous solutions. Organic solvents were believed to destroy the catalytic power of enzymes. Because of the low water solubility of many organic substrates due to their hydrophobic nature, the utilization of enzymes in organic synthesis was thus rather scarce. Water is essential for enzyme activity, because a small number of water molecules act as a lubricant, which is necessary for maintenance of the active and rather flexible conformation of the enzyme. Therefore the pertinent question should not be whether water is required or not but instead how much water is necessary. As long as the hydration shell required for retention of enzyme catalytic activity is preserved, the replacement of the rest of the water with an organic solvent should be possible without lost reactivity of the active conformation. In general, the catalytic activity of enzymes in neat organic solvents is lower than in water. On the other hand, water is not only a poor solvent for most organic compounds but it also can induce many side-reactions such as hydrolysis, racemization, polymerization, and decomposition. In addition, the removal of water from a product mixture is tedious and expensive due to its high boiling point and high heat of vaporization. In the last two decades, synthetic chemists have made enormous progress in their efforts to circumvent these problems. Thus reaction systems have been developed to improve the solubility of hydrophobic substrates. Thus efficient biotransformations displaying high selectivity and activity for hydrophobic substrates in organic solvents have established [Klibanov, 2001; Peng et al., 2004]. The methods adopted nowadays include various solvent systems from pure water to pure organic solvents such as water with an added water-miscible organic cosolvent or the latter neat; biphasic aqueous-organic systems; reverse micellar systems; organic solvents with controlled water activity and nearly anhydrous organic solvents.

2-3-2-1 Organic solvents and solid enzyme In organic solvents, an enzyme in powder form is insoluble and at best it forms a suspension of the solid enzyme in the solvent. In such two-phase systems, lipase CALB has revealed a good performance in hexane or dichloromethane for transesterification of vinyl esters with alcohols (Paper III and IV). In these systems, however, enzyme immobilized on a suitable solid carrier is most commonly used today, particularly because such immobilized enzymes possess a "pH memory” (see paragraph 2-3-4-2). Of course such reaction mixtures must be stirred vigorously and continuously to ensure efficient mass transfer between the solid and the liquid phase.


17

2-3-2-2 Co-solvents By using a co-solvent in water, both the catalyst and all reactants and products i.e. the enzyme, the substrate and the product can be dissolved in a monophasic solution consisting of water and a water-miscrible organic co-solvent. The organic co-solvents are usually polar aprotic solvents such as dimethyl sulfoxide, dimethyl formamide, tetrahydrofuran, dioxane, and acetone. Some alcohols belonging to the protic solvents are also employed, for example, methanol, ethanol or isopropanol. Most co-solvents can be used in concentrations up to ~10% or even more.

2-3-2-3 Biphasic aqueous-organic solutions Reaction systems consisting of two macroscopic phases can be used as reaction medium for biotransformations. Such media usually contains an aqueous phase and a second organic, practically water-immiscible phase. Usually employed are non-polar organic solvents with log P > 4 (the logarithm of the partition coefficient of the solvent between octanol and water) including hydrocarbons, ethers and chlorinated hydrocarbons. The advantage is that the enzyme which is dissolved in the aqueous phase is easy to separate from the organic reactants and products in the organic phase. This technique has been used in our study which provided excellent enantioselectivity, E= 65 ~ 200 [Paper II]. In such systems, however, slow mass-transfer of reactants is a notable problem because the biocatalyst is in a favourable aqueous environment and not in direct contact with the organic solvent, where most of the substrate / product is located. Adequate shaking or stirring of the reaction mixture is very important to enhance the otherwise slow mass-transfer [Wang et al., 2002].

2-3-2-4 Control of water activity (aw)

As mentioned earlier (2-2-2), to enable enzymes to dock a substrate and to adopt their active conformation, a monolayer of water is in most cases needed. Moreover, water molecules are usually an active participant in the reaction. Adequate control of water activity (aw) in a biotransformation is the method of choice to enable a reaction system to maintain the correct water content. Such control can be crucial for improving both enantioselectivity and reaction rate. The dynamic water activity (aw) in organic media can be controlled simply by equilibrating over a saturated solution of a salt or by adding a salt/salt-hydrate pair acting as water activity buffer. The CRL-catalysed esterification of 2-alkanoic acids with 1-dodecanol at temperature 25 °C, gave E = 7 without salts,


18

whereas E = 95 was obtained when Na2SO4 / Na2SO4.10H2O (aw = 0.76; ) was

used and E = 23 with Na2HPO4/ Na2HPO4.2H2O (aw = 0,15) [Högberg et al., 1993].

2-3-2-5 Ionic liquids Ionic liquids are a new class of non-aqueous solvents with ionic character. Ionic liquids are polar organic salts with very low melting points. They have no vapour pressure. Therefore they have been widely investigated as promising "green" alternatives to organic solvents regarding low environmental impact and the possibilility for efficient recycling. Although the first ionic liquid, EtNH3

+•NO3–,

was reported in 1914, imidazolium based ionic liquids such as 1-butyl-3-methyl-imidazolium tetrafluororate (BMIM+•BF4–) are the most commonly used ones in biocatalysis. Remarkable improvement of reactivity, selectivity, and stability of enzymes have been noted [Kragl et al., 2002; Park and Kazlauskas, 2003].

2-3-3 Handling of enzymes

2-3-3-1 Screening of enzymes Numerous enantioselective enzymes are commercially available nowadays. For a given type of transformation, a screen for the correct choice of enzyme(s) and its (their) proper formulation is a necessary step. Indeed, it is a relatively easy way to achieve an improved enantioselectivity. In order to obtain useful E-values for certain substrates, a wider selection of enzymes must often be screened. In such cases, access to large libraries of enzyme or micro-organisms and knowledge in practical biochemistry are vital [Wahler and Reymond, 2001a and 2001b].

2-3-3-2 Immobilization and purity of enzymes Enzymes may suffer from instability due to e.g. spontaneous oxidation, self-digestion, or denaturation. This problem can be minimised by immobilisation of an enzyme onto a suitable carrier support. Sometimes enzyme immobilization is therefore used as a synonym for enzyme stabilization. Moreover, the use of immobilised enzymes presents additional technological advantages such as tolerance to a hydrophobic medium, easier separation from the reaction mixture, and the possibility of reusing the immobilized enzyme. A great variety of immobilization methods are available and these can be grouped into categories: immobilization by entrapment; immobilization by non-covalent binding such as by adsorption, by affinity; immobilization by enzyme crystallization and covalent crosslinking; immobilization by covalent binding to properly functionalised carrier materials [Magnin et al., 2001; Wang et al., 2002; Bagi et al., 1997;


19

Ghanem and Schurig, 2003]. A large number of enzymes immobilised by different bonding techniques onto various carriers are nowadays produced and commercially available, some even in large quantities. In general a crude preparation of an enzyme exhibits a lower selectivity than the pure enzyme. Pure enzymes can nowadays be obtained by employing modern purification methods and they can even be obtained in the form of crystals. However, pure enzymes in quantities large enough for preparative applications are not always readily accessible. Hence, many less pure but effective formulations of enzymes have often been used for synthetic purposes and some have resulted in better enantioselectivity. Thus isoforms of enzymes or their isomeric fractions can be used; stabilized enzymes are often obtained by lyophilization particularly onto a salt that provides a pH-memory (paragraph 2-3-4); by suspension or emulsion in a buffer; by treatment with organic solvents etc [Colton et al., 1995; Lundell et al., 1998; Adam et al, 1998].

2-3-3-3 Protein engineering Rapid progress in genetic engineering has nowadays brought about a revolution in protein science and enzymology. This has enabled modification of an enzyme at its DNA level. Especially notable progresses are the discovery of the polymerase chain reaction (PCR), the advance in the recombinant DNA technology, the rapid development of computational chemistry in molecular modelling, and the entrance into high-throughput (HTP) technology. All this advances have allowed the facile modification of protein structures by mutation of their genes [Bornscheuer, 2001; Hult, 2003; Arnold, 2001; Khosla, 2001; Jaeger, 2004; Stewart, 2001]. In protein engineering rationally designed mutagenesis and directed evolution are the two important recent developments. Rational design is a site-specific mutagenesis of an enzyme, which allows a "logical" change of carefully selected amino acids in the protein sequence to achieve the properties desired. Given a gene for a protein and a means for its expression, one can either change any of its amino acids by changing its code or add new amino acid residues by inserting the corresponding sequences of DNA. Sections of the polypeptide chain can be removed by deleting sections of the gene. In other words, a predetermined set of base pairs in the DNA sequence corresponding to the amino acid sequence of the protein are modified for defined set of new base pairs.


20

Rational protein design by site-directed mutagenesis usually requires both detailed knowledge about the structure of the enzyme and knowledge about the relationships between sequence, structure and function or mechanism. Usually an X-ray structures can supply this information. Recently, great progress has been made in NMR spectroscopy as a tool for solving protein structures and the NMR-methods complement the X-ray diffraction of crystals. Hence, the enormously increasing number of sequences stored in public data bases have been significantly eased to access to data and structures. The use of computational molecular modelling methods has greatly increased the possibility to predict how to enhance the selectivity, activity, and stability of enzymes. Even if there are no structural data available for a specific enzyme, the structures of homologous enzymes can be used as models in such computations. In sharp contrast, directed evolution commonly gives less control in mutations. Directed evolution involves either random mutagenesis of the gene encoding the catalyst e.g. by error-prone PCR, or recombination of gene fragments e.g. derived from DNA-ase degradation, the staggered extension process or random priming recombination. When detailed knowledge of the structure of a selected enzyme is lacking, these methods can often be used at advantage. These methods combined with gene expression and high-throughput screening technology are nowadays widely employed in efforts to discover new enzymes or to optimise enzyme activity and selectivity. Libraries of enzyme clones created in these ways are then usually assayed by testing the different clones of enzymes in arrays to identify new or improved variants of the original enzyme. An impressive development is the combination of both directed evolution and rational protein mutagenesis, which has successfully been carried out with good performance and have led to enzymes with dramatically improved selectivity, activity, stability and yield. In addition, microorganisms living under extreme conditions of temperature, pH, salinity, or pressure are often difficult to grow in laboratory cultures but they provide an important source of new enzymes with desirable properties. Advanced DNA technology, particularly PCR, enables one to express enzymes from these organisms into other vectors that can be cultured under controlled conditions such as Escherichia coli. Thus a large number of new and diverse enzymes can be produced.


21

2-3-3-4 Chemical modification Unlike the previously described methods in protein engineering, which are typically limited to just the 20 primary proteinogenic amino acids, chemical modification of enzymes enables one to introduce a variety of functional groups by reactions with the functional group in the side chains of a particular amino acid along the protein chain. However, chemical reactions directed at a specific type of amino acid will not be site selective but will often occur at several or all sites where this amino acid is present in the protein chain [Fersht, 1997; Davis, 2003]. Although this methodology was initiated mainly for investigating the mechanisms of enzymatic catalysis, chemical modifications have for a long time been employed for the study of enzyme activity and selectivity. As an example, modification of Candida arctica lipase B (CAL-B) was carried out via tresyl chemistry. 59% of the accessible amine groups in the enzyme were attached to polylethylene glycol (PEG) by a chemical reaction. As a result the enantioselectivity of the CAL-B catalysed esterification of 3-methylbutan-2-ol was increased from E=214 to E=277 [Davis, 2003; Overbeeke et al. 2000].

2-3-3-5 Coenzymes In many enzymatic reactions, the presence of coenzymes is a prerequisite. Such reactions include enzymatic resolutions via e.g. oxidation-reductions, carboxylations, and the nature of the coenzyme considerably influences the reaction performance. Common coenzymes are flavin adenine (FAD/FADH2), nicotinamide adenine (NAD/NADH, NADP/NADPH) and biotin. For an organic chemist using biocatalysis as a synthetic tool, the use of enzymatic reactions requiring coenzymes is generally not attractive. Either whole cell systems, e.g. using microrganisms, or enzymes, e.g. lipases, working without cofactors are preferred.

2-3-3-6 Metal ions in enzymes Metal ions are essentials for maintaining the activity of some biocatalysts in vitro, and their presence may result in enhancements of selectivity, activity as well as stability. This is because the metal ions can function as bridging entities and even as cofactors when coordinated with various side chains in the amino acid residues of an enzyme. Metal ions, such as Zn2+, ferrous ion can simply be added into the reaction medium in form of an inorganic salts [Theil and Thorp, 2005; Sasaki and Kise, 1999; Ke and Klibanov et al., 1999].


2-3-4 Optimization of the reaction condition

2-3-4-1 Temperature Increased temperature usually results in increase in the velocity of an enzyme catalysed reaction. However, enzymes normally exert its highest performance in the temperature range of its physiological, natural environment. Once the temperature exceeds this range the enzyme is prone to an irreversible denaturation leading to loss of activity. On the other hand, the enantioselectivity of an enzyme can be expected to be significantly affected by a change of temperature (T) within the natural, environmental boundaries, see Equation 2-9.

∆∆G ≠ = -RT ln E = ∆∆H ≠ -T∆∆S ≠ Equation 2-9

For a racemate without enantiomeric discrimination, E = 1 and ∆∆G≠ = 0, Racemic temperature, Trac is thus defined as below.

≠

≠

[ ]]

∆∆∆∆=

SHTrac Equation 2-10

2-3-4-2 pH The catalytic efficiency of enzymes is pH-dependent. This is because of ionizations of side chain groups in some amino acids residues in an enzyme affect enzyme properties. This is especially important for those groups (acidic or basic) that can form ions at physiological pH and that are involved in the active site as they are crucial for catalysis. Based on the Michaelis-Menten mechanism, the catalytic strength and pH-dependence can thus be given as described in Equation 2-11, where Kα is the dissociation constant [Fersht, 1999].

( ) [HKHk

ka

catHcat +

⋅= Equation 2-11

Regulation of pH in an organic medium is not an easy task. However, when working with enzymes, a “pH memory” can be achieved in practice by dissolving the enzyme in an aqueous medium with optimal pH, followed by lyophilization or solvent precipitation prior to its use. If pH of a biocatalysis experiment is set beyond the environmental range prevailing in Nature, the enzymes may suffer irreversible denaturation with subsequent loss of activity.

22


2-4 Improvement of the yield in enzyme catalysed resolutions Many enzymatic resolutions are reversible processes. This means that a kinetic resolution applied to a reversible reaction system can only be successful as long as the conversion is low. Hence, the principle for improvement of a reversible reaction in terms of conversion and yield is to drive the thermodynamic equilibrium as far as possible towards the products, in other words, to make the equilibrium constant (K) as high as possible. The best case for this purpose is to convert a reversible reaction into an irreversible one. For example, replacing an alkyl acetate acyl donor by vinyl acetate in a lipase-catalysed resolution usually greatly improves both conversion and selectivity. This is because the vinyl group will leave the enzyme as vinyl alcohol, which will rapidly tautomerise to non-nuclophilic ethanal (acetaldehyde), which cannot act as a nuclophile for the acyl-enzyme. Although kinetic resolution of a racemate is a very efficient technique for the preparation of many optically pure compounds, it has an obvious drawback: The theoretical maximum yield is only 50% counted on the reacemic substrate. However, several approaches have been developed to overcome this problem and these are described in following paragraphs.

2-4-1 Enantioconvergent synthesis Enantioconvergent synthesis is the conversion of both enantiomers in a racemic substrate into one single enantiomer of the desired product. One biocatalytic example includes two independent reaction pathways for the enantiomers of the racemic substrate. As illustrated in Scheme 2-2, due to differences in regioselectivity of the enzyme towards the enantiomers of the substrate, each react to give the same enantiomer of the product [Monterde et al., 2004; Mayer et al., 2002].

O

Cl

O

Cl

S

R

OH

Cl

OH

> 90% e.e.

Solanum tuberosum epoxide hydrolase

Scheme 2-2: Enantioncovergent biocatalysis of racemic styrene oxide derivatives.

23


Another method for achieving an enantioconvergent synthesis is to reverse the configuration of the undesired enantiomer of the product by an additional inversion via e.g. a SN2-reaction (Scheme 2-3).

O

OAc Lipase

H2OO

OAc

O

OH

CaCO3

Scheme 2-3: Example of a convergent synthesis by additional SN2 inversion

2-4-2 Dynamic kinetic resolution A kinetic resolution combined with in situ racemization is called dynamic kinetic resolution. A key feature of this methodology is that while the fast reacting enantiomer of a racemate is converted into an inert enantiomerically pure product by the enzyme catalyst, a transition metal complex simultaneously and rapidly acts as the catalyst for the in situ racemization of the enzymatically slow-reacting enantiomer. So far, two combinations have been developed for the efficient dynamic kinetic resolution of alcohols and amines. They both employ lipase catalysts in combination with either a ruthenium catalyst or a palladium catalyst [Kim, et al. 2002; Jung, et al. 2000]. This approach has also been extended to catalysis of various other asymmetric transformation. In most cases, the combination of enzyme and metal catalysts has provided products in good yields and high optical purity. One limitation of this approach is that only the fast reacting enantiomer can be obtained in this way.

O

OH OH

+

(S)-2 (R)-2

ethyl acetate

lipase

OAc

(R)-399% ee

1

Ru(II) catalyst

89%

Scheme 2-4: The dynamic kinetic resolution.

24


2-4-3 The meso-trick and enantiotopos selectivity Even through a single step enzymatic catalysis, a meso-substrate (a compound with stereogenic centres and with a plane of symmetry in its molecule structure, Scheme 2-5) or a prochiral compound (two identical reactive groups connected to one carbon atom, Scheme 2-6) can be differentiated into chiral non-racemic products. Because both enantiomers are available from the same achiral substrate, the theoretical yield is 100%.

AcO OHS R

AcO OAcS R

HO OAcS R

PPLPLE

Scheme 2-5. Asymmetrization of a meso-diacetate

Ph

OAc

OAc

k1

k2

Ph

OH

OAc

Ph

OAc

OH

Ph

OH

OH

Scheme 2-6. Kinetics of enantiotopos differentiation (prochiral substrate) In the case of a prochiral substrate, the selectivity (α) of the reaction is only governed by the rate ratio of the formation of the two enantiomeric products R and S (Scheme 2-6 and Equation 2-12). Thus the enantiomeric excess (ee) will follow by Equation 2-13.

[[ ]

]SR==

2

1

kkα Equation 2-12

11

αα.e.

+−=e Equation 2-13

25

3 Lipases

3 Lipases Enzymes have been classified into six categories according to the type of reactions they catalyse. These categories are oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Among these, hydrolases, particularly the lipase and esterase families, have been widely studied and their properties are well documented. Their application ranges from ingredients in detergents, various uses in the food, pulp and paper industries, to use as catalysts in the chemical industry and in the manufacture of pharmaceuticals [Bornscheuer et al., 1999; Lloyd et al., 2004; Fessner et al., 2001; Thomas et al., 2002; Gubitz et al., 2003; Panke et al., 2005]. The reasons for the popularity of lipases can be attributed to their relatively wide commercial availability, no need for expensive cofactors, their ability to catalyse reactions in either aqueous or non-aqueous based media, and their recoverability. Among their most admirable advantages is their exquisite stereoselectivity in synthesis, especially today, when resolution of racemates can be coupled with recently developed means to overcome the inherent drawback of the maximum 50% theoretical yield (see section 2-4). Lipases are ubiquitous enzymes that are found in bacteria, fungi, plants and animals. Compared with the esterases, lipases are mostly more robust enzymes that normally operate at a hydrophobic/hydrophilic interface. In such an environment they undergo interfacial activation leading to a large increase in hydrolytic activity. [Schmid et al., 1998; Verger 1997]. Lipases are triacylglycerol ester hydrolases, defined in class E.C. 3.1.1.3 by enzyme nomenclature (see Protein Data Bank, http://www.rcsb.org/pdb). In biological systems they are used for catalysis of the hydrolytic degradation of triglyceride lipids such as fats and vegetable oils into free fatty acids and glycerol or its acyl esters (see Scheme 3-1).

CH

O

O

COR1

COR3

O COR2

lipase

H2OR1 OH

O

+CH

OH

O COR3

O COR2

Scheme 3-1: The original function of lipases. Catalysis of the hydrolysis of triacylglycerides

R1=acyl, R2=acyl or H, R3=acyl or H

26

3 Lipases

27

Lipases belong to the serine hydrolase class of enzyme. In preparative organic chemistry the main application of lipases is kinetic resolutions. Although the structures of the different lipases can vary to a large extent, all lipases share a common mechanistic active site, which contains the so called catalytic triad, that is Ser-His-Asp/Glu (Scheme 3-2). The catalytic triad aids the nucleophilic serine to attack the acyl donor, thus providing the basic catalytic power of the lipases. The nucleophilicity of the serine hydroxyl is enhanced by the charge relay formed with properly located

O

O

NN

H

His

Asp/GluOH

Ser

O R

O

R'

O

O

NN

H

His

Asp/GluO

Ser

OH OR'

R

Acylation

Substrate 1Acyl-Enzyme

Deacylation

O

O

NN

H

His

Asp/GluO

Ser

OO

H

RH

Substrate 2

O

O

NN

H

His

Asp/GluO

Ser

O R or HH ro R'

Transation-state complex, tetrahedral interediate

H

O

O

O

NN

H

His

Asp/GluHO

Ser

OR

O

E + SES

E + P

ES*

EP*

Scheme 3-2: Catalytic mechanism of lipase. Acid/base-catalysed Ping-Pong mechanism through procedues 1 5, where E is the enzyme, ES* and EP* are the substrates-enzyme and product-enzyme complexes

1 2

3

45

3 Lipases

28

aspartate or glutamate carboxyl groups together with the proton acceptor, the imidazol group of a histidine. The reaction mechanism has been characterised as Ping-Pong bi-bi mechanism. (Scheme 3-2) [Fersht, 1999]. It is a two-step reaction. In the first step the active site serine of a lipase is acylated with the carboxyl group of a substrate 1, called the acyl donor, forming a tetrahedral intermediate (ES*). Collapse of this releases the alcohol R'OH (or water if R’=H) to give the acyl-enzyme (ES). The second step is the deacylation of the acyl-enzyme complex (ES) caused by attack of a nucleophile e.g. a hydroxyl of substrate 2, which can be water or an alcohol. This yields a second tetrahedral intermediate (EP*), which decomposes to give the free enzyme (E) and the acyl product (P). Thus, two reactive intermediate complexes (ES* and EP*) are involved in the mechanism. An ester as substrate in a hydrolysis reaction acts as substrate 1 in the formation of the acyl enzyme. Therefore, the formation and collapse of the first tetrahedral intermediate (ES*) determines the outcome and selectivity of a lipase-catalysed hydrolysis. In kinetic resolutions of alcohols, however, the formation and collapse of the second tetrahedral intermediate (EP*) is the important one, because the alcohol acts as substrate 2 in the formation of the EP* complex in the transition state. Lipases from Candida antarctica (e.g. CALA and CALB), from Pseudomonas sp. e.g. P. cepacia lipase (PCL), from Candida rugosa (CRL) and porcine pancreas lipase (PPL) are the most popular ones for use in organic synthesis. For the resolution of alcohols CALB and PCL are the favoured ones, and we have used them both with a certain success, as will be shown in more detail in the following paragraphs. 3-1 Candida antarctica lipase B form, CALB From the yeast Candida antarctica, originally found in the Antarctic, two distinct lipases (E.C. 3.1.1.3 triacylglycerol hydrolases) have been isolated named CAL-A (or CALA) and CAL-B (or CALB). The 3D-structures of the free CALB have been determined by X-ray crystallography in 1994. In the Protein Data Bank (PDB) structures are also found for this enzyme with inhibitors of transition-state analogues located in the active site, e.g. 1LBS, 1LBT, 1TCB and 1TCC. However, except for the presence of the inhibitors, almost no conformational differences were observed among these structures. The Candida antarctica lipases are now produced by recombinant techniques.

3 Lipases

CALB is a globular protein with an α/β hydrolase fold [Ollis et al., 1992]. It consists of 317 amino acids and the molecular weight is 33 kDa. The active site is buried in the core of the enzyme and the binding site has a funnel-like shape. The active site consists of the catalytic triad plus an oxyanion hole. The catalytic triad is composed of Ser105–His224–Asp187, while an oxyanion hole functions as a stabiliser of the oxyanion formed in the transition state. Thus, the oxyanion hole provides three stabilising hydrogen bonds with the oxyanion, one with Gly106 and two with Thr40 (see Fig. 3-1) [Ottosson et al. 2002; Uppenberg, et al. 1994 and 1995; Lutz, 2004].

187Asp

O

O

H N N

H

O

O

Ser105

OH

HH

O-Thr40N-Thr40

N-Gln106

His224

H

M

L

Entrance

Protein surface

Figure 3-1: The active site of CALB and its stereospecificity pocket for a sec-alcohol

Alco

hol s

ite Acyl

site

CALB has been proven to be a particularly useful biocatalyst. It can catalyse a diverse range of reactions with high regio- and enantioselectivity, which includes reactions with alcohols, amines, acids, esters, thioesters, carbonates and carbamate and various other nuclophiles. A comprehensive review describing the use of this enzyme in organic synthesis has been published [Anderson, et al. 1998].

29

3 Lipases

The enantiopreference of CALB catalysis towards secondary alcohols follows an empirical rule suggested by Kazlauskas et al. According to this rule, the fast reacting enantiomer is predicted to have the configuration shown in Figure 3-2, where the large size substituent L should not be smaller than a propyl group and the medium size group M should not be larger than ethyl [Kazlauskas, et al. 1991; Carlqvist, et al. 2005]. The favourable configuration binding in the transition state is also outlined in Figure 3-2. Hence, one can imagine that if the configuration of the alcohol substrate is altered, i.e. the L and M groups are exchanged, this substrate will not dock in the binding site in a proper way and hence reacts much slower.

LM

OHH

Figure 3-2: Empirical rule for predicting the fast reacting enantiomerof a secondary alcohol in a CALB-catalysed acylation. M is the

medium size substituent; L is the large substutuent. In our work, CALB was employed in kinetic resolutions of secondary esters and alcohols. Many of the compounds we used as substrates, had an ester group which was hydrolysed in the reaction, a methyl group as the M group and a bromine or sulphur atom attached to the large substituents L. Good to excellent enantioselectivities were obtained with these substrates (Table 3-1). Transesterification of vinyl esters with 2,3-butanol (Entry 2), however, gave poor selectivity. This is probably because the large size group in this case is a 1-hydroxylethyl, which is smaller than propyl. The fast reacting enantiomers all have the R-configuration.

30

3 Lipases

31

Table 3-1: Enantioselectivities (E) of CALB-catalysed kinetic resolutions of secondary

esters and alcohols bearing vicinal stereocenter

Entry Substrate Reaction E-value Paper1 d,l-2,3-diacetoxybutane hydrolysis > 40a I 2 d,l-2,3-butandiol transesterification 4 a I 3 anti-2-acetoxy-3-

bromobutane hydrolysis 65 II

4 syn-2-acetoxy-3-bromobutane hydrolysis 110 II 5 anti-3-bromo-2-butanol transesterification 46 b III 6 syn-3-bromo-2-butanol transesterification 72 b III 7 threo-3-methyl-4-

(phenylsulfanyl)2-butanol transesterification >500 c IV

a E = E1 where E1 is the E-value of the first step in the two-steps sequential kinetic

resolution (Paper I) b Transesterification of diethyl malonate.c Transesterification of vinyl acetate 3-2 Pseudomonas cepacia lipases, PCL Pseudomonas cepacia lipases (PCL) lipase, nowadays called Burkholderia cepacia lipase consists of 320 amino acid residues and has a molecular weight of 33 kDa. The X-ray crystal structure of the open conformation of this enzyme was published in 1997 [Kim, 1997]. Because the names of the bacterial species have been changed over time and with a wide array of manufactures, this enzyme has been given different names and abbreviations such as Burkholderia cepacia lipase, Pseudomonas cepacia lipases (PCL), Lipase PS, Lipase P, Amano P, Amano PS, Amano P-30, PFL (Pseudomonas flourescens lipase). Nevertheless all of these names represent the same lipase that is isolated from the bacterial microorganism ATCC21808 [Kazlauskas, 1998]. Like other lipases, PCL is a α/β hydrolase. The amino acid residues Ser87–His286–Asp264 form the catalytic triad, while the amide hydrogen of Leu17 and Gln88 also contribute to catalysis by forming the oxyanion hole which provides hydrogen bonds to the oxyanion intermediate (see Figure 3-3) [Tuomi et al., 1999]. The enantiopreference of PCL towards a secondary alcohol follows the same empirical rule as CALB does (see Figure 3-2). This means that PCL also

3 Lipases

recognises enantiomers of an alcohol substrate based on the size of the substituents [Lundell et al., 1998;Tuomi et al., 1999]. Unlike what is the case for CALB, researchers have found that PCL not only demonstrates high performances in kinetic resolution of secondary alcohols but it can in addition be highly enantioselective in resolutions of some primary alcohols [Weissfloch 1995; Kazlauskas 1997; Guieysse, et al. 2003].

Asp264

OO

H N N

H

O

O

H

OR H

H

286His

Figure 3-3: The active site of PCL towards an ester at transition state

87Ser N

O

Gln88

N OLeu17

In our studies, PCL was applied in kinetic resolutions via hydrolysis of esters or transesterifications of vinyl esters with secondary alcohols. These reactions exhibited moderate enantioselectivities (Table 3-2).

Table 3-2: Enantioselectivities of PCL-catalysed kinetic resolutions of secondary esters

and alcohols bearing two vicinal stereocenters Entry Substrate Reaction E-value Paper 1 d,l-2,3-diacetoxybutane hydrolysis 39a I 2 anti-2-acetoxy-3-

bromobutane hydrolysis 37 b II

3 d,l-2,3-butandiol transesterification 13 a I a E = E1 where E1 is the E-value of the first step in the two-steps sequential kinetic

resolution (Paper 1); b Amano PS

32

4 Chemoenzymatic preparations of sec-alcohols bearing two vic-stereocenters

4 Chemoenzymatic preparations of secondary alcohols bearing two vicinal stereogenic centres 4-1 Building blocks for synthesis of chiral natural products Every year large areas of forest are defoliated by the larvae of various insects. One of the most important insect groups causing such damage on pines (Pinus spp.) is the members of the family Diprionidae (conifer or pine sawflies). In order to decrease the damage and to avoid the use of insecticides, which usually causes contamination of the natural environment and is a threat to human health, scientists have been striving to improve the monitoring and control methods of pine sawfly populations. This includes exploiting the chemical communication system of the insects, e.g. by using species specific pheromones. Pheromones are chemical signals between individuals of the same species. Insect sexual attractants, sex pheromones, can be used to control some pest insects. Thus, the use of synthetic pheromones allows control of the populations of some important forest insect pests, either by trapping or by a technique called "attract and kill" [Anderbrant et al., 1989; Byers et al., 1989; Schlyter and Anderbrant 1989]. Many pheromone components are chiral compounds. Sex pheromones isolated from many pine sawfly species were first identified as esters of various stereoisomers of 3,7-dimethyl-2-pentadecanol, diprionol (Figure 4-1) [Jewett et al. 1976; Kraemer et al 1981]. They are acetates or propanoates of long chain, secondary alcohols of varying chain lengths. In all of them, stereogenic centres bearing branching methyls are commonly located at carbons 2, 3 and 7. In some pheromones, an additional stereogenic centre with a methyl group occurs at carbon 9 or 11.

* * * * *

OH

Figure 4-1 The common structure of the alcohols found in various species of female pine sawflies. (The dashed bonds means the methyls can be missed in somecase. The asterisks indicate configuration of either R or S)

33


The synthesis of chiral pheromone components can be accomplished by using various strategies (see paragraph 1-3). The group at Mid Sweden University (http://www.kep.mh.se) has long been devoted to the synthesis of natural products, especially pheromones. Thus, various synthetic strategies have been developed for the preparation of pure stereoisomers of many pine sawfly pheromones. [Byström et al. 1981; Högberg et al. 1990; Hedenström et al. 1992; Bergström et al. 1995; Karlsson et al. 2000 and 1999; Larsson et al. 2001]. In particular the approaches represented by a-d, b and c in Figure 4-2 have been very useful when extremely high stereochemical purity was required.

* * *

O

Figure 4-2 Retrosynthetic strategies for the preparation ofstereoisomerically enriched pine sawflies pheromone

components.Z = OH, OCOR', Br or various substitutents, R= CH3 or C2H5

O

* *

R

OHa

* *

O

O

c

OH

Z

b

d fI

II

III

IV

V

R

O

**

**

e

Compounds of type IV containing the stereochemically pure 3-methyl-2-butanol moiety (highlighted in Fig. 4-2) is the key target in the synthetic strategy. Via path a, the target skeleton of IV can be obtained through a ring-opening reaction of enantiopure trans-2,3-epoxybutane (II) with a nucleophile, such as a higher order dialkylcuprate, RCH2CH2Cu(CN)Li2. Via route d, the stereopure enantiomers of 2,3-epoxybutane (II) can be produced from the relevant isomers of a 3-halo-2-butanol (e.g. I when Z =Br) or esters thereof. It is important to note that path a cannot be used to prepare stereoisomerically pure IV from the achiral meso-form of the epoxide (cis-epoxide).

34

http://www.kep.mh.se/


35

In theory, one should be able to use properly functionalised I to get to the target IV, via route b, by using a proper nucleophile e.g. a primary alkylmetal, RCH2Metal. The third alternative, c, to obtain the stereopure target IV is to employ a pure stereoisomer of 3,4-dimethyl-γ-butyrolactone, III. This can indeed be accomplished by reaction with an alkyllithium (RLi). Although the two enantiomers of the cis-lactone III can be obtained from the enantiomers of the trans-epoxide II via route e, the two trans-enantiomers of lactone III cannot, because the cis-epoxide II is achiral (meso-isomer) and will lead to racemic trans-lactone III. Provided that it would be possible to develop a working procedure proceeding via route f, properly functionalised, stereoisomerically pure, derivatives of type I become important. To sum up, highly diastereomerically and enantiomerically pure 2,3-epoxybutane (II) and 3,4-dimethyl-γ-butyrolactone (III) are important starting materials that can lead to the key pine sawfly pheromone precursors of type IV with the desired stereochemistry. Both 2,3-epoxybutane (II) and 3,4-dimethyl-γ-butyrolactone (III) can be derived from 3-functionalised 2-butanol derivatives of type I. In case of the lactone III, all four stereoisomers could in theory be obtained. Hence, each and every pure stereoisomer of properly functionalised derivatives of I are desired building-blocks. By means of combined chemical and enzymatic approaches, we have studied the preparation of isomerically pure secondary alcohols having a vicinal stereocentre, e.g. alcohols of types I and IV (Figure 4-2). The compounds of this type that have been investigated in this thesis include 2,3-butanediol (Paper I) and 3-bromo-2-butanol (Paper II). Our attention has also been drawn to the development of preparative procedures for all four isomers of 3,4-dimethyl-γ-butyrolactone (III) from suitably functionalised building blocks of type I via route f in Figure 4-2, e.g. by making a malonate ester of 3-bromo-2-butanol, which when subjected to an intramolecular enolate alkylation (Paper III and V) will hopefully ring-close to a lactone III. Moreover, the γ-lactone (III) can also be successfully prepared by using a novel procedure from the appropriate stereoisomers of 3-methyl-4-(phenylsulfanyl) butane-2-ol (Paper IV).


36

4-2 2,3-Butanediol (BD), Paper I

4-2-1 Chirality of BD and the synthetic strategy 2,3-Butanediol (BD) possesses two stereogenic centres on C2 and C3. Therefore there are two forms of diastereomers, i.e. dl and meso (Figure 4-3). Since the RS-form is identical to the SR-isomer in meso-BD, there are thus three stereoisomers, i.e. RR, SS and meso (RS = SR) . The pure enantiomers of 2,3-butanediol (BD) are useful reagents in asymmetric synthesis. They can be used as auxiliaries and be transformed into various chiral building blocks, such as isommerically enriched epoxides, vicinal hydroxy-tosylates, or halohydrins, and cyclic sulfites. Reduction of 2,3-butanedione or acetoin etc [Kolb et al. 1994; Tiecco et al. 2003; Scaros et al. 1997] furnishes butanediol isomers. However, BD can also be made industrially via biochemical methods, such as microbial transformation of starch [Afscher et al. 1993]. In addition, BD is one of the major components of side products in the courses of wine fermentation [Bartowsky et al. 2004; Romano et al. 2003]. BD are in most case produced as a mixture of the meso- and dl-isomers. Usually one of the enantiomers, mostly the RR-one is obtained in excess over the other. Hence, the separation of the mixture into each single enantiomer is a challenge, which has attracted many biologists and chemists [Scaros et al. 1997; Caron and Kazlauskas 1993; Mattson et al. 1993; Utille and Boutron 1999]. We have developed an efficient and convenient method, shown in Scheme 4-1, for the separation of isomeric, commercially available mixtures of BD. For dl-BD the procedure involves two crucial steps, epimerisation and enzyme catalysed kinetic resolution, whereas meso-BD was obtained by crystallisation of crude commercial meso-enriched BD. The first step is the separation of the meso- and dl-diastereomers from a mixture of BD. In terms of obtaining a highly pure meso-BD, it is necessary to selectively use mixtures with diastereomeric ratio dr (meso/dl) over 60/40 (see paragraph 4-2-4). Thus meso-BD can be obtained with dr up to 99/1 via recystallisation at 4 ºC. On the other hand, the dl isomer can be obtained from any mixture of BD through an epimerisation of the meso isomer. The efficient epimerisation of meso-BD can probably be explained by lower thermodynamic stability at the transition state of the mono-sodium salt, in comparison to that of the dl-form. In this way, dl-BD isomer was normally provided in dr up to 96/4 (paragraph 4-2-2). If not satisfied, the diacetate derived from the dl-BD can easily be purified by


recrystallisation furnishing dl-2,3-butanediacetate in dr (dl/meso) 99.5/0.5. dl-BD can then be handily obtained with identical dr by hydrolysis.

HO

OH

ZO

OZ

HO

OH

meso:dl >60%

meso or RS/SRd.r. 99/1

dl d.r. 99.5/0.5

HO

OH

HO

OH

Scheme 4-1: Strategy for preparation of each and every enantiomers of 2,3-butanediol (BD)

Z = H or -OCOEt

Recrystallisation EpimerisationRecrystallisation

Step 1Seperation of diastereomers

Step 2Kinetic resolution

SSe.e. 99.5%

RRe.e. 100%

Enzyme-catalysed resolution

The racemates obtained as described above were separated into the enantiomers by lipase catalysed resolutions. Both hydrolysis of dl-2,3-diacetoxybutane and transesterification of dl-BD were investigated. Excellent enantiomeric purity (ee >99.9%) of each enantiomer i.e. RR- and SS-isomer of BD as well as their diacetates were achieved. Because RR-BD is relatively easy to obtain via fermentation, our interest was mainly focused on a simple preparative method for SS-BD. The standard method for obtaining the latter is a multistep and very time-consuming and tedious synthesis starting from L-(+)-tartaric acid [Byström et al. 1981].

4-2-2 Thermodynamically favoured epimerisation In many syntheses of chiral pine sawflies pheromones access to both enantiomers of 2,3-butanediol (BD) are required. However, for reasons given above, we were most interested in the preparation of SS-BD. Although lipase catalysed resolutions of dl-BD have been studied earlier, the low diastereomeric purity of

37


38

the staring BD was a major obstacle for obtaining pure products. Thus, in order to succesfully make the pure enantiomers of BD, we need an efficient method for the preparation of highly diastereomerically pure dl-BD. Therefore the first step in our synthetic strategy becomes the epimerisation of meso-BD in the available BD-mixtures into its dl-isomer. Epimerisation is an interconversion of diastereomers by change of configuration at one or more (but not all) stereogenic centres in a molecule possessing more than one such centre. Diastereomers such as meso-and dl-BD differing in configuration at just one stereocentre are often called epimers. A tiny note in Italian [Bottari et al. 1965] describes that meso-BD can be epimerised into the dl-isomer when it is treated with sodium. Independent of the dr of the starting mixture, we found that by using one equivalent of sodium, an equilibrium between the meso- and dl-epimers was usually reached after 24 hours of refluxing in toluene, which provided dl-BD in dr up to 96/4. For such epimerisation of meso-BD into its dl-form, the difference in free energy between the two epimers (∆G) can thus be calculated with equation below.

∆G = Gdl − Gmeso = − RT lnK = − RT ln([dl] /[meso]) [meso] and [dl] are the concentrations of meso- and dl-BD, respectively, at equilibrium state. In our investigation, the product was repeatedly isolated in dr = [dl] /[meso] = 96/4, thus the equilibrium constant Ka

387 = 96/4 = 24. Entering this in the equation above ∆G = 10.2 kJ / mol = 2.5 kcal/mol. Although the mechanism of the epimerisation is unclear, we tentatively suggested that the mono-salts of the dl- and meso-BD were gave a ring containing a chelated a sodium ion (Figure 4-3) [Von E. Doering 1953].


OH3C

H H

H

H3C

Na

O

OH3C

H H

H3C

H

Na

O

Ka

dl-saltmeso-slat

Figure 4-3. Equilibrium of dl- and meso-2,3-butanediol mono salts in their chelated gauche forms.

The energy difference between the two forms of the salts was assessed by simple MM2 calculations using Chem3DProTM [Paper I]. The calculations indicated that the cyclic mono sodium salt from the dl-form was more stable than the one from meso-form by 0.8 kcal/mol.

4-2-3 Screening of lipases for the kinetic resolutions On the basis of their proven efficiencies in the resolutions of diastereomeric mixtures of 2,3-butanediol, three lipases were selected for study in kinetic resolutions via either a transesterification of dl-BD, or hydrolysis of dl-2,3-diacetoxybutane (DAB) [Scaros et al. 1997; Caron and Kazlauskas 1993; Mattson et al. 1993; Utille at al. 1999]. These were Pseudomonas cepacia lipase (Amano PS, PCL), Porcine pancreatic lipase (PPL), and Candida antarctica lipase B (Novozyme 435). According to the previous studies mentioned, the three lipases all show R-preference. In other word, the R,R-DB- and DAB-enantiomers react faster than the S,S-ones. The kinetic resolutions of the racemic BD and its racemic diacetate, DAB, are two- step, sequential reactions. The total E-value ET was usually high, since ET = 0.5 x E1 x E2 , where E1 and E2 are the enantioselectivities in each of the sequential steps [Ranchoux, M. et al, 1998; Adam, W. et al, 1998]. However, monitored with GC-MS, the products from the second step were in many cases almost absent. This indicated that the second step was rather slow. Thus the two-steps-sequential reactions can be regarded as an one-step resolution, i.e. E ≈ E1. In order to facilitate the comparison of the different enzymes, only the E-values in the first step are used in Table 4-1.

39


40

Table 4-1: Enzymatic resolution of dl-BD or its acetate with PPL, PCL and CALB

Lipase Reaction Time (h) c % c ees % d E1e

Transestericfication a 7.5 85 97 4 CALB Hydrolysis b 4 58 99.8 40 Transestericfication a 50 75 >99 13 PCL Hydrolysis b 24 56 99 39 Transestericfication a 150 57 >98 40 PPL Hydrolysis b 200 67 47.5 7

a Enzyme catalysed kinetic resolution of racemic dl-2,3-butanediol by acylation via transesterification of vinyl propanoate in TBME at ambient temperature. b Lipase catalysed hydrolysis of dl-2,3-diacetoxibutane in the two-phase system hexane / water at ambient temperature c c = conversion of starting material (S,S-enantiomer) d ee of remaining substrate (S,S-enantiomer). e E-values were estimated fitting the experimental data with those generated by the program SeKiRe17

As we can see in Table 4-1, in the cases of transesterification of dl-BD, PPL provided the best enantioselectivity (E1 =40) while CALB gave the worst (E1=4). In case of hydrolysis, on the other hand, CALB revealed the highest performance (E1=40) while PPL showed the poorest behaviour (E1=7). PCL showed moderate E-values in both types of resolutions (E1=39 to hydrolysis and E1=13 to transesterification). In our practices by a compromise with the reaction times, PCL was employed in transesterification of vinyl esters with dl-2,3-butanediol (BD) while CALB was used for the hydrolysis of dl-2,3-diacetoxybutane (DAB) in large scale preparations. The rather low enantioselectivity observed for the resolution of particularly dl-BD was common to a variety of lipases. According to the empirical rule described in paragraph 3-1, fairly good selectively can be achieved only when the large size substituent is not smaller than a propyl group [-CH2CH2CH3]. In the case of dl-BD, the large size group of the substrate is a 1-hydroxyethyl [-CH(OH)CH3]. It is probable that the enzyme perceives this as a smaller group than propyl. In addition, the presence of the polar hydroxyl group in the 1-hydroxyethyl moiety [-CH(OH)CH3] might also affect the preference. Thus, several explanations can be given for the rather low selectivity of CALB against


41

dl-2,3-butanediol. In case of hydrolysis of the diacetate, due to the large relatively non-polar 1-acetoxyethyl group, the first step can be expected to be more selective, which was also found. The docking models for the two types of kinetic resolutions are shown in Figure 4-4. In our hypothesis, CALB (green) possesses the biggest docking pockets for both the large-size group L and the medium-size group M while PPL (red) has the smallest ones. PCL (blue) have docking pockets in the middle size between CALB and PPL. In the case of transesterification (Reaction 1), dl-2,3-butanediol was the substrate reacting with vinyl propanoate. Thus, M is a methyl and L is 1-hydroxyethyl [ -CH(OH)CH3], a group with a size which does not appear to be larger than propyl. As we can see RR-BD will fit well into the docking pockets of all three lipases (A) while the SS-enantiomer completely misfits in PPL, hardly fit in PCL, but is probably reasonably well docked in CALB (B). As a result, PPL exhibited the best specificity (E1 =40) because only the RR-enantiomer can be catalysed by the lipase. However the reaction was very slow (150 hours required to reach 57% conversion). This may be attributed to the small pockets of PPL, which lead to a hindered docking of both enantiomers of BD. In contrast, CALB gave the poorest E-value (E1 =4) because it accelerates the transformation of both RR- and SS-BD (7.5 hours to 85% conversion). CALB catalysed transesterification of vinyl butyrate with 2-butanol also has a low E-value (E=9) and in this case the large group L is an ethyl group [Rotticci et al. 1998]. While PCL mainly favours reaction with RR-BD, it does also partly catalyse the transformation of SS-BD. An intermediate enantioselectivity (E1 =13) was thus found. In contrast to CALB, the lower reactivity of PCL (50 hours to obtain a conversion 75%) may be caused by the smaller free space available for RR-BD to dock into the active site. Reaction 2 of Figure 4-4 illustrates the docking models in hydrolyses of dl-2,3-diacetoxybutane (dl-DAB). Although the medium size group M is still a methyl, the large one, L, is changed to -CH(OCOCH3)CH3 , a much bigger group than propyl. As shown in (C) Figure 4-4, the RR-diacetate can dock well into the pockets of both CALB and PCR while it hardly fits in PPL. In the latter case, the size of L is probably larger than that of the docking pocket. On the other hand, SS-DAB displayed misfits to all three lipases (D). As a consequence, CALB and PCL displayed good selectivities (E1 =40 and 39 respectively, >99%ee for the


OH

CH3

O

O

Alkyl

Serine

R'OCH3

Figure 4-4. Docking Pockets of CALB, PPL and PCL in kinetic resolutions

-OR' = -OCOCH3CALB: with green docking pocketsPCL: with blue docking pockets

O

O

O

Alkyl

Serine

OR'CH3H3C

(C) docking model of R-configuration (D) docking model of S-configuration

H

OH

CH3

O

O

Alkyl

Serine

HOCH3

Reaction1. Ttransesterification

O

O

O

Alkyl

Serine

OHCH3H3C

(A) docking model of R-configuration (B) docking model of S-configuration

H

Reaction2. Hydrolysis

PPL: with red docking pockets

42


43

remaining SS-diacetate). The difference in their reactivities (4 h to reach c = 58% for CALB and 24 h to c = 56% for PCL) may well be the consequence of variations in the size of their docking pockets. Because both RR- and SS-DAB misfit to PPL, the enzymatic process was extremely slow (200 h to reach c = 67%). Under such reaction condition the spontaneous hydrolysis could be a serious side reaction. The selectivity of PPL was quite poor (E1 =7) too.

4-2-4 Binary liquid-solid phase equilibrium – recrystallisation In our synthetic procedure for the preparation of the 2,3-butanediol (BD) isomers, recrystallisation has been used three times in crucial steps in the preparation of highly stereoisomerically pure BD (see Scheme 4-1). The first time it was employed was in the separation of meso-2,3-butanediol directly from a mixture containing both meso- and dl-BD. The second and third application was the recystallisations of the diacetate derived from the epimerised dl-DB and of the final SS-diacetate. Recystallisation was used in a straightforward way to make meso-BD from a commercially available diastereomeric mixture of BD. In order to obtain highly pure meso-BD, as was emphasised in paragraph 4-2-1, for efficient crystallisation of meso-BD it is important to select a mixture with dr (meso/dl) over 60/40. In order to give an interpretation of this critical dr for the starting mixture, a hypothetical diagram of the binary liquid-solid phase equilibrium is shown in Figure 4-5. E is the eutectic point, represented a mixture with the concentration in % of component B, XBE. At the eutectic point E three phases coexist, namely solid A, solid B and liquid, or A(s) +B(s) + l in writing. Pure crystals of B (meso-DB in this case) can usually be obtained in a cooling recrystallization process only when it is prone to precipitate prior to crystals of A (dl-DB in this case). This means that the starting point (q) should have a composition (XBM) located on the right side of the point E, i.e. XBM ≥ XBE. The slightly modified (relative to Paper I) separation by crystallisation experiment can be described as follows: A mixture (ca 800 g, dr = meso/dl = 85 / 15) was placed in a refrigerator (4 °C). After 24 hours the mixture was subjected to a long-term filtration in a cold room (4~5 °C). The sticky colourless crystals of meso-BD (approximately 500g) was obtained with dr = meso/dl ≈ 100/0 (GC).


0% 20% 40% 60% 80% 100%

MPA

MPB

EM N

BA

l +A(s)

l +B(s)

A(s)+B(s)

XB

liquid

q

Figure 4-5: Binary liquid-solid phase equilibrium

Thus, with the starting mixture (point M or q), we know that the gross weight WM = 800 g, XBM = dr = meso/dl = 85% implying the length of MN =15. From the obtained crystal meso-BD (point N), the weight WN = 500, the length EN = EM + MN =EM + 15. Based on Lever rule, the calculation is proceeding as follow:

ENEM=

M

N

WW or

15800500

+=

EMEM

Therefore EM = 25 and EN = 15 + 25 = 40

Thus XBE = 60 (%)

The eutectic point E is thereby located at XBE = 60%. Hence X BM ≥ 60%, in other words, in order to obtain pure meso-BD, one must select a starting mixture with a dr (meso/dl) ≥ 60/40.

44


4-3 3-Bromo-2-butanol and its derivatives (Paper II and III)

4-3-1 Objectives and the synthetic route As mentioned earlier, both enantiomerically pure 2,3-epoxybutane and 3,4-dimethyl-γ-butyrolactone are very useful building blocks for preparation of pheromone components of pine sawflies (paragraph 4-1). Figure 4-6 illustrates that the epoxide can be easily derived from 3-bromo-butanols by treatment with base and the reaction takes place with Walden inversion of the configuration at the carbon atom carrying the bromine in the starting material [Golding et al. 1973]. The absolute configurations of the epoxide can thus be deduced from these bromohydrins, e.g. the syn-(2S,3S)-bromoalcohol will give the achiral (2S,3R)-epoxide i.e. the meso-epoxide [Due to a mirror plane the (2S,3R)-epoxide is identical with the (2R,3S)-epoxide], whereas each enantiomer of the anti-bromoalcohols will give one enantiomer of the C2-symmetric epoxide.

Br

HOBr

HO

Br

HO

syn-3-bromo-2-butanol

(2S, 3S)-syn

(2R,3R)-syn

Br

HOBr

HO

Br

HO

anti-3-bromo-2-butanol

(2S, 3R)-anti

(2R, 3S)-anti

HH

O

(2S, 3R)

HH

O

(2R, 3S)

HH

O

meso-cis-2,3-epoxybutane

Achiral

H

H

O

(2R, 3R)

H

H

O

(2S, 3S) H

H

O

dl-trans-2,3-epoxybutane

Chiral

Figure 4-6 Bromohydrins as the precursors of the three 2,3-epoxybutane isomers.

identical with

is nonidenticalenantiomer of



If the four pure stereoisomers of 3-bromo-2-butanol were available it may be possible to utilise them for the preparation of the four pure stereoisomers of 3,4-dimethyl-γ-butyrolactone. As shown in Scheme 4-2, a likely synthetic route could be formation of 3-bromo-2-butyl ethyl malonate followed by

45


intramolecular enolate alkylation [Paper III and V]. In the latter reaction, a configuration inversion was expected to occur at the stereogenic centre originally bearing the bromine atom via a SN2 mechanism. Because the Cahn-Ingold-Prelog-priorities of the substituents change while the methylene group has replaced bromine in the final, desired γ−lactone product, it only appears as if no change in absolute configuration has occurred.

O

O

O

O

O

O

NaOEt / ethanolintramolecular enolate alkylation

1) hydrolysis2) decarboxylation

2R, 3S-3 3S, 4R-4

3S, 4R-5

Br

OO

OO

2R3S 4R3S

Scheme 4-2. Proposed of synthetic route to 3,4-dimethyl-γ-butyrolactone.

At this point the challenge for us was the preparation of each of the enantiomer of each of the two diastereomers of 3-bromo-butanols or its esters. We therefore projected a synthetic route shown in Scheme 4-3. As an example, dl-2,3-butanediol was treated with a saturated solution of hydrogen bromide in acetic acid (4.1 M, step A). Due to Walden inversion, (±)-anti-2-acetoxy-3-bromobutanes (anti-2) was furnished in 75 - 90% yield. In the same way, the meso-diol produced (±)-syn-2-acetoxy-3-bromobutane (syn-2). The two diastereomers can handily be converted to the corresponding alcohol (step B). In order to obtain each and every enantiomer in a high purity, we found that lipase CALB with its R-configurational preference displayed enormous efficiency in enzyme-catalysed kinetic resolutions. All four enantiomers of 3-bromo-2-butanol were obtained in ee>95% either as such or as their acetates. [Step C or D. also see Table 1 in Paper II]

46


Br

OAc

Br

OH

HO

OH

HBr-HOAc

Br

OAc

Br

OH

Br

OH

Br

OAc

C D E

O OO O

A B

Scheme 4-3. Example of synthetic routes to a variety of enantiomers starting from anti-3-bromo-2-butanol

FH2C COCl

COOEt

O OO OBr

75% cons. HCl

2. anti-Br-acetate 3. anti-Br-alcohol1. dl-BD

(2S,3R)-2 (2R,3S)-3 (2S,3R)-3(2R,3S)-2

75% cons. HCl

Br

(2R,3S)-4

G

(2S,3R)-4

Similarly, from the diastereomers of 3-bromo-2-butanol (anti-3 and syn-3), the two 2R-isomers of 3-bromo-2-butyl ethyl malonate were obtained straightforwardly from lipase catalysed kinetic esterification of diethylmalonate (step E). The 2S-isomers, can be obtained from the remaining substrates, the two (2S)-3-bromo-2-butanols via acylation with the acid chloride of malonic acid monoethyl ester (step G). Thus the four 3-bromo-2-butyl ethyl malonic esters were obtained in good ee (84% ~ 99.4%) and dr (92/8 ~ 99.5/0.5) [see Paper III].

4-3-2 The vicinal bistereogenic centres of the substrates Candida antarctica lipase B has been employed as the catalyst in kinetic resolution of the diastereomers of the bromohydrins or their esters. This lipase has exposed its excellent enantioselectivity, tolerance of a variety of substrates and diversity in hydrolysis as well as in esterification. Figure 4-7 shows the E-values registered in CALB catalysed resolutions of the various substrates we have used. The syn-diastereomers (Grey) generally exhibit better enantioselectivity than its anti-isomers (White). This means that the energy differences in the transition state are larger for the two enantiomers of the syn-isomers than for those of the anti-isomers.

47


0

50

100

150

200

2 3 4

synanti

Figure 4-7: E-values of CALB-catalysis

2 hydrolysis of 3-bromo-2-acetoxybutane, 3 transesterification of 3-bromo-2-butanol with vinyl butyrate; 4 esterification of 3-bromo-2-butanol with diethyl malonic ester

The vicinal bichiral stereogenic centres in the substrate structures seem to influence the interaction with the enzyme. When compared with substrates bearing a single stereocentre, the second vicinal chiral centre in our substrates appears to considerably affect the binding between the enzyme and substrate even though it does not directly participate in the catalytic triad involved in the resolution. In the cases of 3-bromo-2-butanol and its esters, it appears that the E-value is higher when the chirality at the second stereocentre (i.e. on the carbon bearing the bromine) is same as that of the first one (i.e. the carbon bearing the oxygen), e.g. the RR-, SS-enantiomers; whereas the E-value becomes lower when the chiralities on the two vicinal stereogenic centres are different, e.g. RS-, SR-enantiomer. In Table 4-2 CALB-catalysed kinetic resolutions of some secondary alcohols possessing one stereogenic centre are compared with our substrates. The substrates selected for comparison were similar to 3-bromo-2-butanol. The syn- and anti- 3-bromo-butanols have a large-size group L = CHMeBr and the medium-size group M=Me. The difference between L and M is larger in our substrates than in 1-bromo-2-butanol (entry 3) which has L= CH2Br, M= CH2Me, although these molecules have the same molecular weight. As we can see, the E-value of 1-bromo-2-butanol (81) was in between that of syn- and anti-3-bromo-butanol (E=160 and 53, respectively). Moreover, the two substrates of entry 4 and 5 have the same M as in syn- and anti-3-bromo-2-butanol M=Me, but smaller L (L= CH2Br or CH2Cl), i.e. the differences between L and M in the former two are smaller than the ones in 3-bromo-2-butanols. However, the

48


former display higher E-values (371 and 160, respectively). In conclusion, the second vicinal stereogenic centre in our substrates seems to influence the docking into the active site in a more complicated way than can be explained only by steric bulk.

Table 4-2: CALB catalysed resolutions of secondary alcohols bearing vic. halogen

substituted , with vinyl butyrate as the acyl donor

Entry Substrate Structure E-value Reference 1 anti-3-bromo-2-butanol B r

O H 53 Paper II

2 syn-3-bromo-2-butanol B r

O H

160 Paper II

3 1-bromo-2-butanol BrOH

81 Rotticci et al. 1997 and 1998

4 1-bromo-2-propanol BrOH

371 ibid.

5 1-chloro-2-propanol ClOH

160 ibid.

4-4 Intramolecular enolate alkylation Exploratory

experiments, Paper V With the intent to synthesise each and every of the four isomers of 3,4-dimethyl-γ-butyrolactone (paragraph 4-2-2-1), we projected a synthetic route involving ring closures by intramolecular enolate alkylation of the four isomers of 3-bromo-2-butyl ethyl malonate ( e.g. compound 3 in Scheme 4-4). Thus, (2R,3S)-ester 3 on treatment with base was supposed to form 2-ethoxycarbonyl-(3S,4R)-trans-dimethyl-γ-butyrolactone (4). The desired γ-butyrolactone (5) should then be produced through further hydrolysis and decarboxylation.

49


OO

OO

HOOO

O

OEt

OO

O O

Br

OO

OO

O

O

O

O

O

O

34

Scheme 4-6. Attempted base-catalysed intramolecular enolate alkylation

5

2S3R4S3R

Desired path

Actual outcome

Tentative structure

and / or

6 7

Spontaneoushydrolysis on

work-up

8

2S3S

Not isolated

A variety of bases were investigated as catalysts for the intramolecular enolate alkylation. By GC-MS monitoring of the reactions, a new product was identified and this was the same with all the bases studied. This new product was isolated and purified. Structure characterisation, particularly with 1D and 2D NMR were carried out (Table 4-3). Unfortunately, the spectra did not match with the expected γ-lactone ester 4. Instead the spectrum of the compound revealed that it had one acidic proton (OH) and one methylene (CH2) group in addition to that of the ethyl ester group. Therefore, a tentative structure of the compound obtained seemed to be 8, which displayed better agreement with the spectra [Paper V]. Instead of an enolate C-alkylation desired, O-alkylation(s) in the molecule of 3 seemingly occurred, which gave compound 6 and /or 7. Compound 8 may be then formed from the hydrolyses of 6 and /or 7 under the alkaline condition followed by acidification.

50


51

Table 4-3: 1D and 2D NMR of the new compound derived from the intramolecular enolate alkylation of 3-bromo-2-butyl ethyl malonate ester 3

1H NMR 13C DEPT 135 1H-1H COSY 1H - 13C

COSY δ 1.25 (3H, d, J=6.4 Hz) 1.30 (3H, d, J=6.3 Hz) 1.34 (3H, t, J=7.1 Hz) 2.1(1H moved with D2O) 3.36-3.55 (2H, dd, J=16 Hz) 3.80 (1H, quintet, J=6.5 Hz) 4.26 (2H, q, J=7.1Hz), 4.88 (1H, quintet, J= 6.4 Hz)

δ 14.4 16.6 18.9 42.1 62.2 70.4 77.1 166.5167.6

↑(CH, CH3): 14.4(CH3) 16.6(CH3) 20.2 (CH3)

70.4 (CH) 76.9 (OCH) ↓ (CH2 ):

42.2 and 62.2

1.25 (CH3)↔3.80 (CH) 1.30 (CH3)↔4.88(OCH) 1.34(CH3)↔4.26(OCH2)

3.80 (CH) ↔ 4.88 (OCH)

1.25 (3H) ↔18.9 C

1.30 (3H) ↔16.6 C

1.34 (3H) ↔14.4 C

3.36 (2H) ↔ 42.1 C

3.80 (1H) ↔ 70.4 C

4.26 (2H) ↔ 62.2 C

4.88 (1H) ↔ 77.1C

4-5 3-Methyl-4-(phenylsulfanyl)butan-2-ol, Paper IV A satisfying alternative for preparation of the highly stereoisomerically pure trans-3,4-dimethyl-γ-butyrolactone enantiomers (Scheme 4-6) has been recently developed by us [Paper IV]. This synthetic approach is a combination of diastereoselective methylmetal addition and biocatalysis, involving a compound incorporating the relevant diastereomeric form of the 3-methyl-2-butanol moiety (IV, Figure 4-2). This approach involves two major steps: (1) Via diastereoselective Lewis acid-catalysed methylmetal addition, the threo-isomer of 3-methyl-4-(phenylsulfanyl) butan-2-ol (threo-11) was produced from racemic 2-methyl aldehyde (10) in fairly high diastereomeric purity; (2) By consecutive lipase catalysed acylations, the two enantiomers were obtained in high enantiomeric purities (Scheme 4-5). When the synthesis started with each of the enantiomers of the aldehyde 10 separately, the final products after lipase catalysed purification were obtained in >99.9% ee and a dr >200/1.


S

OH

SS

OH

threo-11

OCOC2H5

(2S,3S)-1198% e.e; 95/5 d.r.

S H

O

rac-10

*

S

OH

erythro-11

Me2Zn, TiCl4

+

CALB, vinyl acetate c = 49%

(2R,3R)-1298% e.e; 94/6 d.r.

S

OH

(2S,3S)-1198% e.e; 98/2 d.r.

CALB, vinyl acetate c = 13%1. HO, EtOH2. CALA, vinyl acetate, c=41%

S

OH

(2R,3R)-1199% e.e; 98/2 d.r.

Scheme 4-5. Preparation of isomerically pure 3-methyl-4-(phenylsulfanyl)butan-2-ol from the racemic aldehyde 10.

threo-/erythro- : 95:05

The first step is a Lewis acid-catalysed nucleophilic addition of a methylmetal reagent to a carbonyl group. An asymmetric carbonyl compound normally possesses diastereotopic faces, and diastereoselectivity can be achieved by kinetic control. This is usually in accordance with Cram/anti-Cram rule [Cram et al. 1952; Cornforth et al. 1959; Karabatsos 1967]. In the proposed mechanism, the aldehyde (10) was used as a substrate bearing a suitably placed sulfur atom. When titanium tetrachloride was present as a Lewis acid, a chelate was formed between the carbonyl oxygen and the sulfur. Upon addition of a methylmetal

52


53

reagent, a six-membered ring transition state formed, in which an asymmetric addition with dimethylzinc occurred from the side of the substrate with less steric hindrance. In this way, threo-3-methyl-4-(phenylsulfanyl) butan-2-ol (threo-11) as the anti-Cram product was favourably obtained in high diastereomeric ratio (dr = threo / erythro = 95/5) [Larsson and Högberg 2001]. The second step consists of a series of lipase-catalysed resolutions. Immobilised Candida antarctica lipase B (CALB) and Novozyme SP 525 (CALA) were used in the consecutive acylations with vinyl acetate. Thus, both enantiomers (2R,3R)-11 and (2S,3S)-11 were obtained in high stereopurity, 98/2 dr and >98% ee (Scheme 4-5). Our purpose in choosing the intriguing compound containing a phenylsulfanyl group (PhS) is not only to affect chelation control in the diastereoselective addition with dimethylzinc, but also to facilitate the preparation of the corresponding enantiomers of trans-3,4-dimethyl-γ-butyrolactone (16 in Scheme 4-6). Thanks to the phenylsulfanyl group (PhS), the adjacent methylene carbon can be transformed into an electrophilic one by conversion of the PhS into a good leaving group, whereas, when it is converted to a phenylsulfonyl group (PhSO2) by oxidation, the same methylene carbon can be rendered nucleophilic after deprotonation. The preparation of enaniomerically pure lactone precursor 15 and lactone16 was not described in paper IV, as the procedure for making these in racemic form was described earlier [Larsson and Högberg 2001].


54

S

OH

(2S,3S)-11

S

OTBDMS

(2S,3S)-13

S

OTBBMS

(2S,3S)-14

O O

S

OTBBMS

(2S,3S)-15

O O

OO

O

O

(2S,3S)-16

SOTBBMS

(2S,3S)-17

O

O

n-C11H23

OH

(2S,3S)-18

i. Na-Hg,MeOH, Na2HPO4ii. TBAF, acetyl chloride/pyr.iii. H2 / RaNi, KOH/MeOH

i. DMPU /THF, -40 °C, n-BuLiii. 1-Iodoundecane, THF

m-CPBA

TBDMSCl

DMF imidazole

i. n-BuLi /THF, -78 °Cii. ethyl chloroformate

i. Li, NH3, -78 °Cii. PhCO2Naiii. MeOH, HCl

Scheme 4-6. Preparations of the isomerically pure lactone 16 and the natural alcohol 18 isolated from pine sawflies

55

5 Conclusion In terms of developing novel and efficient methods for synthesis of chiral natural products, such as pheromone components, isomerically pure 3-methyl-2-alkanols segment and 3-methyl-2-butanol moiety are contrived as the key synthetic intermediates. Thus a number of secondary chiral alcohols bearing one vicinal stereocentre have been studied. With the combination of chemical and enzymatic approaches, each and every isomer of 2,3-butandiol have been successfully prepared in high isomeric purity. Starting from the diastereomeric dl- and meso-2,3-butanediol, the four stereopure isomers of 3-bromo-2-butanol are prepared. Wanted as the precursors of 3,4-dimethyl-γ-butyrolactone, four stereoisomers of 3-bromo-2-butyl ethyl malonate ester were additionally furnished. However the desired lactone failed to be produced when anti-3-bromo-2-butyl ethyl malonic ester was treated with a base in order to achieve an intramolecular alkylation. Alternatively, the enantiomers of threo-3-methyl-4-(phenylsulfanyl)butane-2-ol can be prepared and used for the preparation of the enatiomers of otherwise difficult to obtain trans-3,4-dimethyl-γ-butyrolactone.

56

6 Acknowledgements I appreciate all of my advisors, colleagues, friends and relatives who have encouraged me, help me to make this work possible. In particular, I'd like to express my sincere gratitude to the following people: Professor Hans-Erik Högberg, my supervisor, for having accepted me in Organgrupp, for your knowledgeable supervision and valuable guidance, for enabling and bolstering the work transferred in KTH, for the standing to sense and for so many significant supports and unforgettable cares along my way. Professor Torbjörn Norin (emeritus) in the Department of Chemistry, KTH Stockholm, for signing me as a PhD student tills the final striving for the doctorate. Associate professor Per Berglund, for introducing me into the biocatalysis group at KTH, for impressive instruction of enzymes and laboratory works. Professor Karl Kult, Biochemistry KTH, for welcoming me to be one of the Biocats, for enlightening me in protein science, for the enjoyable, open scientific atmosphere, and for your precious time in examination of the thesis. The members of Organgruppen along my way in Sundsvall. Ove and Carin, the genius and nice colleagues who were not only good in chemistry are deeply thanked for helps in many matters; Helen, Marica and Linda, my office-mates for helps and friendship not only on job; Olof and Dan for patient guidance to the state-of-the-art of NMR and many other things; Michael, Ba-Vu and Staffan for sharing the lab tips and collaboration, Carina, Kristina and Erik for friendly helps. Håkan and Torborg for always being the nice and helpful people with generous support in apparatus, chemicals and many other issues. Siw, Maria for various administrative supports. The former members of Biocats during my time at Department of Biotechnology, KTH. Didier for stimulating discussion and constructive suggestions; Fridrik for being a nice lab neighbour and the help-desk; Linda for impressive demonstration of protein modeling, Jenny for helpful GC-MS assistance. Associate professor Ulla Jacobsson in the Department of Chemistry, KTH for NMR support and fruitful discussion. Ingvor and Vera for smooth handling of the administrative works.

57

Finance support from Mid Sweden University, the Swedish Natural and Technical Sciences Research Council (NFR), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning are gratefully acknowledged. All my Chinese friends and colleagues in Sweden, for having fun, exercise together, for taking care and substantial helps. Especially Ting-Ting and You-Zhi for the “first aids” when I started to work in Sweden. My parents, my sister, brother and all in-laws for forever love, support and encouragement. My father in-law for making the amazing Chinese paintings. Finally, my dear husband and my beloved son. Yun for sharing the tears and happiness, for always support with endless love and for undoubtedly accompanying wherever ultima Thule. My sweet heart San-San, the sunshine of my life, for bringing a huge glad and brightness into my life, and now for making me as a happy and proud mom.

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