Applications of aldolases in organic synthesis - Schoevaart Thesis 2000

Applications of aldolases

in organic synthesis

Rob Schoevaart

Front cover:

Sun set in the Mediterranean sea

Er is niets in het bewustzijn dat niet door zijn eigen zelf is.

Phenomenologie des Geistes, Hegel

Applications of aldolases

in organic synthesis

Toepassingen van aldolasen in organische synthese

Proefschrift

ter verkrijging van de graad van doctor

aan de Technische Unversiteit Delft

op gezag van de Rector Magnificus, Prof. ir. K.F. Wakker

ingevolge het besluit van het College voor Promoties

in het openbaar te verdedigen op

dinsdag 23 mei 2000 om 13:30

door

Willem Robert Klaas Schoevaart

geboren op 28 januari 1969 te Coevorden

Dit proefschrift is goedgekeurd door de promotor: Prof. dr. R.A. Sheldon.

Samenstelling promotiecommissie:

Rector Magnificus Technische Universiteit Delft

Prof. dr. A. Bruggink DSM Geleen / KU Nijmegen

Prof.dr.ir. J.J. Heijnen Technische Universiteit Delft

Prof. dr. A.P.G. Kieboom Universiteit Leiden / DSM-Gist

Prof. dr. B. Zwanenburg KU Nijmegen

Dr. W. Fitz ICI

Dr.ir. F. van Rantwijk Technische Universiteit Delft

Dr. ir. F. van Rantwijk heeft als begeleider in belangrijke mate aan de

totstandkoming van dit proefschrift bijgedragen.

ISBN 90-9013780-7

Copyright© 2000 W.R.K. Schoevaart

Printed by Print Partners Ipskamp

This work was carried out as part of the Innovation Oriented research Programme on

Catalysis (IOP Katalyse, no. IKA94010) sponsored by the Netherlands Ministery of

Economic Affairs.

Contents

1............................................................................................................. 1General introduction ......................................................................... 1

IOP Catalysis.......................................................................................................2Enzymatic aldol reactions....................................................................................3The aldol reaction................................................................................................4Aldol reactions with aldolases .............................................................................5Dihydroxyacetone phosphate dependent aldolases ............................................8Pyruvate- and phosphoenolpyruvate-dependent aldolases...............................10Glycine-dependent aldolases ............................................................................112-Deoxyribose-5-phosphate aldolase................................................................12Transketolase....................................................................................................13The objectives and justification of the thesis .....................................................15

General ..........................................................................................................15Specific ..........................................................................................................16

References........................................................................................................16

2........................................................................................................... 21Aldol reactions with DHAP dependent aldolases ......................... 21

Abstract .............................................................................................................21Introduction........................................................................................................22Acceptor substrate specificity of FruA, FcuA and RhuA ....................................23Properties of FruA from different sources..........................................................24

FruA stability under reaction conditions. ........................................................27A 5 mmol scale reaction.................................................................................27

Conclusion.........................................................................................................28Experimental .....................................................................................................28

General ..........................................................................................................28Aldol reaction .................................................................................................28FruA activity assay.........................................................................................29DHAP-assay ..................................................................................................29Enzyme recovery ...........................................................................................30Synthesis of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose ............................31

References........................................................................................................31

3........................................................................................................... 35The stereochemistry of DHAP aldolases....................................... 35

Abstract .............................................................................................................35Determination of stereoselectivity with chiral GC ..............................................36Determination of stereoselectivity with an enzymatic assay..............................39

Comparison of methods.................................................................................41Stereospecificity of FruA and RhuA ...............................................................42

Experimental .....................................................................................................45General ..........................................................................................................45Acetylation of GC-samples ............................................................................46Preparation of product samples .....................................................................46Aldol reaction .................................................................................................46

Retro aldol reaction assay .............................................................................47References........................................................................................................47

4........................................................................................................... 51In situ generation of DHAP: cascade catalysis ............................. 51

Abstract .............................................................................................................51Synthesis of dihydroxyacetone phosphate ........................................................52Integral in situ reaction ......................................................................................55Sequential one-pot reaction...............................................................................60Figure 6. ............................................................................................................60

Synthesis of L-glycerol-3-phosphate..............................................................61Effects of glycerol concentration ....................................................................62Selectivity.......................................................................................................63Synthesis of DHAP from L-glycerol-3-phosphate...........................................64Aldol reaction and dephosphorylation............................................................64Chemical phosphorylation of glycerol ............................................................65


General ..........................................................................................................66Assay for DHAP and L-glycerol-3-phosphate ................................................67DHAP synthesis from glycerol .......................................................................67Aldol reactions ...............................................................................................68Chemical phosphorylation of glycerol ............................................................69

References........................................................................................................69

5........................................................................................................... 73DHAP analogues ............................................................................. 73

Abstract .............................................................................................................73Hydroxyacetone thiosulfate as DHAP analogue................................................74Dihydroxyacetone arsenate as DHAP analogue ...............................................76Results and discussion......................................................................................78

Optimal conditions .........................................................................................79Use of cosolvents ..........................................................................................80Acceptor specificity with DHAAs ....................................................................83Effect of arsenate on the stereoselectivity .....................................................85


General ..........................................................................................................86Preparation of hydroxyacetone thiosulfate (HATS)........................................87Preparation of 2,3-dihydroxypropane thiosulfate ...........................................88Assay of aldehydes with alcohol dehydrogenase ..........................................88Assay of DHA ................................................................................................89Aldol reaction with dihydroxyacetone arsenate..............................................89Stereoisomer assay .......................................................................................90Preparation of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose .........................91

References........................................................................................................91

6........................................................................................................... 95Transketolase versus...................................................................... 95fructose-1,6-bisphosphate aldolase .............................................. 95

Abstract .............................................................................................................95

Introduction........................................................................................................96α-Hydroxyaldehydes .........................................................................................98Cross-linked enzyme crystals..........................................................................100Conclusions.....................................................................................................102Experimental ...................................................................................................103

General ........................................................................................................103Transketolase catalysed reactions...............................................................103FruA catalysed reactions .............................................................................103Hydroxypyruvate assay................................................................................104Synthesis of D,L-lactaldehyde .....................................................................104Synthesis of α-hydroxybutanal.....................................................................105Synthesis of α-hydroxypentanal...................................................................106Synthesis of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose ..........................106Crystallization and cross-linking..................................................................106

References......................................................................................................107

7......................................................................................................... 1112-Deoxyribose-5-phosphate aldolase (DERA)............................. 111

Abstract ...........................................................................................................111Introduction......................................................................................................112Donor substrates .............................................................................................113Acceptor substrates.........................................................................................113Sequential aldol additions ...............................................................................114Results and discussion....................................................................................116

Stability in cosolvents...................................................................................117Activity in cosolvents....................................................................................119

Conclusion.......................................................................................................121Experimental ...................................................................................................121

General ........................................................................................................121Preparation of DERA ...................................................................................121Activity assay ...............................................................................................122Stability test .................................................................................................122

References......................................................................................................122Summary........................................................................................ 124Samenvatting................................................................................. 127Dankwoord..................................................................................... 130Publications................................................................................... 133Curriculum vitae............................................................................ 134Index............................................................................................... 135

aan mijn ouders

1

1

General introduction

The presence of multiple functional groups of similar reactivity in molecules poses

problems for classical organic synthetic methods. Synthesizing and modifying

carbohydrates for example, is accomplished only by applying complex synthetic

schemes with many protection and deprotection steps to ensure regio- and

stereoselectivity. A large proportion of the reactions in the armamentarium of organic

chemists is incompatible with aqueous systems and some modifications are

necessary simply to achieve solubility in non-aqueous solvents. The application of

enzymes1 as catalysts in organic synthesis has succeeded in solving synthetic

problems and developments in enzymology have effectively refuted the perception

that most enzymes are fully dependent on natural substrates, costly, unstable and

require expensive cofactors.

Chapter 1

2

IOP Catalysis

Research supported by the Ministry of Economic Affairs, with the ultimate goal of

creating new economic activity (i.e. to create jobs and prosperity), set itself the

demanding objective of providing an essential creative step on the road from

fundamental science to novel, applicable technology. This implied research based on

a vision of its potential applications in an industrial context2.

Catalytic conversions are involved in the manufacture of more than 80% of the

total volume of chemicals. Hence a solid knowledge base on catalysis is of strategic

interest to the Dutch chemical industry. Catalysis offers the opportunity to steer

chemical conversions into a desired direction. This has resulted in the central

chemical theme of a research programme known as the Innovation Oriented

Research Programme (IOP) Catalysis: precision in chemical conversion. This

precision is required both to save energy and feedstocks and to avoid the formation

of undesired by-products and waste. Catalysis is applied on a large scale in

petroleum refining and in the manufacture of bulk chemicals. This is far less the case

in the manufacture of fine chemicals where classical multi-step chemical conversions

play a much larger role. These classical procedures often involve lower selectivities,

the use of undesirable, toxic, or corrosive reagents, the formation of by-products and

large amounts of waste.

Biocatalysis is of particular interest for the manufacture of the fine chemicals,

such as pharmaceuticals and products of the flavour and fragrance industry. In these

branches specific optically active isomers are often required; biocatalyst have shown

promise in such enantioselective synthesis since they compete with chemical

catalysis on e.g. introduction of chirality, selective modification or resolution of


3

racemates. Beyond that, enzymatic conversion of natural compounds can even yield

“natural” products. For example, “natural” benzaldehyde is distilled from plants, but

retro aldol cleavage of abundant cinnamaldehyde is a “grey–zone chemistry” route to

so-called natural benzaldehyde3. Inexpensive, readily available and renewable

natural precursors, such as fatty or amino acids, can be converted to more highly

valued flavours.

Enzymatic aldol reactions

Catalytic asymmetric aldol reactions remain one of the most interesting and

challenging subjects in synthetic organic chemistry and, without a doubt, in

biocatalysis. This attractiveness and ambition originates from the importance of C-C

bond formation. When comparing simple base catalysed aldol reactions with enzyme

catalysed ones a characteristic of the enzymatic reaction is particularly outstanding:

enantioselectivity. Whereas the simple base catalysed reaction is only

enantioselective with (stoichiometric amounts of) chiral starting materials, application

of enzymes can lead to enantiomerically pure products from completely non-chiral

starting materials. For non-enzymatic reactions regioselectivity is more controlable,

but the use of aldehydes or ketones with free hydroxyl or highly activated groups

might be problematic, making protection and deprotection steps necessary.

Enzymes are usually completely regioselective, reducing protective group chemistry

to a minimum, making biocatalysis an important instrument in C-C bond forming

reactions.

Aldolases have good activity towards hydroxylated compounds which is not

Chapter 1

4

suprising considering their role in the carbohydrate metabolism. This property is

narrowly associated with their common medium: water. Aldol reactions catalysed by

enzymes can consequently be conducted under mild conditions (water, pH 7) and do

not carry the burden of obligatory use of strong bases and organic solvents, thus

obviating again the use of protective groups. The mild conditions and their

biodegradability makes them an excellent environmentally acceptable option as well.

The aldol reaction

In the aldol reaction4 the α-carbon of an aldehyde or ketone adds to the carbonyl

carbon of another. This reaction is called the aldol condensation when the product

(in some cases spontaneously) dehydrates in the course of the reaction, because

the new double bond is conjugated with the C=O bond, forming an α,β-unsaturated

product. The base most often used is OH-, but stronger bases are also applied.

Figure 1. The aldol reaction

In principle, five combinations are possible for the aldol reaction, namely 1) two

identical aldehydes, 2) two identical ketones, 3) two different aldehydes, 4) two

different ketones and 5) an aldehyde and a ketone. Combination 1 has a favorable

equilibrium, in contrast with combination 2. Therefore combination 4 is hard to apply.

R2

R1

O

R4R1

OH

R3

O

R2R3

R4

O

H+ base


5

Combination 3 can give as much as four different products when both aldehydes

have an α-hydrogen. The last combination, also called the Claisen-Schmidt reaction,

is particularly straightforward and affords only one product when the aldehyde bears

no α-hydrogen.

The aldol reaction can create two contiguous stereogenic centers and,

consequently, four stereoisomers. Some control over the stereoselectivity can be

obtained by the use of preformed enolates with e.g. lithium5, Ti6, Zr7, Pd8 or Au9.

Normally, metal Z enolates give the two syn products whereas E enolates are mostly

non-stereoselective. Enantioselectivity can only be obtained by using either chiral

enol derivaties, chiral aldehydes or ketones, or both. Recent studies of catalytic

antibodies10 opened ways to obtain enantiomerically pure aldols via resolution.

Aldol reactions with aldolases

One need only to consider the remarkable complexity and variety of molecules

produced within the biosphere to realize that the enzymes which make these

possible would be of immense synthetic utility. Aldolases are part of a large group of

enzymes called lyases and are present in all organisms. They are mainly involved in

the metabolism of carbohydrates, but also of amino acids and hydroxy acids.

Complex carbohydrates play an important role in various types of biochemical

processes like growth, development, immune response, infection, cell adhesion,

metastasis and numerous signal transduction events. They are synthetically

accessible by application of aldolases. Although their function in vivo is often related

to the degradative cleavage of metabolites, the reactions are reversible and by

Chapter 1

6

choosing the proper conditions synthesis becomes favored. A number of compounds

are accessible11 in this way, particularly analogues of sugars like nitro-, amino-,

deoxy-, thio-, aza-, fluoro-, O-acyl, O-alkyl-sugars or aroma compounds and chiral

intermediates. The more than 30 different aldolases with different donor and

acceptor specificity that currently have been identified, are classified on the basis of

their donor into five groups (Table I).

Table I. Five groups of aldolases, adding either a two or a three carbon unit.

Aldolase Donor Example(s)

DHAP-dependent RAMA, FruA from S.carnosus

pyruvate-dependent Sialic acid aldolase(NeuAc aldolase)

phosphoenolpyruvate-

dependent

NeuAc synthetase

glycine-dependent L-threonine aldolase from

Candida humicola

acetaldehyde-dependent DERA from E. coli

Many of these enzymes are restricted more or less to their natural substrates but this

may not pose a limitation in their use since biologically meaningful compounds are

made by nature in the first place with these aldolases. Hence, applying them in the

synthesis of a derivative will be very advantageous since simple modification of one

HO OPO3-2

O

O-

O

O

O-

OPO3-2

O

H2NOH

O

O


7

of the substrates easily leads to modified end products. Selecting the right aldolase

is, therefore, an important stage in designing a synthetic route.

There are two classes12 of aldolases which vary in structure since they

separated early in evolution, although identical reactions are catalysed. Class I

aldolases, which are present in all groups of living organisms, from prokaryotes to

eukaryotes, are characterized by the formation of a Schiff’s base with the donor

substrate (see Figure 2). When the aldolase is treated with sodium borohydride after

the reaction with the donor, enzyme inactivation is observed. Class II aldolases

occur only in prokaryotes and lower eukaryotes such as yeast, algae and fungi. They

require a metal cofactor such as divalent zinc and are inactivated by chelating

compounds such as EDTA.

Remarkably, the binding of an aldehyde via an imine bond was also

successfully applied in a non-enzymatic aldol reaction, using LiN(iso-Pr)2 as the

base13. This is called a directed aldol reaction.

Figure 2. Imine intermediate in the active site of a type I fructose-1,6-bisphosphate

aldolase.

O

CO-

-2O3PO

HO

HO

Arg-148Lys-146Lys-107

Lys-229

Asp-33

+NH3 NH3+

OH

+NH

O

C

H2N+NHH

O-

O

O-

P

Chapter 1

8

Dihydroxyacetone phosphate dependent aldolases

The best studied aldolase is fructose-1,6-bisphosphate aldolase (FruA) from rabbit

muscle (RAMA). It accepts a wide range of aldehydes in addition to its natural

substrate, glyceraldehyde-3-phosphate, allowing the synthesis of many sugar

derivatives. This tetrameric enzyme is limited in its synthetic application by a low

operational stability14 and is now frequently replaced by the stable monomeric FruA

from Staphylococcus carnosus15,16. L-rhamnulose-1-phosphate aldolase46 (L-RhuA)

and L-fuculose-1-phosphate aldolase46,17 (L-FcuA), both from E. coli, are also

commercially available. Tagatose-1,6-bisphosphate aldolase (TagA) is not

commercially available.

Figure 3. The four complementary DHAP dependent aldolases

+

DHAP

1 2

3 4

FruA

OPO32-

O

HO

L-FcuA

L-RhuA

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

RCHO

TagA


9

These four aldolases (Figure 3) catalyse in vivo the reversible asymmetrical aldol

addition of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate or

L-lactaldehyde, but they are expected to be as flexible towards the acceptor

substrate as RAMA. Two other DHAP-utilizing aldolases have been described,

namely erythrulose-1-phosphate aldolase18 which yields only one new chiral centre

since formaldehyde is the acceptor substrate and phospho-5-keto-2-deoxygluconate

aldolase19 which has the same stereospecificity as FruA but is not commercially

available.

The interest in the group of DHAP-dependent aldolases is mainly due to their

enantioselectivity. Not only are these DHAP aldolases enantioselective, the

combination of four different DHAP aldolases opens the possibility to preplan the

synthesis of any one of the four possible stereoisomers (Figure 3) normally

generated in this type of reaction, a degree of control unprecedented in organic

synthesis.

The scope of DHAP-dependent aldolases in organic synthesis is restricted by

three factors: namely 1) the acceptor substrate specificity, 2) the stereoselectivity

and 3) the circuitous and laborious synthesis of the donor substrate DHAP. We have

found that a wide variety of aldehydes can be used as the acceptor (see Chapter 2).

The influence of the structure of the acceptor on the stereoselectivity of the aldolases

needs to be determined since it is of vital importance to their utility as fine chemicals

or specialties (Chapter 3). A new preparation of the donor substrate DHAP from very

cheap starting materials was developed to overcome the disadvantages associated

with existing syntheses. The new method was integrated in an unprecedented four

steps, one-pot reaction (Chapter 4). The investigation of DHAP analogues was

mainly limited to the use of arsenate (Chapter 5).

Chapter 1

10

Pyruvate- and phosphoenolpyruvate-dependent aldolases

Pyruvate-dependent aldolases have catabolic functions in vivo, whereas their

counterparts employing phosphoenolpyruvate as the donor substrate are involved in

the biosynthesis of keto acids. However, both classes of enzymes can be used to

prepare similar keto acid products20.

N-acetylneuraminic acid (NeuAc) aldolase or sialic acid aldolase catalyzes the

reversible aldol reaction of N-acetyl-D-mannosamine (ManNAc) and pyruvate (Figure

4) to N-acetyl-D-neuraminic acid (Neu5Ac). The sialic acids are key cell-surface

determinants of mammalian glycoconjugates. They are found at terminal positions of

glycoproteins and glycolipids, which play an important role in biological recognition

processes. Neu5Ac is the best-known member of this special class of amino sugars.

Derivatives of Neu5Ac have been found to act as inhibitors of sialidases21.

Figure 4. N-acetylneuraminic acid (Neu5Ac) aldolase catalysed aldol reaction of N-

acetyl-D-mannosamine (ManNAc) and pyruvate to Neu5Ac.

In vivo its synthesis is accomplished by Neu5Ac synthetase through the irreversible

condensation of phosphoenol pyruvate (PEP) and N-acetylmannosamine. For the

synthesis of oligosaccharides and enzyme inhibitors Neu5Ac has been previously

isolated in small amounts from natural sources, such as cow’s milk, edible birds’

nests or eggs. Successful production of Neu5Ac was achieved by enzymatic

NeuAc Aldolase

CO2H

O

O

OH

HO

OHCO2H

OH

HO

AcHN

OHO

HO

HONHAc

OHManNAc Neu5Ac


11

synthesis22 from ManNAc and pyruvic acid (space-time yield 470 g / L-1.d-1) with

Neu5Ac aldolase. The retro-aldol reaction is favored and an equilibrium favoring the

aldol reaction product is induced by providing pyruvate in excess (five- to seven-

fold). The excess pyruvate can be decomposed with pyruvate decarboxylase23 in

order to simplify the product isolation. The enzyme, immobilized onto Eupergit-C,

has been used in industrial scale production24 of Neu5Ac.

Glycine-dependent aldolases

Glycine-dependent aldolases catalyze the reversible aldol reaction of glycine with an

aldehyde acceptor to form β-hydroxy-α-amino acids. These compounds are naturally

occurring amino acids (threonine, serine) as well as components of many complex

natural products such as antibiotics and immunosuppressants (e.g. mycestericin D25,

vancomycin, echinocardin D, cyclosporin, katanosin, polyoxin D, empedopeptin etc.).

Figure 5. Selective retro aldol reaction by a L-specific threonine aldolase

D-threoL-threo

L-aldolase

NaOHMeOH

+

+ H2NCH2CO2H

CO2H

OH

NH2X

CO2H

OH

NH2X

CHO

X

Chapter 1

12

Both D- and L-isomers are biologically significant. Threonine aldolases are the best

described glycine-dependent aldolases. Only a few examples are known of its use

in bond-forming reactions26; yields and stereoselectivity are generally low for non-

natural substrates.

Threonine aldolases, with acetaldehyde as their natural acceptor, have been

used extensively in the resolution of racemic β-hydroxy-α-amino acids27 (Figure 5).

2-Deoxyribose-5-phosphate aldolase

The enzyme 2-deoxyribose-5-phosphate aldolase11 (DERA) catalyses in vivo the

reversible aldol reaction of acetaldehyde and D-glyceraldehyde 3-phosphate to form

D-2-deoxyribose-5-phosphate (Figure 6), the sugar moiety of DNA. Consequently

this type I aldolase is widespread in nature. It is the only aldolase that accepts two

aldehydes as substrates.

Figure 6. Production of D-2-deoxyribose-5-phosphate by DERA.

-2O3PO

HO

O

OH

H

O

-2O3PO O

HO

OOH

-2O3PO

OH

DERA

2-deoxyribose-5-phosphate

acetaldehydeD-glyceraldehyde-3-phosphate

+


13

Furthermore, DERA is an item of interest because it needs no phosphorylated

substrates, in contrast to DHAP aldolases. It has been cloned and overexpressed in

E. coli.

Acetaldehyde can be replaced by propanal, acetone and fluoroacetone, making

DERA suitable for production of β-hydroxy aldehydes or ketones. The extent of the

chain elongation is thus not fixed, but either 2- or 3-carbon long. Initial studies28

showed that the acceptor D-glyceraldehyde-3-phosphate can be replaced by other

aldehydes, albeit at a penalty of more than 99% of the reaction rate. Relatively large

amounts of enzyme are consequently required. The stability of the enzyme is one

point of investigation.

Transketolase

Transketolase is not an aldolase but a transferase. It catalyzes the transfer of a

two-carbon ketol moiety from a ketose to an aldose. Transketolase is particularly

attractive as an alternative to FruA, in combination with an appropriate choice of

substrates. When using an α-hydroxyaldehyde as acceptor and hydroxypyruvate as

donor, compounds with structures identical to those produced by FruA are obtained

(Figure 7). This reaction is of key importance in the synthesis of a wide range of

molecules in vitro and the ability to carry out the reaction stereospecifically means

that this biotransformation is of particular commercial interest.

The natural donor substrate, D-xylulose-5-phosphate, can be replaced by β-

hydroxypyruvate. This renders the reaction irreversible, because instead of

glyceraldehyde-3-phosphate carbon dioxide is released. Hydroxypyruvate29 is also

Chapter 1

14

cheaper and more stable than D-xylulose-5-phosphate, which adds to its

attractiveness as donor. The two natural acceptors D-ribose-5-phosphate and

erythrose-4-phosphate can be replaced by a whole range of aldehydes, which will be

discussed in Chapter 6.

Figure 7. Transketolase catalysed reaction of hydroxypyruvate and an α-

hydroxyaldehyde.

Although transketolase requires thiamine pyrophosphate (TPP) and

magnesium(II) ions to function, only catalytic amounts of these cofactors are

required. Moreover, it does not necessarily require phosphorylated compounds as

substrates30 (although the natural substrates are phosphorylated) which is a

significant advantage over FruA. Even though the enzyme is absolute in its

requirement for the (R)-configuration of the hydroxy functionality at C2 of the

aldehyde, there seem to be no other stereochemical restrictions. Of main interest is:

how well does transketolase compete with or complement FruA regarding reaction

rate and conversion and how stereoselective is the enzyme? These questions are

1) FDP-aldolase

2) acid phosphataseOH

OOH

OH

RR

O

HOPO3

2-

O

HO+

OH

OOH

OH

R

CO2

transketolase+O

-O

OH

O

R

O

H

OH


15

addressed in Chapter 6.

The objectives and justification of the thesis

Aldolases doubtlessly constitute a group of catalysts with great synthetic potential. In

order to outgrow the shortcomings in the employment of aldolases in future

commercial processes, generic problems such as the design of non-natural

substrates, the exploration of enzymes from new species, multi-enzyme systems,

enzyme stabilization and application in the synthesis of fine and specialty chemicals

all have to be addressed.

General

The main objective of this thesis is to increase the applicability of enzyme catalyzed

aldol reactions. This implies that the enzymatic reactions have to be adapted for their

use in organic synthesis and economically viable industrial processes. The

development of practical procedures for the synthesis of enantiomerically pure

compounds based on enzyme catalysed aldol reactions will contribute to an

increased atom utilisation factor and, hence, will reduce waste streams and

enantiomeric ballast.

The exploration of enzymatic aldol reactions is justified by the central position

of aldol reactions in organic synthesis. For synthesizing and modifying carbohydrates

for example, the application of aldolases has already demonstrated its potential for

solving synthetic problems.

Chapter 1

16

Specific

The most common aldolases are dependent on dihydroxyacetone phosphate

(DHAP) as their exclusive donor reactant. The use of DHAP as the only donor

substrate restricts the spectrum of products since the wide variety of possible

acceptor substrates is combined with a single donor, whereas it would be preferred

to have a various choice of donor substrates as well. Hence, aldolases that do not

depend on expensive phosphorylated substrates have to be considered as

alternative to DHAP-dependent ones.

The phosphate anion plays a crucial rol in the binding of the reactants and in

the reaction mechanism. The use of DHAP is accompanied by consumption of

expensive organic phosphate that is ultimately transformed into inorganic phosphate,

which is not contributing to an economical proces. The replacement or efficient

preparation of DHAP would increase competitiveness of enyzme catalysed aldol

reactions relative to chemical ones. To investigate the benefits of phosphate free

aldol reactions, 2-deoxyribose-5-phosphate aldolase (DERA) was selected, although

this leads to different products than use of DHAP dependent aldolases. Exploration

of the transferase transketolase as an alternative to DHAP dependent FruA is a very

alternate approach of the DHAP “problem”, however very engaging since identical

products can be made.

References

1 E.J. Toone, E.S. Simon, M.D. Bednarski and G.M. Whitesides, Tetrahedron, 1989

45 (17) 5365-54222 Dr. Ir. J.M. Oelderik, Chairman of the IOP Catalysis, April 3, 1998


17

3 U. Krings, R.G. Berger, Appl. Microbiol. Biotechnol., 1998, 49, 1-84 J. March, Advanced Organic Chemistry, Wiley-Intersience, New York, 1992, 4th

Ed., 937-9455 Arnett, Fisher, Nichols, Ribeiro, J. Am. Chem. Soc. 1990, 112, 8016 Stille, Grubbs, J. Am. Chem. Soc. 1983, 105, 16647 D.A. Evans, L.R. McGee , J. Am.Chem.Soc, 1981, 103, 28768 Nokami, Mandai, Watanabe, Ohyama, Tsuji, J. Am.Chem.Soc. 1989, 111, 41269 Y. Ito, M. Sawamura and T. Hayashi, J. Am. Chem. Soc., 1986, 108, 6405-6406

10 B. List, D. Shabat and C.F. Barbas III, J. Am. Chem. Soc., 1999, 121, 7283-729111 For reviews see: Gijsen, H.J.M.; Qiao, L.; Fitz, W.; Wong, C.-H.; Chemical

Reviews 1996, 96(1), 443-469; M.D. Bednarski, E.S. Simon, ACS Sypmposium

Series 1990 466 Chapter 1 and 2; Wong, C.-H, Halcomb, R.L., Ichikawa, Y. and

Kajimoto, T., Angew. Chem. Int. Ed. Engl. 1995, 34, 412-432; Wang, P.G., Fitz,

W.; Wong, ChemTech, 1995, april, 22-3212 Gefflaut, T., Blonski, C., Perie, J. and Willson, M., Prog. Biophys. Molec. Biol.

1995, Vol. 63, 301-340,13 Wittig, Frommeld, Suchanek, Angew. Chem. Int. Ed. Engl. 1963, 2, 68314 Brockamp, H.; Kula M. Tetrahedron Letters, 1990, 31 (49), 7123-7126.15 Witke, C.; Götz, F. J. Bacteriol., 1993, 175 (22), 7495-7499.16 Brockamp, H.; Kula, M.; Goetz, F. U.S. 5.162.221, 10 Nov. 1992.17 Ozaki, A.; Toone, E.J.; Von der Osten, C.H.; Sinskey, A.J.; Whitesides, G.M. J.

Am. Chem. Soc. 1990, 112, 4970-497118 G.C. Charalampous, Methods Enzymol. 1962, 5, 28319 W.A. Anderson, B. Magasanik, J. Biol. Chem. 1971, 246, 566220 D.G. Comb and S. Roseman, J.Biol. Chem. 1960, 235, 2529-253721 E. Zbiral, H. Brandstetter, R. Christian and R. Schauer, Liebigs Ann. Chem. 1987,

781-78622 U. Kragl, M. Kittelmann, O. Ghisalba and C. Wandrey, Ann. N.Y. Acad. Sci.1995,

750, 300-30523 C.-H. Lin, T. Sugai, R.L. Halcomb, Y. Ichikawa, C.-H. Wong, J. Am. Chem. Soc.

1992, 114, 1013824 M. Mahmoudian, D. Noble, C.S. Drake, R.F. Middleton, D.S. Montgomery, J.E.

Chapter 1

18

Piercey, D. Ramlakhan, M. Todd and M.J. Dawson, Enzyme Microb. Technol.

1997, Vol. 20, april, 393-40025 K. Shibata, K. Shingy, V.P. Vassilev, K. Nishide, T. Fumita, M. Node, T. Kajimoto,

C.-H. Wong, Tetrahedron Letters 1996, 37 (16), 2791-2794.26 T. Kimura, V.P. Vassilev, F.-J. Shen and C.-H. Wong, J. Am. Chem. Soc. 1997,

119, 11734-1174227 Celgene Corporation, US patent No. 5346828, 199428 C.F. Barbas III, Y.-F. Wang and C.-H. Wong, J. Am. Chem. Soc. 1990, 12, 2013-

201429 Large scale synthesis: K.G. Morris, M.E.B. Smith, N.J. Turner, M.D. Lilly, R.K.

Mitra and J.M. Woodley, Tetrahedron: Asymmetry, 1996, 7 (8), 2185-218830 U. Schörken and G.A. Sprenger, Biochim. et Biophys. Acta, 1998 1385, 229-243

21

2

Aldol reactions with DHAP dependent aldolases

“Kinetiek is de ethiek van de moderne tijd”

Peter Sloterdijk

Abstract

The synthetic application of enzymatic aldol reactions using the commercially

available DHAP-aldolases, D-fructose-1,6-bisphosphate (FruA), L-fuculose-1-

phosphate (FcuA) and L-rhamnulose-1-phosphate (RhuA) aldolase, was

investigated. All three aldolases were shown to have a broad specificity towards the

aldehyde acceptor. The kinetic properties of FruA from two bacterial and one

mammalian source were compared and appeared to be rather similar, but the

bacterial aldolases, were much more stable. The addition of dihydroxyacetone

phosphate to butanal catalyzed by FruA from Staphylococcus carnosus was

performed on a 5 mmol scale, demonstrating the applicability of aldolase catalysed

reactions.

Chapter 2

22

Introduction

The dihydroxyacetone monophosphate (DHAP) dependent aldolases (Figure 3, page

8), which in their natural role perform a retro-aldol reaction, have mainly been used

to synthesize a wide variety of modified carbohydrates.31,32,33,34 With their natural

substrates they exhibit essentially absolute control over the two newly created

stereogenic centers,35 each aldolase being specific for one of the four possible

stereoisomers. However, in order to be a useful synthetic tool aldolases must not

only function with a wide variety of aldehyde substrates but should also exhibit the

same degree of stereospecificity as is observed with their natural substrates. In this

chapter we have investigated the substrate spectrum of commercially available class

I DHAP aldolases.

Fructose-1,6-bisphosphate aldolase (FruA) from Staphylococcus carnosus36,37

and Staphylococcus aureus38 are known to be very heat and pH stable.39 The former

organism is a safer production host than the pathogenic S. aureus and, hence, its

aldolase has been studied more intensively. L-Rhamnulose-1-phosphate aldolase40

(RhuA) and L-fuculose-1-phosphate aldolase40,41 (FcuA), both from E. coli, were also

available. Tagatose-1,6-bisphosphate aldolase (TagA) is not commercially available

and was not investigated.

The best studied DHAP-depending aldolase is FruA from rabbit muscle (RAMA.

It accepts a number of unnatural aldehydes42 as substrate, but its practical

application is hampered by a low operational stability43. The substrate spectrum of

FruA from S. carnosus suggests that it has a similarly relaxed acceptor

specificity,43,44 but its kinetic properties have not been studied in any depth.


23

Acceptor substrate specificity of FruA, FcuA and RhuA

The three commercially available DHAP aldolases FruA∗, FcuA and RhuA were

compared in their ability to accept a number of non-natural aldehyde substrates.

Conversions (see Table I) show that FruA and RhuA have very similar properties.

With the exception of chloroacetaldehyde, methylglyoxal, phenylacetaldehyde and

pivaldehyde (10 to 20% difference), almost equal values were found. With FcuA

consistently lower conversions were found. Surprisingly glycolaldehyde, which

resembles the natural substrate (L-lactaldehyde) most, showed only a conversion of

22%.

Table I. Conversions of aldolases (determined by DHAP consumption)

acceptor FruA FcuA RhuA

Formaldehyde 81 26

Acetaldehyde 84 57 88

Propanal 67 60 76

Butanal 78 61 81

i-Butyraldehyde 78 69 81

Pivaldehyde 85 68 56

Glycolaldehyde 96 22 99

Glyoxal 98 43 98

Chloroacetaldehyde 76 68 92

Methylglyoxal 86 29 99

Phenylacetaldehyde 66 31 79

∗ From Staphylococcus carnosus.

Chapter 2

24

Similarities in kinetic properties are expected since all the class I DHAP

aldolases bind the same donor substrate by involving equivalent amino acid

residues. Consequently, the tolerance towards unnatural acceptor substrates is

expected to vary only within certain limits.

Properties of FruA from different sources

The FruAs from rabbit muscle (RAMA), S. carnosus and S. aureus were compared

for activity in the aldol reaction as well as the retro-aldol reaction. The cleavage of

the natural reactant –D-fructose-1,6-bisphosphate- was used to assay the retro-aldol

activity. The catalytic activity in the synthetic direction was measured with DHAP as

donor; D-glyceraldehyde-3-phosphate (GAP) and propanal were used as acceptors.

The reactions were monitored by a coupled enzymatic assay45 of DHAP. In this

assay DHAP is reduced to glycerol-3-phosphate with NADH using glycerol-3-

phosphate dehydrogenase. The decrease in absorption at 340 nm is proportional to

the amount of DHAP present.

The results of the assays are tabulated in Table II. All three aldolases

converted GAP and propanal at similar rates. Under the reaction conditions used,

the specific synthesis activity of the bacterial aldolases was approximately 50% of

their cleavage activity, whereas RAMA had the same activity in both reactions.


25

Table II. Specific activities of FruA from different sources.

aldolase

splitting

Fruc-1,6-bisphosphate

U/mg enzyme

synthesis

propanal + DHAP

U/mg enzyme

synthesis

GAP + DHAP

U/mg enzyme

S. carnosus 26.7 12.2 12.2

S. aureus 16.8 7.2 8.5

RAMA 22 24 22.4

The acceptor specificity of the three aldolases was investigated by incubating DHAP

and a 100% excess of acceptor aldehyde with 1 PAU* of aldolase. Propanal was

chosen instead of the conventionally used glyceraldehyde-3-phosphate because of

better reproducibility, comparable kinetic properties and ease of use.

The relative initial rates and conversions of the reaction of DHAP with the

different aldehydes are displayed in Table III. The kinetic parameters of RAMA are

comparable with those of the FruA from S. carnosus or S. aureus, as might be

expected on the basis of the similarity in structure of the active sites. Conjugated

aldehydes such as benzaldehyde and acrolein (Vini = 0) are not substrates, with the

exception of pyridinecarboxaldehydes43. Longer alkyl chains result in a lower

(relative) Vini and conversion. Aldehydes bearing an electron-withdrawing group such

as chloroacetaldehyde have an activated carbonyl group, which increases the initial

rate. Bulky and aromatic groups result in lower rates and conversions. Hence, we

conclude that the generalizations made for the substrate spectrum of RAMA42 also

apply to bacterial type I FruA.

* 1 PAU (propanal aldolase unit) converts propanal at an initial rate of 1 µmol per minute.

Chapter 2

26

Table III. Relative initial reaction rates and conversions of aldol reactions with DHAP.

Aldehyde Relative Vini (%)

carnosus aureus RAMA

Conversion (% (t ))

carnosus aureus RAMA

formaldehyde 133 57 43 81 (2h) 16 (2h) 31 (3h)

acetaldehyde 108 141 103 84 (2.5h) 64 (3h) 87 (2h)

propionaldehyde 100 100 100 67 (4h) 68 (2h) 67 (3h)

butanal 37 72 23 78 (4h) 53 (4h) 69 (3h)

pentanal 36 27 20 64 (3h) 50 (4h) 67 (4h)

hexanal 32 22 22 62 (5h) 56 (4h) 63 (3h)

heptanal 8 11 9 a

octanal 2 <1 <1

isobutyraldehyde 8 16 10

isovaleraldehyde 7 9 8

glyoxylic acid 17 13 14 94 (4h) 96 (6h) 97 (5h)

glyoxal 65 21 16 98 (5h) 83 (6h) 89 (6h)

methylglyoxal 35 32 9 86 (6h) 64 (5h) 69 (6h)

glycolaldehyde 253 354 145 96 (0.5h) 95 (3h) 94 (0.7h)

chloroacetaldehyde 308 282 275 76 (1.5h) 85 (4h) 87 (0.5h)

phenylacetaldehyde 33 93 28 66 (2h) 37 (1h) 22 (1h)

2-pyridinecarboxyaldehyde

6 9 2


7 2 3


6 3 <1

[a] Undisplayed conversions could not be determined because reaction rates were slower then the

hydrolysis of DHAP


27

FruA stability under reaction conditions.

The reaction of DHAP with butanal was used to compare the stability of RAMA

(tetramer, 160 kD) and S. carnosus FruA (monomer, 40 kD). After the maximum

conversion was reached, the biocatalysts were concentrated and washed with buffer

in Amicon centripreps (cut-off 10 kD). The aldolase from S. carnosus proved to be

the most stable enzyme, exhibiting 45% of its initial activity compared with 0.1% for

RAMA. With glyoxylic acid as acceptor aldehyde, 100% of the initial activity was

observed with the bacterial FruA and 91% with RAMA. This yield of RAMA is

consistent with its reported44 rate of deactivation under these conditions of 2.47% per

h, which corresponds to 89% residual activity after 4.5 h. The washed and

concentrated aldolase from S. carnosus could readily be reused.

A 5 mmol scale reaction

In order to test the validity of the figures from Table I and III for scale-up of reactions,

the reaction of butanal and DHAP was conducted on a 5 mmol scale. The

concentration of DHAP was increased from 20 to 50 mM. The conversion of DHAP

was 78%. Treatment with wheat germ acid phosphatase (WGAP) followed by

acetylation gave 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose in 53% overall yield.

Hence we conclude that the values form a good basis for performing the reactions

on a larger scale.

Chapter 2

28

Conclusion

All the aldolases tested accepted a broad palette of aldehydes as substrate

analogues. Comparison of FruA, FcuA and RhuA showed that FcuA functions less

well when non-natural substrates are used, while FruA and RhuA both performed

well and with similar profiles. More than twenty aldehydes were found to be

substrates for the aldolases from rabbit muscle, S. carnosus and S. aureus. The

bacterial aldolases were more stable than RAMA, especially with more apolar

aldehydes. Reaction rates and chemical yields were comparable. Isolation of

enzyme from the reaction mixture is feasible with high recovery of activity for S.

carnosus aldolase and makes it more efficient than RAMA.

Experimental

General

1H and 13C spectra were recorded in CDCl3 at 300 Mhz on an Oxford NMR 300. UV

spectroscopy was performed with a Varian Cary 3 Bio equipped with a Cary

temperature controller. DHAP was prepared from its ethyl hemiacetal dimer barium

salt (Fluka).

Aldol reaction

Aldol reactions were performed in 1 ml 50 mM TRIS buffer pH 7.6 containing 20 mM


29

DHAP and 40 mM aldehyde, reactions were initiated by adding 1.0 U aldolase. At

certain intervals 50 µl aliquots were taken and quenched with 15 µl 7% perchloric

acid. After 30 min 10 µl 1M NaOH was added followed by 175 µl of 50 mM TRIS pH

7.6. This neutralized mixture was then assayed for DHAP. The reaction was followed

until only background hydrolysis of DHAP was measured (0.0047 mM/min).

FruA activity assay

The reaction products, DHAP and GAP were assayed with a coupled enzyme

system. To 1.95 ml 50 mM Tris-buffer pH 7.6 containing 0.16 mM NADH in a 2 ml

cuvette, were added 20 µl 190 mM fructose-1,6-bisphosphate, 20 µl of a mixture

containing 1.25 unit D-glyceraldehyde-3-phosphate dehydrogenase and 12.5 unit

triose-1-phosphate isomerase. Then 50 µl diluted aldolase was added and

absorption monitored at 25 °C. 1 unit (U) aldolase converts 1 µmol fructose-1,6-

bisphosphate per minute.

DHAP-assay

DHAP was assayed with a coupled enzyme system: reduction of DHAP with NADH-

consuming glycerol-3-phosphate dehydrogenase14. From a diluted DHAP solution,

50 µl was added in a quartz cuvette containing 1.95 ml 50 mM TRIS pH 7.6, 0.16

mM NADH, 1.25 U D-glycerol-3-phosphate dehydrogenase and 12.5 U triose-1-

phosphate isomerase. The absorption was monitored at 340 nm at 20 °C. The molar

Chapter 2

30

adsorption coefficient taken was 6.22 l.mmol-1cm-1. Reaction rates were calculated

with the method of least squares. Relative rates were calculated by deviding Vpropanal

by Valdehyde and multiplying by 100.

At intervals of 0.5 - 5 minutes during 3 - 30 minutes 50 µl aliquots were taken

and quenched with 15 µl 7% perchloric acid. After 30 min 10 µl 1M NaOH was added

followed by 175 µl of 50 mM TRIS pH 7.6. This neutralized mixture was then

assayed for DHAP.

Enzyme recovery

In a 25 ml roundbottomed flask equipped with a magnetic stirrer which contained 10

ml 50 mM Tris-buffer pH 7.6, 50 mM DHAP and 100 mM aldehyde, 10 U FruA

(lyophilized RAMA and S. carnosus FruA) were added and stirred for 3 h at RT. After

this the reaction mixture was cooled to 4 °C, poured into (10 or 30 kDa) Amicon

centripreps, centrifuged, washed with 2 x 10 ml Tris-buffer and concentrated to 0.5

ml. The residual activity was determined by adding 10 µl concentrated recovered

enzyme to 1.95 ml 50 mM Tris-buffer pH 7.6 and 0.16 mM NADH in a 2 ml cuvet.

Subsequent addition of 20 µl mixture of 1.25 U glyceraldehyde-3-phosphate and

12.5 U triosephosphate isomerase exposed any residual DHAP or product if present.

Then the reaction was started by addition of 20 µl 190 mM fructose-1,6-bisphosphate

and absorption was monitored at 25 °C.


31

Synthesis of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose

A 50 ml solution containing 50 mM Tris pH 7.6, 50 mM DHAP, 100 mM butanal and

48 U S. carnosus aldolase was stirred at room temperature. Conversion was 78%

(based on consumption of DHAP) after 4 h. Treatment with wheat germ acid

phosphatase (WGAP) overnight at pH 5.5 gave after extraction with ethylacetate

214 mg (c.y. 53% yield) product. Acetylation with 20 ml pyridine and 10 ml acetic

anhydride and subsequent column chromatography (EtOAc/Hexane 1:3) yielded 250

mg (67%) of colorless oil. [α]D25 = 25.0° (c 1.05, CHCl3).

1H-NMR (300 Mhz, CDCl3)

δ=4.75 (d, H-1a), 4.88 (d, H-1b); 5.31 (d, H-3); 5.37 (m, H-4); 1.55 (m, H-5a), 1.33

(m, H-5b); 1.55 (m, 2H-6); 0.94 (t, 3H-7); 2.22 (s, 3H-1’); 2.16 (s, 3H-3’); 2.078 (s,

3H-4’).

13C-NMR (300 Mhz, CDCl3)

δ=66.79 (C-1), 198.41 (C-2), 77.15 (C-3), 71.52 (C-4), 32.38 (C-5), 18.51 (C-6),

13.71 (C-7), 170.07 (CO-1’), 20.76 (CH3-1’), 170.19 (CO-3’), 20.42 (CH3-3’), 169.81

(CO-4’), 20.36 (CH3-4’)

References

31 J. Peters, H. Brockamp, R. Minuth, M. Grothus, A. Steigel, M. Kula and L.

Elling, Tetrahedron: Asymmetry 1993, 4 (6), 1173-1182

32 C.-H Wong and G.M. Whitesides, J. Org. Chem. 1983, 3199-3205.

Chapter 2

32

33 J.R. Durrwachter, D.G. Drueckhammer, K.. Nozaki, H.M. Sweers and C.-H. Wong,

J. Am. Chem. Soc. 1986, 108, 7812-7818.

34 K.K.-C. Liu, T.Kajimoto, L. Chen, Z. Zhong, Y. Ichikawa and C.-H. Wong, J. Org.

Chem. 1991, 56, 6280-6289.

35 H.J.M. Gijsen, L. Qiao, W. Fitz and C.-H. Wong, Chemical Reviews 1996, 96(1),

443-469

36 C. Witke and F. Götz, J. Bacteriol. 1993, 175 (22), 7495-7499.

37 H. Brockamp, M. Kula and F. Goetz, U.S. 5.162.221, 10 nov. 1992.

38 F. Götz, S. Fischer and K.-H.Schleifer, Eur. J. Biochem. 1980, 108, 295-301.

39 C. De Montigny and J. Sygusch, Eur. J. Biochem. 1996, 241, 243-248.

40 W.-D. Fessner, G. Sinerius, A. Schneider, M. Dreyer, G.E. Schulz, J. Badia and J.

Aguilar, Angew. Chem. 1991, 103, 596-599

41 A. Ozaki, E.J. Toone, C.H. Von der Osten, A.J. Sinskey and G.M. Whitesides, J.

Am. Chem. Soc. 1990, 112, 4970-4971

42 M.D. Bednarski, E.S. Simon, N. Bischofberger, W.-D. Fessner, M.-J. Kim, W.

Lees, T. Saito, H. Waldmann and G.M. Whitesides, J. Am. Chem. Soc. 1989, 111,

627-635.

43 H. Brockamp and M. Kula, Tetrahedron Letters, 1990, 31 (49), 7123-7126.

44 H. Brockamp and M. Kula, Appl. Microbiol. Biotechnol., 1990, 34, 287-291.

45 H.U. Bergmeyer, Methods of enzymatic analysis, Verlag Chemie, Mannheim 1984;

Vol. IV p. 342-350

35

3

The stereochemistry of DHAP aldolases

Abstract

The four DHAP dependent aldolases – D-fructose-1,6-bisphosphate (FruA), L-

fuculose-1-phosphate (FcuA), L -rhamnulose-1-phosphate (RhuA) and D-tagatose-

1,6-bisphosphate (TagA) aldolase - constitute a complementary toolkit for

predesigning chirality in carbohydrates. Since the stereochemical outcome may differ

when substrates in the aldol reaction are modified, it is necessary to find

unambiguous evidence for the configuration and optical purity of the products. We

used chiral GC and developed a new enzymatic assay to determine the

stereoselectivity of the aldolases towards a number of acceptor substrates.

Determination of isomer formation revealed that D-fructose-1,6-bisphosphate

aldolase from S. carnosus and L-rhamnulose-1-phosphate aldolase from E. coli are

highly stereospecific.

Chapter 3

36

Determination of stereoselectivity with chiral GC

The stereochemical preferences of aldolases have never been investigated in detail.

Kinetic studies (Chapter 2) have demonstrated that almost any aldehyde is accepted

with a preference for α-hydroxyaldehydes or activated ones such as

chloroacetaldehyde. The selectivity of the aldolase for each of the four possible

stereoisomers remains an unanswered question, however, although it is of crucial

importance in the context of synthetic application. The reaction of butanal with DHAP

catalysed by FruA from S. carnosus was chosen to investigate the steric course of

the aldol reaction in detail. The reaction products were examined, after

dephosphorylation and acetylation, by means of chiral GC analysis. Previously46,

diastereomers were identified by NMR, but chiral GC has the advantage that

enantiomers can also be discriminated.

Figure 1. Aldol reaction of butanal and DHAP catalysed by S. carnosus FruA.

Selectivities are in parentheses.

OAc

OAc

OAc

O

OAc

OAc

OAc

O

OAc

OAc

OAc

O

OAc

OAc

OAc

O

1 (3S,4R) (90%) 2 (3R,4S) (< 0.5%)

3 (3S,4S) (6%) 4 (3R,4R) (4%)

+

+

+

HO OPi

O

CHO

1. FruA S. carnosus (78% yield)

2. WGAP (53% yield)3. Ac2O/pyridine (67% yield)


37

The reaction of butanal and DHAP, catalyzed by FruA afforded a major (90%

selectivity) product to which we assigned the (3S,4R) configuration 1 (Figure 1) on

the basis of the known steric preferences of the catalyst.47 Two minor, presumably

stereoisomeric, products were formed with 6% and 4% selectivity.

In order to elucidate their structures we synthesized two of the remaining three

possible stereoisomers of the aldol adduct independently. Reaction of butanal and

DHAP catalyzed by RhuA46 afforded the (3R,4S) product 2; FcuA 46,48 afforded the

(3R,4R) stereoisomer 4 (Figure 2). The (3S,4S) stereoisomer 3 could not be

synthesized directly, because no D-tagatose-1,6-bisphosphate aldolase was

available. However, epimerisation of 2 with sodium acetate in methanol at room

temperature afforded, after 4 h, an equilibrium mixture of 45% 2 and 55% of its 3-

epimer 3. Epimerisation presumably proceeds via enolisation and is also observed

under acidic conditions (p-TosOH was added). Hence, all four aldol adducts of

butanal and DHAP could be resolved by chiral GC.

To reconfirm the structural assignments, 1 was similarly subjected to

epimerisation; after 2 h at room temperature an equilibrium mixture of 60% 1 and

40% of its 3-epimer 4 was obtained. On the basis of chiral GC the minor products of

FruA from S. carnosus were identified as the anti stereoisomers 3 (6% selectivity)

and 4 (4 % selectivity). Because the stereogenic centre at C-4 is not readily

susceptible to epimerisation, the formation of 3 must reflect a lack of complete

stereoselectivity at C-4, In contrast, epimerisation of 1, either during the reaction of

the work-up, could contribute to the observed formation of 4. NMR analysis of the

(phosphorylated) reaction products suggests that S. carnosus aldolase forms 8.5%

of the anti stereoisomers 3 and 4. Hence, the remaining 1.5% presumably results

from epimerisation during the work-up.

Chapter 3

38

Figure 2. Structural assignments of the four possible aldol adducts.

The use of GC requires derivatisation of reaction products. Unfortunately, this

method turned out to be rather inflexible and labor intensive. For every single

aldehyde the derivatisation method must be altered, because of too short or too long

retention times resulting in diminished peak separation. Also, new product samples

with specific configuration must be synthesized each time. However, in the case of

butanal, for the first time, a complete resolution of DHAP aldolase products was

established.

OAc

OAc

OAc

O

OAc

OAc

OAc

O

OAc

OAc

OAc

O

OAc

OAc

OAc

O

1 2

34

1) FruA2) WGAP3) Ac2O /pyridine

NaOAcMeOH

CHO

OPO32-

O

HO

1) L-FcuA2) WGAP3) Ac2O/pyridine

1) L-RhuA2) WGAP3) Ac2O/pyridine

NaOAcMeOH


39

Determination of stereoselectivity with an enzymatic assay

To bypass the limitations encountered with chiral GC, a new coupled enzymatic

method, based on the reversibility of the aldol reaction, was developed to detect the

stereoisomers formed by DHAP aldolases. This simple coupled enzymatic assay

makes use of the known stereopreference46,47 of the commercially available

aldolases in retro-aldol reactions (Figure 3). Three of the four stereoisomers could be

detected directly, the fourth one was calculated. The method does not require

derivatisation or complete work-up of the product samples, only removal or

inactivation of the biocatalyst.

Figure 3. Retro aldol reactions and reduction of DHAP

The assay is as elementary as the aldol reaction itself and incorporates three steps

(Figure 3): 1) removal of aldolase, 2) reduction of any residual DHAP and 3) isomer

+

DHAP

1 2

3 4

FruA

OPO32-

O

HO

L-FcuA

L-RhuA

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

ROPO3

2-

OH

OH

O

RCHO

TagA

NADHGDH

OPO32-

OH

HO

Chapter 3

40

detection. Steps 2) and 3) are repeated for each stereoisomer. First, after the initial

synthetic aldol reaction has reached its maximum conversion the aldolase activity is

quenched by addition of acid or membrane filtration, which was, in our hands, the

fastest way to remove the aldolase activity. Secondly, a sample of the aldolase-free

mixture containing the aldol adducts is added to a suprasil cuvette containing NADH.

Addition of glycerol-3-phosphate dehydrogenase (GDH) mediates reduction of any

residual DHAP49 present in the mixture (∆A1, Figure 4A). Third, one of the aldolases

is added to selectively cleave one of the four possible products to a corresponding

amount of DHAP, which is reduced in situ by NADH in the presence of GDH (∆A2,

Figure 4A). The reduction step renders the retro-aldol reaction irreversible, hence,

the amount of reduced DHAP is equal to the amount of NADH consumed, which is

monitored by UV.

A B

Figure 4. Example of detection of the butanal-DHAP adduct made with RhuA. (A)

The absorption of NADH is measured at 340 nm. Addition of GDH yields ∆A1 which

is correlated to the amount of residual DHAP. Subsequent addition of aldolase yields

∆A2 which is correlated to the amount of stereoisomer with the fructose

configuration. (B) Distinction between two isomers can be made because of different

retro aldol reaction rates. ∆A2 is now extrapolated from the graph.

0.50.60.70.80.9

11.11.21.31.4

0 10 20 30 40t (min)

abso

rptio

n ∆A1

∆A2

GDH added

FruA added

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

0 10 20 30t (min)

abso

rptio

n ∆A1

∆A2

GDH added

FcuA added


41

It may seem paradoxical to distinguish between stereoisomers using the

same enzyme which produced them, but since the minor isomers are cleaved

sluggishly (see Figure 4B), there is a clear distinction in the assay between the four

aldol adducts. Since absorption is measured versus time, the concentration is easily

extrapolated from an absorption-time plot.

Since TagA is not commercially available, only three of the four possible

stereoisomers could be detected via this retro aldol reaction. With FruA, FcuA and

RhuA the corresponding isomers are detected; the missing part, based on consumed

DHAP from the synthetic aldol reaction, is equal to the product with tagatose

configuration. Otherwise, this information can be obtained by allowing the initial

aldolase to convert all the four isomers it produced, thus determining the sum of

concentrations.

In practice this method of analyzing stereoisomers is very convenient, since

no work-up of the reaction product or derivatisation is necessary and small samples

can be taken during the synthetic reaction and analyzed in minutes. In fact, even

when only one aldolase from the set of four is available, this method would prove

itself useful since – besides the significant importance of product formation

monitoring – it is still possible to determine the fraction of the main isomer produced

by this enzyme, based on the total DHAP consumption.

Comparison of methods

The results of the enzymatic assay were compared (see Table I) with those

obtained by stereoisomer detection with gas chromatography and NMR.46

Chapter 3

42

Examination of the distribution of stereoisomers found for the reaction of butanal and

DHAP with GC on the one hand and the enzymatic assay on the other revealed only

small differences. These are probably due to some epimerisation taking place during

the derivatisation necessary for GC analysis and underline the strength of the

enzymatic assay. According to our retro-aldol assay the reaction of acetaldehyde

with DHAP catalyzed by RhuA yields about 2% less of the syn isomers than found by

NMR analysis. For butanal this discrepancy is 1%. We note that NMR analysis also

requires work-up of the samples which might lead to a certain deviation in values.

Table I. Enzymatic assay versus GC and NMR analysis. Configurations can be

found in the Figures 1, 2 and 3

Configuration (%)Acceptor Analysis Aldolase

1 2 3 4

Conversion

butanal GC FruA 90 0 4 6 78%

enzymatic FruA 89.7 0 2.6 7.7 80%

acetaldehyde NMR RhuA 69 31 84%

enzymatic RhuA 2 65 21 12 88%

butanal NMR RhuA 83 17 82%

enzymatic RhuA 14 68 0 18 81%

Stereospecificity of FruA and RhuA

The steric course of the FruA mediated aldol reaction of butanal has been elucidated

by gas chromatography. The steric preferences of RhuA and FcuA were studied with

a number of acceptors46, but since NMR was used only proportions of


43

diastereoisomers could be measured. With our new method it was possible to

quickly scan a range of substrates, delineating a more general picture of the

properties of aldolases regarding stereospecificity.

Table II. Stereochemical preferences of DHAP aldolases. Bold figures

refer to the configurations in Figures 1, 2 and 3.

RCHO FruA RhuA

R 1 2 3 4 1 2 3 4

CH3 88 0 3.5 8.5 1.5 64 21.5 13.0

CH2CH3 96 0 0 4.0 5.0 81 13 1

CH2CH2CH3 90 0 2.5 7.5 14 68 18 0

CH(CH3)2 92.5 0 0 7.5 5.0 68.5 10 16.5

C(CH3)3 96 0 0 4.0 9.5 90.51 0.0 1

CH2OH 96 0 0 4.0 0.3 99 0.2 0.5

CHO 991 0 1.0 1 0.5 99.51 0.0 1

COCH3 95.51 0 4.5 1 3.0 89.5 0.0 7.5

CH2Cl 94 0 4 2.0 0.0 96.5 1.5 2.0

CH2Ph 97.5 0 1.5 1.0 3.0 75.0 20.5 1.5

1 The retro-aldol reaction was too slow for the separate determination of the Tag-stereoisomer

The steric course of the FruA mediated reaction of DHAP and ten different

aldehydes, was investigated using our enzymatic assay (Table II). We found that the

FruA from S. carnosus is highly, but far from absolutely, stereospecific for products

with the fructose configuration, which were formed for > 95% in the majority of the

cases. The specificity for the fructose configuration was less pronounced for the

aldol reactions of acetaldehyde and butanal. However, a high selectivity for the

Chapter 3

44

fructose configuration was observed with related non-polar aldehydes, such as

trimethylacetaldehyde (pivaldehyde) and phenylacetaldehyde.

The amounts of the major, “fructose” stereoisomers in the aldol adducts of

glyoxal and methylglyoxal could not be determined directly owing to the extremely

low rate of the corresponding retro-aldol reactions. The cleavage of the adduct of

glyoxal, for example, was 250 times slower than that of the phenylacetaldehyde

adduct. The sluggish retro-aldol reactions prevented a reliable observation of the

transition from cleavage of the major stereoisomers to that of the minor ones.

However, the stereoisomers with L-rhamnulose and L-fuculose configuration could be

measured and from these the sum of the products with fructose and tagatose

configuration was calculated.

The minor products from the FruA catalyzed reactions mainly had the tagatose

configuration, in particular with acetaldehyde, butanal and dimethylacetaldehyde.

The optical antipodes of the main product, the stereoisomers with the L-rhamnulose

configuration, were not detected at all.

The RhuA catalyzed aldol reactions of the same range of aldehydes were in

general only moderately stereoselective. A high selectivity for the “rhamnulose”

product was only observed with glycolaldehyde and glyoxal. We note that in the

latter case, as well as 2,2-dimethylpropanal, the “rhamnulose” stereoisomer could

not be determined directly because the retro-aldol reaction was too slow.

Consequently, only the sum of the products with L-rhamnulose and tagatose

configuration could be calculated for these reaction products. Contrary to FruA, the

RhuA catalysed reaction also formed detectable amounts (up to 9.5%) of the optical

antipode of the major stereoisomer (with fructose stereochemistry).


45

Conclusion

GC analysis showed that the reaction of butanal with DHAP catalysed by FruA from

S. carnosus afforded the (3S,4R) stereoisomer 1 as the major product (90%)

together with 6% of the (3S,4S) isomer 3 and 4% of the (3R,4R) isomer 2. In contrast

to this method the enzymatic assay is a direct and flexible procedure for the

quantitative determination of stereoisomers formed in aldol reactions catalysed by

DHAP dependent aldolases. It obviates the need for work-up and/or derivatisation; is

much less labor-intensive and does not entail any risk of product epimerisation. The

procedure is universal for DHAP aldol adducts, consequently a whole range of

substrates can be investigated. This proved unambiguously, for the first time, that

the steric preference of FruA and RhuA for the newly created stereogenic center at

C-(3) is far from absolute. The method is also useful for fast monitoring of aldolase

catalysed syntheses. Its main limitation is when the retro-aldol reaction is too slow to

discriminate reliably between cleavage of the major and minor products.

Experimental

General

1H and 13C spectra were recorded in CDCl3 at 300 Mhz on an Oxford NMR 300.

UV spectroscopy was performed with a Varian Cary 3 Bio equipped with a Cary

temperature controller. Chiral gaschromatography was performed with a

diacetyltertbutylsilyl-β-cyclodextrine (50 m x 0.25 mm, df 0.25 µm) column. DHAP

was prepared from its ethyl hemiacetal dimer barium salt (Fluka).

Chapter 3

46

Acetylation of GC-samples

Acetylation prior to GC-injection was conducted by solving 2 to 3 mg of analyte in 0.1

ml pyridine in a 2 ml sample bottle. After addition of 50 µl acetic anhydride the

mixture was shaken for 10 sec at room temperature. Immediately 1 ml diethylether

and 0.3 ml 4N HCl was added to neutralize the mixture and extract the acetylated

compound. The water layer was removed and the organic layer was dried with

sodium sulfate. This solution was then ready for injection. Under these conditions no

racemisation occurred, only after subjecting the analyte for more than 5 min to the

acetylation mixture epimerisation was observed.

Preparation of product samples

Butanal and DHAP were reacted following the procedure of the preparation of 1,3,4-

tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose with 50 U L-fuculose-1-P aldolase or 50 U L-

rhamnulose-1-P aldolase. After extraction with ethylacetate the products were

acetylated according to the acetylation of GC-samples.

Aldol reaction

Aldol reactions were performed in 0.5 ml 50 mM TRIS buffer pH 7.6 containing 20

mM DHAP and 50 mM aldehyde and were initiated by adding 5 U aldolase. After

complete conversion the reaction mixture was centrifuged in an eppendorf


47

centrifuged and transferred into Microcon Centrifugal filter (cutoff 10 kD) and again

centrifuged for 10 minutes. The filtrate contained no aldolase activity. Alternatively,

the complete mixture can be quenched with 20 µl 70% perchloric acid and allowed to

stand for 30 minutes. Precipitate was centrifuged off. Neutralization was not

necessary.

Retro aldol reaction assay

This assay is a modification of the DHAP coupled enzyme assay. DHAP was

assayed by reduction with NADH-consuming glycerol-3-phosphate dehydrogenase.

From the deproteinated reaction mixture 10 to 20 µl was added in a quartz cuvette

containing 1.97 ml 50 mM TRIS pH 7.6, 0.16 mM NADH, 1.25 U glyceraldehyde-3-

phosphate and 12.5 U triose-1-phosphate isomerase. The absorption was monitored

at 340 nm at 20 °C. The molar adsorption coefficient taken was 6.22 l.mmol-1cm-1.

Reaction rates were calculated with the method of least squares. After complete

conversion of residual DHAP, retro aldol reactions were initiated by adding 10 µl

aldolase (5 units). This was repeated for each of the three aldolases.

References

46 W.-D. Fessner, G. Sinerius, A. Schneider, M. Dreyer, G.E. Schulz, J. Badia and J.

Aguilar, Angew. Chem. 1991, 103, 596-599

47 H. Brockamp and M. Kula, Appl. Microbiol. Biotechnol. 1990, 34, 287-291.

Chapter 3

48

48 A. Ozaki, E.J. Toone, C.H. Von der Osten, A.J. Sinskey and G.M. Whitesides,

Am. Chem. Soc. 1990, 112, 4970-4971

49 H.U. Bergmeyer, Methods of enzymatic analysis, Verlag Chemie, Mannheim 1984;

Vol. IV p. 342-350

51

4

In situ generation of DHAP: cascade catalysis

Abstract

A total of four enzymatic steps were combined, in a one-pot reaction, to synthesize

carbohydrates starting from glycerol. This approach avoided restraints normally

associated with the preparation of DHAP. First, phosphorylation of glycerol by

reaction with pyrophosphate in the presence of phytase at pH 4.0 in 95% glycerol

afforded racemic glycerol-3-phosphate in 100% yield. The L-enantiomer of the latter

underwent selective aerobic oxidation to dihydroxyacetone phosphate (DHAP) at pH

7.5 in the presence of glycerolphosphate oxidase and catalase. Subsequently, D-

fructose-1,6-bisphosphate aldolase catalyzed the aldol reaction of DHAP with

butanal. Finally, dephosphorylation of the aldol adduct was mediated by phytase at

pH 4 affording 5-deoxy-5-ethyl-D-xylulose in 57% yield from L-glycerol-3-phosphate.

Switching off the activity of the phosphatase by a pH change during oxidation and

aldol reaction resulted in better results than integral in situ phosphorylation.

Chapter 4

52

Synthesis of dihydroxyacetone phosphate

Enzyme catalyzed aldol addition is a powerful methodology for building carbohydrate

derivatives.50,51 Aldolases, which in their natural function perform a retro-aldol

reaction, are capable of catalyzing a large variety of carbon-carbon bond constituting

reactions52. The group of aldolases which depend on dihydroxyacetone phosphate53

(DHAP) as the donor substrate is particularly attractive as total control over the

stereochemical outcome of the aldol reaction is obtained (see Figure 1, page 8).

They are hampered, nevertheless, in their practical application by the lack of cheap

and efficient access to DHAP.

DHAP was first prepared53 by enzymatic dismutation of D-fructose-1,6-

bisphosphate (Figure 1, below). D-Fructose-1,6-bisphosphate aldolase (FruA)

mediates its retro aldol reaction to equal amounts of DHAP and glyceraldehyde-3-

phosphate. A second aldehyde can then be coupled to DHAP by the same aldolase.

The overall equilibrium may however not be favorable for synthesis and the workup

is complicated by residual substrate54.

It is possible to phosphorylate dihydroxyacetone directly54 with POCl3 but this

results in complex reacton mixtures. A nine step synthesis was first developed from

3-chloro-1,2-propanediol55, with an overall yield of only 15%. The synthesis was

improved by switching to dihydroxyacetone as starting material (4 steps). The

present-day preparation of DHAP proceeds via the dimer of dihydroxyacetone, the

form in which it excists in non-aqueous media, through ketalisation with

triethylorthoformate to 2,5-diethoxy-p-dioxane-2,5-dimethanol56. The phosphorylation

of this intermediate has been extensively investigated and can be performed in

different ways but, because deprotection of 2,5-diethoxy-p-dioxane-2,5-diphosphate


53

to DHAP is at best 66%, overall yields remain low. By improving the work-up

conditions for 2,5-diethoxy-p-dioxane-2,5-dimethanol higher yields of DHAP were

obtained (Colbran57 27%, Effenberger58 35%, Pederson59 55%, Jung60 61%). An

efficient final hydrolysis step is possible by using 3-acetoxy-2,2-dimethoxypropyl

phosphate61 (95%), but this compound requires a five-step synthesis with an overall

yield of only 54%. The hydrolysis of 3-bromo-2,2-dimethoxy-1-propyl dibenzyl

phosphate62 yields only 56% DHAP. Summarizing, the chemical synthesis of DHAP

involves lengthy procedures and modest over-all yields.

Figure 1. Two chemical and four enzymatic routes to DHAP.

The enzymatic preparation63 from dihydroxyacetone was accomplished for the

HO OPO3-2

H3CO OCH3

O

O OPO3-2

OEt

EtO-2O3PO

HO OH

O

HO OPO3-2

OH

O

OPO3-2HO

FDP aldolaseTPI

glycerol kinaseATP

GPO

catalase

2,5-diethoxy-p-dioxane-2,5-diphosphate

HO OH

OH

pyrophosphatephytaseGPO/catalase

DHAP

H+

H+

O-2O3PO

OH

OH

OH

OPO3-2

Chapter 4

54

first time by phosphorylation with ATP54 catalysed by glycerol kinase, in combination

with a regeneration system composed of acetyl phosphate and acetate kinase in

83% yield. A method that is very efficient as regards cost, number of steps and

chemical yield proceeds via the oxidation of L-glycerol-3-phosphate mediated by

glycerol phosphate oxidase (GPO) 64. Accummulation of hydrogen peroxide is

suppressed by adding catalase. Racemic glycerol-3-phosphate can be used,

notwithstanding that GPO exclusively oxidizes the L-enantiomer, making the process

more cost-effective than with pure L-glycerol-3-phosphate. Although conversions run

up to 95%, inhibition of GPO by DHAP is even in the best case substantial (20%

residual activity at [DHAP] = 100mM). However, under these conditions, enzymatic

aldol reactions can be performed in situ, thus avoiding inhibition of GPO and making

yields of up to 96% feasible, the record for DHAP synthesis. The enzymatic

phosphorylation of glycerol cannot be coupled to this system, because of the low

oxygen tolerance of glycerol kinase.

Phosphorylation with a phospholipase was also reported65. The alcohol

function in dihydroxyacetone can replace the polar choline head group of the natural

reactant in a phospholipase D mediated phosphorylation. Subsequent hydrolysis by

phospholipase C then yields DHAP and the corresponding 1,2-diacyl glycerol. This

phosphorylation has an efficiency of only 72% and thus cannot compete with the

other enzymatic methods.

A simple and economical route to DHAP – and therefore to carbohydrates –

should involve a limited number of steps from inexpensive starting materials. The

reaction(s) should preferably be enzymatic as this would obviate the need for

protection and deprotection steps inherent in chemical phosphorylation.66 Moreover,


55

chemical phosphorylation is likely to be incompatible with subsequent enzymatic

steps. Owing to the high energy content of the phosphate ester bond, DHAP

synthesis generally requires an organic phosphate. Alternatively, DHAP can be

prepared by enzymatic oxidation of L-glycerol-3-phosphate which is, in vivo

produced by glycerol kinase catalysed reaction of glycerol with ATP which would

again call for a source of organic phosphate for regeneration. These obstacles can,

in principle, be circumvented by using a phosphatase as the phosphorylation catalyst

and pyrophosphate as an inexpensive source of phosphate.67,68

Combination of such a phosphorylation of glycerol with in situ oxidation to

DHAP and subsequent enzymatic aldol condensation and dephosphorylation would

provide a one-pot procedure from glycerol to carbohydrates (Figure 2).

Integral in situ reaction

Initially, we examined the production of DHAP in a mixture of pyrophosphate,

glycerol / water, phosphatase and GPO / catalase / oxygen. This combination can be

coupled to an aldolase and an acceptor aldehyde. To make sure that only

dephosphorylation of the aldol adduct takes place, oxidation (and subsequent aldol

reaction) must be fast compared to the dephosphorylation rate of DHAP. This implies

slow phosphorylation as well. To avoid base catalysed background hydrolysis of

glycerol phosphate and DHAP, acid phosphatases were the obvious choice.

Oxidation and aldol reaction are optimally performed at neutral pH, but the enzymes

display activity under mildly acidic conditions as well.

Chapter 4

56

Figure 2. One pot preparation of carbohydrates from glycerol.

The acid phosphatases used were phytase69 from Aspergillus ficuum, which is a

cheap and readily available industrial enzyme and bovine intestinal mucosa

phosphatase (BIMP). The effect of the pH on the phosphorylation of glycerol

mediated by phytase and BIMP was examined (see Figure 3). With both enzymes

the productivity increased with the glycerol concentration. The synthesis of DHAP

was performed at up to 55% glycerol. The pH optimum of the system was 5.3 with

HO OH

OH

HO OH

OPO32-

HO OPO32-

OH

HO OPO32-

OH

PO43-

+ +

O2

H2O2

H2O

HO OPO32-

O

HO OH

O

R H

O

OPO32-

O

OH

OH

OH

O

OH

OH

pyrophosphate

Phosphatase

GPOCatalase

FruA

Phosphatase

1/2

PO43-

(DHAP)

PO43-

Phosphatase

Phosphatase

PO43-


57

phytase and 6.8 with BIMP. More DHAP was produced when BIMP was used. The

pH optima of phytase and GPO are more distant than those of BIMP and GPO,

which may account for the lower production of DHAP by the system with phytase.

Figure 3. DHAP synthesis with pyrophophate and phosphatase and GPO/catalase

after overnight reaction. s phytase, l BIMP, ...... 0.5 M glycerol, ___ 55% glycerol.

At lower glycerol concentrations pyrophosphate as well as DHAP are hydrolysed but

it was not clear how much DHAP was hydrolysed to dihydroxyacetone. Thin layer

chromatography showed in each case large amounts of this hydrolysis product,

increasing with decreasing glycerol concentration. DHAP could not be detected with

the coupled enzymatic assay for DHAP by adding DHA to a solution containing

pyrophosphate, BIMP phosphatase, NADH, GDH/TPI, hence, phosphorylation of

DHA back to DHAP does not occur. Similarly, no phosphorylation could be detected

in 30% glycerol. Increasing the amount of glycerol is, thus, the only effective way to

suppress undesired hydrolysis. Unfortunately, the concentration of glycerol should

not exceed 55%, since the activity of GPO decreases dramaticly above this value

(Figure 4). Only swift consumption of DHAP by the aldolase can in principle prevent

0

5

10

4,8 5,8 6,8 7,8pH

mM

DH

AP

Chapter 4

58

untimely dephosphorylation.

Figure 4. Oxidation of L-glycerol-3-phosphate to DHAP by GPO in varying

concentrations of glycerol at pH 7.5. Conversion was measured after 1h. Reaction

rates were expressed relative to the highest rate obtained at 55% glycerol.

Preliminary to the integration of the aldolase in the reaction system, the rate of

DHAP hydrolysis by BIMP and aldol reaction were examined with equal initial

concentration of DHAP (10 mM). Both reactions depend on the glycerol

concentration (Figure 5). At 55% glycerol hydrolysis decreased to zero, while the

aldol reaction still took place (rates expressed in mM consumption per hour). Under

this condition the aldol reaction can be conducted without unwanted DHAP

hydrolysis. The activities are no more than guidelines, since they depend heavily on

the substrate concentration, which will be different in the one-pot reaction. With the

same amount of enzymes combined in the one-pot reaction starting from glycerol,

pyrophosphate and an aldehyde, predictably low concentrations of DHAP were

found, as a result of the direct consumption of in situ produced DHAP by the

aldolase.

0

20

40

60

80

100

0 20 40 60 80 100

glycerol (%)

GP

O V

rel (

%)


59

Figure 5. Aldol reaction and hydrolysis of DHAP (10 mM initially) with 1 mg bovine

intestinal mucosa phosphatase (BIMP) per ml. The “one pot” shows DHAP

concentration after overnight incubation.

Finally, the mixture must be dilluted to initiate the dephosphorylation of the

adduct. The dephosphorylation rate depends on the acceptor (Table I) used; in all

the cases the phosphatase displayed good activity.

Table I. Hydrolysis of the aldol adduct (initial concentration 10mM) by BIMP.

acceptor mM/h

acetaldehyde 3.13

propionaldehyde 2.00

butanal 2.29

Unfortunately, after combining all the components of the multi-enzyme reaction,

only dihydroxyacetone was found in the mixture as the final product. No conditions

were found where the aldol reaction prevailed over the dephosphorylation of the

aldol adduct. This outcome reveals, that in this dynamic system of four enzymes, the

0

5

10

15

0 10 20 30 40 50 60% glycerol

mM

DH

AP

/ h

aldol reactionhydrolysisone pot

Chapter 4

60

equilibrium is unfavorable for the dephosphorylated aldol adduct. Since the aldol

reaction is reversible, the phosphatase can perpetually hydrolyse DHAP released in

the retro aldol reaction. From experimental observation it is obvious that DHAP is a

better substrate than the aldol adduct. The main product from this multienzyme

reaction is therefore dihydroxyacetone.

Sequential one-pot reaction

The oxidation and aldol reactions are optimally performed at pH 7.5; hence, to avoid

interference by the phosphatase its hydrolytic activity should be zero at neutral pH.

This restricts the choice of phosphatase to the acid phosphatases, because the use

of an alkaline phosphatase would introduce the problem of base catalysed

background hydrolysis of glycerol phosphate and DHAP. The phosphatase of choice

was phytase from Aspergillus ficuum, which is a cheap and readily available

industrial enzyme used in animal feed. It has two pH optima for the hydrolysis of its

natural reactant, phytic acid: pH 2.2 and 5.

Figure 6.

Hydrolysis (2h) of L-glycerol-3-

phosphate (50mM) catalysed

by phytase (1 mg / ml) in 0 and

50% glycerol at different pH.0

20

40

60

80

100

2 3 4 5 6 7 8pH

L-G

-3-P

hyd

roly

sis

(%) 0%

50%


61

The pH profile of the hydrolysis of L-glycerol-3-phosphate (see Figure 6) shows an

optimum at pH 3 - 4. At pH 7.5 phytase is stable, but unreactive towards either L-

glycerol-3-phosphate or DHAP, which makes it possible to combine the

phosphorylation, oxidation and aldol addition steps into a one-pot procedure by pH

control. Phytase exhibited the same hydrolytic activity towards DHAP as to L-

glycerol-3-phosphate.

Synthesis of L-glycerol-3-phosphate

The production of L-glycerol-3-phosphate by phytase-mediated reaction with

pyrophosphate was pH dependent with a very broad optimum (see Figure 7). At pH

2 phosphorylation was substantial and it decreased above pH 4 to become zero at

pH 7. The pH optimum shifted with increasing glycerol concentration from pH 3 to 4,

but as long as the pH was kept between these values good results were obtained.

No change in pH was observed during the reaction.

Figure 7. Production of L-

glycerol-3-phosphate catalysd

by phytase (1 mg / ml) in 10, 50

and 85% glycerol at different

pH after 2.5 h incubation with

150 mM pyrophosphate.0

10

20

30

2 3 4 5 6 7 8pH

L-G

-3-P

(mM

)

85%50%10%

Chapter 4

62

Since only L-glycerol-3-phosphate can be oxidized to DHAP, the D-isomer (which is

presumably produced in equal amounts) will remain unused in solution. In the final

dephosphorylation step it is converted back to glycerol and phosphate. This also

implies that the yield of the reaction, based on pyrophosphate, cannot surpass 50%.

Effects of glycerol concentration

The time to reach maximum conversion, based on the amount of

pyrophosphate, was hardly affected by glycerol concentrations between 10% and

85% (v/v), but at 95% the rate had dropped by a factor of ten (data not shown). At

still lower water concentrations the enzyme activity decreased to zero in pure

glycerol, although some activity still could be observed when only 0.25% water was

present.

Figure 8. Phosphorylation of glycerol with pyrophosphate (150 mM) by phytase (1

mg / ml) at pH 4.0 in glycerol / water mixtures. Racemic glycerol-3-phosphate is

obtained in 100% yield in 95% glycerol after 24h.

0

20

40

60

80

100

0 20 40 60 80 100glycerol (%)

phos

phor

ylat

ion

(%)

4.5h

24h


63

The yield of D,L-glycerol-3-phosphate increased with increasing glycerol

concentration (see Figure 8), presumably because the competing hydrolysis of

pyrophosphate and glycerol-3-phosphate is suppressed at low water concentrations.

At 95% glycerol concentration a quantitative conversion of pyrophosphate into

glycerol-3-phosphate was obtained after 24h reaction time.

Selectivity

Phytase is believed to be non-stereoselective in the phosphorylation of

glycerol.67 Since only L-glycerol-3-phosphate is a substrate for GPO, the production

of D-glycerol-3-phosphate cannot be monitored by this enzymatic assay. The

consumption of pyrophosphate and production of phosphate and glycerol phosphate

could be monitored using 31P NMR, however. A reaction mixture with 95% glycerol

and an initial pyrophosphate concentration of 150 mM was analyzed for 31P before

100% conversion was reached. This revealed that the phosphate concentration was

100 mM whereas the pyrophosphate concentration was 50 mM, a decrease of 100

mM (67% conversion). This observation demonstrates that, under these conditions,

pyrophosphate was converted to one equivalent of glycerol phosphate and one

equivalent of phosphate, without any hydrolysis of pyrophosphate or glycerol-3-

phosphate. The concentration of D,L-glycerol-3-phosphate therefore was 100 mM.

Enzymatic detection of L-glycerol-3-phosphate with GPO and GDH gave a

concentration of 50 mM, exactly matching the NMR results and confirming that

phytase is not stereoselective. But, since glycerol-2-phosphate was absent in the

reaction mixture phytase is completely regiospecific.

Chapter 4

64

Synthesis of DHAP from L-glycerol-3-phosphate

After complete conversion of the pyrophosphate, the pH was raised to 7.5 to

suppress the hydrolysis of L-glycerol-3-phosphate (and DHAP) and maximize GPO’s

activity. The concentration of glycerol must also be adjusted since GPO activity is

low at 95 %. To achieve maximum space-time yields a balance must be found

between high L-glycerol-3-phosphate concentration and compatibility with GPO

activity. At pH 7.5 the oxidation rate of L-glycerol-phosphate was increased fiftyfold

by lowering glycerol concentration (Figure 4) from 95% to 55%, which convincingly

shows that dilution is highly advantageous.

A 1.5 mmol (pyrophosphate) reaction was carried out in 95 % glycerol. The

time to reach full conversion at this concentration was one day. After phosphorylation

was completed the mixture was diluted with water to 55% glycerol and the pH was

adjusted to 7.5. Addition of GPO/catalase and entrainment with oxygen for three

hours afforded DHAP in a concentration of 43 mM corresponding to 50% yield based

on pyrophosphate.

Aldol reaction and dephosphorylation

Aldol reactions can be performed with the in situ prepared DHAP in glycerol. To

this end we used the D-fructose-1,6-bisphosphate aldolase (FruA) from

Staphylococcus carnosus to prepare the aldol adduct of DHAP and butanal.70

Butanal and aldolase were added to the mixture containing DHAP, phytase,

glycerolphosphate oxidase, catalase and 55% glycerol. In fact, it is not necessary to


65

do this step separately; the oxidation and aldol reaction can be carried out

simultaneously64, thus saving hours of reaction time. The consumption of DHAP was

used to monitor the course of the reaction. Under these conditions the aldolase

displayed normal activity and modification of the mixture to enhance its performance

was therefore unnecessary. We expect that the combination of the relaxed acceptor

specificity of DHAP dependent aldolases with this new method opens the way to the

synthesis of a wide spectrum of possible structures.

The addition of extra phosphatase for removal of the phosphate group is not

necessary since phytase is still present and active. After oxidation and aldol reaction,

phytase-mediated dephosphorylation of the butanal-DHAP adduct was carried out by

simply lowering the pH to 4.0. Apparently phytase exhibits a wide substrate

specificity in the hydrolysis reaction and, hence, is expected to be generally

applicable for a broad range of substrates in this final step. Extraction afforded 5-

deoxy-5-ethyl-D-xylulose in 57% yield from from DHAP (or 29% from

pyrophosphate).

Chemical phosphorylation of glycerol

Alternatively, glycerolphosphate can be made chemically from glycerol and

phosphoric acid by simply heating glycerol with phosphoric acid. Under these

conditions 1.2 M of D,L-glycerolphosphate can be obtained. Problematic however is

the high salt concentration, which requires substantial dilution. This lowers the final

DHAP concentration to about 20 mM.

Chapter 4

66

Conclusion

Phytase-catalyzed phosphorylation of glycerol with pyrophosphate afforded D,L-

glycerol-3-phosphate in quantitative yield. L-glycerol-3-phosphate underwent GPO-

catalyzed oxidation to DHAP after adjustment of the pH to 7.5. The D-isomer was not

converted but its presence had no effect on the subsequent steps. Under the

conditions used for oxidation and aldol reaction (pH 7.5) phytase did not hydrolyze L-

glycerol-3-phosphate or DHAP. Manipulation of pH thus gives total control over

phytase’s activity; it can be “switched off” by increasing pH from 4 to 7.5 and

“switched on” again by lowering back to pH 4.0.

This alliance of four different enzymes in a one-pot cascade of four enzymatic

transformations provides an attractive procedure for performing aldol reactions with

DHAP aldolases starting from the cheap, readily available glycerol and

pyrophosphate. Combined with the broad substrate specificity of DHAP aldolases

towards acceptor substrates it may constitute a simple procedure for the synthesis of

a wide variety of carbohydrates from glycerol.

Experimental

General

31P spectra were recorded at 300 Mhz on an Oxford NMR 300 with phosphoric

acid as external standard. UV spectroscopy was performed with a Varian Cary 3 Bio

equipped with a Cary temperature controller. The aldolase and oxidase were


67

obtained from Roche Diagnostics. All other enzymes and chemicals were purchased

from Sigma.

Assay for DHAP and L-glycerol-3-phosphate

DHAP was assayed with a coupled enzyme system: reduction with NADH-

consuming glycerol-3-phosphate dehydrogenase enables determination of DHAP by

measuring NADH concentration with UV-spectroscopy. From a diluted DHAP

solution, 50 µl was added in a quartz cuvette containing 1.95 ml 50 mM TRIS pH 7.6,

0.16 mM NADH, 1.25 U glyceraldehyde-3-phosphate and 12.5 U triose-1-phosphate

isomerase. The absorption was monitored at 340 nm at 20 °C. Blank DHAP: 0.0047

mM / min. The molar adsorption coefficient taken was 6.22 l.mmol-1cm-1.

L-glycerol-3-phosphate was detected by oxidizing it to DHAP with GPO and

subsequent assay for DHAP. Samples of 125 µl containing L-glycerol-3-phosphate

were diluted four-fold with 200 mM Tris buffer, pH 8.0. This buffer neutralizes acidic

samples thus preventing hydrolysis by phytase. In a 8 ml vial with gastight Teflon

cap 5 µl GPO/catalase mixture (25 units / 250 units) was added and oxygen was

applied for 30 seconds while the vial was shaken vigorously. After one-hour

incubation at room temperature the mixture was assayed for DHAP.

DHAP synthesis from glycerol

To a 10 ml solution containing 95% (v/v) glycerol and 150 mM pyrophosphate pH 4.0

Chapter 4

68

(adjusted with 2 N HCl) freeze-dried phytase was added (10 mg ). The mixture was

incubated for 24h at 37 °C in a 30 ml flask with Teflon cap and shaken gently. After

cooling down to room temperature the pH was raised to 7.5 by addition of 2.0 N

sodium hydroxide. Water was added (7.4 ml) to obtain a final glycerol concentration

of 55%. Then 100 µl GPO/catalase mixture (50 units / 500 units) was added and

oxygen was applied for three minutes. This was repeated after 30 and 60 minutes.

The flask was shaken at room temperature. Oxidation was stopped after 3h. The

yield of DHAP (from pyrophosphate) was 50% (0.75 mmol). Since phytase produces

equal amounts of the enantiomers the yield of D,L-glycerol-3-phosphate is 100%.

Aldol reactions

To a 10 ml mixture containing 43 mM DHAP 88 µl butanal (100mM) and 12.5 units of

fructose-1,6-bisphosphate aldolase from Staphylococcus carnosus were added.

Conversion was 78% after four hours. Dephosphorylation by phytase still present in

the mixture started by lowering the pH to 4 with 2N HCl and stirring overnight. The

glycerol/water mixture was extracted with ethylacetate, the ethylacetate layer with

water and dried with sodium sulfate. After evaporation of the solvent 39 mg 5-deoxy-

5-ethyl-D-xylulose was isolated (analysed using 1H NMR spectra of authentic

samples70). The yield was 56% from L-glycerol-3-phosphate.


69

Chemical phosphorylation of glycerol

Synthesis of L-glycerol-3-phosphate from glycerol and phosphoric acid (85%)

was achieved by heating an equimolar mixture (17.8 ml) overnight at 110 °C and

subsequently adding 1 equivalent water followed by stirring for 5h at 80 °C. This

resulted in an final concentration of 0.6 M L-glycerol-3-phosphate (8.4% yield). When

equal amounts of α- and ß-glycerolphosphate are formed, phosphate concentration

is about 4.7 M.

References

50 H.J.M. Gijsen, L. Qiao, W. Fitz and C.-H. Wong, Chem. Rev. 1996, 91 (1), 443-

46951 W.-D. Fessner and C. Walter, Angew. Chem. Int. Ed. Engl. 1992, 31 (5), 614-61652 P.G. Wang, W. Fitz and C.-H. Wong, Chemtech 1995, april, 22-2353 O. Meyerhof and K. Lohmann, Biochem. Z. 1934, 271, 8954 C.-H. Wong and C.M. Whitesides, J. Org. Chem. 1983, 48, 3493-349755 C.E. Ballou and H.O.L. Fischer, J. Am. Chem. Soc. 1956, 78, 165956 H.O.L. Fischer and H. Mildbrand, Ber. Dtsch. Chem. Ges. 1924, 57, 71057 R.L. Colbran, J.K.N. Jones, N.K. Matheson and I. Rozema, Carbohyd. Res. 1967,

4, 355-35858 F. Effenberger and A. Straub, Tet. Lett, 198728 (15), 1641-164459 R.L. Pederson, J. Esker and C.-H. Wong, Tetrahedron 1991, 47 (14/15), 2643-

264860 S.-H Jung, J.-H. Jeong, P. Miller and C.-H. Wong, J. Org. Chem. 1994, 59, 7182-

718461 M.-L Valentin and J. Bolte, Bull. Soc. Chim. Fr. 1995, 132, 1167-117162 T. Gefflaut, M. Lemaire, M.-L Valentin and J. Bolte, J. Org. Chem. 1997, 62, 5920-

Chapter 4

70

592263 D.C. Crans and G.M. Whitesides, J. Am. Chem. Soc. 1985, 107, 7019-702764 W.-D. Fessner and G. Sinerius, Angew. Chem. Int. Ed. Engl. 1994, 33 (2), 209-

21265 P. D’Arrigo, V. Piergianni, G. Pedrocchi-Fantoni and S. Servi, J. Chem. Soc.

Chem. Commun. 1995, 2505-2506

66 a) R.L. Colbran, J.K.N. Jones, N.K. Matheson and I. Rozema, Carbohyd. Res.

1967, 4, 355-358, b) F. Effenberger and A. Straub, Tet. Lett. 1987, 28 (15), 1641-

1644, c) R.L. Pederson, J. Esker and C.-H. Wong, Tetrahedron 1991, 47 (14/15),

2643-2648, d) S.-H Jung, J.-H. Jeong, P. Miller and C.-H. Wong, J. Org. Chem.,

1994, 59, 7182-7184

67 A. Pradines, A. Klaébé, J. Périé, F. Paul and P. Monsan, Tetrahedron 1988, 44

(20), 6386-638668 A. Pradines, A. Klaébé, J. Périé, F. Paul and P. Monsan, Enzyme Microb. Technol.

1991, 13, 19-2369 J. Dvorakova, Folia Microbiol. 1998, 43 (4), 323-33870 R. Schoevaart, F. van Rantwijk and R.A. Sheldon, Tetrahedron: Asymmetry 1999,

10 (4), 705-711

73

5

DHAP analogues

Abstract

Hydroxyactone thiosulfate was prepared as a substrate analogue for DHAP, but it

was not converted by DHAP utilizing enzymes. The scope of aldol reactions with in

situ formed dihydroxyacetone arsenate with different aldehydes catalysed by

fructose-1,6-bisphosphate aldolase was examined. The use of inorganic arsenate

and dihydroxyacetone afforded higher yields than dihydroxyacetone phosphate and

precluded the normal, elaborate preparation of phosphorylated dihydroxyacetone.

Cosolvents which were applied to increase the solubility of hydrophobic aldehydes

also increased their reaction rates and stabilised the enzyme as well. The

stereospecifity is somewhat higher with the natural donor substrate.

Chapter 5

74

Hydroxyacetone thiosulfate as DHAP analogue

The possible replacement of dihydroxyacetone phosphate (DHAP) as donor in aldol

reactions catalysed by DHAP depending aldolases is of considerable synthetic

significance. The first DHAP analogue (1, Figure 1) reported71 was acetol phosphate,

with a relative activity of only 1%. 3-Chloro-, 3-bromo- and 3-iodoacetol phosphate72

(2) were synthesised from 3-chloro-1,2-propanediol, to investigate their capability to

bind irreversibly in the active-site of aldolases, triose phosphate isomerases and

glycerolphosphate dehydrogenase. The iodo analogue of DHAP inactivated FruA

rapidly, while the others had no effect. It is unclear if the chloro and bromo

compounds were substrates, however, it is known that 3-fluoroacetol phosphate73 is

not. They did act as effective reversible inhibitors, which demonstrates their ability to

bind in the active site.

The first active substrate analogue was 4-hydroxy-3-oxobutylphosphonic

acid74,75 (3), which was synthesized in six steps from acrylic acid. Its reaction with

FruA was 10% of that of DHAP. 1,3-Dihydroxy-2-butanone 3-phosphate76 (4),

prepared in seven steps from butyl-2-hydroxyacetate, had the same activity but the

very similar phosphonomethyl glycolate (5) was not active at all. The phosphoramide

(6) and phosphorothioate (7) analogues of DHAP prepared from bromoacetyl

chloride or 2-chloro-2-propen-1-ol77 both showed a relative activity of 10%. These

two compounds hydrolyse readily under the reaction conditions. For example, 6 was

hydrolysed fifty times as fast as DHAP. Consequently, also considering their reduced

activity, the synthetic utility of 6 and 7 is quite limited, even though they are relatively

stable at pH 9. The synthesis of 3, 6 and 7 could also be achieved by the GPO

mediated oxidation of the corresponding glycerol-3-phosphate analogues64.

DHAP analogues

75

Figure 1. DHAP analogues

The crucial role of the phosphate group was demonstrated by dihydroxyacetone

monosulfate76 (8), which was completely inactive. This shows the narrow limits

between which the aldolase tolerates changes in the donor substrate, in contrast

with the quite relaxed acceptor specificity.

Summarising, no effective replacement for DHAP has yet been found. In view of the

lack of a theoretical framework for selecting promising reactant candidates, such

replacements can only be identified by experiment. We decided to explore the

capabilities of the thio analogue of 8, dihydroxyacetone thiosulfate (HATS), which we

synthesised via the haloacetol phosphate72 route (Figure 2). In the final step the

chlorine moiety of hydroxyacetone chloride is exchanged for thiosulfate. Afterwards a

shorter route was devised, namely from dihydroxyacetone which had a slightly

higher overall recovery, although only two in stead of six steps were employed.

OPO3-2

O

2 8

4 7

63

5

1

X = Cl, Br, I

X OPO3-2

O

HO OSO3-

O

HO OPO3-2

O

HO SPO3-2

O

HO NHPO3-2

OHO

O

PO3-2

HOO

O

PO3-2

Chapter 5

76

Figure 2. Synthesis of the DHAP analogue hydroxyaceton thiosulfate (HATS)

In an aldolase catalysed reaction with propanal no product could be discovered. The

reduction of HATS with NADH mediated by glycerophosphate dehydrogenase did

not proceed either, although this test is very sensitive. The use of triosephosphate

isomerase in combination with hydrazine did not give an indication that any aldehyde

was formed. Finally, when crude 2,3-dihydroxypropane thiosulfate prepared from

thiosulfate and 3-chloro-1,2-propanediol was exposed to GPO and oxygen no HATS

was found. Sofar, it is has become clear that HATS is not a substrate for any DHAP

utilizing enzyme, which reconfirms the importance of the phosphate moiety.

Dihydroxyacetone arsenate as DHAP analogue

In many phosphate-depending enzymatic reactions arsenate esters can mimic

phosphate esters78. This approach obviates the use of organic phosphates that

require kinases and ATP, as well as ATP regeneration systems for their synthesis.

Because arsenate esters are formed spontaneously and reversible in aqueous

12% overall

8% overall

(HATS)

Na2SSO3

PhCOClOH

O

Ph C O OOHOH O

ClOH O

SSO3NaOH O

OHOH Cl 5 steps

Na2SSO3

DHAP analogues

77

solutions - in contrast with phosphate esters - experimental procedures are

simplified. The synthetic application of the in situ reversibly formed dihydroxyacetone

arsenate (DHAAs) has been demonstrated in the preparation of uncommon sugars79.

In that case DHAAs replaced dihydroxyacetone phosphate (DHAP) in reactions

catalysed by DHAP-dependent aldolases (see figure 3). This group consists of four

enzymes which all have a unique specificity for one of the four possible

stereoisomers. They are named according to specificity D-fructose-1,6-bisphosphate

(FruA), L-fuculose-1-phosphate (FcuA), L-rhamnulose-1-phosphate (RhuA) and D-

tagatose-1,6-bisphosphate (TagA) aldolase.80

Figure 3. Arsenate catalysed aldol reaction with DHAP-aldolases

With FruA from Staphylococcus carnosus as aldol reaction catalyst we explored the

use of many unnatural acceptor substrates, cosolvents and the effects of arsenate

on the stereoselectivity of the enzyme.

HOAsO32-

R

OH

OH

O

OAsO32-

HO OAsO32-

O

HO OH

O

aldolase

R

OH

OH

O

OH

RCHO

Chapter 5

78

Results and discussion

Monitoring the progress of the aldol reaction requires the detection of either

dihydroxyacetone (DHA) or the aldehyde. Enzymatic detection of DHA would require

its phosphorylation to DHAP with ATP, mediated by a kinase81, preliminary to

measuring DHAP by monitoring its reduction with NADH mediated by

glycerolphosphate dehydrogenase (GDH). The phosphorylation step would delay the

analysis by hours and is not always complete. The NADH / GDH system can also be

used to detect DHAAs, however, since its Km is hundred fold higher78 than the one of

DHAP, DHAAs reacts very slowly rendering the system unsuitable for routine

analysis. To circumvent this problem DHA can be reduced with borohydride to

glycerol, which in its turn is detected by phosphorylation with ATP coupled to an ADP

detection system. As this approach requires four steps, we opted for a more

straightforward approach by means of detecting the aldehyde through reduction with

NADH mediated by yeast alcohol dehydrogenase (ADH). This method is fast, but

cannot detect all aldehydes, since the substrate spectrum of alcohol dehydrogenase

is more limited than the one of FruA. For example formaldehyde, i-butyraldehyde,

pivaldehyde, phenylacetaldehyde, methylglyoxal, glyoxal and glyceraldehyde were

found to be substrates for FruA but not for ADH. These aldehydes can alternatively

be detected by reaction with hydrazine, which is monitored at 240 nm, although this

would require all the individual molar extinction coefficients to be the determined

first.

In order to study the scope of DHAAs as a substrate analogue, propanal was

chosen as an example acceptor substrate, since it dissolves readily in water and is a

good substrate for both FruA and ADH.

DHAP analogues

79

Optimal conditions

Two factors which are considered to have a major influence on the enzymatic aldol

reaction with DHAAs were first examined: the pH and the arsenate concentration.

When the pH was varied between pH 6 and 9 the enzymatic aldol reaction took

place, with an optimum at pH 7.6. At values above pH 9 or below pH 6 no aldol

reaction was observed. The base catalysed reaction – at a pH above neutral – was

never detected.

Although arsenate is catalytically79 involved in the reaction, a large excess is

needed to compensate for the reduced binding of the substrate by the enzyme: Km is

fivefold higher and Vmax eightfold lower78 for DHAAs compared to DHAP when FruA

is used as a catalyst. With an arsenate concentration being as low as a few mol

percent, aldol reactions could still be observed. However, with 20 mM DHA present,

the reaction rate reached a maximum at about 120 mM arsenate, a sixfold excess

(see figure 4). Practically speaking this means that arsenate can be used to buffer

the reaction medium. Higher arsenate concentrations gradually slowed down the

aldolase, presumably by the high salt concentration. The reduced activity could, in

principle, be compensated by increasing the amount of enzyme. For example, the

use of DHA and arsenate in concentrations of up to 0.5 M has been reported79, but

this required a fifty fold increase of the amount of aldolase. Because FruA is

inactivated by propanal at concentrations above 200 mM, which effect increases with

less hydrophilic aldehydes, the aldehyde concentration should be kept as low as

practicable. As a compromise, we performed the reaction at 20 mM DHA, 120 mM

arsenate and 50 mM aldehyde.

Chapter 5

80

Figure 4. Optimal arsenate concentration with 20 mM dihydroxyacetone, 50 mM

propanal and 1 unit fructose-1,6-bisphosphate aldolase, conversion after 24h.

Use of cosolvents

The more hydrophobic aldehydes that we used as acceptor, were sparingly soluble

in water. Because the resulting inhomogeneous reaction system provoked

deactivation of the biocatalyst and hampered representative sampling, the use of

organic cosolvents became an attractive proposition.

The effect of a number of cosolvents on the activity of FruA from S. carnosus

was assessed by monitoring the aldol reaction of DHAAs and propanal (Table I). The

addition of 25 % cosolvent in some cases, such as DMSO, DMF and tert. butyl

alcohol, slightly activated the enzyme compared with reaction in aqueous medium. In

other cases a reduction in activity of up to 50% was observed. The lowered activity in

ethanol is most likely caused by ester formation of arsenate and the cosolvent, thus

lowering the effective arsenate concentration. In the case of tert. butyl alcohol the

0

20

40

60

80

100

0 50 100 150 200 250mM arsenate

% c

onve

rsio

n

DHAP analogues

81

effect of unwanted ester formation presumably is reduced by steric hindrance.

Addition of cosolvent lead in many cases to increased enzyme activity, most likely

provoked by withdrawal of water from the protein causing structural changes82. The

effect of 25 % cosolvent (v/v) on the 24-h stability of FruA from S. carnosus as well

as those from Staphylococcus aureus and rabbit muscle (RAMA) was measured via

a standard FruA assay (Table I). It is apparent that the bacterial aldolases from S.

carnosus and S. aureus are superior in stability compared with the traditionally used

fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA). Some general

trends could be observed: in all cases dimethylformamide (DMF) increased stability

whereas acetonitrile and tert. butyl alcohol had the opposite effect. FruA from S.

carnosus is remarkably stable in DMSO. When it was added to pure DMSO, shaken

for 1 hour and centrifuged, the pellet contained 85% of the original activity while the

remaining 15% was found in the supernatant.

Table I. Activity and stability of aldolases from different sources in cosolvents.

Vrel1 stability2

cosolvent (25%) S. carnosus S. carnosus S. aureus RAMA

DMSO 104 151 146 66

1,2-Dimethoxyethane 107 134 125 65

Ethanol 50 116 99 92

Dioxane 87 114 123 60

DMF 118 112 139 106

Acetonitrile 92 98 91 0

t-BuOH 121 81 86 18

Water 100 100 100 96

1 Based on conversion of propanal after 2 hours relative to the activity in water.

Chapter 5

82

2 Cleavage of fructose-1,6-bisphosphate after 1 day incubation at room temperature

relative to the rate in water at t = 0.

Because FruA is activated as well as stabilized in the presence of 25% 1,2-

dimethoxyethane or DMF, the time to reach maximum conversion is reduced,

making these solvents an attractive additive, even with accptor aldehydes that

readily dissolve in water. The preferred cosolvent will depend on the best stability /

activity combination as well as on other restrains, such as work-up procedures. For

example, activity in DMSO is only slightly increased, but stability is excellent. With

tert. butyl alcohol the activity is much higher, but the stability is lowered. Ethanol has

only half the activity, but it has a good stability and is of course attractive because it

is easy to separate from the reaction mixture.

Figure 5. Use of DMSO as a cosolvent for arsenate catalysed aldol reaction of

DHA with propanal.

The effect of DMSO was studied in more detail. Propanal was, for practical

0

20

40

60

0 10 20 30 40 50 60 70 80% DMSO (v/v)

Con

vers

ion

(%)

6h

DHAP analogues

83

reasons, used as the acceptor. Strictly spoken this aldehyde would not require a

cosolvent, but its use allowed us to study the effect of the cosolvent without

interference by heterogeneity issues. The conversion of propanal and DHAAs in the

presence of FruA from S. carnosus slightly increased up to 50% (v/v) of DMSO and

dropped sharply above 60%.

Acceptor specificity with DHAAs

Aldol reactions of DHAAs with a number of aldehydes were performed. Since our

method of analysis restricted the use of aldehydes, the conversion of aldehydes that

could not be beasured by the ADH assay: formaldehyde, i-butyraldehyde,

pivaldehyde, phenylacetaldehyde, glyoxal and methylglyoxal were monitored by

assaying DHAP assay after (partial) phosphorylation of the unconverted DHA. The

hydrophobic acceptors hexanal, heptanal, i-butyraldehyde, pivaldehyde, 3-

phenylpropionaldehyde required the addition of 25% (v/v) DMSO as cosolvent for

reliable sampling.

All the aldehydes employed were found to be acceptor substrates (Table II).

The enzyme specificity found when DHAAs was used as donor is in agreement with

expectations based on aldol reactions with DHAP83,84, in which aldehydes with an α-

hydroxyl group as well as activated aldehydes are normally good substrates.

However, DHAAs gave higher products yield with hydrophobic aldehydes than with

hydrophilic ones, which runs against the general trend observed with DHAP. This

manifestation is a great advantage of the usage of arsenate, although it entails a

penalty as regards reaction rate, leading to an increase in reaction time in all cases.

Chapter 5

84

Under optimized conditions and with the same amount of enzyme, non-polar

aldehydes reacted approximately six times slower, and apolar ones twenty times

slower, with DHAAs than with DHAP.

Table II. Conversion of DHAAs and DHAP by FruA

RCHO Donor

R DHAAs DHAP

H 67 (48h) a 81 (2h)

CH3 78 (48h) 84 (2.5h)

CH2CH3 79 (24h) 67 (4h)

(CH2)2CH3 86 (24h) 78 (4h)

(CH2)3CH3 95 (24h) 64 (3h)

(CH2)4CH3b 76 (24h) 62 (5h)

(CH2)5CH3b 54 (24h) 30 (16h)b

CH(CH3)2 b 83 (48h) a 78 (16h)b

C(CH3)3 b 69 (48h) a 85 (16h)b

(CH2)2Phb 83 (24h) 65 (16h) b

CH2Ph 77 (48h) a 66 (2h)

CHO 83 (48h) a 98 (5h)

COCH3 73 (48h) a 86 (6h)

CH2Cl 63 (24h) 76 (1.5h)

CH2OH 83 (24h) 96 (0.5h)

a) Detected by phosphorylation of DHA (error ±10% ).

b) 25% DMSO was added

DHAP analogues

85

Effect of arsenate on the stereoselectivity

To analyse the stereochemistry of the products, phosphorylation is necessary to

perform a stereoisomer assay85. This assay is based on DHAP detection in the retro

aldol reaction for each of the four possible stereoisomers, catalysed by the

appropriate DHAP dependent aldolase. The concentration of each single

stereoisomer can be detected in this way, giving full insight in the stereoselectivity of

the aldol reaction. The phosphorylation of the aldol adducts, preliminary to the retro-

aldol assay, was performed using a glycerol kinase and ATP. It could not be carried

out to complete conversion, since aldol adducts are even worse substrates for

glycerol kinases than is DHA. But, since glycerol kinase did not seem to discriminate

between the different stereoisomers – which was demonstrated by using RhuA

instead of FruA as the aldol catalyst - this method is nevertheless suited for

determining the ratios of the stereoisomers.

Table III. Stereochemistry of aldol adducts.

RCHO DHAP DHAAs

R Fru Rhu Fcu “Tag” Fru Rhu Fcu “Tag”

CH3 88 0 3.5 8.5 90a 10 0 a

CH2CH3 96 0 0 4.0 93a 5 2 a

CH2CH2CH3 90 0 2.5 7.5 95a 0 5 a

CH(CH3)2 92.5 0 0 7.5 86a 0 14 a

a Since phosphorylation was incomplete, the proportion of the product with the

tagatose configuration could not be calculated

Chapter 5

86

When comparing the steric course of the FruA mediated aldol reactions of DHAAs

and DHAP, the high stereospecificity of the aldolase is evident in both cases (Table

III). The fraction of products with D-fructose (“Fru”) and D-tagatose (“Tag”)

configuration is somewhat lower for DHAAs, however. Moreover, the products with

the L-rhamnulose (“Rhu”) configuration, which were never detected in aldol adducts

of DHAP84, were found with acetaldehyde and to a lower extend also with propanal

as the acceptor substrate. Hence, the use of arsenate instead of phosphate clearly

has some effect on the stereoselectivity of the aldol reaction.

Conclusion

In situ formed dihydroxyacetone arsenate (DHAAs) can advantageously replace the

traditionally used DHAP in D-fructose-1,6-bisphosphate aldolase catalysed aldol

reactions. The procedure obviates the elaborate separate preparation of the donor

and gives improved yields with hydrophobic acceptor aldehydes. The use of DHAAs

somewhat reduces the stereoselectivity of the aldol reaction. Cosolvents can be

used with a triple advantage: increased solubility of hydrophobic aldehydes, faster

reaction and stabilisation of the enzyme.

Experimental

General


DHAP analogues

87

temperature controller. Bacterial aldolases were obtained from Roche Diagnostics as

a gift. DHAP was prepared from its ethyl hemiacetal dimer barium salt (Fluka). All

other enzymes and chemicals were purchased from Sigma. Thin-layer

chromatography (TLC) on pre-coated aluminium foil of silica gel (Merck) was

performed with butanol / acetic acid / water (3:2:5). Detection was effected by

dipping in a solution containing 24 g ammonium molybdate, 1 g of cerium(III) sulfate

and 10% (v/v) H2SO4 per 500 ml and subsequent heating.

Preparation of hydroxyacetone thiosulfate (HATS)

Method 1. 3-Chloro-1-hydroxyacetone was synthesised according to Hartman72. A

stirred solution of this product (110 mg, 1.01 mmol) in water (1 ml) and ethanol (1.5

ml), was treated with sodium thiosulfate pentahydrate (320 mg, 2eq), and then

allowed to stir for 3 hours. The resultant mixture was concentrated in vacuo and the

residual material was solved in dry ethanol, filtrated and again concentrated in

vacuo giving the titel compound as a white powder (25 mg, 12%).

Method 2. In a 250 ml flask 2 g dihydroxyacetone (22.2 mmol) was solved in 80 ml

of dry pyridine containing 100 mg 4-dimethylaminopyridine (0.8 mmol). At –5 °C 2.31

ml (1 eq) of benzoylchloride was added dropwise. After the addition was complete,

the mixture was stirred for one-half hour at room temperature. Subsequently, sodium

thiosulfate pentahydrate (11 g, 2eq) was added and the mixture was stirred for two

hours. The pyridine was evaporated (with toluene), water was added and the

solution was filtered. Then the water was evaporated (with ethanol) and 660 mg

Chapter 5

88

(14% yield) of solid material was isolated. TLC indicated that both methods yielded

the same product, which was used without additional purification in enzymatic

assays.

Preparation of 2,3-dihydroxypropane thiosulfate

A stirred solution of 3-chloro-1,2-propanediol (1g, 9 mmol) in water (8ml) was treated

with sodium thiosulfate pentahydrate (1g, 0.75 eq) and the mixture was stirred

overnight at room temperature and then heated at 85 °C for 5 hours. Ethanol was

added and the crude product was concentrated in vacuo. This yielded a very viscous

residue (1g, 53 %) which was used without further purification in enzymatic assays.

Assay of aldehydes with alcohol dehydrogenase

The aldehydes were assayed with alcohol dehydrogenase (ADH) from yeast. To

1.97 ml of a 50 mM Tris-buffer pH 7.6 containing 0.16 mM NADH in a 2 ml cuvette,

10 µl solution containing 5 units ADH was added. Then a 20 µl sample of tenfold

diluted reaction mixture was added and the absorption was monitored at 25 °C. The

molar absorption coefficient taken was 6.22 l.mmol-1cm-1.

DHAP analogues

89

Assay of DHA

After maximum conversion the reaction mixture was centrifuged in an eppendorf

centrifuge and transferred into a Microcon Centrifugal filter (cutoff 10 kD) and again

centrifuged for 10 minutes. The filtrate contained no aldolase activity. To 1 ml of this

aldolase free mixture containing a maximum amount of 4 mM dihydroxyacetone, 5.5

mg ATP (10 mM) and 10 units glycerol kinase from Pseudomonas species were

added. After shaking for 6 hours at pH 7.5 the mixture was assayed for DHAP.

DHAP was assayed with a coupled enzyme system. To 1.95 ml 50 mM Tris-

buffer pH 7.6 containing 0.16 mM NADH in a 2 ml cuvette, 10 µl of a mixture

containing 1.25 unit D-glycerol-3-phosphate dehydrogenase and 12.5 unit triose-1-

phosphate isomerase were added. Then 40 µl of the phosphorylation mixture was

added and the absorption monitored at 25 °C.

Aldol reaction with dihydroxyacetone arsenate

In a gastight bottle dihydroxyacetone (1,8 mg, 20 mM) and aldehyde (50 mM) were

added to 1 ml of 120mM arsenate buffer pH 7.6. Then 0.5 ml was taken out to

monitor the background reaction. To the other half, 1 unit of fructose-1,6-

bisphosphate aldolase from Staphylococcus carnosus was added. After shaking for

24 hours the mixtures were analyzed.

Chapter 5

90

FruA activity assay

Two aldol reactions - one with 25 % cosolvent (v/v) - were performed as described

above. After incubating at room temperature for two hours, the amount of propanal

converted in both samples was assayed with ADH. The conversion in water was set

at 100%.

FruA stability assay

To 1.95 ml of a 50 mM Tris-buffer pH 7.6 containing 0.16 mM NADH in a 2 ml

cuvette, were added 20 µl of a 190 mM fructose-1,6-bisphosphate as well as 20 µl of

a mixture containing 1.25 unit D-glycerol-3-phosphate dehydrogenase and 12.5 unit

triose-1-phosphate isomerase. Then 50 µl diluted incubation mixture containing the

aldolase was added. The reduction of the reaction products DHAP and GAP by

NADH was monitored at 25 °C. 1 unit aldolase converts 1 µmol fructose-1,6-

bisphosphate per minute.

Stereoisomer assay

The reaction mixture containing the aldol adducts and DHA was deproteinated and

partially phosphorylated as described above. 20 to 40 µl of this reaction mixture was

then added to a quartz cuvette containing 1.97 ml of a 50 mM TRIS buffer pH 7.6,

0.16 mM NADH, 1.25 unit GDH and 12.5 units triose-1-phosphate isomerase. The

DHAP analogues

91

absorption was monitored at 340 nm at 25 °C. After complete conversion of residual

DHAP, the retro-aldol reaction was initiated by adding 10 µl aldolase (5 units). The

amount of DHAP released from this reaction is equal to the amount of adduct with

the configuration corresponding with the used aldolase. For each of the three

aldolases a new sample was taken from the deproteinated solution.

Preparation of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose

In a flask dihydroxyacetone (36 mg, 20 mM) and 88 µl butanal (50 mM) were added

to 20 ml of 120mM arsenate buffer pH 7.6 containing 25% DMF. To initiate the

reaction, 40 units of fructose-1,6-bisphosphate aldolase from Staphylococcus

carnosus was added. After stirring for 24 hours conversion was 86% (assay) the

mixture was extracted with diethylether. This afforded after drying and evaporation of

the solvent, 100 mg of product and unreacted aldehyde. Acetylation in 2 ml pyridine

and 1 ml acetic anhydride yielded 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose (65%

yield) (analysed using 1H NMR spectra of authentic samples).

References

71 I.A. Rose and E.L. O’Connell, J. Biol. Chem, 1969, 244, 12672 F. C. Hartman, Biochemistry, 1970, 9 (8), 1776-178273 R. Ducan, Thesis, Stanford University, 199574 D. Stribling, Biochem. J. 1974, 114, 72575 H.-L. Arth and W.-D. Fessner, Carbohydrate Res. 1998, 305, 313-321

Chapter 5

92

76 N. Bischofberger, H. Waldmann, T. Saito, E.S. Simon, W. Lees, M.D. Bednarski

and G.M. Whitesides, J. Org. Chem. 1988, 53, 3457-346577 R. Duncan and D.G. Drueckhammer, Tet. Lett., 1993, 34 (11), 1733-173678 J.R. Durrwachter, D.G. Druekhammer, K. Nozaki, H. Sweers and C.-H. Wong, J.

Am.Chem.Soc. 1986, 108, 781279 D.G. Drueckhammer, J.R. Durrwachter, R.L. Pederson, D.C. Crans, L. Daniels

and C.-H. Wong, J. Org. Chem. 1989, 54, 70-7780 H.J.M. Gijsen, L. Qiao, W. Fitz and C.-H. Wong, Chem. Rev. 1996, 91 (1), 443-

46981 C.-H. Wong and C.M. Whitesides, J. Org. Chem 1983, 48, 3493-349782 Ö. Almarson and A.M. Klibanov, Biotechnology and Bioengineering 1996, 49, 87-

9283 M.D. Bednarski, E.S. Simon, N. Bischofberger, W.-D Fessner, M.-J Kim, W. Lees,

T. Saito, H. Waldmann and G.M. Whitesides, J. Am. Chem. Soc. 1989, 111, 627-

63584 R. Schoevaart, F. van Rantwijk, R.A. Sheldon, Tetrahedron: Asymmetry 1999, 10

(4), 705-71185 R. Schoevaart, F. van Rantwijk, R.A. Sheldon, submitted

95

6

Transketolase versus

fructose-1,6-bisphosphate aldolase

Abstract

Two approaches were compared for the synthesis of optically pure polyhydroxy

ketones via asymmetric enzymatic C-C bond formation. D-Fructose-1,6-

bisphosphate aldolase (FruA) catalyzes the aldol reaction of an aldehyde with

dihydroxyacetone phosphate (DHAP). Transketolase, on the other hand, catalyzes

the formation of the same product, together with CO2, by irreversible reaction of an

α-hydroxyaldehyde with β-hydroxypyruvate. When the α-hydroxyaldehyde is racemic

the reaction is a kinetic resolution, i.e. both enantio- and diastereoselective.

Transketolase catalyzed reactions proved to be excellent alternatives to FruA-based

synthesis, with the added advantages that transketolase is readily available from a

recombinant E. coli and it does not require a phosphorylated substrate. Cross-linked

enzyme crystals of tranketolase were more stable but substantially less active than

the native enzyme.

Chapter 6

96

Introduction

Transketolase86 catalyzes the reversible transfer of a ketol group between D-

xylulose-5-phosphate and D-ribose-5-phosphate (Figure 1) or D-xylulose-5-

phosphate and erythrose-4-phosphate in the pentose phosphate pathway. Together

with transaldolase it creates a reversible link between two main metabolic pathways,

the pentose phosphate pathway and glycolysis.

Figure 1. Natural substrates of transketolase

Transketolase requires vitamin B1 derived thiamine pyrophosphate (TPP)87 and

magnesium(II) ions to function, but in only catalytic amounts. The enzyme is a dimer

with the cofactor and the active site located at the interface between the two identical

subunits88. A variety of sources for transketolase have been described; however,

commercially available samples are generally derived from yeast89 or spinach

leaves.90 More recently, enzyme obtained from an over-expressed E. coli

transformant91,92 has attracted considerable interest. This source is more suitable for

Sedoheptulose7-phosphate

Glyceraldehyde3-phosphate

Ribose5-phosphate

Xylulose5-phosphate

transketolase

C

OH

OPO3-2

O HC

OH

OH

OPO3-2

OH

O H

CH2OH

O

HO

OH

OH

OH

OPO3-2

CH2OH

O

HO

OH

OPO3-2

Transketolase versus fructose-1,6-bisphosphate aldolase

97

large scale production of transketolase. The properties93 of all three are very similar,

but the specific activity of the E. coli transketolase is about ten fold higher.

When β-hydroxypyruvate94,95,96 is used instead of D-xylulose-5-phosphate as

the ketol donor substrate the reaction becomes irreversible. Moreover, with the

appropriate choice of α-hydroxyaldehydes (Figure 2) products identical to the ones

made by D-fructose-1,6-bisphosphate aldolase (FruA) mediated aldol reactions are

obtained.

Figure 2. Transketolase and FruA can make identical products.

Hydroxypyruvate decomposes slowly in solution, but it is stable enough in Tris

and glycylglycine buffers97. The use of a pH autotitrator gives the best results since

the progress of the reaction is accompanied by a pH increase caused by the

consumption of hydroxypyruvic acid, which can simply be fed on demand98 to control

the pH. Using this system, transketolase can be applied on a large scale99 with high

productivity (space-time yield of 45 gL-1d-1 for L-erythrulose100,101,102).

Over sixty aldehydes103 have been identified as acceptor substrates.

1) FDP-aldolase

2) acid phosphataseOH

OOH

OH

RR

O

HOPO3

2-

O

HO+

OH

OOH

OH

R

CO2

transketolase+O

-O

OH

O

R

O

H

OH

Chapter 6

98

Transketolase is rather flexible towards the acceptor substrate, although its

requirement for the (R) configuration of any hydroxy functionality at C-2 is absolute.

This feature has been used to prepare optically pure L-2-hydroxyaldehydes by

means of kinetic resolution of racemic α-hydroxyaldehydes104. Aldehydes without a

hydroxyl group at C-2 are also converted. A variety of products is accessible via the

transketolase route, e.g. N-phenylglycolhydroxamate105, natural ketoses and

analogues106, labeled xylulose107, the pheromone (+)-exo-Brevicomin108 and the

glycosidase inhibitor Fagomin109, 1,4-dideoxy-1,4-imino-D-arabinitol110, aromatics

from D-glucose111 and D-sedoheptulose112. The half-life of the enzyme is strongly

influenced by the specific aldehyde. Especially non-polar aldehydes such as

acetaldehyde and aromatic aldehydes are detrimental to the enzyme stability and

cause a decrease in the half-life from 24 hours to under one hour.

Transketolase also provides access to compounds that cannot be obtained

using DHAP aldolases. For example α,β-unsaturated aldehydes are not substrates

for FruA, but α-hydroxy-β,γ-unsaturated aldehydes are substrates for transketolase.

Transketolase can even accommodate α,β-unsaturated aldehydes as substrates,

once again demonstrating its versatility. Mutants of transketolase have been

prepared to study its active side structure and the mechanism of the reaction113,114,

but also to broaden its substrate spectrum115.

α-Hydroxyaldehydes

Because the use of α-hydroxyaldehydes in transketolase catalyzed reactions affords

products identical with those made by FruA, we were particularly interested in this


99

group of acceptors. The synthesis of many α-hydroxyaldehydes has been

described98,116. A few strategies can be distinguished: ozonolysis of allylic alcohols

with reductive workup, addition reaction of glycidaldehyde dimethyl acetal,

chlorination at the α-position of an aldehyde followed by hydrolysis117 and synthesis

via silyl protected 2-hydroxyesters. Chiral α-hydroxyaldehydes can be

enantiomerically pure or racemic. Because transketolase is absolute in its

requirement for the (R) configuration of the hydroxy functionality at C-2, racemic

aldehydes also give enantiomerically pure products via a kinetic resolution. Acetals

are commonly used for stable storage of aldehydes and their hydrolysis is

accomplished under acidic aqueous conditions. Storage of aldehydes as aqueous

solutions is likewise convenient.

We chose a small group of substrates for transketolase for comparison with the

synthesis of the same products using FruA (Table I). Two commercially available α-

hydroxyaldehydes were glycolaldehyde and D,L-glyceraldehyde. Lactaldehyde was

synthesized via the reduction of methylglyoxal 1,1-dimethylacetal118. Chlorination at

the α-position of butanal and pentanal followed by hydrolysis was used to prepare α-

hydroxybutanal and α-hydroxypentanal. The corresponding aldehydes for the FruA-

catalyzed syntheses were formaldehyde, glycolaldehyde, acetaldehyde, propanal

and butanal, which are all commercially available.

With the exception of the last entry (Table I) the overall reaction times were

quite similar, even when initial reaction rates differed. However, the conversions of

the transketolase catalysed reactions, with the exception of glyceraldehyde, were

almost quantitative, which is significantly higher than those of FruA. α-

Hydroxyaldehydes are also particularly good substrates for FruA, but only reactions

generating an identical product are considered here. Hence, the use of transketolase

Chapter 6

100

was in most cases superior on account of the high yield. The exception was the

synthesis of D-xylulose mediated by FruA which gave a higher reaction rate and

conversion than the corresponding reaction of transketolase.

In practice, however, the complete synthetic route must be taken into account.

Transketolase requires other aldehydes than FruA and some might have to be

synthesized while others are commercially available. This has to be considered for

each different end product.

Table I. Comparison of transketolase versus FruA synthesizing identical products.

FruA acceptor =RCHO

TK acceptor =R(CHOH)CHO

R Vini conversion Vini conversion

H 100 81 (2h) 100 100 (0.5h)

CH3 81 84 (2.5h) 21 99 (2h)

CH2CH3 75 67 (4h) 58 97 (2h)

(CH2)2CH3 28 70 (4h) 23 96 (4h)

CH2OH 190 96 (0.5h) 12 75 (6h)

Reaction conditions: 25 mM donor, 50 mM (100 mM racemic) aldehyde and 1

unit enzyme.

Cross-linked enzyme crystals

In order to improve the stability of transketolase, the application of cross-linked

transketolase crystals was examined. Stabilization with lipids119,120 was not

successful and immobilization on Eupergit-C had already been accomplished121.


101

Crystallization to collect X-ray crystallographic data from E. coli transketolase had

opened the possibility to make cross-linked enzyme crystals122. Cross-linking

crystals of FruA from rabbit muscle had also been accomplished123,124, but the

stability was not better than bacterial FruA, which happened to be very stable. Cross-

linked crystals generally have increased stability, especially when cosolvents are

applied.

Crystals of transketolase were prepared in 35% activity yield and subsequently

cross-linked with glutaraldehyde. with a yield of 13%. Upon cross-linking more than

62% activity was lost, resulting in an overall activity yield of only 13%.

Figure 3. Activity of transketolase crystals after cross-linking with glutaraldehyde.

The most active cross-linked transketolase crystals were obtained at exactly 1.6 mM

glutaraldehyde (Figure 3). Any deviation from this concentration resulted in a

dramatic reduction in activity. It is not clear why the reduction in activity is so abrupt.

The stability of cross-linked transketolase crystals was tested in glycylglycine

0

10

20

30

40

50

0 1 2 3 4 5Glutaraldehyde (mM)

TK a

ctiv

ity (%

)

Chapter 6

102

buffer and compared with the native enzyme (Figure 4). It was found that the crystals

were more stable, but since 87% activity was lost during their preparation, this

increased stability is not beneficial when normal buffers are used. Addition of more

native enzyme would be far more efficient.

Figure 4. Stability of native and cross-linked TK.

Conclusions

Transketolase is capable of competing with FruA as far as reaction rate and

conversion of the corresponding acceptor substrates is concerned. In order to

assess the best way to synthesize a target molecule both the accessibility of the

different acceptor aldehydes and their activity as a substrate have to be considered.

In cases where it is not possible to use certain aldehydes, e.g. α,β-unsaturated

aldehydes with FruA, transketolase is the automatic choice. In other examples the

use of transketolase could lead to higher yields, making it competitive with FruA. In

0

20

40

60

80

100

120

0 5 10 15 20 25t (days)

TK a

ctiv

ity (%

)

native TKTK-CLEC


103

the preparation of polyhydroxy ketones, use of either one should always be

considered.

Experimental

General


temperature controller.

Transketolase catalysed reactions

In a gastight bottle hydroxypyruvate (HPA, 6.6 mg Li-salt, 25 mM) and an α-

hydroxyaldehyde (50 mM for glycolaldehyde and 100 mM for racemic ones) were

added to 1 ml of TRIS buffer pH 7.6. Then 0.1 ml was withdrawn to monitor the

background reaction. The reaction was initiated by adding transketolase (1 unit). At



7.6. The neutralized mixture was then assayed for HPA by lactate dehydrogenase

mediated reduction with NADH.

FruA catalysed reactions

Aldol reactions were performed in 1 ml 50 mM TRIS buffer pH 7.6 containing 25 mM

DHAP and 50 mM aldehyde, reactions were initiated by adding aldolase (1 unit). At

Chapter 6

104



7.6. This neutralized mixture was then assayed for DHAP. The reaction was followed

until only background hydrolysis of DHAP was measured (0.0047 mM/min).

Hydroxypyruvate assay

50 µl aliquots were taken from the reaction mixture at intervals and quenched with 15

µl 7% perchloric acid. After 30 minutes 10 µl 1M NaOH was added and 175 µl of 50

mM TRIS pH 7.6. From this neutralized and diluted mixture a 40 µl sample was

added to a quartz cuvette containing 1.95 ml 50 mM TRIS pH 7.6 and 0.16 mM

NADH. After the addition of 1.7 units lactate dehydrogenase (10 µl of 1 mg in 1 ml

sat. A2S) the absorption was monitored at 340 nm at 25 °C. The molar absorption

coefficient taken was 6.22 l.mmol-1cm-1.

Synthesis of D,L-lactaldehyde

To a three-necked flask containing 7.2 g LiAlH4 (190 mmol) in 150 ml Et2O 20.4 g

(172 mmol) methylglyoxal 1,1-dimethylacetal was added dropwise under a nitrogen

atmosphere. After the addition of 75 ml Et2O the mixture was refluxed for 30 minutes

and then quenched with saturated sodium chloride solution. After drying with sodium

sulfate the solvent was evaporated and the resulting oil distilled (bp 62-67 °C 30 mm

Hg). Deprotection was effected by heating a 0.7 M solution pH 1.5 at 60 °C for 1

hour. This solution was ready for use after neutralization and could be stored at 5 °C

for months. The exact concentration of the aldehyde was determined by reaction

with hydrazine monitored at 240 nm. The molar absorption coefficient taken was


105

2.73 l.mmol-1cm-1.

Synthesis of α-hydroxybutanal

In a flask equipped with a stirrer and reflux condenser were placed 67 ml (0.75 mole)

of butyraldehyde and 30 ml of methylene chloride. After the solution was cooled to

10 °C in an icebath, a mixture of 72 ml (0.75 mole) of sulfuryl chloride (SO2Cl2) and

10 ml of methylene chloride was slowly added maintaining the temperature between

15 and 40 °C. After completion of the addition, the reaction was stirred for one-half

hour. After addition of 20 ml of methylene chloride the mixture was refluxed for one

hour. All the volatile material was then distilled rapidly at reduced pressure.

Distillation through a 20 cm Vigreux column afforded 24.9 g product (25%) (b.p. 104-

110 °C).

To avoid polymerisation, acetalyzation should be conducted immediately. To 77 ml

of dry methanol was added slowly 5.2 g of sodium, the temperature was maintained

below 20 °C and 24.9 g of α-chlorobutyraldehyde was added. The ice-bath was

removed and the reaction mixture was stirred for one hour. To neutralise any

unreacted sodium water was carefully added. Then anhydrous sodium sulfate and

ethyl acetate was added. After filtration the solvents were rapidly distilled off at

reduced pressure. Distillation at reduced pressure (1 mm Hg) 13.9 g (46%) of α-

hydroxybutyraldehyde dimethyl acetal (b.p. 39-42 °C).

Deprotection was accomplished in 2 hours at pH 1.5 and 60 °C.

Chapter 6

106

Synthesis of α-hydroxypentanal

Using the same procedure as for the preparations of a-chlorobutyraldehyde, the α-

chloropentanal (b.p. 103°C) could be prepared affording 25 g (30% yield) α-

hydroxypentanal dimethyl acetal (b.p. 95 °C, 25 mm Hg).

Deprotection was accomplished in 3 hours at pH 1.5 and 70 °C.

Synthesis of 1,3,4-tri-O-acetyl-5-deoxy-5-ethyl-D-xylulose

A 171 ml solution containing 50 mM Tris pH 7.8, 25 mM β-hydroxypyruvate (HPA, 50

mM 2-hydroxypentanal and 171 units transketolase was stirred at room temperature.

Conversion was 99.4% (based on consumption of HPA) after 16 h. Extraction with

ethylacetate afforded 1.412 g (c.y. 90% yield) product and L-2-hydroxypentanal.

Acetylation with pyridine and acetic anhydride yielded 1,3,4-tri-O-acetyl-5-deoxy-5-

ethyl-D-xylulose (analysed using 1H NMR spectra of authentic samples.

Crystallization and cross-linking

Crystallization of transketolase A from recombinant Escherichia coli K12 was

performed in an eppendorf reaction vessel, sealed in a jar containing CaCl2 and kept

at 4 oC. In each vessel 90 ml of transketolase solution (17 mg / ml) was placed in

120 ml 50 mM glycylglycine buffer pH 8.0 containing 25% PEG 1450. To initiate

crystallization 15 ml of sonificated transketolase crystals were added. After 4 days

crystals were collected by centrifugation and suspended in 560 ml 500 mM

triethanolamine-buffer pH 7.6 also containing 25% PEG and divided into smaller

portions. Varying amounts of 50 mM glutaraldehyde were added for cross-linking

and the solutions were shaken for one hour at 4 oC. The insoluble TK-crystals were


107

washed with water and assayed for activity.

References

86 P. Srere, J.R. Cooper, M. Tabachnik and E. Racker, Archiv. of Biochem. and

Biophysics, 1958, 74, 295-30587 G. Schneider and Y. Lindqvist, Bioorganic Chem. 1993, 21, 109-11788 Y. Lindqvist, G. Schneider, U. Ermler and M. Sundström, The EMBO Journal,

1992, 11 (7), 2373-237989 G. De La Haba, I. Leder and E. Racker, J. Biol. Chem. 1955, 214, 40990 J. Villafranca and G. Axelrod, J. Biol. Chem., 1971, 246, 3126-313091 G.R. Hobbs, M.D. Lilly, N.J. Turner, J.M. Ward, A.J. Willets and J.M. Woodley, J.

Chem. Soc. Perkin Trans. 1, 1993, 165-16692 M. Gyamerah and A.J. Willetts, Enzyme Microb. Technol., 1997, 20, 127-13493 G.A. Sprenger, U. Schörken, G. Sprenger and H. Sahm, Eur. J. Biochem, 1995,

230, 525-53294 Large scale synthesis: K.G. Morris, M.E.B. Smith, N.J. Turner, M.D. Lilly, R.K.

Mitra and J.M. Woodley, Tetrahedron: Asymmetry, 1996, 7 (8), 2185-218895 HPLC analysis: R.K. Mitra and J.M. Woodley, Biotechnology Techniques, 1996, 10

(3), 167-17296 Enzymatic assay: A.W. Holldorf, Methods of enzymatic analysis, 1966, 3th Ed.,

Vol. 6, 614-61897 V. Dalmas and C. Demuynck, Tetrahedron: Asymmetry, 1993, 4 (11), 2383-238898 Y. Kobori, D.C. Myles and G.M. Whitesides, J. Org. Chem., 1992, 57, 5899-590799 J.M. Woodley, R.K. Mitra and M.D. Lilly, Ann. N. Y. Acad. Sci, 1996, 799, 434-445100 R.P. Chauhan, J.M. Woodley and L.W. Powell, Ann. N. Y. Acad. Sci, 1996, 799,

545-554101 R.P. Chauhan, L.W. Powell, J.M. Woodley, Biotech. Bioeng., 1997, 56 (3) 345-

351102 J. Bongs, D. Hahn, U. Schörken, G.A. Sprenger, U. Kragl and C. Wandrey,

Biotechnolgy Lett., 1997, 19 (3), 213-215

Chapter 6

108

103 U. Schörken and G.A. Sprenger, Biochimica et Biophysica Acta, 1998, 1385, 229-

243104 F. Effenberger, V. Null and T. Ziegler, Tetrahedron Lett., 1992, 33 (36), 5157-

5160105 M.D. Corbett and B.R. Chipko, Biochem. J., 1977, 165, 263-267106 J. Bolte, C.Demuynck and H. Samaki, Tetrahedron Lett., 1987, 28, 5525-5528107 C.Demuynck, J. Bolte, L. Hecquet and H. Samaki, Carbohydrate Research, 1990,

206, 79108 D.C. Myles, P.J. Andrulis III and G.M. Whitesides, Tetrahedron Lett. 1991, 32 (37)

4835-4838109 F. Effenberger and V. Null, Liebigs Ann. Chem. 1992, 1211-1212110 L. Hecquet, M. Lemaire, J. Bolte and C. Demuynck, Tetrahedron Lett., 1994, 35

(47), 8791-8794111 K.M. Draths, D.L. Pompliano, D.L. Conley, J.W. Frost, A. Berry, G.L. Disbrow,

R.J. Staversky and J.C. Lievense, J. Am. Chem. Soc. 1992, 114, 3956-3962112 V. Dalmas and C. Demuynck, Tetrahedron: Asymmetry, 1993, 4 (6), 1169-1172113 U. Nilsson, L. Meshalkina, Y. Lindqvist and G. Schneider, J. Biol. Chem., 1997,

272 (3) 1864-1869114 G. Schneider and Y. Lindqvist, Biochimica et Biophysica Acta, 1998, 1385, 387-

398115 C. French and J.M. Ward, Ann. N. Y. Acad. Sci, 1996, 799, 11-18116 C. André, J. Bolte and C. Demuynck, Tetrahedron: Asymmetry, 1998, 9, 1359-

1367117 C.L. Stevens, E. Farkas and B. Gillis, J. Am. Chem. Soc., 1954, 2695-2698118 J.R. Durrwachter, D.G. Drueckhammer, J. Am. Chem. Soc., 1986, 108, 7812-119 Y. Okahata, Y. Fujimoto and K. Ijiro, J. Org. Chem., 1995, 60, 2244-2250120 Y. Okahata and T. Mori, Tibtech, 1997, 15, 50-54121 S.P. Brocklebank, R.K. Mitra, J.M. Woodley and M.D Lilly, Ann. N. Y. Acad. Sci,

1996, 799, 729-736122 J. Littlechild, N. Turner, Acta Cryst. 1995, D51, 1074-1076123 J. Sygusch and D. Beaudry, Journal of Biological Chemistry, 1984, 259 (16)

10222-10227


109

124 S.B. Sobolov, A. Bartoszko-Malik, T.R. Oeschger and M.M. Montelbano,

Tetrahedron Letters, 1994, 35 (42), 7751-7754

111

7

2-Deoxyribose-5-phosphate aldolase (DERA)

Abstract

D-2-Deoxyribose-5-phosphate aldolase (DERA) is the only aldolase which accepts

two aldehydes as substrates. The natural donor acetaldehyde can be replaced by

e.g. acetone, making DERA suitable for synthesizing both β-hydroxyaldehydes and

β-hydroxy ketones by chain elongation with 2- or 3-carbons, respectively. Low

reaction rates for non-natural acceptor substrates however, call for long reaction

times and large amounts of enzyme making DERA a rather inefficient catalyst. The

stability could be improved by addition of cosolvent.

Chapter 7

112

Introduction

Whereas other aldolases use ketones as aldol donors, D-2-deoxyribose-5-phosphate

aldolase (DERA) accepts two aldehydes as substrates, namely acetaldehyde and D-

glyceraldehyde-3-phosphate. These two aldehydes undergo reversible C-C bond

formation to give 2-deoxyribose-5-phosphate (Figure 1). The newly generated chiral

center always has the (S) configuration.

Figure 1. Synthesis of 2-deoxyribose-5-phosphate by 2-deoxyribose-5-phosphate

aldolase (DERA) from D-glyceraldehyde-3-phosphate and acetaldehyde.

DERA plays a key role in the biosynthetic pathway125 of the sugar moiety of DNA.

Obviously, this enzyme can provide a route to a wide range of potentially biologically

active compounds, in particular the synthesis of deoxysugars such as deoxyriboses,

2-deoxyfucose analogs, dideoxyhexoses, trideoxyhexoses, deoxythiosugars and

13C-substituted D-2-deoxyribose-5-phosphate. It also affords a route to a variety of

chiral aldehydes. Synthesis of N-heterocycles which are used as building blocks for

-2O3PO

HO

O

OH

H

O

-2O3PO O

HO

OOH

-2O3PO

OH

DERA

2-deoxyribose-5-phosphate

acetaldehydeD-glyceraldehyde-3-phosphate

+


113

antihypertensive drugs is possible via azide substituted acceptors.

This type I aldolase126 has been isolated from different sources, but is

commonly derived from the E. coli gene, overexpressed in the same microbe. DERA

from E. coli is a dimer of identical subunits. It is not commercially available, but the

organism containing the gene, which is suitable for preparing the enzyme on a large

scale127 can be obtained from the ATCC.

Donor substrates

The natural nucleophilic reactant, acetaldehyde, can be replaced by propanal,

acetone or fluoroacetone128. Introducing a methyl group at C-2 makes it possible to

synthesize chiral β-hydroxyketones, besides β-hydroxyaldehydes. In this case two

new stereogenic centers are formed, having the 2(R) and 3(S) configuration. The

use of fluoroacetone as a donor results in the regioselective addition at the non-

fluorinated carbon. Acetol, chloroacetaldehyde, pyruvonitrile and glycolaldehyde are

not donor substrates for DERA, presumably due to steric hindrance in the active site.

Hence, at C-2 of this compound, hydrogen can only be substituted by a methyl

group.

Acceptor substrates

A wide variety of aldehydes, including aldose sugars128, can be used as acceptor for

DERA, but the reaction rates with unnatural acceptor substrates are relatively low

Chapter 7

114

(Table 1) and a relatively large amount of enzyme is required. The adducts of 2-

chloroaldehydes can be converted to epoxy aldehydes and epoxy ketones. N- and

S-heterocycles129 are obtained by the use of 3-azido- or 3-thiolaldehydes. The need

for a phosphate group at the 3-hydroxy position is reflected in the reaction rate; use

of D- or L-glyceraldehyde results, in both cases, in a 250-fold reduction in reaction

rate compared with D-glyceraldehyde-3-phosphate.

Table 1. Relative rates for DERA substrates (references 128 and 129).

Acceptor Vrel

D-glyceraldehyde-3-P 100

L-glyceraldehyde-3-P 5

D-glyceraldehyde 0.4

L-glyceraldehyde 0.4

acetaldehyde 0.03

chloroacetaldehyde 0.3

propionaldehyde 0.07

butanal 0.03

Sequential aldol additions

A problem associated with DERA is its competing self aldol reaction of acetaldehyde.

In principle each acceptor will react with the donor acetaldehyde, but when the

reaction rate is near or lower than the rate (Table I) with acetaldehyde as acceptor,

selfcondensation130,131 of acetaldehyde will predominate. This limits the number of

useful acceptor substrates. However, it has been used to advantageously synthesize


115

2,4,6-trideoxyhexoses, which are precursors for cholesterol lowering agents132. This

compound is created by a three-substrate aldol reaction (Figure 2).

Figure 2. Sequential three-substrate aldol additions with DERA.

The yields vary widely (3-80%) and long reaction times (2 weeks with 2500 units

DERA / mmol) are necessary. With DERA having a specific activity of 58.1 units per

mg, 4.3 g of pure enzyme per liter would be needed. Having an activity less than

10% makes it almost impossible to perform these reactions on a reasonable scale.

Even low yields of reaction products from a four-substrate aldol reaction131 can be

detected.

Figure 3. Sequential one-pot aldol reaction catalysed by DERA and RAMA.

The combination of DERA with D-fructose-1,6-bisphosphate aldolase from rabbit

muscle using a 2-substituted acetaldehydes (see Figure 3) allowed the synthesis of

mixtures of 5-deoxy ketoses133 in low yields (10-50%) after long reaction times (6

DERA,RAMA+HR

O

H

O

+ HO OPO32-

O

O

OH

OH

OH

OH

R

H

ODERA

+HR

O

H

O

+

O OHR

OHR=Me, 22%R= (CH2)2COOH, 80%

Chapter 7

116

days). Furthermore the combination of DERA with N-acetylneuraminic acid aldolase

(NeuAc aldolase) is possible, leading also to 2,4,6-trideoxyhexoses.

Summarizing, DERA shows flexibility towards both donor and acceptor substrate

analogues, making it possible to synthesize a variety of β-hydroxyaldehydes or

ketones. The very low reaction rates for non-natural acceptor substrates requires

sometimes as much as 250.000 units of the enzyme per liter, while the reaction time

is in the order of weeks. DERA is therefore a rather inefficient enzyme.

Results and discussion

A possible solution to the problem of low activity towards acceptor substrates that is

associated with the use of DERA could be the use of cross-linked enzyme crystals to

increase activity and/or long term stability during the reaction. Crystallization

procedures have been developed for acquisition of x-ray crystallographic data of

DERA crystals134. However, presumably because of impure enzyme preparations, no

DERA crystals could be obtained in our laboratory leaving the possible benefits of

cross-linking unexplored.

In a second approach DERA was covalently immobilized on Eupergit C 250L.

with 45% activity retention of activity. Immobilization provided a simple procedure to

separate the enzyme from the reaction mixture, but no improvement in the

operational stability compared with the native enzyme was observed.


117

Stability in cosolvents

To conclude our investigation of DERA the use of cosolvents was investigated. Since

the acceptor substrates in DERA catalyzed reactions are often quite hydrophobic,

solubility can pose a significant obstacle. Incomplete mixing of substrates with the

aqueous medium causes enzyme deactivation as well as a reduced rate of reaction.

To increase the solubility of hydrophobic substrates the use of cosolvents was

examined. Shorter reaction times would be possible in view of the higher

concentration of the acceptor substrate by complete mixing with the reaction

medium. These cosolvents can furthermore increase enzyme stability because no

liquid-liquid interface is present, thereby reducing deactivation of the enzyme during

the reaction. The stability of DERA can be defined as the residual activity after

incubation in water / solvent mixtures. In Table 2 the residual activities of DERA after

one day incubation in different water / solvent combinations (75:25, v/v) are

tabulated. It is apparent that DERA can be combined rather well with cosolvents. For

example DMSO and DMF increased its activity and the long term stability of the

aldolase under these conditions is remarkable as well: after one week of incubation it

was still a completely active. Acetonitrile is detrimental for the stability since it

rendered the enzyme completely inactive after one day already. The behavior of

DERA in ethanol is perplexing: with 25% solvent the residual activity is zero after one

week, whereas with 20% solvent an initial reduction of activity is seen after which it

increases to become 130% of the original activity after one week.

Chapter 7

118

Table 2. Stability (expressed as residual activity) of DERA in 25% cosolventa.

Residual activity (%)

Cosolvent (25%) 1 day 1 week

Ethanol 73 0

DMSO 150 115

Dioxane 85 0

Acetone 55 55

DMF 128 128

t-BuOH 63 0

Acetonitrile 0 0

a Residual activity in the cleavage of D-2-deoxyribose-5-phosphate

To examine the effect of cosolvent under reaction conditions, that is with the donor

aldehyde present, the stability of DERA was determined in 100 mM acetaldehyde

(Table 3) and 10% ethanol or DMSO. This cosolvent concentration represents a

compromise between enzyme activity (see next paragraph) and stability.

Acetaldehyde clearly has a negative effect on the stability of DERA during

incubation. After one week the enzyme retained only 21% activity, whereas in the

presence of ethanol and DMSO 33 and 36% was found, respectively. Since large

amounts of catalyst are normally required, even modest improvements in stability are

significant as many units of enzyme are preserved. Hence, the use of these

cosolvents in DERA-catalyzed aldol reactions clearly has practical benefits.


119

Table 3. Stability of DERA in 100 mM acetaldehyde and 10% cosolvent.

acetaldehyde % activity (1 day) 7d

- 100 99

+ 38 21

+/10%ethanol 71 33

+/10% DMSO 58 36

Activity in cosolvents

Besides the need for stability of the catalyst during the reaction its activity under the

specified conditions is also important. Determination of the activity of DERA in 25%

cosolvent can be performed in two ways: analysis of the synthetic reaction or

analysis of the cleavage (retro-aldol reaction) of D-2-deoxyribose-5-phosphate, which

is the standard DERA activity test. To follow the progress of the synthetic reaction

sampling is required at time intervals. With the standard activity test a single

measurement is sufficient. We chose this last approach for a quick assessment of

the activity. For the standard DERA activity assay the enzymes triose-3-phosphate

isomerase (TPI) and glycerol-3-phosphate dehydrogenase (GDH) are required. TPI

isomerises D-glyceraldehyde-3-phosphate liberated by DERA from 2-deoxyribose-5-

phosphate to dihydroxyacetone phosphate which in its turn is reduced by NADH

catalysed by GDH. These two enzymes were found to be stable and active in up to

30% ethanol or DMSO (determined with D-glyceraldehyde-3-phosphate).

Chapter 7

120

Figure 4.

Activity and stability (1

week incubation) of DERA

in 25% ethanol.

Figure 5.

Activity and stability (1 week

incubation) of DERA in 25%

DMSO.

The activity of DERA in water / ethanol mixtures immediately drops to become

zero at 25 % ethanol (Figure 4). The stability was excellent at concentrations of up to

20%. Its activity in DMSO was much better (Figure 5); between 0 and 20% cosolvent

the activity of DERA rose to about 150%. At 30% cosolvent three quarters of the

activity remained. The enzyme is very stabile in this cosolvent. These findings are in

favor of the use of DMSO as an additive in DERA catalyzed aldol reactions.

0

25

50

75

100

125

150

175

0 10 20 30% DMSO

% a

ctiv

ity

activitystability

0

20

40

60

80

100

120

140

0 5 10 15 20 25% EtOH

% a

ctiv

ity

activitystability


121

Conclusion

The ability of DERA to catalyze the synthesis of either β-hydroxyaldehydes or β-

ketones would broaden the scope of aldolases in organic synthesis. However, DERA

is a rather inefficient enzyme. The very low reaction rates for non-natural acceptor

substrates requires, in some cases, 250.000 units of the enzyme per liter often with

reaction times of weeks and only moderate yields. Improvement of the stability by

addition of cosolvents compensated to some extent the poor performance of this

aldolase but its synthetic utility remains very limited.

Experimental

General


temperature controller

Preparation of DERA

The E. coli strain DH5α (ATCC 86963), with the deo C system containing plasmid p

VH17, was grown in LB broth containing ampiciline at 37 °C on a 0.7 liter scale.

DERA was purified by centrifugation, cell disruption and ammonium sulfate

fractionation. This provided 400 units of DERA (should be 4000 units according to

literature).

Chapter 7

122

Activity assay

The reaction product, GAP was assayed using a coupled enzyme system. To 1.95

ml 50 mM Tris-buffer pH 7.6 containing 0.16 mM NADH in a 2 ml cuvette, were

added 20 µl 190 mM 2-deoxyribose-5-phosphate, 20 µl of a mixture containing 1.25

unit D-glycerol-3-phosphate dehydrogenase and 12.5 unit triose-1-phosphate

isomerase. Then 50 µl diluted aldolase was added and absorption monitored at 25

°C. 1 unit (U) aldolase converts 1 µmol 2-deoxyribose-5-phosphate per minute.

Stability test

Conditions: 1 unit enzyme per 1 ml assay containing the appropriate amount of

cosolvent (v/v), the remainder is 50 mM TRIS-buffer pH 7.6. After incubation a

sample was taken (typically 50 µl which diluted when activity is high) and the activity

is measured with the assay described above.

References

125 E. Racker, J. Biol. Chem. 1952, 196 (1), 347-365126 B.L. Horecker, S. Pontremoli, C. Ricci and T. Cheng, Proc. Natl. Acad. Sci. U.S.A.

1961, 47, 1942127 C.-H. Wong, E. Garcia-Junceda, L. Chen, O. Blanco, H.J.M. Gijsen and D.H.

Steensma, J. Am. Chem. Soc. 1995, 117, 3333-3339128 C.F. Barbas III, Y.-F. Wang and C.-H. Wong, J. Am. Chem. Soc. 1990, 112,

2013-2014


123

129 L. Chen, D.P. Dumas and C.-H. Wong, J. Am. Chem. Soc. 1992, 114, 741-748130 H.J.M. Gijsen and C.-H. Wong, J. Am. Chem. Soc. 1994, 116, 8422-8423131 H. Gijsen and C.-H. Wong, J. Am. Chem. Soc. 1995, 117, 7585-7591132 R. Rosen and C.H. Heathcock, Tetrahedron 1986, 42, 4909133 H.J.M. Gijsen and C.-H. Wong, J. Am. Chem. Soc. 1995, 117, 2947-2948134 E.A. Stura, S. Ghosh, E. Garcia-Junceda, L. Chen, C.-H. Wong and I.A. Wilson,

Proteins: structure, function and genentics 1995, 22, 67-72

Applications of aldolases in organic synthesis

124

Summary

Biocatalysts have shown promise in enantioselective synthesis since they compete

with chemical catalysis on e.g. introduction of chirality, selective modification or

resolution of racemates. Aldolases constitute a group of catalysts with many

potential applications in carbon-carbon bond forming reactions. In order to overcome

the shortcomings concerning their employment in future commercial processes,

generic problems like design of non-natural substrates, exploration of enzymes from

new species, enzyme stabilization and multi-enzyme systems need to be

investigated.

In Chapter 2 the acceptor substrate specificity of the commercially available

dihydroxyacetone phosphate (DHAP) dependent aldolases, D-fructose-1,6-

bisphosphate (FruA), L-fuculose-1-phosphate (FcuA) and L-rhamnulose-1-

phosphate (RhuA) aldolase, was investigated. Enzymatic detection of the donor

DHAP was found to be the fastest and most accurate way of determining the

progress of the aldol reactions and, hence, the kinetic properties of the aldolases.

Comparison of FruA, FcuA and RhuA showed that FcuA functioned less well

when non-natural substrates were used, whereas FruA and RhuA both performed

well and with similar profiles. More than twenty aldehydes were found to be

substrates for the aldolases from rabbit muscle, S. carnosus and S. aureus. The

bacterial aldolases were much more stable than the mammalian one.

Since the stereochemical outcome may differ when substrates in the aldol

reaction are modified, it is necessary to find unambiguous evidence for the

configuration and optical purity of the products. In Chapter 3 chiral

gaschromatography and a new retro aldol reaction assay were used to determine the


125

stereoselectivity of the aldolases towards a number of acceptor substrates. This

proved unambiguously, for the first time, that the steric preference of FruA and RhuA

for the newly created stereogenic center at C-(3) was far from absolute. The

enzymatic method is also useful for fast monitoring of aldolase catalysed syntheses.

In Chapter 4 a one-pot reaction of four enzymatic steps is described. Phytase-

catalyzed phosphorylation of glycerol with pyrophosphate was a key step which

afforded D,L-glycerol-3-phosphate in quantitative yield. Switching off the activity of

the phosphatase by a pH change during oxidation and aldol reaction resulted in

better results than integral in situ phosphorylation. Dephosphorylation was effected

by lowering the pH back to 4. Combined with the broad substrate specificity of DHAP

aldolases towards acceptor substrates it constitutes a simple procedure for the

synthesis of a wide variety of non-natural carbohydrates from glycerol. At the

moment this is the most economical preparation of DHAP.

Chapter 5: Hydroxyacetone thiosulfate was prepared as a substrate analogue

for DHAP, but it was not converted by DHAP utilizing enzymes. In situ formed

dihydroxyacetone arsenate was investigated in depth as analogue for DHAP. Use of

arsenate allows uncomplicated performance of aldol reactions, since synthesis of

phosphorylated donor is avoided. Analysis was afforded by enzymatic detection of

the acceptor substrates. It was found that cosolvents could be used with a triple

advantage: increased solubility of hydrophobic aldehydes, faster reaction and

stabilisation of the enzyme. The stereospecifity is somewhat higher with the natural

donor substrate.

Two approaches for the synthesis of optically pure polyhydroxy ketones (with

transketolase and FruA) were compared in Chapter 6. In order to assess the best

way to synthesize a target molecule both the accessibility of the different acceptor


126

aldehydes and their activity as a substrate were considered. Transketolase was

capable of competing with FruA as regards reaction rate and conversion of the

corresponding acceptor substrates.

In Chapter 7 the use of 2-deoxyribose-5-phosphate aldolase (DERA) is

examined. It is the only aldolase which accepts two aldehydes as substrates. The

ability of DERA to catalyze the synthesis of either β-hydroxyaldehydes or β-ketones

broadens the scope of aldolases in organic synthesis, however, DERA is a rather

inefficient enzyme. Improvement of the stability by addition of cosolvents

compensated to some extent the poor performance of this aldolase but its synthetic

utility remains very limited.

Summarizing, this work has resulted in a new synthesis of DHAP which makes this

important substrate cheap and readily available in large amounts. The

stereoselectivity of the aldol reaction was clarified by developing a “stereoisomer

assay”. The use of stable and active bacterial aldolases was promoted as an

alternative for aldolase from rabbit muscle. Dihydroxyacetone arsenate was shown

to be attractive for facile labscale experimentation. Furthermore, investigation of

other aldolases demonstrated these enzymes to be usefull catalysts.

Rob Schoevaart


127

Samenvatting

Biokatalysatoren zijn veelbelovend voor het gebruik in enantioselectieve syntheses,

omdat ze kunnen concurreren met chemische katalysatoren met betrekking tot de

introductie van chiraliteit, selectieve modificatie of resolutie van racematen.

Aldolasen vormen een groep katalysatoren met vele potentiële toepassingen in

koolstof-koolstof band vormende reacties. Om hun toepassingsmogelijkheden in

commerciële processen te vergroten, moeten algemene problemen zoals het

ontwerpen van niet-natuurlijke substraten, enzym stabiliteit, multi-enzym systemen

onderzocht worden. Tevens moeten enzymen uit nieuw gevonden organismen

uitgeprobeerd worden.

In Hoofdstuk 2 is de acceptor substraat specificiteit van de commercieel

verkrijgbare dihydroxyaceton fosfaat (DHAP) afhankelijke aldolasen, D-fructose-1,6-

bisfosfaat (FruA), L-fuculose-1-fosfaat (FcuA) and L-rhamnulose-1-fosfaat (RhuA)

aldolase beschreven. Enzymatische detectie van de donor DHAP bleek de snelste

en meest nauwkeurige manier om de voortgang van de aldol reacties en dus de

kinetisch eigenschappen van aldolasen te onderzoeken.

Een vergelijking van FruA, FcuA en RhuA toonde aan dat FcuA minder goed

functioneert als niet-natuurlijke substraten worden gebruikt, terwijl FruA en RhuA

beiden goed presteren met gelijkwaardige eigenschappen. Meer dan twintig

aldehyden werden als substraat voor de aldolasen van konijnespierweefsel,

Staphylococcus carnosus en Staphylococcus aureus gevonden. De bacterieële

aldolasen zijn veel stabieler dan de dierlijke.

Omdat de stereochemische uitkomst van de reactie anders kan zijn als niet

natuurlijke substraten in de aldol reactie gebruikt worden, is het nodig om


128

onomstotelijk bewijs te vinden voor de configuratie en de optische zuiverheid van de

producten. In Hoofdstuk 3 is chirale gaschromatografie en een nieuwe retro aldol

reactie assay gebruikt om de stereoselectiviteit van de aldolasen met een aantal

acceptor substraten te bepalen. Hieruit bleek voor het eerst dat de sterische

voorkeuren van FruA en RhuA voor het nieuwe chirale centrum op C-(3) niet

absoluut is. De enzymatische methode is ook handig voor een snelle bepaling van

de productvorming in aldolase gekatalyseerde reacties.

In Hoofdstuk 4 is een éénpotsreactie beschreven die bestaat uit vier

verschillende enzymatische stappen. Fytase gekatalyseerde fosforylering van

glycerol met pyrofosfaat was de sleutel tot de kwantitatieve bereiding van D,L-

glycerol-3-fosfaat. Het uitschakelen van de aktiviteit van de fosfatase door een pH

verandering (van pH 4 naar pH 7) tijdens de oxidatie en de aldol reactie leverde

betere resultaten op dan een integrale in situ fosforylering. Door de pH weer te

verlagen naar 4 kwam de defosforylering op gang. In combinatie met de brede

substraat specificiteit van DHAP aldolasen voor acceptor substraten vormt de

eenpotsreaktie een eenvoudige procedure voor de synthese van een keur aan

onnatuurlijke koolhydraten uit glycerol. Op dit moment is het ook de meest

economische bereiding van DHAP.

Hoofdstuk 5: Hyroxyaceton thiosulfaat was gesynthetiseerd als substraat

analoog voor DHAP, maar het werd niet omgezet door DHAP-afhankelijke enzymen.

In situ gevormde dihydroxyaceton arsenaat is uitgebreid onderzocht als analoog

voor DHAP. Het gebruik van arsenaat staat het ongecompliceerd uitvoeren van aldol

reacties toe, omdat de synthese van gefosforyleerde donor vermeden wordt.

Analyse van de reactie werd deze keer uitgevoerd door middel van enzymatische

detectie van de acceptor substraten. Cosolvents konden met een drievoudig


129

voordeel gebruikt worden: betere oplosbaarheid van hydrofobe aldehyden, snellere

reactie en stabilisatie van het enzyme. De stereospecificiteit is voor het natuurlijke

donorsubstraat iets hoger.

Twee benaderingen voor de synthese van optisch zuivere polyhydroxy ketonen

(met transketolase en FruA) werden vergeleken in Hoofdstuk 6. Om de beste

synthese route te vinden voor het doel molecuul is het nodig de toegankelijkheid van

de verschillende acceptor aldehydes en hun activiteit als substraat te overwegen.

Transketolase was in staat te concurreren met FruA m.b.t. de reactiesnelheid en

omzetting van de overeenkomstige acceptor substraten.

In Hoofdstuk 7 is 2-deoxyribose-5-phosphate aldolase (DERA) onderzocht. Het

is de enige aldolase die twee aldehyden als substraat accepteert. De mogelijkheid

van DERA om de synthese van β-hydroxyaldehyden or β-ketonen te katalyseren

verbreedt de scope van aldolasen in organische syntheses. Helaas is DERA een

zeer inefficient enzym. Verbetering van de stabiliteit door de toevoeging van

cosolvents compenseerde de slechte prestaties een beetje, maar de synthetische

toepasbaarheid blijft beperkt.

Samengevat heeft dit onderzoek tot een nieuwe synthese van DHAP geleid

waardoor dit belangrijke substraat goedkoop en gemakkelijk in grote hoeveelheden

gemaakt kan worden. De stereoselectiviteit van de aldol reaktie is opgehelderd door

hiervoor een speciale enzymatische assay te ontwikkelen. Voordelen van het

gebruik van stabiele en actieve bacteriële aldolasen boven het gebruik van de

dierlijke uit konijnespierweefsel zijn benadrukt. Dihydroxyaceton arsenaat bleek

aantrekkelijk te zijn voor eenvoudige experimenten op labschaal. Het onderzoek aan

andere aldolasen heeft de bruikbaarheid van deze enzymen aangetoond.

Rob Schoevaart

130

Dankwoord

Dit werk is mede tot stand gekomen dankzij de hulp van vele mensen, waarvan ikeen aantal persoonlijk wil bedanken.

Als eerste natuurlijk Roger Sheldon, die mij de mogelijkheid heeft geboden hetpromotieonderzoek te doen. Ik heb de afgelopen vier jaar met plezier gewerktonder jouw begeleiding. Wat ik zeker geleerd heb is kritisch naar m’n eigen werkte kijken alsof ik nooit gezien had en het ter beoordeling voor me lag. Zoals jevaak tijdens werkbesprekingen zei: “The proof of the pudding is in the eating”.Op zoek naar nieuwe ideeën hoopte ik altijd “the best thing since sliced bread”te vinden.

Fred van Rantwijk, verantwoordelijk voor de dagelijkse begeleiding, heeft eengrote invloed gehad op eigenlijk alle aspecten van het onderzoek. Niet alleenstond je altijd klaar voor vragen (waarbij je altijd instant wist waarover hetging, wat een geheugen!), ook kwam je altijd weer met nieuwe ideeën op deproppen. Verder denk ik graag terug aan alle buitenlandse avonturen.

De heren van HSL, ook ik moet toegeven dat pret omgekeerd evenredig is metde hoeveelheid daglicht. De sporadische behoefte aan zonlicht heeft zich bijjullie vooral vertaald in bijzondere LAT relaties. Luuk “The Force”, schei-kundig,ook in de scheikunde, Delft zou zonder jou niet hetzelfde geweest zijn. Ikherinner me vele kilometers in de Starlet, de verhuizing in 37 minuten, deontbijtkoffie in de “casa Parallelia“ en Gezellig Samen Mobiel. “Hacking”, watentropie in de thermodynamica is, ben jij op het lab en heel ver daarbuiten. Denachtelijke biertjes op het strand van La Grande Motte, alle bezwaarschriften

131

die de centen weer in de juiste portemonnee deden belanden, het afblaffen vanLuuk’s ex-huisbaas en je experimenteerlust kleurden het leven in Delft.

Michel Verhoef, wat kan ik zeggen? Het liefst natuurlijk: dinsdag, klokslagnegen uur, de Wijnhaven. Hadden we maar meteen aandelen in die tent gekocht.Als collega was je toch vooral collega-brouwer.

Rute, ondanks dat HSL LSL is geworden, is er niets veranderd. Ik hoop datik nog eens net zo snel Portugees kan leren als jij Nederlands geleerd hebt.

Esther Geenen heeft als HLO-stagiaire een aantal enzym substratengesynthetiseerd. Dit was vaak vrij lastig, maar je inzet was groot. Goede blondeherinneringen heb ik nog steeds aan het concert van K’s Choice.

Paloma, Gerd-Jan (“louder”), Göran (we moeten weer eens brouwen), Mike,Gerard, Michiel Van Vliet, Margreth, Arné, Martin “Sjaak” (wil je me straffen?),Annemieke, Emrin, Fred van de Velde en nog vele anderen wil ik bedanken vooralle goede contacten die ik altijd met ze heb.

Verder wil ik bedanken Adrie Knol voor de massaspectrometrie, Anton vanEstrik, Anton Sinnema en Joop Peters voor de altijd snelle en kundige acquisitievan NMR spectra en de hulp bij de vragen die dit opriep; Bert van der Hulst enChris van Drongelen voor het bestellen van chemicaliën en anderelaboratoriumbenodigdheden en de gezellige koffiepauzes tijdens de practica;Ernst Wurz voor z’n twee rechterhanden, Leen Maat, Herman van Bekkum; enJan Baas voor alle zorg over de UV (vooral toen we nog OS-2 WARP hadden!).

Mieke van der Kooij bedankt voor de administratieve ondersteuning en veleandere zaken.

Many thanks to Dr. Tischer and Peter Rasor from Roche Diagnostics,Penzberg, Germany for the generous gifts of enzymes. I think it’s obvious thisthesis would not have been the same without good old S. carnosus aldolase. Zumwohl!

Via het IOP ben ik in aanraking gekomen met vele mensen. De leden van de

132

begeleidingscommissie ben ik zeer dankbaar voor het bijwonen van dehalfjaarlijkse vergaderingen, tijdens welke het onderzoek op de rails werdgehouden en geen station overgeslagen werd. De commissieleden waren: TomKieboom (DSM-Gist), Wolfgang Fitz (ICI), dhr. Van Scharrenburg (SolvayDuphar), Gerard Kramer (Unilever Research Vlaardingen), dhr. Ter Burg(Tastemaker ). dhr. J. Frankena (Solvay Pharmaceuticals), dhr. Weenen (ICI),Prof. dr. H.E. Schoemaker (DSM Research), Erik De Vroom (DSM-Gist), dhr.Winkel (ICI) en dhr. Dijkstra (Solvay Pharmaceuticals).

Niels Ouwerkerk, bedankt voor jouw DERA kweek en wie had ooit gedachtdat we nog collega’s zouden worden!

Georg Sprenger and Ulrich Schörken, Institut für Biotechnologie 1,Forschungszentrum Jülich GmbH, Germany; thanks for the crude extract oftransketolase and the assistance with the purification and the initialcrystallizations. Furthermore, thanks for the pleasant company, also during thebeers.

Simon de Vries van Enzymologie ben ik dankbaar voor de hulp bij hetkweken en zuiveren van “DERA”.

Pa, jammer dat je mijn promotie niet meer hebt mogen meemaken, maar ik weetzeker dat je trots op me was.

Karel en Nel bedankt voor het bewaren van de vrede als ik thuis zat te werkenen niet voor de witte haren op m’n zwarte truien en de vlekken in devloerbedekking.

En natuurlijk als laatste, Martine “Tien” bedankt, voor meer dan teveel om op tenoemen en je Shift+F7 tip.

133

Publications

1. R. Schoevaart, F. van Rantwijk and R.A. Sheldon, “Class I fructose-1,6-

bisphosphate aldolases as catalysts for asymmetric aldol reactions”,

Tetrahedron Asymmetry 1999, 10 (4), 705-711

2. R. Schoevaart, F. van Rantwijk and R.A. Sheldon, “Carbohydrates from

glycerol: an enzymatic four-step one-pot synthesis”,

Chemical Communications, 1999, 24, 2465-2466

3. R. Schoevaart, F. van Rantwijk and R.A. Sheldon, “A four-step enzymatic

cascade for the one-pot synthesis of non-natural carbohydrates from glycerol”,

submitted.

4. R. Schoevaart, F. van Rantwijk and R.A. Sheldon, “The stereochemistry of non-

natural aldol reactions catalysed by DHAP aldolases”, submitted.

5. R. Schoevaart, F. van Rantwijk and R.A. Sheldon, “Facile enzymatic aldol

reactions with dihydroxyacetone arsenate”, submitted.

134

Curriculum vitae

Rob Schoevaart werd op 28 januari 1969 geboren te Coevorden. In 1988 behaalde

hij zijn VWO diploma aan de Rijksscholengemeenschap in Coevorden. In hetzelfde

jaar begon hij met de studie scheikunde aan de Katholieke Universiteit Nijmegen.

Daar werd Organische Chemie onder leiding van Prof. dr. B. Zwanenburg en dr.

G.J.F. Chittenden als hoofdvak gekozen en Microbiologie onder leiding van Prof. dr.

ir. G.D. Vogels als bijvak. In 1994 werd het doctoraal diploma behaald en in 1995

werd begonnen met het promotieonderzoek bij de vakgroep Organische Chemie en

Katalyse aan de Technische Universiteit Delft onder begeleiding van Prof. Dr. R.A.

Sheldon. Momenteel is de auteur werkzaam als post-doc bij het Leids Instituut voor

Chemisch onderzoek onder leiding van Prof. Dr. A. Kieboom.

135

Indexα-hydroxyaldehydes .............................97, 113α-hydroxybutanal........................................ 105β-hydroxyketones ....................................... 113α-hydroxypentanal...................................... 106β-hydroxypyruvate ........................................ 97

2

2-deoxyribose-5-phosphate..........................12

3

3R,4R............................................................373R,4S ........................................................8, 373S,4R ............................................................373S,4S ............................................................37

A

acceptor substrate specificity........................23alcohol dehydrogenase

assay of aldehydes................................... 88aldehydes

hydrophobic.............................................. 80aldol adduct

hydrolysis of ............................................. 59aldol reaction

general........................................................4aldol reactions

enzymatic ...................................................3aldolase...........................................................5

2-deoxyribose-5-phosphate aldolase12, 111Class I.........................................................7Class II........................................................7dihydroxyacetone phosphate dependent

aldolases................................................8from rabbit muscle...................................... 8from Staphylococcus aureus ....................22from Staphylococcus carnosus ..................8fructose-1,6-bisphosphate aldolase ......... 21glycine-dependent.................................... 11L-fuculose-1-phosphate aldolase...............8L-rhamnulose-1-phosphate aldolase.......... 8phosphoenolpyruvate-dependent............. 10pyruvate-dependent ................................. 10RAMA .........................................................8specific activities....................................... 25stability................................................27, 81tagatose-1,6-bisphosphate aldolase.......... 8threonine aldolases .................................. 12

alsolaseFruA activity assay ................................... 29

arsenateesters........................................................76

B

biocatalysis..................................................... 2

C

carbon dioxiderelease of ................................................. 13

cascade catalysis ......................................... 51catalase ........................................................ 54catalytic antibodies......................................... 5chemical phosphorylation............................. 65chiral GC....................................................... 36Claisen-Schmidt reaction ............................... 5Class I............................................................. 7contents .......................................................... 5cosolvents..................................................... 80curriculum vitae .......................................... 134

D

dankwoord .................................................. 130dephosphorylation........................................ 64DERA.................................................... 12, 111

acceptor substrates................................ 113activity in cosolvents .............................. 119stability in cosolvents ............................. 117

D-glyceraldehyde-3-phosphatedehydrogenase ........................................ 24

DHAassay of.................................................... 89

DHAAsacceptor specificity................................... 83conversion by FruA .................................. 84

DHAP.................................... 22, 25, 52, 54, 65analogues................................................. 73assay of .................................................... 29In situ generation of.................................. 51

dihydroxyacetone arsenate.......................... 76dihydroxyacetone monophosphate.............. 22dihydroxyacetone phosphate

synthesis of .............................................. 52DNA synthesis.............................................. 12donor .............................................................. 6D-ribose-5-phosphate................................... 96D-xylulose-5-phosphate ......................... 13, 96

E

enolates .......................................................... 5entropie....................................................... 130enzyme recovery .......................................... 30epimerisation ................................................ 37erythrose-4-phosphate ................................. 96Eupergit-C .................................... 11, 100, 116

136

F

Force...........................................................130

G

general introduction ........................................ 1glutaraldehyde ............................................ 101glyceraldehyde-3-phosphate..................8, 112glycerol phosphate oxidase..........................54glycolaldehyde.............................................. 23glycolysis ......................................................96

H

hydroxyacetone thiosulfate...........................74Hydroxypyruvate........................................... 13

I

Imine intermediate .......................................... 7in situ oxidation ............................................. 55inhibitors .......................................................74initial rates.....................................................25IOP Catalysis ..................................................2

K

Kinetiek .........................................................21

L

L-FcuA ......................................................8, 22L-lactaldehyde .............................................. 23L-RhuA......................................................8, 22lyases..............................................................5

M

ManNAc ........................................................10

N

N-acetyl-D-mannosamine.............................10N-acetylmannosamine.................................. 10N-acetylneuraminic acid ...............................10NADH....................................................24, 104Neu5Ac synthetase....................................... 10NMR

31P NMR..................................................63O

objectives and justification............................15

One pot preparation of carbohydrates ......... 56one-pot ......................................................... 55one-pot reaction

integral in situ ........................................... 55sequential................................................. 60

P

pentose phosphate pathway......................... 96phosphatase

from Aspergillus ficuum............................ 56from bovine intestinal mucosa ................. 56phospholipase C ...................................... 54phytase..................................................... 56wheat germ acid phosphatase................. 27

phosphoenolpyruvate................................... 10POCl3............................................................ 52propanal.................................................. 23, 24publications................................................. 133pyrophosphate.............................................. 55

R

reaction rates................................................ 26retro aldol reactions...................................... 39retro-aldol reaction.................................. 22, 24

Ssamenvatting .............................................. 127Schiff’s base ................................................... 7self aldol reaction........................................ 114sialic acid aldolase........................................ 10

sialic acids .................................................... 10stereoselectivity

effect of arsenate ..................................... 85enzymatic assay....................................... 39NMR analysis ........................................... 42

structural assignments ................................. 37summary..................................................... 124

T

thiamine pyrophosphate......................... 14, 96transaldolase ................................................ 96transferase.................................................... 13transketolase

from E. coli ............................................... 97from spinach leaves ................................. 96

transketolase .......................................... 13, 95cross-linked enzyme crystals ................. 100from yeast ................................................ 96versus FruA............................................ 100

Applications of aldolases in organic synthesis - Schoevaart Thesis 2000

Documents

Transcript of Applications of aldolases in organic synthesis - Schoevaart Thesis 2000