ENZYME-CATALYZED SYNTHESIS OF CARBOHYDRATES: SYNTHETIC POTENTIAL OF TRANSKETOLASE

Article abstract of DOI:10.1016/S0040-4039(00)93434-8

The synthetic potential of yeast or spinach transketolases was studied, using hydroxypyruvate as a donor substrate and 31 aldehydes as acceptors.All of them react, including aromatic, heteroaromatic and α,β unsaturated aldehydes.

Full text of DOI:10.1016/S0040-4039(00)93434-8

Talrahedron Letters, Vol.32, No.38, pp 5085-5088, 1991
Printed in Great Britain
0040-4039/91 $3.00 + .00
Pergamon Press plc
ENZYME-CATALYZED SYNTHESIS OF CARBOHYDRATES : SYNTHETIC POTENTIAL
OF TRANSKETOLASE
Colette Demuynek*, Jean Bolte, Laurence Hecquet and Val~rie Dalmas
Laboratoire de Chimie Organique Biologique associ~ au CNRS, Universit6 Blaise Pascal,
63177 Aubi~re, France.
Abstract - The synthetic potential ofyeast or spinach transketolases was studied, using
hydroxypyruvate as a donor substrate and 31 aldehydes as acceptors. All of them react,
including aromatic, heteroaromatic and ~,fl unsaturated aldehydes.
During the last few years, an increasing number of papers dealing with enzymatic syntheses of
monosaccharides by aldol reactions has demonstrated their usefulness1. Transketolase (TK) [EC 2.2.1.1], a
transferase involved in vivo in the oxidative pathway of pentose phosphates, catalyzes the reversible transfer of
a ketol group from a suitable donor (xylulose 5-phosphate in vivo) to an aldehyde (ribose 5-phosphate in vivo).
Thiamine pyrophosphate and magnesium ions are required as cofactors. Transketolase has been used to
stereospecitically extend the chain of several aldoses by two carbon units2-4.
The use of hydroxypyruvate as the source of a ketol group makes the reaction irreversible, leading to
complete aldehyde transformation, since carbon dioxide is the by-product3 (scheme 1).
Scheme 1
CH2OH
CH2OH
I
C = O
~ --O
I
HO-C-H + CO2
H
~
--~
+
\ C = O
The aim of this work was to extend the synthetic potential of this enzyme by the use of new acceptors.
Commercial Transketolase from yeast, as well as TK from spinach leaves have been used for this study. The
present report deals with the TK-catalyzed reaction of 31 aldehydes with hydroxypyruvate : we show that even
the poorest substrates lead to the corresponding ketoses.
Substrate specificity of yeast transketolase
Table I lists the aldehydes that have been assayed as TK substrates, together with their initial relative
velocities (Vrel) expressed as the percent of the initial velocity of glycolaldehyde, the best substrate found. The
specific activity of the enzyme for glycolaldehyde is 8.6 gmole/min/mg of protein. Initial velocities have been
measured at different aldehyde concentrations. The concentrations reported table I are those giving maximal
velocities : they are rather high, indicating a poor affinity of the enzyme for these substrates. Some aldehydes
5085

5086
react with the amino groups of the enzyme, leading to activity loss : the half-life of the enzyme has been
determined in some cases. At the concentrations indicated in the table I, the tl/2 is > 24 hours for glyceraldehyde
and glycolaldehyde 1, 3 hours for acetaldehyde 2 and 3-methoxybutyraldehyde 13 and about 30 rain for the
heterocyclic aldehydes 27, 28, 29.
Table I. Relative reactivities of aldehydes (RCHO) with hydroxypyruvate in yeast transketolase-catalyzed a
carbon-carbon bonds formation.
Conc.
(raM)
Conc. Vrel
(raM)
R b
VreI
R b
HOCH2-
1
2
100
150
100
"
1
CH2OH-(CHOH)2-
(D-erythrose)
1 6
17
60
"
0.84
CH3-
0.25
0.32
0.11
0.44
0.47
0.20
0.19
0.11
0.29
0.43
0.45
0.3
CH3OCH2-
(CH30)2-CH -8
CR,S) CH3-CHOH-
3
CH2OH-(CHOH)2-
(L-threose)
0.39
4
5
60
"
D-ribose
18
19
150
100
"
0.3
(R,S) CH2F-CHOH-
(R,S) CH30-CH2-CHOH-
CH3-CO-
6
2-deoxy-D-ribose
(CH3)2C=CH-
OHC-(CH2)3-CHOH-
2,5-Dimethoxy-3
tetrahydrofuranyl
Phenyl
0.16
0.11
0.27
0.13
7
80
100
"
2 0
2 1
2 2
8
"
CH3-CH2-CH2-
9
"
(R,S) CH3-CHOH-CH2-
10
"
(R,S) CH2OH-CHOH-CH2-6 1 1
(R) CH2OH-CHOH-CH2-6 12
(R,S) CH3-CH(OCH3)-CH2- 13
"
2 3
2 4
"
"
< 0.1
"
2-Hydroxy-phenyl
0.28
0.32
0.11
0.21
0.11
0.32
0.13
0.13
"
2,3-Dihydroxy-phenyl 2 5
"
(R,S) CH2-CH-CH2-6
14
15
200
0.11
Benzoyl
2 6
2 7
2 8
2 9
30
150
100
"
O
O
2-Pyrrolyl
2-Furanyl
2-Thiophenyl
2-Pyridyl
3-Pyridyl
k
/
__,'C\
CH3 CH3
"
(R,S) CH3-CH(OBzl)CH2-7
100
0.14
150
"
3 1
a Experimental conditions : thiamine pyrophosphate (0.2 mM), MgC12 (0.9 raM), hydroxypyruvate (7.5 raM), transketolase (1
U/ml) in glycylglycine buffer 0.1 M, pH 7.5. The disappearance of hydroxypyruvate was monitored by enzymatic assay10.
b The references describe the preparation ofaldehydes that were not commercially available.
Aliphatic aldehydes are good substrates and the presence of an hydroxyl group or an oxygen atom at Ca
or/and C~ has a positive effect : for example, glycolaldehyde 1 is a better substrate than acetaldehyde 2 and
compounds 3 and 10 are more reactive than butyraldehyde 9. We have already shown that yeast TK displays
some enantioselectivity for racemic 0~-hydroxyaldehydes3. Steric hindrance near the aldehyde group reduces the
reactivity, as shown by the activities of compounds 14 and 15 compared to those of compounds 11 and |0,
respectively.

,
5087
~,13 Unsaturated aldehydes are accepted as substrates : 3-methyl crotonaldehyde 20, benzaldehyde 23,
hydroxy-benzaldehydes 24, 25 and heterocyclic aldehydes 27-31. Benzaldehyde itself is a poor substrate but
the presence of ortho-hydroxyl groups leads to higher activities (24, 25). Heterocyclic aldehydes are better
substrate than benzaldehyde.
Synthesis of 4-deoxy-L-erythrulose
For the preparative scale reactions, we choose spinach leaves as an inexpensive source of TK. In all
cases tested, enzymatic activities of the yeast and spinach enzymes were very similar. We choose acetaldehyde
to demonstrate the high synthetic potential of TK even with a poor substrate, which furthermore is able to
denaturate the enzyme. The synthesis was carried out on a 5 mmole scale. The loss in enzyme activity was about
75 % after 8 hours.
TK was extracted from spinach leaves and partially purified by ammonium sulfate precipitation4. The
procedure is particularly easy and inexpensive • 300 U of enzyme were obtained from 300 g of spinach leaves.
The experimental procedure is as follows : a 30 ml solution of glycyl-glycine buffer 0,05 M, pH 7.5,
containing hydroxypyruvate (150 mM), thiamine pyrophosphate (2 raM), MgC12 (2mM) and spinach trans-
ketolase4,5 (340 U), was deoxygenated with a stream of argon and then, acetaldehyde (150 raM) was introduced
with a syringue. After complete disappearence of hydroxypyruvate (24 h), methanol (50 ml) was added and the
precipitate discarded3,4. The sugar was isolated and purified by chromatography on cation exchanger H+. The
fractions that contains the sugar were combined, ajusted to pH 6, and concentrated. Flash chromatography on
silicagel (elution with chloroform-methanol, 4:1) of the residue gave 4-deoxy-L-erythrulose (168 mg, 36 %) as
a syrup. [~]25D + 4 (0.59 methanol).
1H NMR (300 MHz) CD3OD 5 : 1.3 (d, J = 4.8 Hz, 3H) ; 4.24 (q, J = 4.8 nz, 1H) ; 4.41 (s, 2H). 13C NMR
(300 MHz) CD3OD ~ : 21.0, 67.1, 73.5, 215.3.
4-Deoxy-L-erythrulose diacetate was obtained as an oil by action of acetic anhydride4 and purified by
chromatography on silicagel (elution with pentane-ether 1:2) [cx]25D = - 33 (1, CHC13).
1H NMR (300 MHz), CDCI3 8 : 1.47 (d, J -- 7.2 Hz, 3H, C~!3-CH(OAc)') ; 2.15 (s, 3H, -CO-CH3) ; 2.18 (s,
3H, CO-C_H_3) ; 4.83 (q, J = 16 Hz, 1H, CO-CH2-OAc) ; 5.22 (q, J = 7.2 Hz, 1H, CO-CH-OAc-CH3)~
The optical purity of the diacetate determined by NMR in presence of Eur(tfc)3 at 4.83 ppm signal was
80%.
The relatively low stereospecificity of transketolase concerns only the two carbon substrates and was not
observed in the synthesis of D-xylulose and 5-deoxy-D-xylulose (manuscript in preparation).
Conclusion
The Transketolization reaction is useful for the efficient and easy synthesis of natural and unusual
ketoses and can be compared with rabbit muscle aldolase catalysis9. It accepts a fairly broad range of aldehydes
as acceptor substrate, particularly aldehydes without hydroxyl group at Ca which lead to 4-deoxy ketoses, these
compounds cannot be obtained with aldolase. Furthermore c~,[3-unsaturated and aromatic aldehydes arc TK
substrates, while they do not (or scarcely) react with aldolasesl,9a. TK is obtained by a simple and inexpensive
process from spinach leaves or yeast,5 ; its stability is good. The donor substrate, hydroxypyruvate, is
inexpensive and leads to irreversible transformations. Non-phosphorylated ketoses are obtained in a one-step
reaction.

5088
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(Received in France 1 July 1991)