Carbohydrates from glycerol: An enzymatic four-step, one-pot synthesis

Article abstract of DOI:10.1039/a907874f

A novel one-pot procedure, involving a cascade of four enzymatic steps, for the synthesis of carbohydrates from glycerol and an aldehyde is reported.

Full text of DOI:10.1039/a907874f

Carbohydrates from glycerol: an enzymatic four-step, one-pot synthesis
Rob Schoevaart, Fred van Rantwijk and Roger A. Sheldon*
Laboratory of Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The
Netherlands. E-mail: r.a.sheldon@tnw.tudelft.nl
Received (in Cambridge, UK) 30th September 1999, Accepted 1st November 1999
A novel one-pot procedure, involving a cascade of four
enzymatic steps, for the synthesis of carbohydrates from
glycerol and an aldehyde is reported.
become zero at pH 7. The yield of glycerol-3-phosphate (at pH
4) increased with increasing glycerol concentration, presumably
because the competing hydrolysis of pyrophosphate and
glycerol-3-phosphate is suppressed at low water concentrations.
At 95% glycerol 75 mM l-glycerol-3-phosphate (correspond-
ing to the theoretical yield of 50%) was formed according to the
Dihydroxyacetone phosphate (DHAP)-dependent aldolases cat-
alyze the highly stereoselective synthesis of a wide variety of
natural and non-natural carbohydrates1 via the aldol reaction
of DHAP with an aldehyde acceptor. C–C bond formation can
produce four different stereoisomers and it is possible, by an
appropriate choice of aldolase, to selectively produce any one of
–3
31
assay, after 24 h reaction time. P NMR analysis confirmed that
under these conditions the conversion of pyrophosphate into dl-
glycerol-3-phosphate was 100%. Glycerol-2-phosphate was
absent in the reaction mixture, demonstrating that phytase is
completely regiospecific.
the four possible stereoisomers. For example, the fructose-
4
1
,6-bisphosphate aldolase (FruA) used in this study produces
The concentration of glycerol was adjusted to 55%, because
the activity of glycerol phosphate oxidase (GPO) is low in 95%
glycerol. Moreover, the pH was increased to pH 7.5, which
corresponds with the optimum of GPO and catalase (and that
of the aldolase as well) and renders the phytase inactive,
thus preventing undesirable hydrolysis. After quantitative
oxidation of l-glycerol-3-phosphate to DHAP, fructose-1,6-bi-
an aldol adduct with 3S,4R stereochemistry. Chemical methods
for preparing DHAP are circuitous and/or require expensive
reagents.5 Known enzymatic methods require the use of
6,7
expensive enzymes (kinases) and regeneration of ATP. We
reasoned that the use of a phosphatase8 as the phosphorylation
catalyst would enable the use of inexpensive inorganic
pyrophosphate as the phosphate source.
,9
sphosphate aldolase (FruA) from Staphylococcus carnosus and
butanal1
4,15
were added to start the aldol addition (78%
Our synthetic scheme embodies a cascade of four enzymatic
steps: kinetically controlled phosphorylation of glycerol by
inorganic pyrophosphate, glycerol phosphate oxidase (GPO)-
catalysed aerobic oxidation of l-glycerol-3-phosphate to DHAP
coupled with catalase mediated decomposition of hydrogen
peroxide,10 aldol reaction of DHAP with an aldehyde acceptor11
and, finally, enzymatic dephosphorylation of the aldol adduct
conversion after 4 h). Lowering the pH to 4 initiated the
dephosphorylation of the butanal–DHAP adduct, affording
5-deoxy-5-ethyl-d-xylulose14 in 57% yield from l-glycerol-
3-phosphate. The addition of extra phosphatase for removal of
the phosphate group was not necessary since phytase was still
present and active.
(
see Scheme 1). The key to its success depends on the judicious
The combination of four different enzymes and four
enzymatic transformations in one pot provides an attractive
procedure for performing aldol reactions with DHAP aldolases
starting from the cheap, readily available glycerol and pyr-
ophosphate. Combined with the broad substrate specificity of
DHAP aldolases towards acceptor substrates, a wide variety of
carbohydrates is readily accessible using this method.
Generous gifts of enzymes by Roche Diagnostics (Penzberg,
Germany) are gratefully acknowledged. This work was finan-
cially supported by the Innovation Oriented Program Catalysis
(IOP-catalysis).
use of pH control to switch the activities of the various enzymes
on and off during the cascade.†
The phosphatase of choice was phytase12 from Aspergillus
ficuum, which is a cheap and readily available industrial
enzyme. Its production of dl-glycerol-3-phosphate is pH
dependent with a very broad optimum. For quick analysis l-
glycerol-3-phosphate is detected by oxidation to DHAP and
subsequent coupled assay13 (equal amounts of the d-isomer are
assumed to be formed). At pH 2 phosphorylation [glycerol
>
10% (v/v)] was substantial and it decreased above pH 4 to
Scheme 1
Chem. Commun., 1999, 2465–2466
This journal is © The Royal Society of Chemistry 1999
2465

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6
7
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Notes and references
†
Optimal conditions (per ml): phosphorylation was carried out in 95%
glycerol with 150 mM pyrophosphate and 1 mg phytase at pH 4.0. After
shaking at 37 °C for 24 h the pH was adjusted to 7.5 and the glycerol
concentration was reduced to 55% by dilution. GPO (5 units) and catalase
8 A. Pradines, A. Klaébé, J. Périé, F. Paul and P. Monsan, Tetrahedron,
1988, 44, 6386.
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(50 units) were added and oxygen was applied for 3 h at room temperature.
Then the mixture was shaken for 4 h with FruA (1.25 units) and butanal (100
mM). Lowering the pH to 4, stirring overnight and extraction with EtOAc
afforded 5-deoxy-5-ethyl-d-xylulose (57% from l-glycerol-3-phosphate).
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11 Oxidation and aldol reaction can be conducted in situ, see ref. 10.
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1
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2
1
1
4
5 Butanal was used as acceptor because we had already fully charac-
terized the corresponding final product, 5-deoxy-5-ethyl-d-xylulose.
6
(ref. 14).
2
5
Communication 9/07874F
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Chem. Commun., 1999, 2465–2466