Enzymatic synthesis of aldonic acids

Article abstract of DOI:10.1016/j.carres.2006.05.023

Several aldonic acids (d-mannonic, d-galactonic, d-xylonic, 2-deoxy-d-arabinohexonic (2-deoxy-d-gluconic)) were prepared on a scale of several grams by a simple oxidation catalyzed by glucose oxidase in pure water.

Full text of DOI:10.1016/j.carres.2006.05.023

Carbohydrate Research 341 (2006) 2290–2292
Note
Enzymatic synthesis of aldonic acids
F. Pezzotti and M. Therisod*
Laboratoire de Chimie Bioorganique et Bioinorganique, UMR 8182, ICMMO, Universite´ Paris-Sud, 91405 Orsay, France
Received 6 April 2006; received in revised form 19 May 2006; accepted 25 May 2006
Available online 27 June 2006
Abstract—Several aldonic acids (D-mannonic, D-galactonic, D-xylonic, 2-deoxy-D-arabinohexonic (2-deoxy-D-gluconic)) were pre-
pared on a scale of several grams by a simple oxidation catalyzed by glucose oxidase in pure water.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Aldonic acids; Oxidation; Glucose oxidase; Bioconversion; Green chemistry
Polyhydroxy acids and mostly sugar acids have been
proved to be useful in a wide range of practical applica-
tions, especially as sequestering agents for metal ions
(Zn, Fe, Cu, Ca in the presence of borate).1,2 These
metal–aldonate complexes have found industrial, medical
and agricultural applications.3,4 Their structures and
physicochemical properties have been extensively stud-
ied.1 Xylonic acid has recently been shown to be a valu-
able precursor for an industrial synthesis of 1,2,4-
butanetriol through microbial fermentation.5
The most common methods of preparation of aldonic
acids use stoichiometric amounts of bromine, copper or
silver hydroxides in totally non-ecological processes,
and their chemical synthesis have been reviewed
recently.6,7 Alternatively, some aldonic acids like gluconic
acid and their metabolites have been prepared by
fermentation.8 However, the products must then be
purified from complex aqueous media. An efficient
synthesis of D-xylonic acid from D-xylose (70%) by oxi-
dation using fermentor-controlled cultures of a particu-
lar strain of Pseudomonas fragi has been reported.5
Glucose oxidase (GO; EC 1.1.3.4) has the reputation
of being extremely specific for D-glucose to the point
of being considered as its single substrate. This charac-
teristic is commonly used in analytical biochemistry
for the selective estimation of glucose in complex biolog-
ical fluids (e.g., blood).9 Early studies10,11 indicated that
other sugars were oxidized only at a very slow or negli-
gible rate as compared to glucose, ranging from 0.5% for
galactose, 1% for mannose, 2% for 2-amino-2-deoxy-D-
glucose to 20% for 2-deoxy-D-arabino-hexose (2-deoxy-
glucose). GO therefore appeared to be an unusable
enzyme for the preparative bioconversion of sugars,
except glucose itself. Chemists using enzymes in organic
synthesis more often have to deal with non-natural sub-
strates, on which enzymes have low activity. They over-
come this difficulty by extending the reaction time
(several days) if the enzyme is stable enough and/or
commercially available in notable quantities.
GO from Aspergillus niger is a robust enzyme now
produced on an industrial scale and marketed in large
quantities at reasonable prices from several companies
like Novozymes Biotech, Inc., and Amano Enzyme,
Inc. (Besides its use in biochemical analysis, it is an
important additive in bakery products.) This opens
new prospects for the use of this biocatalyst. As an
example, we report herein our results relative to the pre-
parative oxidation by GO of ‘inert’ aldoses, as a contin-
uation of our work previously reported on the
enzymatic preparation of 2-amino-2-deoxy-D-gluconic
acid from 2-amino-2-deoxy-D-glucose (glucosamine).12
Oxidation was performed by simple dissolution of the
sugar in water, followed by addition of glucose oxidase
and catalase under vigorous agitation [catalase (E.C.
1.11.1.6) was added in order to decompose the hydrogen
peroxide formed as a byproduct, which is known to have
a deleterious effect on GO; Scheme 1]. The aldonic acid
*
Corresponding author. Tel.: +33 16915 6311; fax: +33 16915 7281;
e-mail: therisod@icmo.u-psud.fr
0008-6215/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.05.023

F. Pezzotti, M. Therisod / Carbohydrate Research 341 (2006) 2290–2292
2291
Scheme 1. Synthesis of aldonic acids (e.g., xylonic acid) catalyzed by a two-enzyme system of glucose oxidase and catalase.
formed during oxidation was continuously neutralized
with NaOH, added by means of a pH-stat. As a result,
the pH of the medium was maintained constant during
the reaction, and the volume of added NaOH was di-
rectly proportional to the amount of sugar being oxi-
dized (Fig. 1). The reaction mixture was then filtered
through a column of Dowex 1 (AcOꢀ form), from which
the aldonic acid was eluted with 1 M aqueous HCl.
NMR (D2O, Na2CO3) indicated that this simple purifi-
cation gave a pure product, free from the enzymes and
from any unreacted substrate. We report a complete
assignment of the 1H and 13C NMR signals of the
aldonic acids (salts), which has not been published
before.
Not surprisingly, the reaction rate was dependent on
temperature (Fig. 2). When conducting the reaction
under an atmosphere of pure oxygen, the reaction rate
was also enhanced, but incomplete oxidation occurred,
probably as a result of an oxidative denaturation of
the enzyme.
Figure 2. Galactose oxidation dependence on temperature and oxygen
atmosphere.
Oxidations of up to 100% could be obtained within
40 h (e.g., with D-mannose). As long as few grams of
products are needed, it is not worth recycling the
enzymes, considering their low costs. For large-scale
syntheses, however, immobilization of GO and catalase
can be considered, for which number of established pro-
cedures are available.13
In conclusion, we have devised a very simple proce-
dure for the preparative synthesis of various aldonic
acids from the corresponding aldoses. This ‘green chem-
istry’ process takes advantage of the availability of
cheap, robust industrial enzymes.
1. Experimental
1.1. Enzymes
Glucose oxidase from A. niger (GluzymeÒ, from Novo-
zymes) had a specific activity of 2 U/mg. Catalase (Cata-
zymeÒ, from Novozymes) had a specific activity of
25 kU/mL.
1.2. Oxidation procedure
Figure 1. Oxidation of various aldoses into the corresponding aldonic
acids catalyzed by glucose oxidase at room temperature (estimated
from the volume of NaOH delivered by a pH-stat).
The aldose (11.1 mmol) was dissolved in water to a final
concentration of 0.5 M (0.6 mmol and 0.12 M for

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F. Pezzotti, M. Therisod / Carbohydrate Research 341 (2006) 2290–2292
2-deoxy-D-glucose) and subjected to oxidation by addition
of 200 mg of glucose oxidase (400 U) (10 mg for 2-
deoxy-D-glucose) and a large excess of catalase (1 mL,
25 kU). The mixture was vigorously stirred under air,
and the pH was kept constant at pH 7.5 by means of
a pH-stat adding continuously 1 M NaOH (0.1 M for
2-deoxy-D-glucose). Conversion was directly calculated
considering the volume of added NaOH since 1 mol of
NaOH neutralizes 1 mol of aldonic acid formed. The
reaction mixture was then filtered through a Dowex 1
(AcOꢀ) column to eliminate the enzymes and any resid-
ual substrate. Aldonic acids were then recovered by elu-
tion with 1 M aq HCl. After evaporation in vacuo, the
identity and purity of each product were confirmed by
1H and 13C NMR spectroscopy (D2O–Na2CO3). The
1H and 13C NMR signals were assigned using two-
dimensional NMR COSY and HMQC experiments.
3.62 (dd, 1H, J 11.7, J 3.9, H50), 3.71 (m, 1H, H4),
3.76 (dd, 1H, J 5.9, J 2.5, H3), 3.99 (d, 1H, J 2.55,
H2). 13C NMR (90 MHz, D2O): d 62.72 C5, 72.54 C3,
73.09 C2, 73.19 C4, 179.19 C1. Lit.: 13C NMR (D2O):
d 63.9, 73.9, 74.1, 74.3, 180.1.19 [a]D 7.05 (c 10, H2O).
Lit. [a]D 7.4.20
Acknowledgements
We are grateful to Novozymes Biotech., Inc., for the
kind supply of glucose oxidase. This work was sup-
ported by a studentship to F.P. from Mexican Govern-
ment CONACyT (National Council for Science and
Technology).
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