Reaction pathways of glucose oxidation by ozone under acidic conditions

Article abstract of DOI:10.1016/j.carres.2009.05.012

The ozonation of d-glucose-1-¹³C, 2-¹³C, and 6-¹³C was carried out at pH 2.5 in a semi-batch reactor at room temperature. The products present in the liquid phase were analyzed by GC-MS, HPAEC-PAD, and ¹³C NMR s

Full text of DOI:10.1016/j.carres.2009.05.012

Carbohydrate Research 344 (2009) 1303–1310
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Reaction pathways of glucose oxidation by ozone under acidic conditions q
Olivier Marcq a, , Jean-Michel Barbe a, Alain Trichet b, Roger Guilard a,
*
a ICMUB-LIMRES (UMR 5260), Université de Bourgogne, Faculté des Sciences Mirande, F- 21100 Dijon, France
b Air Liquide—Centre de Recherche Claude-Delorme, Les Loges en Josas, BP 126, F-78350 Jouy en Josas, France
a r t i c l e i n f o
a b s t r a c t
The ozonation of D
-glucose-1-13C, 2-13C, and 6-13C was carried out at pH 2.5 in a semi-batch reactor at
Article history:
Received 17 March 2008
Received in revised form 22 April 2009
Accepted 13 May 2009
room temperature. The products present in the liquid phase were analyzed by GC–MS, HPAEC-PAD,
and 13C NMR spectroscopy. Common oxidation products of glucose have also been submitted to identical
ozonation conditions. For the first time, a pentaric acid was identified and its formation quantitatively
correlated to the loss of C-6 of glucose in the form of carbon dioxide. Potential mechanisms for the for-
mation of this pentaric acid are discussed. The well-accepted pathway involving the anomeric position in
glucose, gluconic acid, arabinose, and carbon dioxide is reinvestigated. The origin of small molecules such
as tartaric, erythronic, and oxalic acids is clarified. Finally, new reaction pathways and tentative mecha-
nisms consistent with the formation of ketoaldonic acids and smaller acids are proposed.
Ó 2009 Elsevier Ltd. All rights reserved.
Available online 18 May 2009
Keywords:
Ozone
D-Glucose
Uronic acids
Keto acids
Oxidation mechanism
1. Introduction
Table 1). Upon reviewing the literature, it became obvious that
there was not a clear consensus in the literature as far as the rela-
While desired in many cases such as for water treatment,1 car-
bohydrate oxidation with ozone is also often the undesired result
of processes aimed at degrading or modifying other companion
materials (e.g., delignification2–5 of paper pulp, feedstock, or cotton
stalk). Understanding the reaction mechanisms or identifying the
reaction pathways is a key step toward a better control of the pro-
cesses involved. Identification of stable intermediates and final
products is therefore essential. Classically, techniques such as
HPAEC-PAD, GC–MS, and more recently NMR spectroscopy have
been used to identify products in the liquid phase. Site-specific iso-
topic labeling and molecular modeling are very useful tools also.
Several studies on carbohydrate oxidation resorted to isotope-la-
beled substrates.6 More recently, this approach was successfully
tive reactivity of carbon atoms in hexoses or polysaccharides is
concerned, we therefore set out to reinvestigate the oxidation of
D-Glucose at various pH.
We previously published an analysis of the gas phase resulting
from the ozonation of 1-13C, 2-13C, and 6-13C labeled
D-glucose
molecules at pH 2.5 and 10.6,11,12 To the best of our knowledge
no other study of the ozonation of labeled carbohydrates has been
reported. The equal contribution of carbon atoms C-1 and C-6 of
the glucose molecule in the production of carbon dioxide at pH
2.5 raised anew the question of the reactivity of carbon atom C-
6.13–19
Oxidation at C-1 is considered as the main pathway to lower
products from glucose.13,14,16,20–25 Because of the formation of both
arabinose and carbon dioxide, observed in several cases, this aspect
of the ozonation reaction has been compared25,26 to the Ruff deg-
radation27 reaction.à
Here, quantitative and isotopic analyses of the liquid phase and
correlation with the analysis of the gas phase, allowed us to iden-
tify, for the first time, a 5-carbon atom molecule corresponding to
the loss of C-6 from glucose.12,28 We also confirmed the high reac-
tivity of the anomeric position. The well-accepted pathway involv-
ing gluconic acid, arabinose and carbon dioxide is reinvestigated.
The origin of small molecules such as tartaric, erythronic, and
oxalic acids is also clarified. Finally, new reaction pathways and
applied to the study of the oxidative degradation of
D-glucose in
hot alkaline solution7 and to the Maillard reaction.8–10
Our research focused on the action of ozone on cellulose and re-
lated compounds as part of an industrial collaboration on paper
pulp bleaching. Numerous reports also studied the ozonation of
mono-, oligo-, and polysaccharides often as part of research on
ozone as a bleaching agent or oxidation agent for the removal of
organic substances in water (for a selection of publications, see
q
For a report on the analysis of the gas phase in ozonized 13C-labeled
D-glucose
compounds, see Ref. 6.
* Corresponding author.
E-mail address: rguilard@u-bourgogne.fr (R. Guilard).
à
In the presence of hydrogen peroxide and ferric ions, aldonic acids are oxidized to
Present address: Wyeth Vaccines, 401 N Middletown Rd, Pearl River NY, USA.
give CO2 and an aldose with one less carbon atom.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.05.012

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O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
Table 1
Identification and quantitative and isotopic analyses of the oxidation products in the liquid phase (at 1.5 equiv of ozone consumed per mole of glucose)
Identified product
Number of carbon
atoms
Mmol at 1.5 equiv
of ozone consumed
Contains
Previously reported in Ref.
Gluconic acid
6
6
6
6
6
0.27
0.03
0.05
0.05
0.06
C-1, C-2, C-6
C-1, C-2, C-6
C-1, C-2, C-6
C-1, C-2, C-6
C-1, C-2, C-6
16,20–26,29,30,33,34,37,42–44
11,12,16,20,21,23
12,16,23,30
13
2-Ketogluconic acid
5-Ketogluconic acid
Hexodialdose
Glucuronic acid
From glucose, see: 13,16,18,20,23,30,43 and
from methyl glucoside see: 13,15,23,26,31,37,45
Glucaric acid
Arabinose
Arabinonic acid
Xylaric acid
Tartaric acid -1
Tartaric acid -2
Erythronic acid
Glycaric acd
2-Ketoglycaric acid
Glyceric acid
Glycolic acid
Oxalic acid
0.01
C-1, C-2, C-6
C-2, C-6
C-2, C-6
C-1, C-2
C-1, C-2
C-2
C-6
C-1, C-2
C-6
16,23,24,29
13,20,22,29,33,34
—
5
4
3
2
0.017
0.016
0.061
0.016
ꢀ0
New
13,16,21,24,29,30,46
0.011
Traces
Traces
0.009
0.129
0.031
30. For erythrose, see Ref. 16
20
30
—
C-6
C-6
C-1, C-2
16,20,23
tentative mechanisms consistent with the formation of ketoaldonic
acids and smaller acids are proposed. The variety of possible
pathways led us to investigate the ozonation of commercially
available oxidation products to support our discussions on mecha-
nisms.§ In this paper, ‘C-1’, ‘C-2’ and ‘C-6’ will refer to the carbon
atoms in the parent glucose molecule.
However this product is not observed during the ozonation of
glucose described here. Rather, a pentaric acid was identified by
comparison of its mass spectrum (Fig. 1) with that of persilylated
arabinaric acid reported by Peterson.36 Erythrose, the precursor of
erythronic acid, has been detected by HPAEC under similar exper-
imental conditions.16,23 Two peaks were identified by GC–MS as
tartaric acid. Considering concentration (see Table 1), we focused
on the main peak in the rest of this article. This main peak con-
tains mainly both C-1 and C-2 atoms. Three acids with 3 carbon
atoms were identified: glyceric, glycaric (hydroxypropanedioic),
and 2-ketoglycaric acids. The latter was identified as its trim-
ethylsilylketal. The products containing 3 carbon atoms are obvi-
ously formed by different pathways as indicated by the presence
of C-1 in the glycaric acid and by the presence of C-6 in 2-keto-
glycaric and glyceric acid. An increasing concentration of glycolic
and oxalic acids (compounds containing 2 carbon atoms) was
observed as already reported by our group16,23 and Holen20 dur-
ing ozonation of cellulose model compounds. Oxalic acid appears
to contain C-1 and C-2 atoms but not C-6 according to the anal-
ysis by 13C NMR.– No formaldehyde has been identified and the
production of formic acid remains below carbon dioxide
production.6,11
2. Results
For convenience, we summarize in Table 1 qualitative, quantita-
tive and isotopic analyses of products found in the liquid phase at
pH 2.5. We also indicated in the last column relevant literature ref-
erences where the same product was also reported. The third col-
umn gives the amount of product present after consumption of
1.5 equiv of ozone per mole of glucose. The fourth column indi-
cates the presence of the original carbon atoms C-1, C-2, or C-6
in the product. The tracking of glucose carbon atoms in the oxida-
tion products is based on the isotopic analyses conducted during
the respective ozonation of 1-13C, 2-13C and 6-13C labeled glucose
molecules.
The products identified are in agreement with reports by other
groups. As expected, products corresponding to an oxidation at C-
1 represent the majority of the 6-carbon atom compounds. Hexo-
dialdose was reported once before under batch and free pH con-
ditions13 but never under semi-batch conditions. Its oxidation
product, glucuronic acid is classically identified. Glucaric acid is
known to form at high concentrations of ozone.16,24,29 Ketoaldonic
acids have been reported during the oxidation of glucose or alkyl
glucosides.16,20,21,23,30–32 As expected, the absence of C-1 carbon
atom is evidenced in the products identified as arabinose and ara-
binonic acid. Arabinose, identified by HPAEC and GC–MS, is ob-
NMR monitoring showed that, for up to 2 equiv of ozone con-
sumed per mole of glucose, the limited amount of formic acid pro-
duced is not formed from the C-2 carbon atom and that the
concentration of HC(1)OOH is 4 times as large as that of HC(6)OOH.
To the contrary, the amount of C(1)O2 is similar to that of C(6)O2
indicating that these two one carbon atom products are produced
via different mechanisms.
2.1. Mass balances
served in
a rather low concentration in agreement with
literature data.13,20,22,23,29,33,34 It is noteworthy that the ozonation
of arabinose leads to the formation of a ketopentonic acid identi-
To assess the reliability of our quantitative analyses, we com-
puted mass balances for each of the labeled positions (C-1, C-2,
and C-6). For that purpose, we used the quantitative and isotopic
analysis summarized in Table 1 for the liquid phase, and the
quantitative and isotopic analysis of the gas phase given in our
fied as
D-arabino-pent-4-ulosonic acid by mass spectrometry
(molecular ion at 452 Da for the silylated derivative),11,12 congru-
ent with the results of Parthasarathy and Peterson on xylose.35
§
–
We conducted direct ozonation of commercially available samples of D-glucu-
However other combinations (C-2/C-3, C-3/C-4, C-4/C-5) cannot be excluded.
ronic acid, D-gluconic acid, D-glucurono-1,4-lactone, 2- and 5-keto-D-gluconic acids, L-
arabinose, and polygalacturonic acid (a summary of the gas chromatography data is
given as Supplementary data). Results of these reactions are mentioned when needed
throughout the text.
Indeed, under the conditions applied for 13C NMR analysis, only labeled carbon atoms
were detectable. Nevertheless, by integrating the 13C NMR signal, we could determine
that oxalic acid bearing glucose C-1 and C-2 carbon atoms represents a constant ratio
in the overall oxalic acid produced.

O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
1305
Spectrum A
Abundance
550000
Ozonation of 1-13C glucose 408409
73
526
(average 22.84 to 22.93 min)
450000
300000
250000
150000
50000
436
423
425
468
466
400 420 440 460 480 500 520 540
147
293
189
334
45
408
322
221
526
102
436
172
245
380
18
468
0
m/z-->
0
50
100
150
200
250
300
350
400
450
500
Spectrum B
526
Abundance
60000
73
Ozonation of 2-13C glucose 408409
(average 22.96 to 22,98 min)
436
50000
40000
30000
20000
10000
468
425
424
400 420 440 460 480 500 520 540
293
147
408
334
322
190
221
45
526
172
246
103
380
468
18
0
m/z-->
0
50
100
150
200
250
300
350
400
450
500
Abundance
30000
Spectrum C
73
407
Ozonation of 6-13C glucose
(average 22.8 to 22.9 min)
25000
20000
15000
10000
5000
525
409
421
435
467
466
292
400 420 440 460 480 500 520 540
147
189
407
217
323133
45
102
525
171
245
379
435
467
18
0
m/z-->
0
50
100
150
200
250
300
350
400
450
500
Figure 1. Typical mass spectra of xylaric acid sampled during the ozonation of 1-13C glucose (spectrum A, averaging between 22.84 and 22.93 min), 2-13C glucose (spectrum
B, averaging between 22.96 and 22.98 min), and 6-13C glucose (spectrum C, averaging between 22.80 and 22.90 min).
previous report.6 Based on 8 points between 0.4 and 3 equiv of
ozone consumed, the recoveries were 106% (% CV = 5.1) for C-1,
109% (% CV = 5.0) for C-6, and 103% (% CV = 3.9) for C-2. Hence,
the present analysis can be considered reliable and the quantita-
tive analysis as representative of the actual content of the
samples.
ation of 1-13 -glucose (Fig. 1A) and 2-13
C D C D-glucose (Fig. 1B).
Identification of this acid as xylaric acid is therefore esta-
blished.
2.3. Isotopic content over time
The isotopic enrichment of xylaric but also tartaric acids was
calculated|| and the results are consistent with constant pathways
for their formation. Xylaric was found to contain between 73% and
80% of C-6 and C-2 depending on the peak studied (292 or 321 Da)
and tartaric acid was found to contain 72% of C-1 and 99% of C-2.
The higher C-2 content in tartaric acid is explained by a high ratio
of the C-1 through C-4 segment and a small proportion of the C-2
through C-5 segment in this acid.
2.2. Identification of xylaric acid
Figure 1 features the mass spectra of the silylated pentaric
acid sampled during the ozonation of 1-13C
D
-glucose (Fig. 1A),
-glucose (Fig. 1C), respec-
tively. The absence of 13C-labeled fragments in the acid (Mw
2-13C -glucose (Fig. 1B), and 6-13
D C D
540 g/molÀ1) formed during ozonation of -glucose-6-13C is obvi-
D
ous. One can consider especially peaks at m/z 525 [M–CH3]+Å, 435
[M–CH3–Me3SiOH]+Å, 408, and 292 (Fig. 1C) which appear respec-
tively at m/z 526, 436, 409, and 293 for the acid formed by ozon-
||
Available as Supplementary data.

1306
O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
dioxide.29,34 Our report on carbon dioxide origin6 actually gave a di-
3. Discussion
rect evidence for the early participation of the C-1 carbon atom in
the production of CO2 and formic acid. As expected, arabinose and
arabinonic acid do not contain the C-1 atom (see Table 1). Arabi-
nonic acid can simply result from further oxidation of arabinose
as we observed during the ozonation of arabinose. The formation
of arabinonic acid has also been attributed to the decarboxylation
3.1. Oxidation at position C-1, subsequent oxidation at C-2 and
corresponding products
The high concentration of gluconic acid in the reaction
mixture provides, here again,
a clear evidence of the high
of
D
-arabino-hex-2-ulosonic acid (2-ketogluconic acid).13,14,16,23
reactivity of the C-1 position (hemiacetal) as often reported be-
fore.20,25,26,29,30,33,34,37 To evaluate the significance of oxidation at
C-1 in the formation of other oxidation products, we need to con-
sider potential oxidation products of gluconic acids. For instance,
formation of arabinose13,14,16,20,22,23,25,29 has been explained in
several cases as the result of the loss of C-1 in form of carbon
However, ozonation of the commercially available hemicalcium
salt of 2-ketogluconic acid did not lead to the expected arabinonic
acid. This suggests that 2-ketogluconic acid might be a minor or rel-
atively stable product, rather than the precursor to arabinose or
arabinonic acid at pH 2.5. Based on a mechanism (Scheme 1)
C
C
C
δ+
C
C
H
O
H
C
O
H
C
C
O
O
H
H
δ−
δ−
O
O
H
O
-O
O
O
δ+
O
O+
- O2
C
O
H
C
O
C
+ H2O
C
OOH
+
C
O
H
C
C
O
O
Scheme 1. Formation of acetone and ethyl acetate during ozonation of diethyl ether.38
OH
6
O
1
O
OH
O
O+
HO
2
H
-O
HO
OH
6
O
1
OOH
O
OH
OOH
HO
2
O
OH
OH
OH
OH
H
O
6
6
O
H
O
See Scheme 3D
OH
OH
O
2
HO
HO
OH
HO
1
OH
HO
O
6
1
O
O
2
O
1
OH
O
OH
2
HO
2-Ketogluconic acid
Scheme 2. Proposed mechanism leading to both 2-keto derivatives and lower aldonic acids.
O

O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
A. Nucleophilic addition of ozone
1307
O
HO
HO
HO
HO
O3
H
H
HO
H
O
OH
O
O
HCO2H
HO
O2
CHO
B. Ozone reaction with enol resulting in formic acid
HCO2H
+
O
HO
O3
H
HO
HO
H
HO
O
H
C. Ozone insertion in C-H bond of a hemiketal
O
O
O
2
O3
OH
OH
O
OH
HO
HO
HO
O
OH
O
CO2
OH
OH
HO
OH
HO
O
O2
CHO
O
D. Reaction of ozone with a free carboxylic acid
O
O
O
O3
OH
OH
HO
HO
HO
OH
O
O
OH
HO
O
O
Carbonic acid
CO2 + H2O
Scheme 3. Proposed mechanisms leading to carbon dioxide or formic acid formation from aldehyde, hemiketal, or carboxylic acid intermediates.
proposed to explain the formation of acetone and ethyl acetate dur-
ing ozonation of diethyl ether,38 we now suggest that under acidic
conditions, an unstable intermediate leading to 2-ketogluconic acid
(from gluconic acid), can also decompose into arabinonic acid and
carbonic acid (or carbon dioxide). This is summarized in Scheme
2.
The formation of both erythronic acid and oxalic acid (contain-
ing C-1 and C-2 carbon atoms) could also be explained by further
oxidation of gluconic acid by oxidative cleavage of the C-2/C-3
bond. Such a carbon–carbon bond cleavage has been previously
suggested.39 As shown in Scheme 2, we propose that the same
intermediate leading to either 2-ketogluconic acid or arabinose
and carbon dioxide can decompose into a C-1–C-2 fragment (oxalic
acid) and a C-3 through C-6 fragment (erythrose or erythronic
acid). Erythronic acid (trihydroxybutyric acid) can obviously be
also the result of oxidation of erythrose (observed under similar
conditions23).
3.2. Oxidation at position C-6 and subsequent decarboxylation
While highlighted in a few studies,11,13–15,18,19,23,28,31 oxidation
at C-6 has never been as well established as oxidation at C-1. As
others, we do observe a relatively low concentration of 6 carbon
atom products oxidized at the C-6 position (namely, glucuronic
acid and hexodialdose), compared to the concentration of gluconic
acid. Conversely, we found a substantial involvement of the C-6
carbon atom in the production of carbon dioxide.6 We monitored
the production of carbon dioxide during ozonation of gluconic acid,
gluconic acid-1,5-lactone, glucuronic acid, and glucaric acid-1,4-
lactone. The decarboxylation of gluconic acid appears to be limited
in its lactone form, predominant under acidic conditions. We were
able to conclude to an enhanced sensitivity of uronic acid groups
toward ozone under acidic conditions.11,12,28,40 Their tendency to
decarboxylate in the presence of ozone certainly contributes to
both the presence of carbon atom C-6 in the carbon dioxide formed
and the low concentration of products oxidized at C(6). However,
these data alone are not sufficient to allow to conclude that the
newly identified xylaric acid is the result of the decarboxylation
of glucuronic acid.
While radical processes cannot be excluded, the mechanisms proposed in this
article (Schemes 2 and 3) focus on molecular ozone and are consistent with the
absence of detectable amounts of hydroxyl radicals at this pH. (see Ref. 16).

1308
O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
O
O
6
OH
6
H
O
O
1
1
OH
OH
OH
OH
HO
2
HO
2
OH
Hexodialdose
OH
Glucuronic acid
See Scheme 3D
via enol
See Scheme 3A
See Scheme 3B
OH
H
O
H
O
H
O
OH
O
1
OH
1
HO
2
HO
2
OH
OH
Y
X
OH
O
OH
1
O
OH
HO
2
OH
Xylaric acid
Scheme 4. Reaction pathway to xylaric acid after oxidation at C-6.
To establish a correlation, we calculated the ratio of carbon
involves the formation of the least stable radical intermediate. 5-
Ketogluconic acid (observed) however is likely to form via the
mechanism leading to 2-ketogluconic acid by oxidation at position
2. Additionally, if ozonation of glucuronic acid does lead to xylaric
acid, the ozonation of gluconic acid does not lead to any xylaric
acid. Ozonation of 5-ketogluconic acid, gives no detectable amount
of xylaric acid either. Therefore, we favor a mechanism such as that
in Scheme 3D. We summarize this path in Scheme 4 with interme-
diate Y giving carbon dioxide from glucuronic acid.
Glucaric acid which can be formed from hexodialdose or oxida-
tion of gluconic acid (directly or via guluronic acid), is not likely to
be a key intermediate in the formation of xylaric acid. Further
oxidation of such an aldaric acid would potentially lead to the for-
mation of both xylaric and arabinaric acids but the latter was
detected.
dioxide formed from C-6 to the amount of xylaric acid formed.
We found that this ratio remains close to 0.9 up to 2.5 equiv of
ozone consumed. The same ratio determined for the C-1 position
based both on the production of carbon dioxide and on the produc-
tion of arabinose, arabinonic, and tartaric acids gave only an aver-
age value of 0.76. Carbon dioxide formation from C-6 is therefore
quantitatively correlated to the formation of xylaric acid whereas
that of carbon dioxide form C-1 is the result of multiple reactions.
If xylaric acid is the result of a decarboxylation, what is the
mechanism and is the decarboxylation actually occurring on glucu-
ronic acid? It is noteworthy that no aldehyde precursor of xylaric
acid is identified whereas arabinose, the potential aldehyde pre-
cursor to arabinonic acid is detected. Still, xylaric acid could be
formed by the fast oxidation of an aldehyde precursor not stabi-
lized as a hemiacetal. By analogy with suggestions given by others
for gluconic acid,22,25,29,34 a dialdehyde intermediate X (Scheme 4)
could be derived from glucuronic acid by decarboxylation or from
hexodialdose by deformylation of carbon 6. Mechanisms given in
Scheme 3A and B could account for that.
3.3. Formation of products with less than 5 carbon atoms
Tartaric acid unexpectedly contains mainly the C-1 through C-4
segment and to a lesser extent, the C-2 through C-5 segmentàà
The formation of xylaric acid could also be explained by a mech-
anism similar to that given in Scheme 2. This mechanism, applied
to the carbon 5 of glucuronic acid, would lead either to xyluronic
acid and carbon dioxide or to 5-ketoglucuronic acid. Neither xylu-
ronic nor 5-ketoglucuronic acids have been observed in this study
or elsewhere. The same mechanism, when applied to C-5 of
gluconic acid, would actually give xylaric acid (observed) but also
àà
Tartaric acid containing the C-2/C-5 segment forms in a very limited amount
according to the isotopic analysis. Such a diacid could be the result of a double
decarboxylation of glucaric acid leading to the formation of both C6O2 and C1O2.
Decarboxylation of xylaric acid can also explain the formation of both C-1 through C-4
or C-2 through C-5 tartaric acids. The slow decrease in concentration of xylaric acid
after 2 equiv of ozone consumed could account for such a degradation.

O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
1309
throughout the reaction. In previous reports based on HPLC16,23–25,29
or NMR spectroscopy20,21 during the ozonation of unlabeled glucose,
the formation of tartaric acid could not be attributed to the oxidation
of a specific precursor. We believe that oxidation at position 5 of glu-
conic acid leads mainly to 5-ketogluconic acid. And, we propose that
glycolic acid and tartaric acid would result from an oxidation at po-
sition 4 of gluconic acid or glucuronic acid. There is evidence of oxi-
dation at the C-4 position.15 The isotopic analysis of the products
containing 3 carbon atoms also supports an oxidation at position
4. They are formed in very small amounts, but we were able to show
that these compounds contain either the C-1–C-3 or C-4–C-6 seg-
ments implying a cleavage of the C-4–C-3 bond. By applying once
more the mechanism given in Scheme 1 to carbon C-4, one could ex-
plain formation of 4-keto derivatives (not observed here) and forma-
tion of fragments with 3 carbon atoms in addition to fragments with
2 and 4 carbon atoms as mentioned earlier.
sulfuric acid and maintained by automatic titration with NaOH
(250 mmol). For more details on experimental conditions and mea-
surement of ozone concentration see Ref. 6. Ozone consumption
was determined by the difference between the concentrations of
ozone before and after the reactor. Throughout the article, ozone
consumption is expressed in mole of ozone consumed per mole
of glucose introduced (i.e., equivalent of ozone).
D
-Glucose-1-13C,
were purchased from Omicron Biochemical—USA.
Commercially available -glucuronic acid, -gluconic acid,
glucono-1,4-lactone, -glucaric acid, 5-keto- -gluconic acid, 2-
keto- -gluconic acid hemicalcium salt, -arabinose, and pectic acid
D D
-glucose-2-13C and -glucose-6-13C, 99% 13C
D
D
D-
D
D
D
L
(Aldrich cat # 37,474-1) were also submitted to the same oxidation
conditions.
5.1.2. Analysis of neutral sugars by HPAEC (
-arabinose)
HPAEC (High Performance Anion Exchange Chromatography)
D-Glucose,
L
4. Conclusion
analyses16,23,24,29,41 were performed with a Dionex system (DX-
500) using a 250 Â 4 mm anion exchange column (Carbopac PA1)
fitted with a pre-column (50 Â 4 mm) and a pulsed amperometric
detector (PAD). A ternary eluent was used at a flow rate of 1 mL/
min. Eluents A (water), B (NaOH 150 mMol), and C (500 mmol AcO-
Na and 150 mmol NaOH) were mixed according to the following
gradient. At t 0 s, the eluent was a mixture of A (70%) and B
(30%). At 60 min, the eluent was a mixture of A (30%), B (40%),
and C (30%).
Compounds identification was performed by comparison of
their retention times with those of standard mixtures. Quantifica-
tion was made using calibration curves established by analyzing
standard solutions.
The use of 13C-labeled
D-glucose allowed for a better under-
standing of the origin of carbon dioxide and gave a significantly
better insight in the formation of ozonation products of glucose.
By using isotopic and quantitative analyses, we were able to di-
rectly correlate the formation of a pentaric acid (xylaric acid) to the
loss of carbon C-6. Presence or formation of carboxyl groups in lieu
of exocyclic hydromethyl groups in polyhexoses could be of signif-
icance during treatment by ozone. Their fast decarboxylation
would indeed lead to an ‘open’ residue in the structure. Such mod-
ification would directly affect the physical properties of the poly-
saccharide (critical in paper manufacturing for example) and
could lead to subsequent decrease in molecular weight such as
by b-elimination for example. We indirectly assessed some of these
possibilities by treating pure polygalacturonic acid (pectin) with
ozone at pH 2.5§§ and observed the expected formation of carbon
dioxide and the corresponding pentaric acid.11,40
In the present report, we establish for the first time a defined
path for oxidation on C-6 (Scheme 4) while confirming the well-ac-
cepted formation of arabinose by loss of C-1 and the high reactivity
of C-1. The paths given in Scheme 3A–D account for the observed
formation of both carbon dioxide and formic acid from carbon C-
1 (as aldehyde or hemiketal or carboxylic acid) and mainly carbon
dioxide from C-6 (as uronic acid).
Finally, a set of mechanisms for glucose ozonation under acidic
conditions is proposed that accounts for the majority of the prod-
ucts reported in the literature by explaining: (i) The formation of
arabinose or arabinonic acid and carbon dioxide or formic acid
from gluconic acid or glucose, of oxalic acid and erythronic acid
from gluconic acid and of 2-ketogluconic acid from gluconic acid as
shown in Scheme 2; (ii) The formation of 5-ketogluconic acid;
and (iii) The formation of 4-keto derivatives of glycolic acid and
tartaric acid or fragments with 3 carbon atoms.
5.1.3. Quantification and identification by GC–MS
Analyses with a combined GC–MS apparatus were performed
after silylation of the samples on a Hewlett Packard 5890/5972
MSD instrument. The separation was achieved using an apolar
High Performance Capillary Column (HP1-MS, 30 m length and
0.25 mm of internal diameter). The helium flow rate was set at
1.6 mL/min. Injections were done in the split/splitless mode with
an injector temperature of 100 °C. Solvent delay was set to 5 min.
The temperature was kept at 60 °C for 1.5 min, then a linear tem-
perature program was applied up to 31.5 min to reach 300 °C at
10 °C/min. Finally the temperature was maintained at 300 °C until
36 min.
5.1.4. Silylation procedure
The reaction sample (600
room temperature and dissolved in 700
mixture was placed for 2 min in an ultrasonic bath before addition
of 500 L of a 5% soln of trimethylsilyl chloride (Aldrich 99% purity)
l
L) was concentrated to dryness at
lL of dry pyridine. The
l
in bis(trimethylsilyl)trifluoroacetamide (Aldrich 99% purity). The
sample was then stored 2 h in the dark prior to analysis.
Compounds identification was performed either by comparison
of their mass spectra with those obtained from commercially avail-
able chemicals submitted to an identical silylation process or by
comparison with spectra of compounds in the database of the Na-
tional Bureau of Standards (NBS-USA) or relevant literature.
Before relying on GC integration as a quantitative tool, we took
the additional step of determining a response coefficient of com-
mercially available by-products by reference to the glucose signal
(see Supplementary data).11 The relevance of these coefficients
was then assessed by comparing GC quantification with HPAEC-
PAD (for neutral by-products) or HPAEC-PED (for acidic by-prod-
ucts using a Dionex AS11 column). For experimental samples,
glucose, measured independently by HPAEC-PAD, was used as an
internal GC standard and the respective GC response coefficients
5. Experimental
5.1. Materials and methods
5.1.1. Ozonation reactions
Reactions were carried at room temperature on 250 mg of D-
glucose (1.38 mmol) in 100 mL of deionised water (UHQ water
from an Elgastat apparatus). The soln was magnetically stirred
and ozone introduced (55–60 mg/L and 60 mL/min produced from
oxygen ‘C’ Air Liquide with a Lab-Lox Trailigaz ozonizer) into the
solution through a glass bubbler. The initial pH was set with concd
§§
This will be detailed in a forthcoming paper.

1310
O. Marcq et al. / Carbohydrate Research 344 (2009) 1303–1310
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Amounts of by-products are expressed in moles throughout the
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the true contribution of isotopic enrichment in the signal at M+1
and to calculate a percentage of enrichment. This method enabled
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tartaric acids (for an example see Supplementary data).11
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Acknowledgments
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The ‘Centre National de la Recherche Scientifique’ (CNRS), Air
Liquide Co. and the ‘Conseil Régional de Bourgogne’ are gratefully
acknowledged for the financial support to this study. O.M. thanks
the CNRS and the ‘Conseil Régional de Bourgogne’ for a PhD grant
(B.D.I.). The assistance of Ms. Béatrice Billier in some of the analyt-
ical work during her MS internship is strongly appreciated.
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Supplementary data
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American Chemical Society National Meeting; Boston, MA, 2002; CARB-087.
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