Oxidation of mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass spectrometry

Article abstract of DOI:10.1016/j.carres.2011.09.011

The hydroxyl radicals are widely implicated in oxidation of carbohydrates during biological and industrial processes being responsible for their structural modifications and causing functional damage. The identification of intermediate oxidation products is hampered by a lack of reliable sensible methods for their detection. In this study, the oxidation of two models of galactomannans (Man₃ and GalMan₂) has been studied in reaction with hydroxyl radical generated by Fenton reaction. The oxidation patterns were assessed using preparative ligand-exchange/size-exclusion chromatography (LEX/SEC) coupled with tandem electrospray mass spectrometry (ESI-MS/MS). This allowed the identification of derived oligosaccharides (OS) containing hexuronic, hexonic, pentonic and erythronic acid residues and neutral OS bearing hydroperoxy, hydrated carbonyl moieties and residues from pyranosyl ring cleavage. The depolymerization products have been also detected upon oxidation of oligomers. This study allowed developing a simple, effective 'fingerprinting' protocol for detecting the damage done to mannans by oxidative radicals.

Full text of DOI:10.1016/j.carres.2011.09.011

Carbohydrate Research 346 (2011) 2603–2611
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Oxidation of mannosyl oligosaccharides by hydroxyl radicals as assessed
by electrospray mass spectrometry
a
b
a
c
a
Joana Tudella , Fernando M. Nunes , Rosa Paradela , Dmitry V. Evtuguin , Pedro Domingues ,
a
d
b
a,
⇑
Francisco Amado , Manuel A. Coimbra , Ana I.R.N.A. Barros , M. Rosario M. Domingues
Mass Spectrometry Centre, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
CQ-VR, Chemistry Research Centre, Department of Chemistry, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal
CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
a
b
c
d
QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2011
Accepted 15 September 2011
Available online 22 September 2011
The hydroxyl radicals are widely implicated in oxidation of carbohydrates during biological and indus-
trial processes being responsible for their structural modifications and causing functional damage. The
identification of intermediate oxidation products is hampered by a lack of reliable sensible methods
3 2
for their detection. In this study, the oxidation of two models of galactomannans (Man and GalMan )
has been studied in reaction with hydroxyl radical generated by Fenton reaction. The oxidation patterns
were assessed using preparative ligand-exchange/size-exclusion chromatography (LEX/SEC) coupled
with tandem electrospray mass spectrometry (ESI-MS/MS). This allowed the identification of derived oli-
gosaccharides (OS) containing hexuronic, hexonic, pentonic and erythronic acid residues and neutral OS
bearing hydroperoxy, hydrated carbonyl moieties and residues from pyranosyl ring cleavage. The depo-
lymerization products have been also detected upon oxidation of oligomers. This study allowed develop-
ing a simple, effective ‘fingerprinting’ protocol for detecting the damage done to mannans by oxidative
radicals.
Keywords:
Oligosaccharides
Oxidation
Electrospray ionization mass spectrometry
Galactomannans
Hydroxyl radical
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
carbohydrates.7,14,15 Some of these products, such as uloses, aldo-
nic and uronic acids, tetrose and tetrulose derived from parent
Å
14–19
The hydroxyl radicals (HO ) are recognized highly active species
in oxidation of carbohydrates with oxygen, ozone or peroxides in
hexoses and pentoses have been identified.
The structural
changes in the carbohydrates can modulate significantly their
1
2
Å
many biological and industrial processes. Thus, HO are suspected
to be responsible for the non-enzymatic scission of polysaccharides
in biological systems with hyaluronate,3 chitosan,5 pullulan,
carboxymethylcellulose, welan, scleroglucan,6 xyloglucan, and
HO are accused for the strong depolymerization of poly-
saccharides during oxygen, ozone or hydrogen peroxide bleaching
reaction patterns, as for example hexuloses are more reactive
than the original reducing end units in redox and S
N
reactions2
0,21
,4
22
and hexonic acids are not reactive at all in those reactions.
Scarce structural details exist for the oligo- and polysaccharides
that have been subjected to the radical reactions (oxidation inter-
7
–9
Å
pectin.
7
–9,14,16
mediates) and did not degrade to monomeric units.
Major
10,11
of chemical pulps for the papermaking
and promote the
difficulties in analysing those intermediates, which bring precious
information about reaction pathways are their relatively low
abundance and stability during isolation/derivatization.
Mass spectrometry (MS) has been widely considered during the
last decade for the structural characterization of oligosaccha-
thermal degradation of cellulose.12 These reactive species were
also proposed to be involved in oxidation of galactomannans
during coffee roasting.13
A variety of secondary end products may be formed from
carbohydrate-derived free radical afforded after C–H bond scis-
sion with HO . Major studies on the oxidized intermediates of car-
2
3–25
rides.
Electrospray mass spectrometry (ESI-MS) and tandem
Å
n
mass spectrometry (ESI-MS ) have been recognized particularly
advantageous tool for the structural analysis of low abundance
(up to picomole levels) oligomers possessing an increased molecu-
lar mass without their previous derivatization and(or) partial
depolymerization.
The main goal of this work was to study the oxidation patterns
of two isomeric trisaccharides, Man and GalMan (Scheme 1) in-
duced by the hydroxyl radical generated under conditions of
Å
bohydrates with OH have been carried using monomeric sugars
and much less with oligo- and polysaccharides.1
4,15
The majority
of detected oxidation products are low molecular weight com-
pounds generated from depolymerization and degradation of
2
4,25
⇑
Corresponding author. Tel.: +351 234 370698; fax: +351 234 370084.
E-mail address: mrd@ua.pt (M.R.M. Domingues).
3
2
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.011

2
604
J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
CH2OH
O
OH
OH
CH2OH
CH2OH
CH2OH
O
O
O
O
CH2OH
CH2
OH
OH
OH
O
OH
OH
O
OH
OH
OH
O
O
OH
OH
HO
OH
OH
O
OH
HO
Man3
GalMan2
Scheme 1. Isomeric mannosyl containing trisaccharides, Man
(H O /Fe ).
2 2
3
and GlalMan
2
that were induced to oxidation by the hydroxyl radical generated under Fenton conditions
2+
Fenton reaction. Oxidation products were separated using semi-
preparative ligand-exchange/size exclusion chromatography
for 20 min at 4 °C with solution of triphenylphosphine (TPP)
(1 mg/mL in ethanol).
(
LEX/SEC) and analyzed by electrospray mass spectrometry (ESI-
MS) and tandem mass spectrometry (MS/MS). The results obtained
allowed developing a simple, effective ‘fingerprinting’ protocol for
detecting the damage done to galactomannans by HO radicals,
2.3. Quantification of the hydroperoxides
Å
An aliquot of trisaccharides solution (50
reduction with TPP were incubated with Fox reagent (950
0 min in dark. The Fox reagent was prepared with 100 M XO, fer-
rous sulfate (250 M; 6.95 mg), 25 mM of sulfuric acid (139 L),
and 100 of sorbitol in aqueous solution (total volume
00 mL). The absorbance at k = 560 nm was assessed using a Ther-
mo Fisher Scientific GENESYS 10 Bio UV–vis Spectrophotometer.
l
L) before and after
using the model oligosaccharides Man
3
and GalMan
2
.
lL) for
3
l
l
l
2
2
. Materials and methods
.1. Reagents
lM
1
1
,4-b-
D-Mannotriose (Man
) and 61-
a-D-galactosyl-mannobiose
3
2
.4. Chromatographic separation
(
2
GalMan ) were supplied from Megazyme Comp. (Wicklow, Ire-
land). Ferrous chloride and hydrogen peroxide (30%) were obtained
from Merck Comp. (Darmstadt, Germany). Xylenol orange (XO)
The products from oxidation of oligosaccharides were separated
by semi-preparative LEX/SEC (pump Knauer K-1001) using a
(
Fluka Chem. Comp.), FeSO
4
ꢀ7H
2
O (Merck (Darmstadt, Germany))
Shodex sugar KS 2002 (Showa Denko K.K.) column (300 mm ꢁ
and H SO were used for Fox method. Triphenylphosphine was
2
4
2
H
0 mm) at 30 °C and ultra-pure water (pH 5.8 adjusted with
used for reduction of the hydroperoxydes. All solvents used were
HPLC grade, and the solutions used were prepared with MilliQ high
purity water.
2
SO
a detector RI (Knauer K-2401) were used. The injected sample vol-
ume was 500 L. Approximately 1 mg of oxidation products was
injected per run. Fractions obtained were analyzed by ESI-MS
4
diluted solution) as eluent. A flow rate of 2.80 mL/min and
l
2
.2. Oxidation reaction
2
6,27
and ESI-MS/MS.
In this work, the oxidation of two different trisaccharides Man
3
and Man
generated under Fenton Reaction conditions. Briefly, the trisaccha-
rides (2 mM) were incubated at 37 °C in 200 L of aqueous med-
ium containing FeCl (40 M) and H (50 mM). The reaction
2
Gal (Figs. 1 and 2) was induced by the hydroxyl radical
2.5. Electrosprayionization mass spectrometry (ESI-MS and MS/
MS)
l
2
l
2
O
2
The ESI-MS and ESI-MS/MS were carried out on a Q-TOF2 hybrid
tandem mass spectrometer (Micromass, Manchester, UK). Samples
mixtures were left to react overnight. The hydroperoxide deriva-
tives obtained after the oxidation reaction were degraded while
incubating the solution of oxidized trisaccharides (ca. 50 lg/mL)
were introduced at a flow rate of 10
source. In MS and MS/MS experiments TOF resolution was set to
lL/min into the electrospray
3 2
Figure 1. LEX/SEC chromatograms of oxidized Man (A) and GalMan (B).

J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
2605
(
A) Man3
(B) Man Gal
2
3
65.1
365.2
381.1
100
100
3
47.1
2
03.1
3
81.1
525.2
5
25.2
2
01.1
5
43.2
2
01.1
2
19.1
543.2
3
45.1
203.1 219.1
185.1
275.1
1
85.1
3
05.1
4
65.2
305.1
435.2 495.2
0
m/z
0
m/z
200 250 300 350 400 450 500 550
100 150 200 250 300 350 400 450 500 550
X1 m/z381
m/z219
OH
y
1
m/z 185
OH
X2
m/ z 185
m/ z 365
203
Gal
y2
Gal
Man
O
Man
O
Man
Man
O
Man
O
Man
m/ z 381
O
OH m/ z 365
O
m/ z 381
m/ z 381
m/z185
m/z347
Man
O
Man
OH
Man
O
Man
m/ z 201
m/ z 363
m/ z 185
m/ z 201
Z1
Man
m/ z 381
OH
m/ z 203
m/ z 201
Z2
Gal
OH
O
Man
O
Man
m/ z 381
O
m/ z 365
m/ z 185
m/ z 363
Man
O
Man
m/ z 185
+
Figure 2. ESI-MS/MS spectra of the ion at m/z 543, [Hex
3
+O+Na] , detected in neutral fractions F2 for the oxidized Man
3
2
(A) and Man Gal (B).
approximately 9000. For ESI analysis, oligosaccharides were diluted
in methanol–water–formic acid (50:50:0.1, v/v/v). The cone voltage
was set to 30 V, and capillary voltage was maintained at 3 kV.
Source temperature was at 80 °C and desolvation temperature at
observed with lower mass to afore mentioned [M+Na]+ ions:
(+nO ꢂ 2 Da) at m/z 525, 541, 557, 573, and 589, and (ꢂnO ꢂ 2 Da),
at m/z 539 and 555 (Table 1).
Ions with lower m/z value when compared with the non-modi-
fied trisaccharides were also observed indicating that depolymer-
ization or cleavage of one hexose unit occurred during the
oxidation. These ions observed at m/z 365 and at m/z 203 were as-
1
50 °C. Tandem mass spectra were obtained using Ar as the colli-
sion gas and the collision energy was set between 25 and 45 V.
The data were processed using a MassLynx software (version 4.0).
+
+
signed to the [Hex
2
+Na] and [Hex+Na] , respectively, and their
+O+-
Na] (365 + 16 Da), 397 [Hex+2O+Na] (365 + 32 Da), 219 [Hex+-
corresponding oxidation products were seen at m/z 381 [Hex
2
3
3
. Results and discussion
+
+
+
+
O+Na] , 217 [Hex+Oꢂ2 Da+Na] (Table 1).
.1. Fractionation of oxidation products by LEX/SEC
Other groups of neutral oxidized OS were identified as resulting
from the oxidative ring scission reactions (Table 1). Some of these
oxidation products were identified at m/z 497, assigned to OS bear-
ing a pentose and formed due to oxidative cleavage (decarboxyl-
ation at C1) of the reducing end sugar unit, and at m/z 467,
assigned to OS bearing a tetraose as a result of oxidative cleavage
between C2 and C3 of the reducing end unit. This is a common fea-
ture in hexose oxidation, as it was reported recently for the oxida-
tion of glucose under conditions of Fenton reaction, generating
arabinose and erythrose as oxidation products. These oxidation
products occur in minor abundance. Ions assigned to the
correspondent hydroxyl and hydroperoxy derivatives were also
detected in the MS spectra although at very low relative abun-
dance. All these oxidation products were analyzed by ESI-MS/MS
in order to assess their structure.
3 2
The oxidation of Man and GalMan by hydroxyl radical gener-
ated within Fenton Reaction mechanisms was monitored by ESI-
MS after the preliminary fractionation of diverse oxidation products
using preparative LEX/SEC. LEX/SEC provided effective separation
of acidic and neutral products due to the faster elution of the form-
2
6,27
ers thus allowing a more accurate identification by ESI-MS.
The
elution chromatograms of oxidation products from both trisaccha-
rides are shown in Figure 1.
1
6
According to assays with model compounds and results of pre-
2
6,27
vious studies,
two peaks (fraction F1) with lowest elution
times were assigned to acidic reaction (Fig. 1). The fraction eluted
at ca. 16–20 min (F2) was assigned to oligomeric (dimeric–
trimeric) products; monomeric sugars were eluted at 20–22 min
F3) and low molecular weight oxidation products at 23–24 min
F4) and 25–27 min (F5). Fractions F1 and F2 affording major struc-
(
(
tural information on oxidation intermediates were collected and
analyzed by ESI-MS. OS oxidation products were observed in a
positive mode ESI analysis as [M+Na] ions and were further struc-
3.3. ESI-MS/MS of neutral oxidation products from fractions F2
+
The fragmentation patterns from different ions observed in ESI-
MS/MS of spectra indicated clearly the cleavage of the glycosidic
turally characterized by ESI-MS/MS.
1
3
bonds typical for hexose oligosaccharides. Additionally, the cross
3
.2. ESI-MS of neutral OS (F2)
ring cleavages with loss of 60 Da, typical for (1?4)-linked pyran-
2
osyl units were detected, whereas in the case of GalMan and it
The identification by ESI-MS (Supplementary Fig. 1) of a series
oxidation products, the loss of 60, 90, and 120 Da was observed
of the new ions resultant from the oxidation products of the neu-
tral OS present in fraction F2 is resumed in Table 1. Several oxida-
tion products show higher m/z value when compared with the non
oxidized OS at m/z 527, namely ions at m/z 543, 559, 575, and 591
that resulted from the introduction of one, two, three and four oxy-
gen atoms to the molecule, respectively. Other series of ions were
indicated the presence of a (1?6)-linked disaccharide.28
3.3.1. Oxidation products with +nO
The oxidation product ions at m/z 543 and 559, formed respec-
tively by the addition of one (527 + 16 Da) and two (527 + 32 Da)
oxygen atoms could correspond to the formation of additional

2
606
J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
Table 1
Resume of the main neutral oxidation products observed in the ESI-MS spectra generated from the oxidation induced by the hydroxyl radical to the of Man
the assignment of the respective m/z values of the [M+Na] ions and possible identification
3
and GalMan
2
, with
+
3
Man /Gal Man
2
n = 0
n = 1
n = 2
n = 3
n = 4
DP3
OS + nO
527
525
523
543
541
539
559
557
555
575
573
571
591
589
587
OS with one hexulose + nO (ꢂ2 Da + nO)O
OS with two keto groups + nO (ꢂ4 Da + nO)
Depolymerization
DP2
Hex
Hex
Hex
2
2
2
+ nO
365
363
381
379
397
395
393
+ nO ꢂ 2 Da
+ nO ꢂ 4 Da
DP1
Hex + nO
Hex + nO ꢂ 2 Da
203
201
219
217
Ring scission
OS with pentose + nO
OS with pentose + nO ꢂ 2 Da
OS with tetraose + nO
OS with tetraose + nO ꢂ 2 Da
497
495
467
465
513
511
483
481
+
hydroxy and hydroperoxy moieties in OS molecules, similar to that
[Hex+2O+Na] . The presence of dihydroxy derivatives (alternative
to structure with hydroperoxide moieties) was rejected, because
no ions at m/z 219 (Hex+O) and at m/z 201 (Hexres+O) were
detected.
29
30
31
observed during the oxidation of lipids, peptides, or proteins.
The ESI-MS/MS spectra (Fig. 2) of the ion at m/z 543, [Hex +O+-
), showed the ions
3
+
3 2
Na] , for both trisaccharides (Man and GalMan
+
+
at m/z 381 [Hex
2
+O+Na] and 219 [Hex+O+Na] , due to the loss of
In order to confirm the formation of hydroperoxides during
mannosyl oxidation, they were quantified employing Fox method.
The concentration of hydroperoxides was found to be 2.33
Man and 5.30 M for GalMan . Additionally, the reduction of
hydroperoxides has been carried out with triphenylphosphine
(TPP). The characteristic disappearance of hydroperoxides after
this treatment confirmed their initial presence upon OS oxidation.
The other oxidation products with three and four oxygen atoms
corresponded to hydroxy–hydroperoxy derivatives and dihydrop-
eroxy derivatives (data not shown). These molecules included sev-
eral isomers according to the location of introduced OH and OOH
one and two hexose residues from the non-reducing end, (Hex-
res,ꢂ162 Da), and the ion at m/z 347 (due to loss of Hex+O) indicat-
ing that one oxidation product with the additional oxygen in the
reducing end was formed (Fig. 2, structure X1). The ion at m/z
lM for
3
l
2
3
6
3
47 is absent for GalMan
2
, since in GalMan
2
this terminal Man unit
is substituted with Gal. On the other hand, the ion at m/z 365,
formed by the loss of oxidized Hexres (Hexres+O), and the ion at
+
m/z 201 [Hexres+O+Na] (Fig. 2, structures Y1 and Y2), indicate that
an isomer with the additional hydroxyl group in the Hex from the
non-reducing end is also present. This assumption is supported by
the detected ion at m/z 363, due to the loss of a Hex unit (ꢂ180 Da)
from the reducing end. However, as well the location of the OH
group in the middle hexose residue cannot be completely excluded
groups. The ESI-MS/MS spectra of the ions attributed to [Hex1-
+nO+Na] , resultant from Hex
presence of –OH and –OOH derivatives also in these structures
(data not shown).
+
2
3
depolymerization, confirmed the
(
Z1 and Z2). Overall, the oxidation product at m/z 543 may corre-
spond to a mixture of three isomeric oxidation products, depend-
ing on the location of the additional OH group.
3.3.2. Oxidation products of hexulose type (OS-2Da)
To our knowledge, an intermediate containing an additional OH
group attached to the carbohydrate after the oxidation with HO
Oxidation products with the additional loss of 2 Da (OS-2) were
assigned to the formation of OS with a hexulose unit (Table 1). To
confirm this structural feature, the ESI-MS/MS spectra of ions at m/
Å
has never been evidenced previously. The formation of this carbo-
Å
+
+
hydrate derivative can be due to the reaction of free radical (R )
z 525 ([Hex
3
ꢂ2 Da+Na] ) and at m/z 557 ([Hex
3
+2Oꢂ2 Da+Na] )
with hydrogen peroxide or due to the hydroperoxyde cleavage
were acquired and analyzed in details.
(
ROOH) catalyzed by the iron ions, similarly as described for the li-
The ESI-MS/MS spectrum of the ion at m/z 525 (Fig. 4) shows
3
2,33
+
pid hydroperoxides.
the hexose units cannot yet be accurately determined by ESI-MS/
MS.
The position of the new hydroxyl group in
the product ion at m/z 363, assigned to [Hex
2
ꢂ2 Da+Na] due to
loss of a Hexres and indicating that the hexulose residue can be lo-
cated at the reducing end, which is confirmed in turn by the de-
+
+
The ESI-MS/MS of the ion at m/z 559 [Hex
loss of 32 Da, assigned to the loss of O
m/z 527, thus confirming the presence of a hydroperoxy derivative
Fig. 3). Loss of O was found to be a typical fragmentation pathway
3
+2O+Na] , showed a
tected ion at m/z 201 ([Hexꢂ2 Da+Na] ). However, the detected
+
2
, with formation of the ion at
ion at m/z 203 ([Hex+Na] ) indicated the eventual location of this
structural unit in the middle of the chain. A small abundance ion
at m/z 365 due to the loss of (Hexres ꢂ 2 Da) indicates that an iso-
mer with the hexulose at the non reducing terminal might be also
present, although not of the same higher abundance as observed
for the reducing terminal and for the middle units of the chain.
The distinct isomers bearing a hexulose are depicted in Figure 4.
(
2
of hydroperoxide observed previously before in the MS/MS analy-
sis of phospholipids and peptide oxidation products.29,34,35 MS/MS
spectra of this oxidation product from both Man
3
and GalMan
2
+
show the same product ions. The ions at m/z 217 [Hexres+2O+Na]
and at m/z 365, formed due to loss of (Hexres+2O) suggest that the
peroxy moiety is located mainly in the non-reducing end (Man or
Gal residues, depending on the OS involved). The abundant ion at
m/z 277 observed in both spectra was attributed to a ring cleav-
The ESI-MS/MS spectra of the oxidation product with sodium
+
adduct at m/z 557 ([Hex
3
+2Oꢂ2 Da+Na] ) is shown in Figure 5.
Both trisaccharides (Man
3
2
and Man Gal) showed a major loss of
2
32 Da (–O , ion at m/z 525), confirming the presence of hydroper-
oxy derivatives. This fact, however, impedes the identification of an
exact localization of hydroperoxy moiety in the chain. An oxidation
2
,4
24
age
A
2
.
The oxidation product with –OOH at reducing end is
also suggested due to the presence of ion at m/z 235 assigned to

J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
2607
(A) Man3
(B) Man Gal
2
2
77.0
365.1
100
100
2
17.0
1
87.0
2
77.0
1
99.0
363.1
397.1 527.2
235.1
289.0
300
2
17.0
1
87.0
175.0
171.0
3
97.1
3
79.1
3
65.1
305.1
337.1
300
559.2
1
75.0
57.0
379.1
5
27.2541.2
m/z
467.2
500
1
0
1
0
100
m/z
00
200
400
500
200
400
m/z 217
m/z 185
Gal
OOH
Gal
OOH
Man
OOH
m/z 203
m/z 397
m/z 235
m/z 397
m/z 397
O
m/z 397
O
m/z 365
m/z 397
Man
O
Man
O
Man
O
Man
O
Man
Man
m/z 185
O
Man
Man
O
Man
OOH
m/z 185
m/z 347
m/z 185
m/z 379
m/z 185
m/z 185
OOH m/z
Man
Gal
365
m/z 203
OOH m/z 365
Man
O
O
Man
O
Man
m/z 397
O
Man
m/z 217
m/z 379
m/z 217
+
Figure 3. ESI-MS/MS spectra of the ion at m/z 559, [Hex
3
+2O+Na] , observed in the neutral fractions F2 for the oxidized Man
3
2
(A) and Man Gal (B).
(
B) Man Gal
2
(
A)Man3
2
25.1
363.1
100
100
5
25.1
3
63.1
2
69.2
313.2
525.2
1
89.0
365.1
5
25.3
161.0 191.1
261.1
1
45.0
524.6
272.1
465.2
4
01.2
0
1
m/z
0
100 150 200 250 300 350 400 450 500
m/z
00 150 200 250 300 350 400 450 500
m/z 183
m/z 185
GalIns
Gal
m/z 363
m/z 201
m/z 363
m/z 203
m/z 363
O
m/z 363
O
Man
O
Man
O
ManIns
Man
O
ManIns
O
Man
m/z 365
m/z 363
Man
O
Man
Man
O
ManIns
m/z 185
m/z 347
m/z 185
m/z 345
m/z 185
m/z 185
m/z 365
m/z 203
m/z
1
85
Gal
ManIns
O
Man
O
Man
m/z 365
O
m/z 363
m/z 183
m/z 345
ManIns
O
Man
m/z 183
+
Figure 4. ESI-MS/MS spectra of the ion at m/z 525, [Hex
3
ꢂ2 Da+Na] , observed in the neutral fractions F2 for the oxidized Man
3
(A) and Man
2
Gal (B). Manins = Manꢂ2 Da.
+
product possessing the product ion at m/z 363 ([Hex
2
ꢂ2 Da+Na] )
introduced hydroxyl and/or peroxyl group and a hexulose unit
indicates the presence of a hexulose residue in the same chain,
more likely at the reducing end or at the middle of the chain.
Hence, these features allow assignment of this OS to trisaccharide
that bears simultaneously a peroxy group and a hexulose unit.
Similarly to other oxidation products described, the hydroperoxy
groups can be randomly distributed along the mannosyl and galac-
tosyl units, as depicted in Figure 5.
was confirmed. The ions in ESI-MS/MS spectra assigned to
+
[Hex1-2+nOꢂ2 Da+Na] , resultant from Hex
3
depolymerization,
confirmed also the presence of hexulose, OH and/or OOH moieties
in these structures (Table 1).
3.4. MS analysis of acidic OS (F1)
The other oxidation products correspondent to the addition of
three and four oxygen atoms and with the loss of 2 Da (+nO ꢂ 2 Da)
have been analyzed and the presence of several isomers bearing an
The acidic OS recovered in F1 after separation by LEX/SEC
chromatography were identified by ESI-MS (Supplementary
Fig. 2, Table 2). The most abundant ions were observed at m/z

2
608
J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
(
B) Man Gal
2
(A) Man3
5
25.2
363.1
100
100
3
63.1
5
25.2
3
65.1
3
65.2
95.2
3
45.1
3
3
95.2
557.2
m/z
2
03.1
277.0
203.1 275.1 3
557.2
1
85.1
217.0
465.2
465.2
03.1
0
1
0
m/z
00 150 200 250 300 350 400 450 500 550
100 150 200 250 300 350 400 450 500 550
GalIns
m/ z 185
Gal
OOH
Man
OOH m/ z 363
m/ z 203
OOH m/ z 363
O
m/ z 395
m/ z 201
m/ z 397
OOH m/ z 365
O
Man
O
Man
Man
O
O
ManIns
Man
O
ManIns
O
Man
m/ z 395
Manins
O
Man
m/ z 217
m/ z 185
m/ z 379
m/ z 217
m/ z 379
m/ z 217
Gal
O
OOH
m/ z 217
OOHm/ z
OOH
m/ z 365
m/ z 203
363
m/ z 201
m/ z 395
m/ z 363
Man
O
Man
O
ManIns
ManIns
O
Man
O
Man
Man
O
ManIns
m/ z 183
m/ z 361
m/ z 217
m/ z 379
+
Figure 5. MS/MS Spectra of the ion at m/z 557, [Hex
3
ꢂ2 Da+O+Na] , detected in the neutral fractions F2 for the oxidized Man
3
(A) and Man
2
Gal (B). Manins = Man ꢂ 2 Da.
5
43 and 541, assigned to the ions [Hex
3
+O+Na]+ and [Hex
3
+-
mannonic acid after hydrolysis of the corresponding lactone
(Scheme 2). The structure of this oxidation product (ion at m/z
+
37
Oꢂ2 Da+Na] , respectively. The acidic character of these ions sug-
gests the presence of a carboxylic group at C1 of the reducing
end unit or at C6 of any hexose residue (Scheme 2). Ions at m/z
other than with difference of 16 Da (+O) were also observed and
should be formed by the introduction of additional hydroxy groups
543) was confirmed by the analysis of the ESI-MS/MS spectra for
both trisaccharides (Fig. 6) tough some differences in MS/MS spec-
tra patterns of two trisaccharides were observed.
Both spectra showed the loss of one and two Hexres, with forma-
tion of the ions at m/z 381 and 219, respectively. The ion at m/z 219
correspond to the sodium adduct of the mannonic acid. The oxida-
(
Table 2), similarly to that as it was observed for the neutral oxida-
tion products (Table 1). Several acidic OS were detected at m/z 513,
11, and 483 that can be attributed to OS with an acidic unit con-
5
3
tion product arisen from Man shows in ESI-MS/MS spectrum the
taining five and four carbons, possibly resultant from oxidative
cleavages of the sugar residue at the reducing end. Acidic OS with
one or two sugar units were identified (Table 2). These results are
in tune with those reported for the acidic OS formed from galacto-
mannans upon coffee roasting.13
ion at m/z 347 (Fig. 6), formed by the loss of mannonic acid residue
(ꢂ176 Da). No loss of 180 Da from the molecular ion and no ion at
+
m/z 203, [Hex+Na] were observed, confirming the modification of
the reducing terminal hexose unit. The ion at m/z 497 was formed
by the loss of 46 Da (–HCCOH), showing the presence of a carbox-
ylic acid group in the molecule. On the other hand, since in the oxi-
+
The oxidation products with [M+Na] ions at m/z 543, 541 513
and 483 were analyzed by ESI-MS/MS in order to confirm their
structures.
2
dation product from GalMan the mannonic acid is linked to Gal,
no loss of the mannonic unit was observed (absence of the ion at
m/z 347).
Ions correspondent to the acidic OS with mannonic acid and
with one introduced oxygen atom is observed at m/z 559. As it
was shown for the neutral oxidation products, several oxidation
structures emerging diverse combinations of positioning of
COOH/OH groups and scission products could be found among
derivatives containing the mannonic acid residue.
3
.4.1. ESI-MS/MS of acidic oxidation products in C1
The formation of OS containing mannonic acid at the reducing
Å
end is a common product of oxidation with HO induced by proton
Å
abstraction at C-1 of the reducing end. Further reaction with HO
gives primarily the
D-manno-1,5-lactone derivative, producing
Table 2
3
Oxidized acidic derivatives of Man and Gal Man2 detected by ESI-MS and MS/MS
mass spectrometry and respective m/z values of the [M+Na] ions and possible
identification
3
.4.2. ESI-MS/MS of acidic oxidation products at C6
The formation of uronic acids is a common pathway in the
+
HOÅ induced oxidation of carbohydrates in the presence of
oxygen.
tion with HO at C6 of the carbohydrate followed by further
one-electron oxidation to hexodialdose that in turn oxidized to
uronic acid. Oxidation at C6 with formation of mannuronic acid
3
Man /Gal Man
2
n = 0
n = 1
14,15,39
Uronic acids are formed after the proton abstrac-
Å
OS with mannonic acid (ox in C1) + nO
OS with hexuronic acid(ox in C6) + nO
OS with pentonic acid
OS with penturonic acid
OS with erythronic acid
543
541
513
511
483
559
557
527
2
(or galacturonic in the case of GalMan ) was observed and
confirmed due to the formation of the ion at m/z 541, as one
of the major ions in F1 of both studied disaccharides. The
ESI-MS/MS spectra of the ion at m/z 541 are shown in Figure
Depolymerization
DP2
OS with mannonic acid (ox in C1)
OS with hexuronic acid(ox in C6)
381
379
2
7. In the case of GalMan , the formation of a galacturonic acid
DP1
Mannonic acid
Mannuronic acid/galacturonic acid
can also occur.
219
217
The MS/MS spectra of the ion at m/z 541 show the loss of one
Hexres, with formation of the ion at m/z 379. However, in the

J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
2609
(
a)
(b)
COOH
OH
CH2OH
CH2OH
O
OH
O
OH
+
-
H2O
H2O
COOH
OH
OH
OH
OH
O
OH
HO
HO
HO
(I)
(II)
Scheme 2. Proposed structures for the acidic residues in (a) C6 (mannuronic acid) and (b) C1 (mannolactone (I) in equilibrium with the mannonic acid (II)).
(
A) Man3
(
B) Man Gal
2
381.1
3
47.1
3
1
00
100
81.1
2
19.1
2
19.0
1
21.0
64.9
543.1
m/z
5
43.2
1
278.9 305.1
300
363.1
4
97.2
185.0
0
1
m/z
0
00
200
400
500
100 150 200 250 300 350 400 450 500 550
m/ z 381
m/ z 219
m/ z 185
Gal
Man
O
Man
O
ManA-C1
m/ z 381
O
m/ z 185
m/ z 347
m/ z
381
Man
O
ManA-C1
m/ z 185
Figure 6. ESI-MS/MS spectra of the ion at m/z 543, detected in acidic fractions F1, and corresponding to an acidic oxidation product with the acid in C1 detected for the
oxidized Man
3
2
and Man Gal. ManA-C1 represents the mannonic acid due to oxidation in C1 with formation of a carboxylic acid in the terminal position.
(
A)Man3
(
B) Man Gal
2
3
65.1
3
365.1
3
1
00
100
2
03.1
347.1
79.1
79.1
5
41.2
1
85.1
3
45.1
541.1
2
31.1
259.1
495.2
07.2 421.2
203.1
1
85.0
305.1
481.1
4
245.1
407.1
1
43.0
0
1
m/z
0
m/z
00 150 200 250 300 350 400 450 500 550
100 150 200 250 300 350 400 450 500 550
m/z 199
GalA-C6
m/z 379
m/z 217
m/z 379
m/z 203
m/z 379
O
Man
O
Man
O
ManA-C6
Man
O
ManA-C6
O
Man
m/z 365
Man
O
Man
m/z 185
m/z 347
m/z 185
m/z 361
m/z 185
m/z 365
m/z 203
m/z 185
Gal
ManA-C6
O
Man
O
Man
m/z 365
O
m/z 379
m/z 199
m/z 361
ManA-C6
O
Man
m/z 199
3 2
Figure 7. MS/MS Spectra of the ion at m/z 541 detected in acidic fractions F1 for the oxidized Man and Man Gal.
spectrum of Man
only, as expected, due to the formation of [ManA+Na] at m/z
17 upon oxidation at the reducing end. High relative abundance
of the ion at m/z 541 indicated that oxidation at C6 of Gal with
formation of galacturonic acid in GalMan should be a predom-
inant oxidation route. This could be due to steric effects that
turn the 6-linked sugar residue more prone to oxidation by the
hydroxyl radical. The oxidation of Man residue from the non-
reducing end was suggested because of detection a small ion
3
the combined loss of two Hexres was observed
at m/z 199 and in the middle Man residue and by the presence
of characteristic ions at m/z 361 and 185, as depicted in Figure
7. The detected abundant ions at m/z 203 and 365 indicated that
+
2
3
even in the case of Man the oxidation of reducing end seems to
2
occur in less extent than the oxidation at C6 (Fig. 7).
OS with mannuronic acid and plus one (m/z 557) and two oxy-
gen atoms (m/z 573) were also observed and analyzed by MS/MS
thus confirming the presence of hydroxy and peroxy derivatives
among oxidized trisaccharides.

2
610
J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
3
.4.3. ESI-MS/MS of acidic oxidation products resultant from
reaction. These products included keto/aldo, carboxyl and peroxyl
hexose cleavage
The most abundant acidic OS, with molecular mass lower than
the starting trisaccharides, were observed at m/z 513, 511, and
derivatives, as well as products formed by oxidative cleavage of
pyranose ring. The oxidative depolymerization has been also con-
firmed. These degradation pathways are briefly summarized in
Scheme 3.
4
83. These products are formed by oxidative cleavage of one hex-
ose unit accomplishing of a terminal acidic group respectively at
C5 and C4. The oxidation product with the ion at m/z 511 contained
an additional hexulose unit. The proposed structures of these
acidic oxidation products were confirmed by ESI-MS/MS and
exemplified in Figure 8, for the ions at m/z 513 and 483. The anal-
ysis of fragmentation pathways confirm that oxidative cleavage oc-
curred at the reducing end mannose unit. Interestingly, OS with
acidic residues were observed in the mannan OS extracted from
roasted coffee beverage, analyzed by mass spectrometry, namely
the ions at m/z 543 and 513, with similar MS/MS spectra with those
obtained in the present study.13
Being a strong oxidation agent with impaired electron (standard
reduction potential from +1.8 to +2.7 V, depending on pH)38 HOÅ
abstract a proton at different carbons in Man3 and GalMan
generating carbohydrate carbon-centered radicals ( -hydroxyalkyl
radicals, except for the abstraction of the hydrogen radical from C5,
2
thus
a
3
9,40
Scheme 3).
This reaction proceeds exceedingly rapidly (rate-
9
ꢂ1 ꢂ1
constant, k = 1–4 ꢁ 10 M
s ) and is rather non-specific though
3
9
stereo dependent. Apparently the last factor is determinant for
Å
the preferential attack of HO at C1 and C6. The fate of the carbohy-
drate radicals is dependent on their location and conditions of Fen-
ton reaction which may be summarized as follows:
Fe2þ=Fe3þ
ꢀ
3H
2
O
2
!
2HO þ O
2
þ 2H
2
O
ð1Þ
3
.5. Concluding remarks
Å
Å
a
-Hydroxyalkyl radical (HO–C <) reacts fast with HO to form
hemiacetal/hemiketal or with O
the corresponding peroxyl radical (Scheme 3). The
9
ꢂ1 ꢂ1 41
The results presented in this work allowed identifying a major
series of oxidation products of mannosyl trisaccharides formed
during oxidation with hydroxyl radical under conditions of Fenton
2
(k = 2 ꢁ 10 M
s
)
to form
-hydrox-
3
9
a
yalkylperoxyl radicals are spontaneously decomposed with release
Acidic OS with pentanoic acid (m/z 513)
(
B) Man Gal
2
(
A) Man3
3
47.1
351.1
100
100
3
51.1
56.3
283.3
513.1
512.5
513.4
2
1
64.9
1
89.1
62.9
3
65.1
76.9 467.2
1
21.0
278.9 305.1
513.2
m/z
189.0
3
52.1
1
63.3
319.0
2
481.1
3
0
1
0
m/z
00 150 200 250 300 350 400 450 500
100 150 200 250 300 350 400 450 500
m/z 165
Gal
O
H
m/z 189
m/z 351
O
m/z 189
OH
OH O
m/z 351
OH
Man
O
Man
O
Man
O
OH
O
m/z 185
m/z 347 m/z 365
OH
OH
Acidic OS with erythronic acid (m/z 483)
(
B)Man Gal
2
(
A) Man3
3
47.1
321.1
100
100
4
83.2
1
59.0
185.1 249.0 279.0
3
21.1
2
78.9
1
21.0 164.9
365.1
465.2
1
80.9
121.0
451.1 483.2
347.0
0
1
m/z
00 150 200 250 300 350 400 450 500
0
m/z
100 150 200 250 300 350 400 450 500
Gal
m/z 185
O
H
m/z 321
m/z 321
m/z 159
Man
O
OH
m/z 321
Man
O
O
O
OH
Man
O
O
m/z 347
OH
m/z 365
OH
m/z 185
Figure 8. MS/MS Spectra of the ions at m/z 513 and 483 detected in acidic fractions F1 for the oxidized Man
pentanoic acid (m/z 513) and a erythronic acid (m/z 483).
3 2
and Man Gal, as identified as OS with, respectively as OS with a

J. Tudella et al. / Carbohydrate Research 346 (2011) 2603–2611
2611
Supplementary data
H
C OH
m/z 527
OH
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.09.011.
•
2
H O
C1
Pentose + H
m/z 497
2
CO
•C OH
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(
(
project PEst-C/QUI/UI0062/2011), Research Unit in Vila Real
POCTI-SFA-3-616) and RNEM by the Foundation for Science and
Technology (FCT) and COMPETE.