Synthesis and structural investigation of a series of mannose-containing oligosaccharides using mass spectrometry

Article abstract of DOI:10.1039/c7ob02723k

A series of compounds associated with naturally occurring and biologically relevant glycans consisting of α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved mass spectrometry (ERMS), and ¹H nucle

Full text of DOI:10.1039/c7ob02723k

Organic &
Biomolecular Chemistry
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Synthesis and structural investigation of a series of
mannose-containing oligosaccharides using mass
spectrometry†
Cite this: Org. Biomol. Chem., 2018,
16, 228
a
S. Daikoku,‡a R. Pendrill,‡b Y. Kanie, a Y. Ito, c G. Widmalm *b and O. Kanie
*
A series of compounds associated with naturally occurring and biologically relevant glycans consisting of
α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved
mass spectrometry (ERMS), and 1H nuclear magnetic resonance spectroscopy. The CID experiments of
sodiated species of disaccharides and ERMS experiments revealed that the order of stability of mannosyl
linkages was as follows: 6-linked > 4-linked ≧ 2-linked > 3-linked mannosyl residues. Analysis of linear tri-
saccharides revealed that the order observed in disaccharides could be applied to higher glycans. A
branched trisaccharide showed a distinct dissociation pattern with two constituting disaccharide ions. The
estimation of the content of this ion mixture was possible using the disaccharide spectra. The hydrolysis
of mannose linkages at 3- and 6-positions in the branched trisaccharide revealed that the 3-linkage was
cleaved twice as fast as the 6-linkage. It was observed that the solution-phase hydrolysis and gas-phase
dissociation have similar energetics.
Received 6th November 2017,
Accepted 5th December 2017
DOI: 10.1039/c7ob02723k
rsc.li/obc
Introduction
In order to elucidate the glycan structure of naturally occurring
oligosaccharide samples, the use of a logically arranged array
of compounds as a data source is considered advantageous.1–3
Although an array of compounds should provide a complete
set of structural data, it is often very difficult to obtain such a
library. A variety of manno-oligosaccharides exist in nature,
some of which are shown in Fig. 1 with symbolic structures.4–7
A mammalian high mannose-type N-linked glycan is com-
posed of nine mannoses with different anomeric configur-
ations and linkage positions, and it plays an important role in
the folding of glycoproteins in endoplasmic reticulum and is
later reconstructed into complex N-glycans in Golgi
apparatus.8–10 The hybrid type of N-linked glycan is associated
with infectious diseases and immunity.11–15 Other forms of
mannose-containing oligosaccharides i.e., glycosylphosphati-
dylinositol anchored glycoprotein,16 and cell wall polysacchar-
ides of Saccharomyces cerevisiae17,18 and Mycobacterium
Fig. 1 Symbolic representations of naturally occurring glycans consist-
ing of multiple mannosyl residues. Symbols are used as suggested.4–7
Individual linkage isomers are shown with distinctive angle
presentations.4
aDepartment of Applied Biochemistry, Tokai University, 4-1-1 Kitakaname,
Hiratsuka, Kanagawa 259-1292, Japan. E-mail: kanie@u-tokai.ac.jp
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
S-106 91 Stockholm, Sweden
cSynthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
smegmatis19 also exist. Surprisingly, when considering α-linked
mannosyl residues, all the possible linkage isomers, namely
mannose (Man) residues linked to 2-, 3-, 4-, and 6-positions of
adjacent Man, exist in nature.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7ob02723k
‡These authors contributed equally.
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Therefore, it is important to discriminate individual struc- The results provide valuable information about the order of
tures. As the amounts of samples obtainable from natural stability of mannosyl linkages, and the fragmentation analyses
sources are often limited, mass spectral analysis is an excellent of individual ions with different linkages were carried out in
technique for structural investigations. In particular, low- detail. We also showed that the analysis of a mixture of ions
energy collision-induced dissociation (CID) and ion mobility obtained from a branched structure is possible. A comparison
mass spectrometry are suitable techniques for the differen- of the gas-phase CID reaction and acid hydrolysis reaction in
tiation of subtle structural differences.1,20,21
solution was also carried out.
Herein, we perform the mass spectrometric analysis of syn-
thetic compounds useful for investigating larger oligosacchar-
ide structures. A series of disaccharides and trisaccharides,
namely, αMan–(1 → 2)–αMan–OMe (1), αMan–(1 → 3)–αMan–
OMe (2), αMan–(1 → 4)–αMan–OMe (3), αMan–(1 → 6)–αMan–
OMe (4), αMan–(1 → 2)–αMan–(1 → 2)–αMan–OMe (5), αMan–
(1 → 2)–αMan–(1 → 3)–αMan–OMe (6), αMan–(1 → 2)–αMan–
(1 → 6)–αMan–OMe (7), and αMan–(1 → 3)–[αMan–(1 →
6)–]αMan–OMe (8) individually representing partial structures
of biologically relevant glycan structures (Fig. 2) were analyzed.
Results and discussion
Oligosaccharide synthesis
The chemical synthesis of D-mannosyl-containing α-(1 → 4)-
linked disaccharide 3 and αMan–(1 → 2)–αMan–(1 → 6)-linked
trisaccharide 7 and its [6-13C]-isotopologue 7c was carried out
via N-iodosuccinimide/silver triflate promoted glycosylation as
the key reaction (Scheme 1). The coupling of donor 10 with
acceptor 9 in the temperature interval of −50 °C to −30 °C pro-
ceeded smoothly to result in the anticipated α-(1 → 4)-linked
disaccharide 11 in good yield (84%).
The subsequent two-step deprotection procedure employing
hydrogenolysis first with palladium hydroxide on carbon as
the catalyst followed by O-debenzoylation under Zemplén con-
ditions resulted in target disaccharide 3 in 55% yield over two
steps. In the synthesis of trisaccharide 7, the O-benzylated
acceptor 12 was glycosylated by the suitably protected manno-
syl donor 13 with an acetyl group installed at O2, thereby facili-
tating neighboring group participation ensuring that an
α-linkage was formed in the disaccharide product; this was fol-
lowed by the removal of the O-acetyl group using sodium
methoxide in methanol, thereby unmasking the alcohol func-
tion to result in 14 in 53% yield over two steps. Disaccharide
14 was subsequently coupled with donor 10 under the same
experimental conditions as in the previous glycosylation step,
which subsequently resulted in trisaccharide 15 in 67% yield.
Fig. 2 Structures of oligomannosides investigated. The individual
mannose residues are designated as a, b, and c by reducing the end ter-
minus for clarity in the molecular structures. Symbolic representations Employing the same two-step deprotection procedure for the
of the structures are also provided.
above disaccharide resulted in trisaccharide 7, albeit with 31%
Scheme 1 Synthesis of oligosaccharides 3, 7 and 7c.
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yield. Synthesis of the corresponding 4-aminophenyl glycoside diagnose the structures. However, the ratio of these cleavages
of 7 was previously described by Khan et al.22 The 13C-isotopo- can be used for the verification, called a “spectral matching
logue 7c, which has been investigated using nuclear magnetic technique.”27,28 The percentages of these glycosyl cleavages are
resonance (NMR) relaxation experiments,23 was synthesized shown in the table in Fig. 3. As evident from the table, these
using the same protocol; the yields of the first glycosylation cleavage reactions are the major process in each case. A charac-
reaction and deprotection steps were, in this case, improved to teristic feature is the preferential methyl glycoside cleavage in
62% and 63%, respectively.
sodiated compound 2. A clear explanation cannot be provided
for this phenomenon but this can be a diagnostic event.
Compounds 3 and 4 in their sodiated forms provided a similar
fragmentation pattern with a preferential formation of the
Structural details of disaccharides obtained from low-energy
CID experiments
In order to understand the structural details of a series of X-ion series. In these fragmentation reactions, the 3,5X1 ion
oligosaccharides, the analysis of the fragmentation process of (m/z = 319) was formed in the spectrum for 3, and 0,2X1 ion
the corresponding precursor ions was carried out. Prior to the (m/z = 245) and 0,3X1 ion (m/z = 275) were characteristic of 4.
investigation, we decided to analyze sodium adducted mole- We also investigated the fragmentation of sodiated isotopo-
cules in the CID experiments, because they are frequently logue 4c [4c + Na+], in which C6 of the reducing terminus man-
observed without any preparative manipulation and they noside was replaced with 13C. A series of product ions pro-
provide data superior to those of proton adducts in energy- duced during the CID of the sodiated compound was observed
resolved mass spectrometry (ERMS).24,25 The common ions to be virtually identical to that of parent 4. This type of
produced during the CID process of the sodiated precursor evidence might be useful in the investigation of pulse-chase
ions obtained for compounds 1–4 are ions with m/z 185 (B1) experiments.29 Although cross-ring cleavages are useful in
and 217 (Y1), each corresponding to the units b and a. (Fig. 3) identifying linkage isomers, an ion with m/z = 319 which could
(Assignment of ion species was done according to Domon be assigned to be the 2,4X1 ion might be used to identify
and Costello.26) A pair of ions with m/z 185 and 217 corres- 4-linked mannoside only.
pond to the cleavage of a glycosidic bond at the non-reducing
We subsequently analyzed the energy dependence of the
end of the mannosyl residue. As these ions are common in all fragmentation reactions of individual sodiated disaccharides.
disaccharides, the m/z values themselves cannot be used to We focus on the decay process of precursor ions during ERMS
Fig. 3 Fragmentation patterns of a series of sodiated disaccharides under CID conditions. The observed fragments are indicated in structures. The
CID experiments were conducted at the end-cap radio frequency amplitude of 0.7 V. The table summarizes the intensities of individual product ions.
The relative ion intensities are indicated by a red-colored scale.
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can be explained by the rotational freedom around the glycosi-
dic linkage compared to the secondary linkages.2
Dissociation of a series of sodiated linear trisaccharides:
c–b–a–OMe
A series of linear trisaccharides individually consisting of three
distinct glycosidic linkages were subsequently analyzed. At a
glance, dehydration ions with m/z = 523 ([M + Na-H2O]+)
observed for 5 only, 3,5A3 ions with m/z = 451 for 6 and 1,5X2
ions with m/z = 467 for 7 are characteristic of the linkage
isomers in the b–a units. Among a variety of product ions
formed during the CID process, the major cleavage pathway in
all the cases was the glycosyl rupture (Fig. 5). However, the
intensities of signals corresponding to individual glycosyl clea-
vages were different.
The sums of intensities for the glycosyl cleavages between
units b and c (intensity (Int.) for [c + Na]+ + Int. [(b–a) + Na]+)
were 55.4% for αMan–(1 → 2)–αMan–(1 → 2)–αMan–OMe (5),
39.4% for αMan–(1 → 2)–αMan–(1 → 3)–αMan–OMe (6), and
72.9% for αMan–(1 → 2)–αMan–(1 → 6)–αMan–OMe (7). The
intensities of signals for the cleavage between units a and b
(Int. m/z 217 + Int. m/z 347 + Int. m/z 329) were 35.9% (5),
24.6% (6), and 11.4% (7). The intensities associated with the
dissociation of the methyl group were zero (not observed) (5),
28.1 (6), and 1.78% (7). Interestingly, the tendency of reducing
the methyl glycoside cleavage in the ion of (6) can be observed
in the CID data for one of the disaccharides, namely αMan–
Fig. 4 Decay curves of a series of sodiated disaccharides obtained via
+
+
+
ERMS experiments. [1 + Na]+
(
), [2 + Na]
), [3 + Na]
■
○
(
□
( ), [4 + Na]
●
(
), and [4c + Na]+ (★).
(Fig. 4). The decay curves indicate the conversion process of
individual precursor ions into product ions under CID con-
ditions, which cannot be obtained via standard MS/MS experi-
ments. The most labile disaccharide appeared to be the
3-linked compound 2 whereas 6-linked 4 was the most stable.
The difference observed between sodiated 4 and its 13C-labeled
counterpart 4c is believed to reflect the mass difference in the
dissociation reaction. The decay curves of 2- (1) and 4-linked
(3) disaccharides were in between these two disaccharides and
showed similar reactivities under the given conditions. A
visual inspection of the decay process of individual precursor
ions in ERMS indicated the order of stability to be: 6-linked >
4-linked ≧ 2-linked > 3-linked disaccharide. It has been
reported that the primary glycosidic linkage is stable, which
Fig. 5 Fragmentation patterns of a series of sodiated trisaccharides under CID conditions. The CID experiments were conducted at the end-cap
radio frequency amplitude of 0.7 V. The table summarizes the intensities of individual product ions. A designated red intensity scale is also shown.
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(1 → 3)–αMan–OMe (2), which shares the same 3-linked disac-
charide structure.
Since the energy dependence of individual ions is quite
complex and reflects various factors, we attempted to consider
the internal energy as a reference point. The order of ion stabi-
lity of a series of sodiated disaccharides observed under the
CID conditions was as follows: 6-linked > 4-linked ≧ 2-linked >
3-linked, as described above. It is difficult to discuss the
underlying mechanism using the data alone. However, as a
series of linear trisaccharides contained the same disaccharide
structure, we surmised that it would be possible to determine
a correlation between the structure and the observed order of
ion stability. Thus, the non-reducing mannosyl residue (unit c)
is considered to be an internal energy reference because the
position and anomeric configuration of the residue are the
same and furthermore, the structural similarity makes it suit-
able to evaluate other inter-mannosyl linkages between units a
and b. Although methyl glycoside was common to all the com-
pounds, it was not considered to be appropriate as an internal
Fig. 6 MS3 experiments of Y-ions produced from a series of sodiated
trisaccharides under CID conditions. The series of product ions for indi-
vidual precursors are virtually identical to those obtained from the
corresponding disaccharide ions at MS2.
energy reference because the residue was closely associated those of the disaccharide library (Fig. 6). According to our pre-
with the cleavage of linkage a–b.
vious results, the Y-ion species are treated as “pure” ions
Thus, we compared the sum of intensities of signals associ- without any structurally isomeric ions, and thus can be used
ated with individual glycosyl cleavages and obtained a ratio a– for the structural identification at different stages of MSn.25
b/b–c to compare the relative strength of mannosyl linkages Therefore, these types of ion species are suitable for structural
present in a structure. The obtained a–b/b–c values were 0.55 identification. The fragmentation products and their intensi-
(5), 0.56 (6), and 0.10 (7). This indicates that the stability order ties were strikingly similar to those of the corresponding disac-
is 6-linked > 2-linked ≥ 3-linked, which is consistent with the charides indicating that the structures of Y-ions produced
order observed for the stability of disaccharides. Moreover, the from trisaccharides can be deduced from those of library di-
CID of an isotope labeled compound 7c, at C6 of reducing saccharides except for the branched compound. In the current
terminus mannoside, resulted in an almost identical fragmen- experiment, using a library of disaccharides, the CID data were
tation pattern with one atom unit shifts in the product ions used to identify the partial structure of a trisaccharide. When
carrying 13C instead of 12C.
we assume that the structure of a trisaccharide is not known,
the partial structure [b–a + Na]+ is determined at the MS3 stage
by comparing a series of data from a library.
Dissociation of the branched trisaccharide: b–(c–)a–OMe
Naturally occurring manno-oligosaccharides often contain a
Regarding the product ion obtained from the branched tri-
branching structure with α(1 → 3)- and α(1 → 6)-linkages saccharide ion, the structure b–(c–)a-OMe (8) contains both 3-
(Fig. 1). Therefore, compound 8 would serve as a valuable and 6-linked mannosyl residues. As mentioned above, the frag-
information source. The CID analysis of sodiated αMan–(1 → mentation pattern of the produced ion with m/z 379 was
3)–[αMan–(1 → 6)–]αMan–OMe (8) was carried out (Fig. 5). The similar to that of [2 + Na]+ with some minor ions observed in
fragmentation pattern resembled that of sodiated 7 having a the fragmentation of [4 + Na]+, indicating that the formed
6-linkage, rather than that of 6 having a 3-linkage, observed Y-ion may be a mixture of these two product ions.
with an ion at m/z 379 corresponding to the Y-ion structure
We thus carried out an ERMS experiment on the produced
(b–a unit) that lost a non-reducing mannosyl residue (c unit) Y-ion corresponding to the partial structure of trisaccharide 8,
resulting in a prominent product ion. This indicates that the and compared it with those of authentic disaccharides,
relative strengths of glycosidic linkages at the 3- and 6-posi- namely 2 and 4. As shown in Fig. 7, the decay curve obtained
tions are quite different, as clearly shown in the dissociation for the degradation of the Y-ion generated from [8 + Na]+ was
of disaccharides, where 6-linked mannose is more stable observed in between those of individual decay curves of [2 +
(Fig. 4). Therefore, the glycosyl cleavage at the 3-position will Na]+ and [4 + Na]+. This suggested that the Y-ion was a mixture
predominantly occur in sodiated 8.
of these two ions, which is consistent with the result of MS/MS
experiments.
We subsequently estimated the contents of [2 + Na]+ and
[4 + Na]+ in the Y-ion mixture obtained from the branched tri-
MS/MS results of a series of product ions with m/z 379
produced from sodiated trisaccharides
The CID experiments of sodiated disaccharide ions for unit saccharide [8 + Na]+. Accordingly, the ion intensities of individ-
b–a (m/z 379) produced from a series of sodiated trisaccharides ual product ions at the resonant frequency amplitude of 0.7 V
were carried out at the MS3 stage to investigate whether the were used. Consequently, it was estimated that the Y-ion pro-
product ions are identical to the fragmentation patterns of duced was a mixture of [2 + Na]+ (37%) and [4 + Na]+ (63%)
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Fig. 7 Comparison of decay curves of sodiated disaccharides (2 and 4)
+
and Y-ion obtained from sodiated 8 in ERMS. [2 + Na]+
and Y-ion (m/z 379) produced by CID of [8 + Na]+ (◐).
(
(
○
), [4 + Na]
●
),
based on the assumption that there was no loss of ions during
the CID (Fig. 8). Subtle differences could be observed in these
spectra, which were attributed to the different populations of
differently sodiated ion species (different coordination sites).
Oligosaccharide hydrolysis monitored using NMR
At this stage, we considered whether the gas phase CID reac-
tions were related to the hydrolysis reactions. We performed
acidic hydrolyses of compounds 2, 4, and 8 to examine the
1
reactivities of 3- and 6-linked glycosidic bonds under H NMR
monitoring (Fig. 9a). In trisaccharide 8, substitution by α-D-
mannopyranosyl groups occurred at secondary and primary
alcohol groups, i.e., at O3 and O6, respectively. In order to
study the relative stability to hydrolysis at these glycosidic lin-
kages, the trisaccharide was subjected to acid hydrolysis at an
elevated temperature (1 M trifluoroacetic acid, D2O, 70 °C) and
monitored using 1H NMR spectroscopy. As a reference, both
the α(1 → 3)- and α(1 → 6)-linked disaccharides 2 and 4,
respectively, were investigated under corresponding experi-
mental conditions. The progress of the hydrolysis reaction was
Fig. 9 NMR analysis of hydrolysis reactions. (a) Time-course for acid
hydrolysis (1 M TFA in D2O at 70 °C) of the 3,6-disubstituted tri-
saccharide 8, the constituent disaccharides 2 and 4, α-(1 → 3)- and
α-(1 → 6)-linked, respectively, analyzed using 1H NMR spectroscopy with
□
◇
(
) 2, ( ) 4, (+) 2 and 8, (×) 4 and 8, whose fitted data are represented by
solid black, solid red, dashed black, and dashed red lines, respectively;
(b) calculated time-course for the acid hydrolysis of the 3,6-di-
substituted trisaccharide 8 (cyan). The hydrolysis intermediates, i.e., di-
saccharides 2 and 4, are shown as black and red lines, respectively. The
analyzed by monitoring the disappearance of the 1H reso- sum of 2 and 8 is shown by the dashed black line and the dashed red
line corresponds to the sum of 4 and 8.
nances from anomeric protons of the terminal mannopyrano-
syl groups. While this is straightforward for disaccharides 2
and 4, notably, in trisaccharide 8, the anomeric 1H NMR
chemical shifts at the α(1 → 3)-linkage are degenerate for 2
and 8 and this is also the case for the α(1 → 6)-linkages in
compounds 4 and 8. This degeneracy indicates that in the tri-
saccharide, the hydrolysis of 2 and 8 is monitored at the α(1 →
3)-linkage and in the same way, the hydrolysis reaction for the
α(1 → 6)-linkage is followed, but not differentiated, for com-
pounds 4 and 8. In the disaccharides, the α-D-Manp group
linked to O3 showed t1/2 = 5.3 h, whereas when linked to O6,
t1/2 = 10.9 h. When these data were compared to the apparent
hydrolysis rates in the trisaccharide, t1/2 = 5.4 h and t1/2
=
10.7 h, respectively, it became evident that the reactivity in the
trisaccharide is similar to that of its constituent disaccharides.
The experimentally determined half-lives were used to calcu-
late the evolution of the composition of the trisaccharide and
the two disaccharides over the course of the hydrolysis reaction
of the trisaccharide (Fig. 9b). The hydrolysis of 8 (cyan line in
Fig. 9b) is at its initial stage foremost owing to the cleavage of
the α(1 → 3)-linkage resulting in the rapid formation of disac-
Fig. 8 Estimated MS/MS spectrum of the ion mixture of constituting
disaccharide ions and the observed spectrum.
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charide 4 (red line in Fig. 9b). During the later course of the ments. The results obtained herein may provide information
reaction, consequently, its α(1 → 6)-linkage is cleaved, which for elucidating α-glycosidically linked mannosyl structures and
can be observed from the significantly higher concentration of may, in the future, be extended to other sugar residues with
4 compared to that of 2 (red and black lines in Fig. 9b, gluco- or galacto-configurations and β-linked oligosaccharides,
respectively).
some of which have been studied using the ERMS technique.32
The fact that hydrolysis at the α(1 → 3)-linkage occurred
twice as fast as that at the α(1 → 6)-linkage for the mannose-
containing oligosaccharides when using 1 M trifluoroacetic
acid (TFA) at 70 °C is consistent with the previous results of
hydrolysis rates for α(1 → 3)-linked nigerose, which was hydro-
lyzed 4.5 times faster in 0.1 N hydrochloric acid at 80 °C than
α(1 → 6)-linked isomaltose; furthermore, at 99.5 °C, it was
hydrolyzed 3 times faster.30 Notably, the hydrolysis rates at the
α-D-Manp-linkages mirrored the stability of the mannose oligo-
saccharides when subjected to CID and the propensity to frag-
ment as detected using MS. Although our result may be seen
as partially contradictory to the hydrolysis results of
S. cerevisiae mannan where the order of mannose release is
not precisely controlled, the presence of branching mannoside
such as mannosyl residues with the oligosaccharide attached
to both 2- and 6-positions might be highly reactive due to
steric factors.31 Thus the observed phenomena in this investi-
gation cannot be directly applied to the hydrolysis of higher
glycan structures.
Experimental
General
The oligosaccharides methyl α-D-mannopyranosyl-(1 → 2)-α-D-
mannopyranoside (1), methyl α-D-mannopyranosyl-(1 → 3)-α-D-
mannopyranoside (2), methyl α-D-mannopyranosyl-(1 → 6)-α-D-
mannopyranoside (4), methyl α-D-mannopyranosyl-(1 → 6)-α-D-
[6-13C]mannopyranoside (4c), methyl α-D-mannopyranosyl-
(1 → 2)-α-D-mannopyranosyl-(1 → 2)-α-D-mannopyranoside (5),
methyl α-D-mannopyranosyl-(1 → 2)-α-D-mannopyranosyl-(1 →
3)-α-D-mannopyranoside (6) were available from previous
studies33–35 and methyl α-D-mannopyranosyl-(1 → 3)[α-D-manno-
pyranosyl-(1 → 6)]-α-D-mannopyranoside (8) was purchased
from Carbosynth, Compton, UK.
Synthesis
General. All reagents were used as delivered. Column chrom-
atography was performed manually on silica gel with a pore
size of 60 Å or by using a Biotage Isolera flash purification
system with KP-Sil snap chromatography cartridges. TLC was
carried out on silica gel 60 F254 (20 × 20 cm, 0.2 mm thick-
Conclusions
A series of synthetic disaccharides and trisaccharides consist- ness), and monitored with either UV light 254 nm or sulfuric
ing of α-linked mannopyranoside were used to investigate gas- acid 8%. NMR spectra for characterization were recorded at
1
phase CID reactions and acid hydrolyses. Analyses data of all 27 °C on spectrometers operating at a H frequency of 400 or
the possible linkage-isomers of α-mannosyl disaccharides were 500 MHz, except for compounds 3 and 7 that were recorded at
used in the analyses of trisaccharides. As the dissociation reac- 24 °C and 25 °C, respectively. The NMR chemical shifts are
1
tions of ions of trisaccharides produce their partial structures reported in ppm and for H referenced to TMS, sodium 3-tri-
including disaccharide equivalent ions called Y-ions, it was methylsilyl-(2,2,3,3-2H4)-propanoate (TSP), both set to 0 ppm,
confirmed that individual trisaccharides contained the corres- or the residual CHCl3 solvent peak at 7.26 ppm as an internal
ponding disaccharides. This indicates that an array of low-level standard; for 13C the chemical shifts were referenced to 1,4-
glycans is powerful in investigation higher glycan structures. dioxane in D2O, 67.40 ppm, using an external standard or
The linkage isomers of these constituting disaccharides could internally to the CDCl3 solvent signal at 77.16 ppm. For com-
be discriminated using low-energy CID experiments where the pounds 3 and 7 1H chemical shifts and JHH coupling constants
patterns of a series of produced product ions were different. were refined from 1D 1H NMR spectra using a NMR spin simu-
Individual structures could also be differentiated based on lation methodology.36 High resolution mass spectra were
ERMS. The analysis of linear trisaccharides revealed that the recorded on a Bruker Daltonics micrOTOF spectrometer in
order of dissociation of the linkage isomers was the same as positive mode.
that of disaccharides when a common mannosyl cleavage was
Methyl 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1 → 4)-
used as the internal energy reference. Furthermore, a 3- and 2,3,6-tri-O-benzyl-α-D-mannopyranoside (11). Methyl 2,3,6-tri-
6-branched trisaccharide showed a distinctive dissociation O-benzyl-α-D-mannopyranoside (9)37,38 (62.3 mg, 134.1 µmol)
pattern producing a lower number of product ions. As the and ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-α-D-mannopyranoside
branched compound produced two constituting disaccharide (10)39–41 (128.9 mg, 201.2 µmol) were dissolved in DCM (1 mL)
ions, the estimation of their population in the ion mixture was under an argon atmosphere and stirred with 4 Å molecular
possible using the individual spectra of the disaccharides. sieves. After cooling the solution to −50 °C, NIS (60.0 mg,
Furthermore, the reaction progress of the hydrolytic glycosyl 268.2 µmol) was added followed by a catalytic amount of
cleavages of 3- and 6-linked disaccharides and the branched AgOTf. The temperature was allowed to rise to −30 °C over
trisaccharide was monitored using NMR, and compared with 45 min after which TLC indicated completion. After quenching
gas-phase dissociation reactions. The results indicated that the with Et3N (a few drops), the mixture was filtered through Celite
cleavage-reaction rates mirrored those observed in CID experi- and the solvent removed under reduced pressure. Purification
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using silica column chromatography (gradient, toluene → the reaction mixture turned deep red and was quenched with
toluene/EtOAc: 20/1) yielded title compound 11 (117 μg, Et3N. After filtering through Celite, diluting with DCM and
0.112 µmol) in 84% yield. 13C-NMR (CDCl3): δ = 55.1 (OMe), washing with sat. Na2S2O3 (aq.) and brine, the organic phase
62.9–80.2 (10 C2–C6, 3 BnCH2), 98.9, 99.4 (2 anomeric), was dried with MgSO4 and the solvent was removed under
127.6–138.4 (42 aromatic), 165.0–166.2 (4 CO). 1H-NMR reduced pressure. The resulting brown oil was applied to a
(CDCl3): δ = 3.41 (3H, s, OMe), 3.75 (1H, dd, J = 1.9, 3.1 Hz, silica column (6 : 1 toluene : EtOAc) giving methyl 2-O-acetyl-
H2), 3.82–3.91 (2H, m, H6), 3.92 (1H, m, H5), 4.01 (1H, dd, J = 3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1
→
6)-2,3,4-tri-O-
3.1, 9.2 Hz, H3), 4.22 (1H, dd, J = 3.8, 12.2 Hz, H6′pro-R), 4.27 benzyl-α-D-mannopyranoside in 58% yield (632 mg,
(1H, dd, J = 9.2, 9.2 Hz, H4), 4.29 (1H, ddd, J = 2.5, 3.8, 10.0 0.673 mmol). This compound (629 mg, 0.670 mmol) was then
Hz, H5′), 4.40 (1H, dd, J = 2.5, 12.2 Hz, H6′pro-S), 4.56–4.71 dissolved in MeOH and NaOMe was added until pH ≈ 9. After
(6 H, m, PhCH2), 4.79 (1H, d, J = 1.9 Hz, H1), 5.64 (1H, d, J = 21 hours, Dowex-50 (H+-form) was added prior to filtration and
1.9 Hz, H1′), 5.79 (1H, dd, J = 1.9, 3.2 Hz, H2′), 5.82 (1H, dd, J = removal of the solvent under reduced pressure. After purifi-
3.2, 10.0 Hz, H3′), 6.02 (1H, dd, J = 10.0, 10.0 Hz, H4′), cation on silica gel (toluene/EtOAc: 7/3) product 14 45 was
7.02–8.13 (35H, aromatic). ESI-MS: m/z [M + Na]+ calc. for obtained in 53% yield over two steps (547 mg, 0.610 mmol).
C62H58NaO15 1065.3668, found 1065.3681.
13C-NMR (CDCl3): δ = 54.74 (OMe), 66.4–80.2 (10 C2–C6, 6
Methyl α-D-mannopyranosyl-(1 → 4)-α-D-mannopyranoside PhCH2), 98.9 (C1, 1JC1,H1 = 169.8 Hz), 99.7 (C1′, 1JC1′,H1′ = 170.9
(3). Methyl 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1 → 4)- Hz), 127.6–128.5 (30 aromatic), 137.9–138.6 (6 i-Bn). 1H-NMR
2,3,6-tri-O-benzyl-α-D-mannopyranoside
(11)
(70.8
mg, (CDCl3): δ = 2.32 (1H, d, J = 2.9 Hz, HO2′), 3.26 (1H, s, 3H,
67.9 µmol) was dissolved in an equal mixture of EtOAc and OMe), 3.57–4.93 (27H, H1, H2–H6, H2′–H6′, 6 PhCH2), 5.07
EtOH (5 mL) and a catalytic amount of Pd(OH)2 was added fol- (1H, d, J = 1.8 Hz, H1′), 7.13–7.39 (30H, aromatic). ESI-MS: m/z
lowed by a drop of HCl (37% aq.). The mixture was placed [M + Na]+ calc. for C55H60NaO11 919.4028, found 919.4029.
under a hydrogen atmosphere (100 psi) and stirred for
30 hours. After filtering through Celite and rinsing with EtOH, 3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1
Methyl 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1 → 2)-
6)-2,3,4-tri-O-
→
the solvent was removed in vacuo. The resulting debenzylated benzyl-α-D-mannopyranoside (15). A solution of methyl 3,4,6-
material was dissolved in MeOH (10 mL) and NaOMe was tri-O-benzyl-α-D-mannopyranosyl-(1 → 6)-2,3,4-tri-O-benzyl-α-D-
added until the solution was slightly basic (pH ≈ 9). After stir- mannopyranoside (14) (43.3 mg, 48.2 µmol) and ethyl 2,3,4,6-
ring for 24 hours, Dowex-50 (H+-form) was added followed by tetra-O-benzoyl-1-thio-α-D-mannopyranoside (10) (38.7 mg,
filtration, rinsing with MeOH and evaporation of the solvent. 60.4 µmol) in DCM (1 mL) was stirred with molecular sieves
The resulting crude product was purified on a C18 reverse under an argon atmosphere while being cooled to −30 °C. NIS
phase cartridge, eluting with H2O. After lyophilization, the (31.6 mg, 140.4 µmol) was added followed by a spatula tip of
material was purified using gel-permeation chromatography AgOTf. After 30 min, the color had turned to dark red and
on an ÄKTApurifier equipped with a SuperdexTM 30 column, Na2S2O3 (sat. aq.) was added. The solution was filtered
prep. grade gel (GE Healthcare, Uppsala, Sweden) using water through a plug of Celite and extracted thrice with toluene. The
containing 1% n-butanol as the eluent. The title compound 3 combined organic phase was washed with water, followed by
(13.4 mg, 37.6 µmol) was obtained in 55% yield. 13C-NMR brine, dried with MgSO4 and concentrated under reduced
(D2O): δ = 55.5 (OMe), 61.7 (C6′), 61.9 (C6), 67.4 (C4′), 71.1 pressure. The crude product was purified on a column of silica
(C2′), 71.1 (C3′), 71.2 (C2), 71.9 (C3), 71.8 (C5), 74.5 (C5′), 75.1 (gradient, toluene → toluene/EtOAc: 13/1) giving product 15 in
1
1
(C4), 101.5 (C1, JC,H = 171 Hz), 102.3 (C1′, JC,H = 172 Hz). 67% yield (48.3 mg, 32.2 µmol). 13C-NMR (CDCl3): δ = 54.8
1H-NMR (D2O): δ = 3.42 (3H, s, OMe), 3.68 (1H, dd, JH4′,H5′
=
(OMe), 62.9–80.3 (15 C2–6, 6 PhCH2), 98.9–99.8 (3 anomeric),
3
10.0 Hz, H4′), 3.68 (1H, ddd, JH5′,H6′pro-R = 5.7 Hz; JH5′,H6′pro-S 127.5–138.8 (60 aromatic), 165.2–166.3 (4 CO). 1H-NMR
3
3
3
3
= 2.1 Hz, H5′), 3.70 (1H, dd, JH5,H6pro-R = 5.8 Hz; JH5,H6pro-S
2.1, H5), 3.77 (1H, dd, JH6′pro-R,H6′pro-S = –12.3 Hz, H6′pro-R), PhCH2), 4.81–5.24 (3H, 3 × d, J = 1.8, 1.9, 1.9 Hz, H1, H1′,
3.81 (1H, dd, JH3′,H4′ = 9.4 Hz, H3′), 3.81 (1H, dd, JH6pro-R, H1″), 4.88 (1H, d, J = 11.1, PhCH2), 4.92 (1H, d, J = 11.0 Hz,
H6pro-S = –12.1 Hz, H6pro-R), 3.82 (1H, dd, 3JH4,H5 = 10.0 Hz, H4), PhCH2), 5.93 (1H, dd, J = 1.9, 3.2 Hz, H2″), 5.99 (1H, dd, J =
=
(CDCl3): δ = 3.26 (3H, s, OMe), 3.59–4.76 (25H, 15 H2–6, 10
2
3
2
3
3.90 (1H, dd, H6′pro-S), 3.91 (1H, dd, JH2,H3 = 3.6 Hz, H2), 3.91 3.2, 10.1 Hz, H3″), 6.20 (1H, dd, J = 10.1, 10.1 Hz, H4″),
(1H, dd, H6pro-S), 3.92 (1H, dd, JH3,H4 = 9.1 Hz, H3), 4.06 (1H, 7.04–8.17 (50H, aromatic). ESI-MS: m/z [M + Na]+ calc. for
3
3
3
dd, JH2′,H3′ = 3.3 Hz, H2′), 4.78 (1H, d, JH1,H2 = 1.5 Hz, H1), C89H86NaO20 1497.5605, found 1497.5589.
5.25 (1H, d, JH1′,H2′ = 1.9 Hz, H1′). ESI-MS: m/z [M + Na]+ calc.
Methyl α-D-mannopyranosyl-(1 → 2)-α-D-mannopyranosyl-
6)-α-D-mannopyranoside (7). Methyl 2,3,4,6-tetra-O-
2)-3,4,6-tri-O-benzyl-α-D-
3
for C13H24NaO11 379.1211, found 379.1213.
Methyl 3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1 → 6)-2,3,4- benzoyl-α-D-mannopyranosyl-(1
(1 →
→
tri-O-benzyl-α-D-mannopyranoside (14). A solution of methyl mannopyranosyl-(1 → 6)-2,3,4-tri-O-benzyl-α-D-mannopyrano-
2,3,4-tri-O-benzyl-α-D-mannopyranoside (12)37 (538 mg, side (15) (114.8 mg, 77.7 µmol) was dissolved in EtOAc : EtOH
1.16 mmol) and ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-α-D- (1 : 1, 5 mL) and Pd(OH)2/C (spatula tip) as well as a drop of
mannopyranoside (13)42–44 (778 mg, 1.50 mmol) was stirred in HCl were added (37% aq.). The mixture was stirred under H2
DCM (15 mL) with molecular sieves at −30 °C. NIS (311 mg, (110 psi) overnight before being filtered through Celite and the
1.38) and AgOTf (spatula tip) were added. After 15 minutes, solvent being evaporated. The residue was dissolved in MeOH
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and NaOMe was added until pH ≈ 9 before stirring overnight. m/z [M + Na]+ calc. for C8813CH86NaO20 1498.5638, found
Dowex-50 (H+-form) was added and stirred for a few minutes 1498.5584.
before filtering and removing the solvent under reduced
Following the same procedure as that for the unlabeled
pressure. The crude material was applied to a C-18 reversed- compound, methyl 2,3,4,6-tetra-O-benzoyl-α-D-mannopyrano-
phase cartridge eluting with H2O and lyophilized before being syl-(1 → 2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1 → 6)-2,3,4-
purified by gel-permeation chromatography on an tri-O-benzyl-α-D-[6-13C]mannopyranoside (15c) (120.8 mg,
ÄKTApurifier equipped with a SuperdexTM 30 column, prep. 81.8 µmol) gave the title compound 7c in 63% yield (26.7 mg,
grade gel (GE Healthcare, Uppsala, Sweden), using water con- 51.3 µmol). ESI-MS: m/z [M + Na]+ calc. for C1813CH34NaO16
taining 1% n-butanol as the eluent. Compound 7 was obtained 542.1773, found 542.1771.
in 31% yield (12.7 mg, 24.4 µmol). 13C-NMR (D2O): δ = 55.46
(OMe), 61.60 (C6′), 61.79 (C6″), 66.43 (C6), 67.19 (C4), 67.54
(C4″), 67.59 (C4′), 70.55 (C2), 70.63 (C2″), 70.91 (C3′), 70.99
(C3″), 71.37 (C5), 71.42 (C3), 73.43 (C5′), 73.91 (C5″), 79.36
Mass spectrometric analysis of glycans
Instrumentation and data collection. Samples of disacchar-
ides were analyzed using a quadrupole ion trap mass spectro-
meter (QIT-MS) coupled with an electrospray interface
(Amazon ETD) (Bruker Daltonics, GmbH, Bremen, Germany).
Samples dissolved in MeOH (ca. 0.1 μmol mL−1) were intro-
duced into the ion source via infusion (flow rate, 120 μL h−1).
The parameters for the analysis were: (1) dry temperature:
200 °C, (2) nebulizer gas (N2): 10 psi, (3) dry gas (N2): 4.0
L min−1, (4) scan range: m/z 50–750, (5) compound stability:
100%, (6) ion charge control, on/target, 200 000, (7) maximum
acquisition time: 200 ms, (8) average: 5 spectra, and (9)
polarity, positive. In our MSn experiments, the end cap rf
amplitude was raised by 0.02 V increments until the precursor
ion could no longer be detected. Only the end cap rf amplitude
was controlled during the CID experiment. The He pressure
was 4.86 × 10−6 mbar and the CID time was 40 ms. Averages of
15 spectra were used for CID experiments. Isotopic peaks with
[Ii + 1] and [Ii + 2], where In indicates a fragment ion, were
included in the calculations. For the isolation of a product ion,
m/z 2 (w = 2) were isolated and subjected to the CID experi-
ments to include isotopes. Standard MS/MS spectra are the
extracts of this ERMS at a designated amplitude.
1
(C2′), 98.65 (C1′, JC′,H′ = 172 Hz), 101.71 (1JC,H = 171 Hz, C1),
1
103.02 (1JC″,H″ = 171 Hz, C1″). H-NMR (D2O): δ = 3.40 (3H, s,
3
OMe), 3.62 (1H, dd, JH4″,H5″ = 10.0 Hz, H4″), 3.69 (1H, ddd,
3
3JH5′,H6′pro-R = 5.7 Hz; JH5′,H6′pro-S = 2.0 Hz, H5′), 3.69 (1H, dd,
3
3JH4′,H5′ = 9.3 Hz, H4′), 3.72 (1H, dd, JH4,H5 = 9.9 Hz, H4), 3.73
2
(1H, dd, JH6″pro-R,H6″pro-S = –12.3 Hz, H6″pro-R), 3.74 (1H, dd,
3
3
3JH3,H4 = 9.4 Hz, H3), 3.74 (1H, ddd, JH5,H6pro-R = 4.7 Hz; JH5,
2
= 1.6 Hz, H5), 3.75 (1H, dd, JH6pro-R,H6pro-S = –11.7 Hz,
H6pro-S), 3.76 (1H, dd, JH6′pro-R,H6′pro-S = –12.2 Hz, H6′pro-R),
3.77 (1H, ddd, JH5″,H6″pro-R = 6.6 Hz; JH5″,H6″pro-S = 2.2 Hz,
H5″), 3.84 (1H, dd, JH3″,H4″ = 9.7 Hz, H3″), 3.89 (1H, dd,
H6″pro-S), 3.89 (1H, dd, H6′pro-S), 3.93 (1H, dd, JH2,H3 = 3.4 Hz,
H2), 3.95 (1H, dd, JH3′,H4′ = 9.6 Hz, H3′), 3.96 (1H, dd,
H6pro-S
2
3
3
3
3
3
3
H6pro-R), 4.01 (1H, dd, JH2′,H3′ = 3.4 Hz, H2′), 4.07 (1H, dd,
3
3JH2″,H3″ = 3.4 Hz, H2″), 4.74 (1H, d, JH1,H2 = 1.7 Hz, H1), 5.02
3
3
(1H, d, JH1″,H2″ = 1.8 Hz, H1″), 5.14 (1H, d, JH1′,H2′ = 1.8 Hz,
H1′). ESI-MS: m/z [M + Na]+ calc. for C19H34NaO16 541.1739,
found 541.1731.
Methyl α-D-mannopyranosyl-(1 → 2)-α-D-mannopyranosyl-(1
→ 6)-α-D-[6-13C]mannopyranoside (7c). Starting from 2,3,4-tri-
O-benzyl-α-D-[6-13C]mannopyranoside34
(12c)
(120
mg,
Data manipulation. In order to obtain graphs of the ERMS,
the following equations were used. When an ion “IP” produces
a series of product ions, I1, I2, I3,… Ii, the relative ion currents
for individual ions were defined by the equation,
257 µmol) and using the same procedure as that for the
unlabeled compound, methyl 3,4,6-tri-O-benzyl-α-D-mannopyr-
anosyl-(1
→
6)-2,3,4-tri-O-benzyl-α-D-[6-13C]mannopyranoside
(14c) was obtained in 62% yield (143 mg, 159 µmol). ESI-MS:
m/z [M + Na]+ calc. for C5413CH60NaO11 920.4061, found
920.4067. A solution of methyl 3,4,6-tri-O-benzyl-α-D-manno-
i
CI
relC ¼
ꢀ 100
ð1Þ
n
P
P
i
CI
þ
CI
pyranosyl-(1
→
6)-2,3,4-tri-O-benzyl-α-D-[6-13C]mannopyrano-
i¼1
side (14c) (134 mg, 149 µmol) and ethyl 2,3,4,6-tetra-O-
benzoyl-1-thio-α-D-mannopyranoside (10) (120 mg, 184 µmol)
in DCM (2 mL) was stirred with molecular sieves under an
argon atmosphere while being cooled to −30 °C. NIS (67.2 mg,
300 µmol) was added followed by a spatula tip of AgOTf. After
50 min, the color had turned dark red and sat. Na2S2O3 (aq.)
was added. The solution was extracted three times with
toluene, the combined organic phase was washed with brine,
dried over MgSO4 and concentrated under reduced pressure.
The crude product was purified on a column of silica (toluene
where relC indicates the ion current of a given ion among the
i
observed ions in percentage, CI is the observed ion current in
P
focus, and CI is the ion current of a precursor ion. The calcu-
lations were performed using a program we developed with
Excel (Excel 2000 (Microsoft Co.)), which was based on the
DSUM function and programmed to choose a range of isotopes
(w) to be taken into consideration (w = 2 in the experiments).
Hydrolysis of oligosaccharides
→ toluene/EtOAc: 13/1) followed by a second column of silica Acid hydrolysis reactions were carried out in 5 mm NMR tubes
(pentane → pentane/EtOAc: 3/1) yielding methyl 2,3,4,6-tetra- containing compound 2 (1.0 mg), 4 (1.0 mg) or 8 (1.5 mg)
O-benzoyl-α-D-mannopyranosyl-(1
mannopyranosyl-(1
6)-2,3,4-tri-O-benzyl-α-D-[6-13C]manno- progress was monitored by one-dimensional 1H NMR spec-
pyranoside (15c) in 45% yield (99.3 mg, 67.2 µmol). ESI-MS: troscopy at 500 MHz analyzing for the disappearance of perti-
→ 2)-3,4,6-tri-O-benzyl-α-D- using 1 M TFA in D2O (0.5–0.6 mL) at 343.15 K. The reaction
→
236 | Org. Biomol. Chem., 2018, 16, 228–238
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Paper
nent anomeric proton resonances; the approximate degeneracy 13 S. J. Lee, S. Evers, D. Roeder, A. F. Parlow, J. Risteli,
of the anomeric 1H chemical shifts in 8 vs. 2 or 4 was con-
firmed by NMR chemical shift predictions using the CASPER
program.46
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
The authors would like to acknowledge Professor K. F. Aoki-
Kinoshita for her suggestions on symbolic nomenclature. This
work was funded by grants from the Swedish Research Council
(No. 621-2013-4859) and The Knut and Alice Wallenberg
Foundation.
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