Synthesis and MALDI-TOF MS Analysis of Protected Oligosaccharide Components of N-Glycoproteins

Article abstract of DOI:10.1080/07328303.2014.950737

Essential components of N-glycoproteins were synthesized in octyl glycoside form starting from 3-O-allyl-D-glucose derivative. The β-mannosidic linkage was formed by the oxidation reduction method. MALDI-TOF mass spectrometry and its post-source decay (PS

Full text of DOI:10.1080/07328303.2014.950737

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Synthesis and MALDI-TOF MS Analysis of
Protected Oligosaccharide Components
of N-Glycoproteins
Károly Ágostona, Gyöngyi Gyémántb, László Kalmárc, János
Kerékgyártód, Zoltán Szurmaie, Boglárka Dönczőc & András Guttmancf
a RCNS-HAS, Institute of Organic Chemistry H-1117, Budapest,
Magyar Tudósok körútja 2, Hungary
b Institute of Inorganic and Analytical Chemistry, Faculty of
Sciences and Technology, University of Debrecen, Debrecen H-4032,
Debrecen, Egyetem tér 1, Hungary
c Horvath Laboratory of Bioseparation Sciences, University of
Debrecen H-4032, Debrecen, Nagyerdei körút 98., Hungary
d Bio-organic Laboratory, Department of Botany, Faculty of Sciences
and Technology, University of Debrecen H-4032, Debrecen, Egyetem
tér 1, Hungary
e Bio-organic Laboratory, Department of Ecology, Faculty of Sciences
and Technology, University of Debrecen H-4032, Debrecen Egyetem
tér 1, Hungary
f HAS-PE, Translational Research Group, University of Pannonia
H-8200, Veszprem Egyetem ut 10, Hungary
Published online: 26 Aug 2014.
To cite this article: Károly Ágoston, Gyöngyi Gyémánt, László Kalmár, János Kerékgyártó, Zoltán
Szurmai, Boglárka Döncző & András Guttman (2014): Synthesis and MALDI-TOF MS Analysis of
Protected Oligosaccharide Components of N-Glycoproteins, Journal of Carbohydrate Chemistry, DOI:
10.1080/07328303.2014.950737
To link to this article: http://dx.doi.org/10.1080/07328303.2014.950737
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Journal of Carbohydrate Chemistry, 00:1–18, 2014
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2014.950737
Synthesis and MALDI-TOF MS
Analysis of Protected
Oligosaccharide Components
of N-Glycoproteins
1
2
3
´
Ka´ roly Agoston, Gyo¨ngyi Gye´ma´ nt, La´ szlo´ Kalma´ r,
Ja´ nos Kere´kgya´ rto´,4 Zolta´ n Szurmai,5 Bogla´ rka Do¨nczo˝,3
and Andra´ s Guttman3,6
1RCNS-HAS, Institute of Organic Chemistry H-1117, Budapest, Magyar Tudo´sok
ko¨ru´tja 2, Hungary
2Institute of Inorganic and Analytical Chemistry, Faculty of Sciences and Technology,
University of Debrecen, Debrecen H-4032, Debrecen, Egyetem te´r 1, Hungary
3Horvath Laboratory of Bioseparation Sciences, University of Debrecen H-4032,
Debrecen, Nagyerdei ko¨ru´t 98., Hungary
4Bio-organic Laboratory, Department of Botany, Faculty of Sciences and Technology,
University of Debrecen H-4032, Debrecen, Egyetem te´r 1, Hungary
5Bio-organic Laboratory, Department of Ecology, Faculty of Sciences and Technology,
University of Debrecen H-4032, Debrecen Egyetem te´r 1, Hungary
6HAS-PE, Translational Research Group, University of Pannonia, H-8200, Veszprem
Egyetem ut 10, Hungary
Essential components of N-glycoproteins were synthesized in octyl glycoside form start-
ing from 3-O-allyl-D-glucose derivative. The β-mannosidic linkage was formed by the
oxidation reduction method. MALDI-TOF mass spectrometry and its post-source de-
cay (PSD) mode were used for identification and structural elucidation of protected
synthetic oligosaccharides related to N-glycoproteins. Most fragments, identified in the
PSD spectra, were products of the cleavage of glycosidic bonds.
Keywords Oligosaccharide synthesis; β-Mannoside; N-Glycoprotein; MALDI-TOF
MS; Post-source decay (PSD)
Received June 17, 2014; accepted July 29, 2014.
´
Address correspondence to Ka´roly Agoston, RCNS-HAS, Institute of Organic Chem-
istry, H-1117, Budapest, Magyar Tudo´sok ko¨ru´tja 2, Hungary. E-mail: agoston.karoly@
ttk.mta.hu
1

´
K. Agoston et al.
2
INTRODUCTION
Chemical syntheses of the glycan moieties of N-glycoproteins are very impor-
tant because the isolation of such oligosaccharides from natural sources is
often troublesome and usually provides only small amounts of pure glycans.
Synthetic methods can produce highly pure materials in large quantities for
various investigations and offer greater flexibility, especially when the goal is
the production of oligosaccharide analogs of natural N-glycans.
Construction of oligosaccharides with well-defined structures requires the
application of special synthesis strategies including protecting group manipu-
lations and fine-tuned glycosylation approaches. Despite highly sophisticated
NMR techniques, the identification and structural analysis of fully protected
oligosaccharides are rather difficult. MALDI-TOF MS, which was introduced
to carbohydrates by Mock et al.,[1] and its post-source decay (PSD) mode, intro-
duced by Kaufmann,[2] can verify the structure of synthetic protected products
of glycosylation reactions.
The PSD spectra of free, permethylated, or peracetylated oligosaccha-
rides showed that fragments originated mainly from the cleavage of glycosidic
bonds. The first PSD analysis of partially or fully protected oligosaccharides
was published by our group in 2001.[3] Recently, our attention has been fo-
cused on the production of octyl glycosides of glycan components. Octyl spac-
ered oligosaccharides are very useful acceptors in enzymatic glycosylation re-
actions. Because the spacer is sufficiently hydrophobic, the workup procedure
becomes especially simple. The product of the reaction can be easily isolated
by means of reverse-phase C-18 cartridges. Moreover, a carbohydrate array
has been displayed on a microtiter plate using oligosaccharides bearing an
alkyl spacer. The oligosaccharides were bound to the plate based only by phys-
ical attachment. Noncovalent binding between the hydrophobic linker and the
plate resulted in complete retention of the carbohydrate residue on the plate
after aqueous washings.[4]
The structures of our target compounds are α-D-Manp-(1→3)-[α-D-Manp-
(1→6)]-β-D-Manp-[1→Ooctyl] (I), β-D-GlcNAcp-(1→2)-α-D-Manp-(1→3)-[α-D-
Manp-(1→6)]-β-D-Manp-[1→Ooctyl] (II), and β-D-Galp-(1→4)-β-D-GlcNAcp-
(1→2)-α-D-Manp-(1→3)-[α-D-Manp-(1→6)]-β-D-Manp-[1→Ooctyl] (III) in
protected forms.
There are two examples for the preparation of trisaccharide I in the lit-
erature, but both synthetic routes have their disadvantages. The first one is
very simple and nonselective,[5] from the chemical point of view. The other
one is more elegant: forming the β-mannosidic linkage by using the “ulosyl-
bromide” approach.[6] However, the price paid for the elegancy was an unre-
active octyl β-mannopyranoside bearing benzoyl protection groups, which was
not an ideal acceptor for further mannosylations. It is worth noting that a great
number of modified octyl spacered oligosaccharides, as well as compound II,
have been synthesized by Paulsen and his coworkers[7,8] in order to investigate

Protected Oligosaccharide Components of N-glycoproteins
3
glycosyltransferase enzymes. The crucial step, the formation of the β-
mannosidic bonds in these derivatives, was realized by direct mannosylations,
applying heterogeneous promoters.
With the aim of preparing our target compounds, we elaborated on a com-
pletely new reaction sequence strategy. The β-mannosidic linkage was con-
structed by our modified highly selective oxidation reduction protocol.[9] The
stepwise glycosylation strategy was chosen to prepare the target compounds
in which all the required oligosaccharides could be obtained within one re-
action sequence. In this article, we report the chemical synthesis, identifica-
tion, and structural analysis of a few protected oligosaccharides related to N-
glycoproteins as octyl glycosides.
RESULTS AND DISCUSSION
Synthesis of Octyl Spacered Protected Oligosaccharides
The key compound of our synthetic route was octyl 3-O-allyl-2,4-di-O-
benzyl-β-D-mannopyranoside (10). For the formation of the β-mannosidic link-
age in 10, 2,4,6-tri-O-acetyl-3-O-allyl-α-D-glucopyranosyl bromide (1)[10] was
condensed with n-octanol in dichloromethane using HgBr2 as promoter[11] to
give 2 (Sch. 1). The moderate yield can be explained by the fact that under
Koenings-Knorr conditions, the “2-OH” compounds may be formed[12] because
of the rearrangement of the corresponding orthoester. It is likely that the for-
mation of the by-product is typical when the aglycon is highly hydrophobic.[13]
Scheme 1: (a) HgBr2, CH2Cl2, 4 Å molecular sieves, rt, 35%.
Deacetylation of 2 (→3) followed by isopropylidenation resulted in 4. The
oxidation reduction procedure was used to change the configuration of C-2.
Compound 4 was reacted with DMSO and Ac2O. Then, the obtained ulose was
[14]
treated with TBABH4
in THF, giving the manno derivative 5 in a stereos-
elective manner. The formation of the β-mannosidic linkage from β-gluco was
confirmed by NMR spectroscopy. Benzylation of 5 with benzyl bromide in the

´
K. Agoston et al.
4
presence of NaH gave the fully protected mannose derivative 6. Removal of the
isopropylidene acetal with 60% aq. acetic acid afforded the diol 7 (Sch. 2).
Scheme 2: (a) acetone dimethylacetal, pTsOH, 85.7%; (b) i; Ac2O, DMSO, ii; TBABH4, THF,
0◦C, 85%; (c) BnBr, NaH, DMF, 90%; (d) aq 60% AcOH; (e) TrCl, pyridine, DMAP, 0◦C, 70%;
(f) BnBr, NaH, DMF, 83%; (g) aq. 80% AcOH, 92%.
Selective protection of OH-6 with trityl function (→8) using trityl chloride
in pyridine and then benzylation resulted in 9. Removal of the trityl group with
80% aq. acetic acid afforded 10, the glycosyl acceptor for the next step. Conden-
sation of α-acetobromo mannose 11 with 10 in the presence of HgBr2 yielded
disaccharide 12 (74%). The change of the acetyl functions to benzyl persistent
protecting groups gave 13. Compound 13 was converted into 14 with PdCl2,
CuCl in THF, and water (9:1) (Sch. 3). The 1H NMR spectrum of compound 14
clearly showed the absence of the allyl protection of position 3.
Scheme 3: (a) HgBr2, CH2Cl2, 4 Å molecular sieves, rt, 74%; (b) i; NaOMe, MeOH-CH2Cl2 ii;
BnBr, NaH, DMF, 62%; (c) PdCl2, CuCl, THF-water, 64%.
The TMSOTf-catalyzed reaction of the imidate 15[15] with the acceptor 14
resulted in trisaccharide 16, in an acceptable yield (Sch. 4). Zemple´n deacety-
lation of 16 resulted in 17, having only one free OH. The NIS/AgOTf-promoted
coupling reaction of the donor 18 with the acceptor 17 afforded the tetrasac-
charide 19 in good yield (Sch. 5).
Removal of the acetyl function in compound 19 with potassium carbon-
ate in THF/MeOH yielded the tetrasaccharide acceptor 20. Condensation of
α-acetobromo-galactose 21 with compound 20, in the presence of silver triflate
promoter, afforded the target pentasaccharide 22 in an excellent yield (Sch. 6).

Protected Oligosaccharide Components of N-glycoproteins
5
Scheme 4: (a) TMSiOTf, CH2Cl2, 4 Å molecular sieves, –40◦C, 45%; (b) NaOMe,
MeOH-CH2Cl2.
Scheme 5: (a) NIS, AgOTf, MeCN-CH2Cl2, 4 Å molecular sieves, –10◦C, 79%; (b) K2CO3,
MeOH-THF.
Scheme 6: (a) AgOTf, toluene-CH2Cl2, 4 Å molecular sieves, –40◦C, 89%.

´
K. Agoston et al.
6
Figure 1: MALDI-TOF spectra of compound 16.
Structural Analysis of the Oligosaccharides Using MALDI-TOF MS
First, the synthesized protected trisaccharide 16 was investigated. The
MALDI-TOF MS spectra of this compound can be seen in Fig. 1. In the spec-
tra, two fragmentation products were identified. Both impurities were formed
during the MALDI-TOF measurement by losing one or two benzyl groups, re-
spectively. The quasi-molecular ion corresponding to sodium and potassium
cationized species has been observed in monoisotopic resolution using 2,5-
dihydroxybenzoic acid as matrix from spots that contain 1–5 nmol of sample.
The calculated mass accuracy is 0.03%; the resolution is 3073 for Na and 3488
for K adducts.
The recorded PSD spectra of protected tetrasaccharide 19 can be seen
in Fig. 2. The analysis of these fragment ion masses allows obtaining struc-
tural information beyond the mere molecular weight data. The parent ion was
1964.2 Da with the molecular weight accuracy of 0.009%. It was found that all
of the PSD fragment ions were produced by cleavage of glycosidic linkages. We
show the observed peaks and the corresponding species in Table 1 using the
nomenclature of the fragmentation proposed by Domon and Costello.[16]
If the glycosidic bond is cleaved and the charge is retained on the carbo-
hydrate portion, ions containing the aglycone or the reducing sugar unit are
labeled Yj, Zj depending on whether the cleavage results in retention or loss

Protected Oligosaccharide Components of N-glycoproteins
7
Figure 2: MALDI-TOF spectra of compound 19 recorded in PSD mode.
of the linking oxygen atom, respectively, and where j is the number of the in-
terglycosidic bound counted from the aglycone and is numbered 0. Fragments
retaining the charge of the nonreducing terminus are designated as Bi and Ci,
where i represents the number of the glycosidic bound cleaved, counted from
the nonreducing end. The situation is more complicated in the case of branched
oligosaccharides. The carbohydrate portion can be divided into a “core” unit
Table 1: Observed fragments of compound 19
m/z
measured
calculated
Species
Type of fragmentation
926.8
926.12
970.06
C55H66O11Na+
B-C
A-B
Y1α, Y2
969.4
C57H56O12NNa+
C62H73O11Na+
C85H94O18NNa+
C89H101O16Na+
C89H101O16K+
B2
1018.9
1442.1
1451.1
1468.5
1964.2
1981.9
1117.25
1410.67
1449.77
1465.88
1964.33
1980.44
C-D
Y1
Y1α
A-B-C
B-C-D
B-C-D
A-B-C-D
A-B-C-D
Y2
Y2
C119H129O23NNa+
Parent ion
Parent ion
C
119H129O23NK+

´
K. Agoston et al.
8
Figure 3: Definition of the fragments according to Domon and Costello.[16]
and branches called “antennae.” Core refers to the unbranched portion. Each
antenna is represented by Greek letter α, β, γ, with α having the largest molec-
ular weight (Fig. 3).
The PSD spectra verify the expected structure. The intensity of the
1018.9 Da fragment ion indicates that the α mannosidic linkage is much
weaker than the other glycosidic linkages in the PSD fragmentation. The low
intensity of the 926 Da fragment ion could be due to the prerequisite formation
of two independent fragmentations. The MS analysis of the 22 pentasaccharide
was performed in a similar way as trisaccharide 16. The molecular weight data
of compounds 16, 19, and 22 are collected in Table 2.
The structures of the synthesized compounds were verified by NMR spec-
troscopy and MALDI-TOF mass spectrometry. The stereochemistry at the
anomeric position of the mannose units were unambiguously proven by JC1-H1
coupling data. The deprotection of the tri-, tetra-, and pentasaccharide deriva-
tives and the biological studies are in progress. The applied MALDI-TOF mass
spectrometry techniques were used for structural analysis of protected syn-
thetic oligosaccharide libraries prepared by means of the combination of con-
ventional and combinatorial carbohydrate chemistry.[17]
Table 2: The molecular weight data of compounds 16, 19 and 22
m/z
No.
Formula
measured calculated Accuracy% Resolution Matrix
16
16
19
19
22
22
C91H104O17Na
1492.72
1508.11
1964.15
1980.18
2252.68
2269.11
1491.72
1507.72
1964.33
1980.33
2252.58
2268.59
0.03
3073
3488
676
DHB
DHB
DHB
DHB
DHB
DHB
C
C
C
91H104 17
O
K
0.026
0.009
0.002
0.004
0.023
119H125O23N Na
119H125O23N K
695
C131H145O31N Na
623
632
C131 145 31
H
O
N K

Protected Oligosaccharide Components of N-glycoproteins
9
EXPERIMENTAL
General Methods
Optical rotations were measured at rt with a Perkin-Elmer 241 automatic
polarimeter. Melting points were determined on a Kofler hot-stage appara-
tus and were uncorrected. TLC was performed on Kieselgel 60 F254 (Merck)
with detection by charring with 50% aqueous sulfuric acid. Column chro-
1
matography was performed on silica gel 60 (Merck 63–200 mesh). The H
NMR (200, 360, and 500 MHz) and 13C NMR (50.3, 90.54, and 125.76 MHz)
spectra were recorded with Bruker WP-200 SY, Bruker AM-360, and Bruker
1
DRX-500 spectrometers. Internal references: TMS (0.000 ppm for H), CDCl3
(77.00 ppm for 13C). Elemental analyses were performed at the analytical lab-
oratories in Debrecen. The MALDI spectra of oligosaccharide products were
obtained in positive-ion mode using a Bruker Biflex MALDI-TOF mass spec-
trometer equipped with delayed-ion extraction. Desorption/ionization of the
sample molecules was effected with a 337-nm nitrogen laser. Spectra from
multiple (at least 100) laser shots were summarized using 19-kV accelerat-
ing and 20-kV reflectron voltage. External calibration was applied using the
[M+Na]+ peaks of cyclodextrins DP 6–8, m/z: 995, 1157, 1319 Da, respectively.
The spectrum was obtained in 2,5-dihydroxybenzoic acid (DHB) matrix using
the dry-droplet method. The protected oligosaccharides (1 mg) were dissolved
in EtOAc or CH2Cl2 (1 mL). A 10-μL aliquot of this solution was diluted to
0.5 mL with EtOH. A 10-μL sample and 10-μL matrix solution were mixed,
and then 0.5 μL was applied to the sample target and allowed to dry at rt. The
pseudo-molecular ions corresponding to sodium and potassium adducts were
observed in monoisotopic resolution. The calculated values used for compari-
son were derived from IUPAC exact isotopic weights using XMASS 5.0 soft-
ware from Bruker. The resolution was lower for protected pentasaccharide 22,
and therefore the average molecular mass was used for the comparison. The
accuracy and resolution value (R) were also calculated: R = (m/z)/FWHM when
m/z: mass/charge of the ion, FWHM: full width at half maximum.
Octyl 2,4,6-tri-O-acetyl-3-O-allyl-β-D-glucopyranoside (2)
A mixture of 2,4,6-tri-O-acetyl-3-O-allyl-α-D-glucopyranosyl bromide[10] (1;
10 g, 24.4 mmol) and 1-octanol (7.7 mL, 49.1 mmol) in dry CH2Cl2 (70 mL),
˚
containing activated powdered 4 A molecular sieves (10 g), was stirred under
Ar for 20 min. Then, HgBr2 (9.37 g, 26.0 mmol) was added and stirring was
continued at rt overnight. The mixture was diluted with CH2Cl2 (450 mL)and
filtered through Celite, and the filtrate was washed with aq. 5% KI (3 × 50 mL)
and water (3 × 50 mL), dried (MgSO4), filtered, and concentrated. The products
were separated by column chromatography (CH2Cl2:acetone 95:5). The first

´
K. Agoston et al.
10
compound that was eluted was 2 (3.98 g, 35.5% based on 1); syrup; [α]2D5 –16.0
1
(c 0.22, CHCl3); H NMR δ 5.85–5.68 (m, 1H, H2C = CHCH2O), 5.32–5.08 (m,
2H, H2C = CHCH2O), 5.05 (dd, 1H, J4,5 = 9.9 Hz, H-4), 4.97 (dd, 1H, J2,3
=
9.6 Hz, H-2), 4.40 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.23 (dd, 1H, J5,6a = 5.0 Hz,
J6a,6b = 12.2 Hz H-6a), 3.84 (dt, 1H, OCH2), 3.59 (t, 1H, J3,4 = 9.6 Hz, H-
3), 3.60 (m, 1H, H-5), 3.45 (dt, 1H, OCH2), 2.09, 2.08 and 2.07 (3s, each 3H,
3OAc), 1.55 (m, 2H, spacer), 1.40–1.15 (m, 10H, spacer), 0.88 (t, 3H, –CH3).
13C NMR δ 170.64, 169.18, 168.95 (3 C = O), 134.20 (–CH = ), 116.79 (H2C =),
100.97 (C-1), 79.74 (C-3), 72.57 and 69.87 (2 –OCH2), 72.41, 71.88, 69.56 (C-
2,4,5), 62.30 (C-6), 31.68, 29.30, 29.15, 25.71, 22.52 (octyl –CH2–), 20.74 (acetyl
–CH3), 13.96 (octyl –CH3). Anal. Calcd for C23H38O9: C, 60.24; H, 8.35. Found:
C, 60.37; H, 8.34. The next compound eluted was octyl 4,6-di-O-acetyl-3-O-
allyl-β-D-glucopyranoside; [α]D25 –15.3 (c 0.32, CHCl3); 1H NMR δ 5.93–5.77 (m,
1H, H2C = CHCH2O), 5.30–5.13 (m, 2H, H2C = CHCH2O), 5.00 (dd, 1H, J4,5
= 9.9Hz, H-4), 4.27 (d, 1H, J1,2 = 7.4 Hz, H-1), 2.48 (d, 1H, OH, deuterable),
2.09 and 2.08, (2s, each 3H, 2OAc), 1.62 (m, 2H, spacer), 1.45–1.15 (m, 10H,
spacer), 0.88 (t, 3H, –CH3).
Octyl 3-O-allyl-β-D-glucopyranoside (3)
To a solution of 2 (3.90 g, 8.5 mmol) in dry MeOH (50 mL) was added
NaOMe (230 mg, 4.26 mmol). After stirring for 2 d, the solution was neu-
tralized with DOWEX-50 (H+) resin, filtered, and concentrated. Column chro-
matography (hexane:acetone 7:3) of the residue gave 3 (2.12 g, 75%) as an
oil; [α]2D5 –32.8 (c 1.2, CHCl3); 13C NMR δ 135.01 (–CH = ), 117.37 (H2C = ),
102.87 (C-1), 83.44 (C-3), 73.67 and 70.41 (2 –OCH2–), 62.21 (C-6), 31.74, 29.53,
29.31, 29.16, 25.87 and 22.58 (6 -CH2– spacer), 14.03 (–CH3). Anal. Calcd for
C17H32O6: C, 61.42; H, 9.70. Found C, 61.36; H, 9.68.
Octyl 3-O-allyl-4,6-O-isopropylidene-β-D-glucopyranoside (4)
To a solution of 3 (2.5 g 7.5 mmol) in acetone dimethylacetal (15 mL)
was added pTsOH (pH 3). The reaction mixture was kept at rt for 30 min
and then diluted with CH2Cl2 (150 mL), washed with aq. 10% NaHCO3 (3 ×
20 mL) and water (2 × 20 mL), dried (MgSO4), filtered, and concentrated. Col-
umn chromatography (hexane:EtOAc 65:35) of the residue afforded 4 (2.4 g,
85.7%) as a syrup; [α]2D5 –29.0 (c 0.45, CHCl3); H NMR δ 6.01–5.87 (m, 1H,
1
H2C = CHCH2O), 5.34–5.14 (m, 2H, H2C = CHCH2O), 4.36–4.23 (m, 2H, H2C
= CHCH2O), 4.33 (d, 1H, J1,2 = 7.1 Hz, H-1), 2.59 (bs, 1H, OH, deuterable),
1.62 (m, 2H, spacer), 1.49 and 1.41 (2s, each 3H, C(CH3)2), 1.40–1.20 (m, 10H,
spacer), 0.87 (t, 3H, –CH3); 13C NMR δ 135.11 (–CH = ), 116.92 (CH2 = ), 103.23
(C-1), 99.26 (–C(CH3)2), 80.36 (C-3), 73.18 and 70.40 (–OCH2–), 62.15 (C-6),
31.75, 29.55, 29.30, 29.16, 25.86 and 22.59 (OCH2(CH2)6CH3), 29.09, 19.05 and

Protected Oligosaccharide Components of N-glycoproteins
11
14.05 (–CH3). Anal. Calcd for C20H36O6: C, 64.49; H, 9.74. Found C, 64.56; H,
9.71.
Octyl 3-O-allyl-4,6-O-isopropylidene-β-D-mannopyranoside (5)
Acetic anhydride (5 mL) was added to a solution of 4 (2.2 g, 5.9 mmol) in
DMSO (10 mL). The reaction mixture was stirred overnight, then concentrated
and co-concentrated with toluene (5 × 10 mL). Tetrabutylammonium borohy-
dride (3.0 g, 11.7 mmol) was added to a solution of the residue in THF (15 mL)
at 0◦C and stirred for 10 min, then diluted with CH2Cl2 (300 mL), washed with
aq 10% NaCl (2 × 50 mL) and water (2 × 50 mL), dried, filtered, and concen-
trated. Column chromatography (hexane:EtOAc 7:3) of the residue yielded 5
(1.87 g, 85%) as a syrup; [α]2D5 –49.4 (c 0.24, CHCl3); H NMR δ 6.10–5.82 (m,
1
1H, H2C = CHCH2O), 5.36–5.15 (m, 2H, H2C = CHCH2O), 4.48 (d, 1H, J1,2
< 1 Hz, H-1), 2.49 (bs, 1H, OH, deuterable), 1.60 (m, 2H, spacer), 1.51 and
1.42 (2s, each 3H, C(CH3)2), 1.40–1.25 (m, 10H, spacer), 0.88 (t, 3H, –CH3).
13C NMR δ 134.80 (–CH = ), 117.16 (CH2 = ), 100.29 (C-1, JC-1,H-1 156 Hz),
99.60 (–C(CH3)2), 77.23 (C-3), 71.17 and 70.05 (–OCH2–), 70.62, 69.69, 67.75
(C-2,4,5), 62.04 (C-6), 31.67, 29.38, 29.11, 25.83 and 22.51 (OCH2(CH2)6CH3),
29.23 and 19.10 (–C(CH3)2), 13.95 (octyl –CH3). Anal. Calcd for C20H36O6: C,
64.49; H, 9.74. Found C, 64.47; H, 9.69.
Octyl 3-O-allyl-2-O-benzyl-4,6-O-isopropylidene-β-D-
mannopyranoside (6)
To a suspension of NaH (168 mg, 7.0 mmol) in DMF (3 mL) was added
a solution of 5 (1.3 g, 3.5 mmol) and benzyl bromide (833 μL, 7.0 mmol) in
DMF (12 mL). After stirring for 2 h, the reaction was quenched by addition of
MeOH, and then the mixture was diluted with toluene (300 mL), washed with
aq 10% NaCl (3 × 30 mL) and water (3 × 30 mL), dried (MgSO4), filtered, and
concentrated. Column chromatography (hexane:EtOAc 8:2) of the residue gave
6 (1.45 g, 89.8%) as a syrup; [α]D25 –57.3 (c 0.57, CHCl3); 1H NMR δ 7.50–7.11
(m, 5H, aromatic), 5.95–5.75 (m, 1H, H2C = CHCH2O), 5.31–5.15 (m, 2H, H2C
= CHCH2O), 4.91 (q, 2H, PhCH2), 4.41 (bs, 1H, J1,2 < 1 Hz, H-1), 1.58 (m, 2H,
spacer), 1.52 and 1.41 (2s, each 3H, C(CH3)2), 1.45–1.10 (m, 10H, spacer), 0.88
(t, 3H, –CH3). Anal. Calcd for C27H42O6: C, 70.10; H, 9.15. Found C, 70.02; H,
9.12.
Octyl 3-O-allyl-2-O-benzyl-6-O-trityl-β-D-mannopyranoside (8)
A suspension of 6 (1.9 g, 4.1 mmol) in aq. 60% AcOH (30 mL) was boiled
for 10 min, and then the mixture was concentrated and co-concentrated with
toluene. The crude 4,6-diol (7) was dissolved in pyridine (10 mL), cooled to 0◦C,
and then trityl-chloride (1.71 g, 6.13 mmol) and DMAP (75 mg, 0.614 mmol)

´
K. Agoston et al.
12
were added and the reaction mixture was stirred for 48 h. Then, it was diluted
with CH2Cl2 (250 mL), washed with water (3 × 50 mL), dried (MgSO4), fil-
tered, and concentrated. Column chromatography (CH2Cl2:EtOAc 98:2) of the
residue gave 8 as a syrup (1.9 g, 70%); [α]2D5 –81.2 (c 1.9 CHCl3); H NMR δ
1
7.52–7.15 (m, 20H, aromatic), 5.95–5.73 (m, 1H, H2C = CHCH2O), 5.30–5.15
(m, 2H, H2C = CHCH2O), 4.85 (q, 2H, PhCH2), 4.42 (bs, 1H, J1,2 < 1 Hz,
H-1), 3.26 (dd, 1H, J2,3 = 3.0 Hz, J3,4 = 9.4 Hz H-3), 2.86 (d, 1H, OH, deuter-
able), 1.64 (m, 2H, spacer), 1.45–1.22 (m, 10H, spacer), 0.88 (t, 3H, –CH3). 13
C
NMR δ 143.74, 138.78 (aromatics), 134.53 (–CH = ), 128.70–127.00 (aromatics),
117.33 (H2C = ), 101.67 (C-1), 87.00 (Ph3C) 81.39, 74.70, 73.05, 68.86 (skele-
ton carbons), 73.71, 70.27 and 69.84 (3 –OCH2–), 65.00 (C-6), 31.77, 29.67,
29.37, 29.22, 26.15 and 22.61 (6 –CH2– spacer), 14.05 (–CH3). Anal. Calcd for
C43H52O6: C, 77.68; H, 7.88. Found C, 77.73; H, 7.85.
Octyl 3-O-allyl-2,4-di-O-benzyl-6-O-trityl-β-D-mannopyranoside
(9)
To a suspension of NaH (65 mg, 2.7 mmol) in DMF (2 mL) was added a solu-
tion of 8 (900 mg, 1.35 mmol) and benzyl bromide (0.32 mL, 2.7 mmol) in DMF
(8 mL). After stirring for 2 h, the reaction was quenched by addition of MeOH,
and then the mixture was diluted with toluene (150 mL) and washed with aq.
10% NaCl (3 × 30 mL) and water (2 × 30 mL), dried (MgSO4), filtered, and con-
centrated. Column chromatography (hexane:EtOAc 9:1) of the residue gave 9
(850 mg, 83.2%) as a syrup; [α]D25 –38.4 (c 0.4, CHCl3); 1H NMR δ 7.58–7.16 (m,
25H, aromatic), 5.95–5.82 (m, 1H, H2C = CHCH2O), 5.31–5.14 (m, 2H, H2C =
CHCH2O), 4.98 (q, 2H, PhCH2), 4.50 (q, 2H, PhCH2), 4.44 (bs, 1H, J1,2 < 1 Hz,
H-1), 3.30 (dd, 1H, J2,3 = 4.9 Hz, J3,4 = 9.7 Hz H-3), 1.65 (m, 2H, spacer),
1.48–1.20 (m, 10H, spacer), 0.87 (t, 3H, –CH3). 13C NMR δ 144.21, 139.30,
138.26 (aromatics), 134.95 (–CH = ), 128.70–127.00 (aromatics), 116.84 (H2C
= ), 101.66 (C-1), 86.27 (Ph3C) 82.21, 75.60, 74.86, 74.41 (skeleton carbons),
75.14, 73.70, 70.74 and 69.58 (4 –OCH2), 63.09 (C-6), 31.81, 29.79, 29.42, 29.28,
26.29 and 22.64 (6 –CH2– spacer), 14.07 (–CH3). Anal. Calcd for C50H58O6: C,
79.54; H, 7.74. Found C, 79.61; H, 7.73.
Octyl 3-O-allyl-2,4-di-O-benzyl-β-D-mannopyranoside (10)
A mixture of 9 (800 mg, 1.06 mmol) and aq. 80% AcOH (20 mL) was
boiled under reflux for 20 min, then concentrated and co-concentated with
toluene. Column chromatography (hexane:EtOAc 8:2) of the residue afforded
10 (500 mg, 92%) as a syrup; [α]D25 –59.1 (c 0.4, CHCl3); 1H NMR δ 7.50–7.22 (m,
10H, aromatic), 6.00–5.77 (m, 1H, H2C = CHCH2O), 5.35–5.13 (m, 2H, H2C =
CHCH2O), 4.91 and 4.78 (2q, each 2H, 2PhCH2), 4.40 (bs, 1H, J1,2 < 1 Hz, H-1),

Protected Oligosaccharide Components of N-glycoproteins
13
2.21 (bs, 1H, OH, deuterable), 1.64 (m, 2H, spacer), 1.48–1.20 (m, 10H, spacer),
0.89 (t, 3H, –CH3). 13C NMR δ 138.59, 138.32 (aromatics), 134.67 (–CH = ),
128.36–127.41 (aromatics), 116.79 (H2C = ), 101.68 (C-1), 82.25, 75.70, 74.80,
73.65 (skeleton carbons), 75.19, 73.80, 70.53 and 70.20 (4 –OCH2–), 62.58 (C-
6), 31.75, 29.63, 29.33, 29.19, 26.05 and 22.59 (6 –CH2– spacer), 14.03 (–CH3).
Anal. Calcd for C31H44O6: C, 72.62; H, 8.65. Found C, 72.59; H, 8.64.
Octyl (2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-(1→6)-3-O-
allyl-2,4-di-O-benzyl-β-D-mannopyranoside (12)
A mixture of 10 (570 mg, 1.11 mmol), 2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl bromide 11 (660 mg, 1.6 mmol), dry CH2Cl2 (10 mL), and
˚
4 A activated powdered molecular sieves (2 g) was stirred under Ar for
15 min. HgBr2 (635 mg, 1.76 mmol) was added and stirring was continued for
24 h. The mixture was diluted with CH2Cl2 (200 mL); filtered through Celite;
washed with aq. 5% KI (2 × 20 mL), aq. 5% NaHCO3 (2 × 20 mL), and wa-
ter (2 × 20 mL); dried; filtered; and concentrated. Column chromatography
(CH2Cl2:EtOAc 97:3) of the residue gave 12 (693 mg, 73.9%) as a syrup; [α]D25
1
–3.8 (c 0.2, CHCl3); H NMR δ 7.50–7.18 (m, 10H, aromatic), 5.95–5.75 (m,
1H, H2C = CHCH2O), 5.39–5.12 (m, 3H, H-2’,3’,4’), 4.91 (q, 2H, PhCH2), 4.86
(bs, 1H, J1’,2’ < 1 Hz, H-1’), 4.77 (q, 2H, PhCH2), 4.37 (bs, 1H, J1,2 < 1 Hz,
H-1), 4.25 (dd, 1H, J5’,6a’ = 5.1 Hz J6a’,6b’ = 12.8 Hz H-6a’), 2.14, 2.08, 2.01 and
1.97 (4s, each 3H, 4 OAc), 1.63 (m, 2H, spacer), 1.42–1.22 (m, 10H, spacer),
0.88 (t, 3H, –CH3); 13C NMR δ 170.69, 169.93 and 169.76 (C = O), 134.57
(–CH = ), 116.92 (CH2 = ), 101.46 (C-1), 97.34 (C-1’), 74.98, 73.62, 70.38 and
69.98 (–OCH2–), 67.42, (C-6), 62.14, (C-6’), 31.77, 29.71, 29.41, 29.25, 26.16
and 22.62 (OCH2(CH2)6CH3), 20.83, 20.62, (OAc), 14.05 (–CH3). Anal. Calcd
for C45H62O15: C, 64.12; H, 7.41. Found C, 64.08; H, 7.39.
Octyl (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-(1→6)-3-O-
allyl-2,4-di-O-benzyl-β-D-mannopyranoside (13)
To a solution of 12 (400 mg, 0.47 mmol) in 9:1 MeOH-CH2Cl2 (7 mL) was
added NaOMe (pH 9). After stirring for 3 h, the solution was neutralized with
DOWEX-50 (H+) resin, filtered, and concentrated. The residue was dissolved
in DMF (5 mL) and benzyl-bromide (0.3 mL, 2.5 mmol) was added. This so-
lution was added to NaH (82 mg, 3.4 mmol) and the mixture was stirred for
12 h. MeOH was added to decompose the excess of NaH, and then the mixture
was diluted with CH2Cl2 (100 mL), washed with water (3 × 20 mL), dried,
filtered, and concentrated. Column chromatography (CH2Cl2→CH2Cl2:EtOAc
98:2) of the crude product gave 13 (303 mg, 61.7%) as a syrup; [α]2D5 –9.1 (c
1
1, CHCl3); H NMR δ 7.48–7.07 (m, 30H, aromatic), 5.95–5.75 (m, 1H, H2C =

´
K. Agoston et al.
14
CHCH2O), 5.33–5.14 (m, 2H, H2C = CHCH2O), 5.12 (bs, 1H, J1’,2’ < 1 Hz, H-
1’), 4.32 (bs, 1H, J1,2 < 1 Hz, H-1), 1.56 (m, 2H, spacer), 1.40–1.25 (m, 10H,
spacer), 0.88 (t, 3H, –CH3); 13C NMR δ 134.73 (−CH = ), 116.74 (CH2 = ),
101.72 (C-1), 98.36 (C-1’), 75.37, 75.04, 73.84, 73.18, 72.23, 71.07, 70.42 and
69.96 (–OCH2–), 69.19 (C-6’), 66.52 (C-6), 31.81, 29.68, 29.41, 29.24, 26.14 and
22.65 (OCH2(CH2)6CH3), 14.11 (–CH3). Anal. Calcd for C65H78O11: C, 75.41; H,
7.59. Found C, 75.49; H, 7.59.
Octyl (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-(1→6)-2,4-
di-O-benzyl-β-D-mannopyranoside (14)
PdCl2 (4.2 mg, 23.7 μmol) and CuCl (2.3 mg, 23.7 μmol) were added to a
solution of 13 (24 mg, 23.2 μmol) in 9:1 THF-water (1 mL) and the mixture
was stirred at rt. When TLC (hexane:EtOAc 8:2) showed the disappearance
of the starting compound (Rf: 0.45) and the formation of the product (Rf: 0.4),
the reaction mixture was diluted with CH2Cl2 (50 mL), washed with water (3
× 10 mL), dried (MgSO4), filtered, and concentrated. Column chromatography
(hexane:EtOAc 8:2) of the residue gave 14 (14.7 mg, 63.7%) as a syrup; [α]D25
–8.2 (c 0.55, CHCl3); 1H NMR δ 7.41–7.11 (m, 30H, aromatic), 4.42 (bs, 1H, J1,2
< 1 Hz, H-1), 2.45 (bs, 1H, OH deuterable), 1.60 (m, 2H, spacer), 1.40–1.15 (m,
10H, spacer), 0.87 (t, 3H, –CH3); 13C NMR δ 101.72 (C-1), 98.18 (C-1’), 74.98,
74.82, 74.69, 73.22, 72.34, 71.44 and 70.08 (–OCH2–), 69.17 (C-6’), 66.50 (C-6),
31.82, 29.68, 29.40, 29.26, 26.15 and 22.66 (OCH2(CH2)6CH3), 14.11 (–CH3).
Anal. Calcd for C62H74O11: C, 74.82; H, 7.49. Found C, 74.78; H, 7.46.
Octyl (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-(1→6)-[2-O-
acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl]-(1→3)-2,4-di-
O-benzyl-β-D-mannopyranoside (16)
A solution of 15 (15 mg, 23.5 μmol) and 14 (15 mg, 15.1 μmol) in dry
˚
CH2Cl2 (1 mL) containing 4 A molecular sieves (100 mg, pellets) was cooled to
–40◦C under Ar. Then, a solution of TMSOTf (1.3 μL, 7.2 μmol) in dry CH2Cl2
(0.5 mL) was added dropwise and the mixture was stirred for 20 min at –40◦C.
After the addition of pyridine (10 μL) and CH2Cl2 (25 mL), the mixture was
filtered through Celite, and the filtrate was washed with aq. 5% NaHCO3 (2
× 5 mL) and water (3 × 5 mL), dried (MgSO4), filtered, and concentrated. Gel
filtration (Sephadex LH-20) of the residue gave 16 (9.9 mg, 45%) as a syrup;
[α]2D5 +7.3 (c 1, CHCl3). For H NMR and 13C NMR data, see Tables 3 and 4,
1
respectively. Anal. Calcd for C91H104O17: C, 74.36; H, 7.13. Found C, 74.41; H,
7.11. For MALDI-TOF data, see Table 2.

Protected Oligosaccharide Components of N-glycoproteins
15
Table 3: Characteristic 1H NMR data of the protected oligosaccharides
16
19
22
1
4.35 (J1,2 < 1 Hz)
1
2
4.23 (J1,2 < 1 Hz)
1
2
4.23 (J1,2 < 1 Hz)
2
3.98
3.77
3.75
3
3.78
3
3.58
3
3.59
1’
5.16 (J1,2 = 2.0 Hz)
1’
4.96 (J1,2 = 1.5 Hz)
1’
4.96 (J1,2 = 2.1 Hz)
2’
3.98
2’
4.08
2’
4.05
3’
3.89
3’
3.85
3’
3.86
1”
2”
3”
-OAc
-CH3
5.24 (J1,2 = 1.8 Hz)
1”
5.10 (J1,2 = 1.8 Hz)
1”
5.09 (J1,2 = 2.0 Hz)
5.58
4.01
2.10
0.88
2”
3.94
2”
3.96
3”
3.88
3”
3.88
1”’
2”’
3”’
4”’
-OAc
-CH3
5.02 (J1,2 = 8.1 Hz)
1”’
2”’
3”’
1””
2””
3””
-OAc
-CH3
4.97 (J1,2 = 8.7 Hz)
4.37
4.30
5.10
1.98
0.88
4.22
4.11
4.59 (J1,2 = 8 Hz)
5.21
4.88
1.95
0.88
Octyl (4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-
mannopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-
mannopyranosyl)-(1→6)-]-2,4-di-O-benzyl-β-D-
mannopyranoside (19)
To a solution of 16 (70 mg, 47 μmol) in 1:3 CH2Cl2-MeOH (4 mL) was
added NaOMe (pH 9). After stirring for 2 h, the solution was neutralized with
Table 4: Characteristic 13C NMR data of the protected oligosaccharides
16
19
22
1
102.1 (JC1,H1 = 154 Hz)
1
2
102.0
78.1
81.2
99.4
73.4
78.2
98.9
75.2
79.6
96.7
55.6
76.8
72.7
21.3
14.5
1
2
102.0
78.3
81.4
99.3
75.1
78.3
98.9
75.1
79.6
96.5
55.9
76.4
100.7
70.0
71.7
21.1
14.5
2
75.3
3
80.2
3
3
1’
99.0 (JC1,H1 = 170 Hz)
1’
1’
2’
75.4
2’
2’
3’
79.2
3’
3’
1”
2”
3”
-OAc
-CH3
100.0 (JC1,H1 = 170 Hz)
1”
1”
69.1
78.6
21.4
14.5
2”
2”
3”
3”
1”’
2”’
3”’
4”’
-OAc
-CH3
1”’
2”’
3”’
1””
2””
3””
-OAc
-CH3

´
K. Agoston et al.
16
Amberlite IR 120 (H+) resin, filtered, and concentrated. Column chromatogra-
phy (hexane:EtOAc 7:3) of the residue gave 17 (61 mg) as a glass; [α]2D5 +12.3
(c 0.21, CHCl3). NIS (280 mg, 1.24 mmol) and AgOTf (32 mg, 0.125 mmol) in
acetonitrile (4 mL) were added to a solution of ethyl 4-O-acetyl-3,6-di-O-benzyl-
2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside 18 (182 mg, 316 μmol) and
˚
17 (116 mg, 81 μmol), in CH2Cl2 (3 mL) containing 4 A molecular sieves, and
the mixture was kept at –10◦C for 30 min. When TLC (hexane:EtOAc 6:4)
showed the disappearance of the acceptor 17 (Rf: 0.2) and the formation of the
new spot (Rf: 0.25), pyridine (0.2 mL) was added and the reaction mixture was
diluted with CH2Cl2 (50 mL); filtered through Celite; washed with Na2S2O3
(3 × 10 mL), NaHCO3 (3 × 10 mL), and water (2 × 10 mL); dried; filtered; and
concentrated. Column chromatography of the crude product gave 19 (125 mg,
79%) as a syrup; [α]2D5 +14.2 (c 1.42, CHCl3). For 1H NMR and 13C NMR data,
see Tables 3 and 4, respectively. Anal. Calcd for C119H129NO23: C, 73.62; H,
6.70. Found C, 73.57; H, 6.71. For MALDI-TOF data, see Tables 1 (PSD) and 2.
Octyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-(3,6-
di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-
(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→3)-
[2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-(1→6)]-2,4-di-
O-benzyl-β-D-mannopyranoside (22)
Potassium carbonate (15 mg, 109 μmol) was added to a solution of 19
(110 mg, 56.6 μmol) in MeOH/THF (1:1, 4 mL) and the mixture was stirred
for 1 h, then diluted with CH2Cl2 (100 mL) and washed with water (3 ×
20 mL), and the combined washings were extracted with CH2Cl2 (3 × 20 mL).
The combined organic solutions were dried, filtered, and concentrated. Column
chromatography (hexane:EtOAc 7:3) of the residue gave 20 (85 mg, 79%) as a
syrup; [α]2D5 +0.7 (c 0.99, CHCl3). To a stirred solution of 20 (40 mg, 21 μmol)
and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide 21 (41 mg, 0,1 mmol)
˚
in dry CH2Cl2 (3 mL) was added 4 A powdered molecular sieves (100 mg),
and the mixture was stirred under Ar for 15 min. A solution of AgOTf (38 mg,
148 μmol) in dry toluene (2 mL) was added to the mixture over a period of 5 min
at –40◦C. Stirring was continued for an additional 10 min. The mixture was
neutralized with pyridine (0.1 mL), diluted with CH2Cl2 (50 mL), and filtered
through Celite. The filtrate was washed with aq. 10% Na2S2O3 (3 × 10 mL),
aq. 5% NaHCO3 (3 × 10 mL), and water (2 × 10 mL); dried; filtered; and con-
centrated. Column chromatography of the residue gave 22 (42 mg, 89%) as a
syrup; [α]2D5 –0.3 (c 4.5, CHCl3). For H NMR and 13C NMR data, see Tables 3
1
and 4, respectively. Anal. Calcd for C131H145NO31: C, 70.57; H, 6.56. Found C,
70.54; H, 6.59. For MALDI-TOF data, see Table 2.

Protected Oligosaccharide Components of N-glycoproteins
17
FUNDING
´
This research was supported by the TAMOP 4.2.4. A/2-11-1-2012-0001 Na-
tional Excellence Program – Elaborating and operating an inland student and
researcher personal support system convergence program. The project was
subsidized by the European Union and cofinanced by the European Social
Fund. The support of the “Momentum” grant 97101 of the Hungarian Academy
of Sciences (MTA-PE Translational Glycomics) is gratefully acknowledged.
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