Effect of Aglycon Structure on Saccharide Elongation by Cells

Article abstract of DOI:10.1002/cbdv.201400278

Alkyl N-acetyl-β-D-glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N-acetyl-β-D-glucosaminide (GlcNAc-C12), tridecan-7-yl N-acetyl-β-D-glucosaminide (GlcNAc-2C6), and pentacosan-13-yl N-acetyl-β-D-glucosaminide (GlcNAc-2C12) primers were synthesized by glycosylation of dodecan-1-ol, tridecan-7-ol, and pentacosan-13-ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc-C12 was elongated to give NeuAc-Gal-GlcNAc-C12. GlcNAc-2C6 was also elongated to afford Gal-GlcNAc-2C6 and NeuAc-Gal-GlcNAc-2C6. On the other hand, GlcNAc-2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5-times by modifying the aglycon structure of GlcNAc from C₁₂ to 2 C₆ despite having almost the same number of C-units.

Full text of DOI:10.1002/cbdv.201400278

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
239
Effect of Aglycon Structure on Saccharide Elongation by Cells
a
b
a
a
b
by Tamami Kimura ) ), Maria Carmelita Z. Kasuya ), Kenichi Hatanaka* ), and Koji Matsuoka )
a) Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505,
Japan (phone: þ81-3-54526356; e-mail: hatanaka@iis.u-tokyo.ac.jp)
b) Division of Material Science, Graduate School of Science and Engineering, Saitama University, 255
Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
Alkyl N-acetyl-b-d-glucosaminide (GlcNAc primers) with different aglycon moieties were
synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation.
Dodecyl N-acetyl-b-d-glucosaminide (GlcNAc-C12), tridecan-7-yl N-acetyl-b-d-glucosaminide
(
GlcNAc-2C6), and pentacosan-13-yl N-acetyl-b-d-glucosaminide (GlcNAc-2C12) primers were syn-
thesized by glycosylation of dodecan-1-ol, tridecan-7-ol, and pentacosan-13-ol, respectively, with per-
acetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids.
After 48 h incubation, results showed that GlcNAc-C12 was elongated to give NeuAc-Gal-GlcNAc-C12.
GlcNAc-2C6 was also elongated to afford Gal-GlcNAc-2C6 and NeuAc-Gal-GlcNAc-2C6. On the other
hand, GlcNAc-2C12 primer was not elongated. Significantly, the results demonstrated that the amount of
glycosylated product increased 1.5-times by modifying the aglycon structure of GlcNAc from C12 to 2 C6
despite having almost the same number of C-units.
Introduction. – Carbohydrates are known as biomakers. For example, carbohy-
drates play significant roles in generation of cells, canceration, immunity, cell adhesion,
and fecundation [1–5]. We can obtain important information on some vital biological
phenomena by investigating the carbohydrates involved. However, the preparation of
carbohydrates is not simple. The number of OH groups that are found in the structure
makes the carbohydrate synthesis difficult. During carbohydrate synthesis, repeated
introduction and removal of the protecting groups is required. So, we developed an
approach that combines chemical synthesis and biosynthesis for the facile preparation
of carbohydrates.
The strategy combining chemical synthesis and biosynthesis to prepare a saccharide
by using a saccharide primer is called biocombinatorial synthesis [6]. In this method,
various kinds of carbohydrates can be synthesized from one kind of alkyl glycoside
(
primer) and a combination of different cell types. For example, GM3 analog can be
obtained from B16 cells using a lactoside primer. On the other hand, GM3, GM2, and
GM1 can be obtained from the same primer by using MDCK (MadinꢀDarby canine
kidney) cells.
The mechanism of primer elongation involves endocytosis, followed by transport of
the primer to the endoplasmic reticulum (ER) and/or the Golgi where the primer is
elongated by glycosyltransferase. Finally, the product is released from the cells to the
culture medium. Previously, it was reported that a dodecyl aglycon unit was best suited
to obtain elongated products from B16 cells [7]. However, only minute amounts of the
ꢀ
2015 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
desired carbohydrate could be obtained. In this work, the structure of aglycon was
modified to determine the effect of the aglycon structure on cellular elongation.
Results and Discussion. – Synthesis of the Primers. For the synthesis of primers, the
aglycon moieties were prepared by Grignard reaction using HCOOEt and various alkyl
bromides [8]. Glycosylation of the long-chain alkanols (aglycon moieties) with 2-
acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-d-glucopyranose (1) gave the desired prod-
ucts, 2a–2c, as outlined in Scheme [9]. Finally, the protecting group was removed under
Zemplen condition to afford GlcNAc-C12 (3a), GlcNAc-2C6 (3b), and GlcNAc-2C12
1
(
3c). The structures of the synthesized compounds were confirmed by the H- and
C-NMR data.
1
3
Effect of Aglycon Structure. Each primer was administered to mouse melanoma B16
cells [10]. The cells were incubated for 48 h in the absence (control) and presence of
each primer (Fig. 1). The primers did not affect cell morphology. As shown in Fig. 1,
there is no significant difference between the cells cultured in the absence and presence
of primers. Moreover, the primers were not cytotoxic at 50 mm concentration. After
48 h, the medium and cell fractions were collected, and the glycolipids were extracted
using SepPak C18 column and by sonication, respectively, and were monitored on a
Scheme. Chemical Synthesis of GlcNAc-C12 (3a), GlcNAc-2C6 (3b), and GlcNAc-2C12 (3c) Primers
i) Trimethylsilyl trifluoromethanesulfonate (TMSOTf), ClCH CH Cl. ii) Long-chain alkanol, camphor-
2
2
sulfonic acid (CSA), ClCH CH Cl. iii) MeONa, MeOH.
2
2
Fig. 1. Effect of aglycon structure on B16 cells after incubation for 48 h in the absence (a), or in the
presence of GlcNAc-C12 (b), GlcNAc-2C6 (c), and GlcNAc-2C12 (d)

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
241
HPTLC plate. The HPTLC results showed the absence of band corresponding to
GlcNAc-2C12, suggesting that most of the primer was taken up and remained in the
cells (Fig. 2,a). Moreover, HPTLC results confirmed that GlcNAc-2C12 was not
elongated.
On the other hand, HPTLC results in Fig. 2,b, indicated that GlcNAc-2C6 primer
was elongated and afforded two kinds of products that were analyzed by ESI-MS.
Quantification using a densitometer revealed that the amount of the product obtained
from GlcNAc-2C6 primer was larger than that obtained from GlcNAc-C12 primer.
Results evidenced that GlcNAc-2C6 primer was elongated by the addition of a
galactose unit, or by galactose and sialic acid units. Treatment with enzyme confirmed
Fig. 2. HPTLC Profile of lipids obtained after incubation of B16 cells with a) GlcNAc-2C12 or GlcNAc-
C12 and b) GlcNAc-2C6 or GlcNAc-C12

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
that the sialic acid residue of GlcNAc-2C6 is a2,3-linked to the galactose unit as shown
in Fig. 3. This result agreed with previous reports that B16 cells add an a2,3-linked sialic
acid unit to the galactose residue of primers [11]. Comparing GlcNAc-C12 with
GlcNAc-2C6, only GlcNAc-2C6 primer afforded galactosylated GlcNAc-2C6, suggest-
ing that the galactosyl GlcNAc-2C6 is stable.
Administration of a mixture of GlcNAc-2C6 (50 mm) and GlcNAc-C12 primers
(
50 mm) was also studied to determine which primer could easily be glycosylated. As
shown in Fig. 4, only the GlcNAc-2C6 primer was elongated. The amount of products
obtained from the mixture of GlcNAc-2C6 and GlcNAc-C12 was less than that when
the primers were administered separately. This may be due to the cytotoxic effect of the
mixture of GlcNAc-2C6 and GlcNAc-C12 (cf. Fig. 5).
The cells treated with the mixture of GlcNAc-2C6 and GlcNAc-C12 primers were
dead and detached from the substrate after 48 h.
On the other hand, separate administration up to a concentration of 100 mm did not
exhibit cytotoxicity. No significant difference could be observed before and after
treatment of up to 100 mm as shown in Fig. 6,a and b. However, a morphological change
was observed after 48 h treatment with 150 mm GlcNAc-C12 or GlcNAc-2C6.
Moreover, the mixture of primers gave mostly Gal-GlcNAc-2C6 and NeuAc-Gal-
GlcNAc-2C6. Based on these results, GlcNAc-2C6 primer is elongated more than
GlcNAc-C12 primer, when a mixture is administered.
Cellular saccharide elongation occurs via endocytosis, transport to the ER and/or
the Golgi, enzyme reaction, and release of elongated primers from the cells. GlcNAc-
Fig. 3. HPTLC Profile before and after treatment with a2,3-
sialidase. Lane 1: before treatment of NeuAc-Gal-GlcNAc-2C6.
Lane 2: after treatment of NeuAc-Gal-GlcNAc-2C6. Lane 3: Gal-
GlcNAc-2C6; Lane 4: GlcNAc-2C6.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
243
Fig. 4. HPTLC Profile of lipids obtained after incubation of B16 cells with GlcNAc-2C6, GlcNAc-C12,
and a mixture of GlcNAc-2C6 and GlcNAc-C12
Fig. 5. Effect of the mixture of GlcNAc-C12 (50 mm) and GlcNAc-2C6 (50 mm) on B16 cells
2C6 gave 1.5-times more product than GlcNAc-C12, despite the fact that both GlcNAc-
C12 and GlcNAc-2C6 were taken up by B16 cells. Although the aglycon moieties have
almost the same number of C-units, the structure of the aglycon may have a different
effect on transport to ER and/or the Golgi, enzyme reaction, and release of elongated
primers from the cells.
Conclusions. – In this study, biocombinatorial synthesis using saccharide primers
and animal cells was carried out to produce the glycolipid GM3. GlcNAc-C12,
GlcNAc-2C6 and GlcNAc-2C12 primers were chemically synthesized, and were

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Fig. 6. The treatment of different concentrations of a) GlcNAc-C12 and b) GlcNAc-2C6
introduced to mouse melanoma B16 cells to serve as substrate for glycosylation by
cellular enzymes. The primer with dialkyl chain afforded 1.5-times more product than
the primer with a single alkyl chain.
By improving the method for obtaining GM3, the relation between GM3, which is
expressed in nerve cells, and the brain disease such as Alzheimer may be clarified.
Moreover, since GM3 is involved in the infection of the influenza virus, development of
strategy for diagnosis can be achieved when sufficient amount of GM3 is readily
available.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
245
Experimental Part
General. All reactions were monitored by TLC. TLC: Silica gel 60 F-254 (E. Merck, DE-Darmstadt);
visualization by spraying with anisaldehyde/H SO and heating. Column chromatography (CC): silica gel
2
4
1
6
0 (SiO , 230–400 mesh; E. Merck, DE-Darmstadt). H-NMR Spectra: JEOL spectrometer at 600 MHz
2
(
JEOL, Tokyo, Japan).
Chemical Synthesis of Alcohols (aglycon moiety) [12]. Mg (120 mmol) in a flask was dried overnight
under N . THF (50 ml), alkyl bromide (90.2 mmol), HCOOEt (41 mmol) were added, and the mixture
2
was stirred under reflux for 10 min. After cooling to r.t., to the resulting slurry were added H O and 1m
2
H SO . The mixture was filtered using Celite. The desired product was recrystallized from MeCN,
2
4
tridecan-7-ol (72.1%), pentacosan-13-ol (88.6%).
1
Tridecan-7-ol. H-NMR (600 MHz, (D )DMSO): 0.85 (t, J¼6.6, 6 H); 1.20–1.30 (br., 20 H); 3.32–
6
13
3
3
.37 (br., 1 H); 4.10 (d, J¼5.5, 1 H). C-NMR (150 MHz, CD OD): 14.07; 22.61; 25.61; 29.38; 31.84;
3
7.49; 72.02.
1
Pentacosan-13-ol. H-NMR (600 MHz, (D )DMSO): 0.86 (t, J¼7.1, 6 H); 1.22–1.30 (br., 44 H);
6
3
.33–3.38 (br., 1 H).
Chemical Synthesis of Saccharide Primers [13]. 2-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-d-
glucopyranose (1; 1.5 mmol) was mixed with trimethylsilyl trifluoromethylsulfonate (TMSOTf;
.9 mmol) in ClCH CH Cl under N at 08 for 30 min. The soln. was refluxed with stirring overnight.
1
2
2
2
The mixture was neutralized with Et N, the product was washed with sat. NaHCO , H O, and brine, dried
3
3
2
(
Na SO ), and concentrated in vacuo. To the mixture was added the long-chain alkyl alcohol (1.5 mmol)
2 4
and (þ)-10-camphorsulfonic acid (CSA; 2.2 mmol) in ClCH CH Cl (10 ml), and the resulting mixture
2
2
was stirred at 908 for 4 h. After completion of the reaction, CHCl was added, and the mixture was
3
3 2 2 4
washed with sat. NaHCO , H O, and brine, dried (Na SO ), and concentrated in vacuo. The
peracetylated glycosides 2a–2c were purified by CC (toluene/AcOEt 1:1).
Deacylation was carried out in the presence of MeONa (0.22 mmol) in MeOH (5 ml) for 3 h. After
treatment with cation-exchange resin, concentration in vacuo afforded GlcNAc-C12 (3a; 16.5%),
GlcNAc-2C6 (3b; 17.8%), and GlcNAc-2C12 (3c; 10.8%).
Dodecyl 3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-b-d-glucopyranoside (2a). 1H-NMR (CDCl3,
6
00 MHz): 0.83 (t, J¼6.6, 3 H); 1.20–1.25 (br., 18 H); 1.47–1.55 (m, 2 H); 1.90 (s, 3 H); 1.98 (s, 3 H); 1.99
(
s, 3 H); 2.04 (s, 3 H); 3.42 (q, J¼7.1, 7.1, 1 H); 3.66–3.67 (m, 1 H); 3.75–3.83 (m, 2 H); 4.08 (d, J¼12.1,
1
8
2
H); 4.22 (dd, J¼4.4, 12.1 H); 4.65 (d, J¼8.2, 1 H); 5.02 (t, J¼9.6, 1 H); 5.28 (t, J¼9.9, 1 H); 5.66 (d, J¼
13
.8, 1 H). C-NMR (150 MHz, CDCl ): 14.06; 20.60; 20.67; 20.71; 22.62; 23.26; 25.81; 29.28; 29.31; 29.37;
9.57; 29.60; 31.84; 54.79; 62.14; 68.68; 69.92; 71.64; 72.31; 100.62; 169.39; 170.13; 170.72; 170.83.
3
1
Dodecyl 2-(Acetylamino)-2-deoxy-b-d-glucopyranoside (¼GlcNAc-C12; 3a). H-NMR (600 MHz,
CD OD): 0.90 (t, J¼7.2, 3 H); 1.29–1.35 (br., 18 H); 1.52–1.55 (m, 2 H); 1.97 (s, 3 H); 3.25–3.32 (m,
3
13
3
H); 3.42–3.46 (m, 2 H); 3.61–3.69 (m, 2 H); 3.86–3.89 (m, 2 H); 4.38 (d, J¼8.8, 1 H). C-NMR
(
150 MHz, CD OD): 14.45; 22.99; 23.74; 27.16; 30.50; 30.56; 30.68; 30.77; 30.82; 30.85; 33.08; 57.42; 62.80;
3
7
0.58; 72.13; 76.07; 77.95; 102.72; 173.61.
Tridecan-7-yl 3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-b-d-glucopyranoside (2b). 1H-NMR
(
1
1
CDCl , 600 MHz): 0.84 (t, J¼7.1, 6 H); 1.21–1.40 (br., 20 H); 1.89 (s, 3 H); 1.98 (s, 3 H); 1.99 (s,
3
H); 2.03 (s, 3 H); 3.50 (t, J¼5.8, 1 H); 3.64–3.72 (m, 2 H); 4.08 (dd, J¼1.4, 11.3, 1 H); 4.17 (dd, J¼4.9,
13
2.1, 1 H); 4.71 (d, J¼8.3, 1 H); 4.99 (t, J¼9.6, 1 H); 5.32 (t, J¼9.9, 1 H); 5.57 (d, J¼8.8, 1 H). C-NMR
(
150 MHz, CDCl ): 14.02; 14.04; 20.61; 20.67; 22.56; 22.60; 23.26; 25.01; 25.06; 29.28; 29.51; 31.78; 31.86;
3
3
4.00; 34.68; 55.47; 62.46; 69.00; 71.41; 72.35; 81.11; 100.20; 169.44; 169.95; 170.63; 170.80.
Tridecan-7-yl 2-(Acetylamino)-2-deoxy-b-d-glucopyranoside (¼GlcNAc-2C6; 3b). 1H-NMR
(
1
600 MHz, CD OD): 0.90 (t, J¼7.2, 6 H); 1.23–1.40 (br., 16 H); 1.41–1.63 (br., 4 H); 3.20–3.26 (m,
3
H); 3.28–3.36 (m, 2 H); 3.48 (t, J¼9.6, 1 H); 3.53–3.63 (m, 2 H); 3.69 (dd, J¼5.5, 11.6, 1 H); 3.85 (d,
13
J¼11.5, 1 H); 4.46 (d, J¼8.2, 1 H). C-NMR (150 MHz, CD OD): 14.47; 23.13; 23.75; 23.78; 25.94;
3
2
6.21; 30.61; 30.84; 33.04; 33.14; 34.84; 35.80; 57.91; 62.89; 72.14; 75.89; 77.66; 81.48; 102.20; 173.41.
1
Pentacosan-13-yl 3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-b-d-glucopyranoside (2c). H-NMR
(
1
CDCl , 600 MHz): 0.84 (t, J¼7.1, 6 H); 1.14–1.32 (br., 44 H); 1.89 (s, 3 H); 1.98 (s, 3 H); 1.99 (s,
3
H); 2.03 (s, 3 H); 3.49 (t, J¼2.7, 1 H); 3.63–3.73 (m, 2 H); 4.08 (dd, J¼2.8, 12.1, 1 H); 4.17 (dd, J¼4.9,

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1
2.1, 1 H); 4.71 (d, J¼8.3, 1 H); 4.99 (t, J¼9.6, 1 H); 5.32 (t, J¼9.9, 1 H); 5.58 (d, J¼8.8, 1 H). 13C-NMR
(
150 MHz, CDCl ): 14.08; 20.62; 20.68; 22.64; 23.28; 25.09; 29.31; 29.63; 29.64; 29.68; 29.70; 29.88; 31.86;
3
3
3.95; 34.68; 55.44; 62.43; 68.96; 71.38; 72.31; 81.17; 100.20; 169.45; 169.96; 170.64; 170.81.
Pentacosan-13-yl 2-(Acetylamino)-2-deoxy-b-d-glucopyranoside (¼GlcNAc-2C6; 3c). 1H-NMR
(
1
600 MHz, CD OD): 0.90 (t, J¼6.9, 6 H); 1.22–1.40 (br., 40 H); 1.42–1.63 (br., 4 H); 3.22–3.25 (m,
3
H); 3.30–3.35 (m, 2 H); 3.48 (t, J¼9.6, 1 H); 3.55–3.63 (m, 2 H); 3.69 (dd, J¼5.5, 12.0, 1 H); 3.85 (dd,
13
J¼2.2, 12.1, 1 H); 4.46 (d, J¼8.3, 1 H). C-NMR (150 MHz, CD OD): 14.49; 23.17; 23.77; 25.90; 26.22;
3
3
0.52; 30.75; 30.82; 30.90; 31.14; 33.11; 34.75; 35.73; 57.91; 62.89; 72.14; 75.89; 77.67; 81.44; 102.20; 173.40.
General Method for Incubation of B16 Melanoma Cells with GlcNAc Primers. B16 Cells were
obtained from Riken Cell Bank (Tsukuba, Japan). Dulbeccoꢂs Modified Eaglesꢂs Medium/Hamꢂs F-12
D-MEM/F-12) and Insulin-Transferrin-Selenium-X (ITS-X) were from Life Technologies (Carlsbad,
(
CA, USA). Fetal bovine serum (FBS) was from JRH Biosciences (Lenexa, KS, USA). Antibiotic-
antimycotic soln. (100 ꢁ) was from Nacalai Tesque (Kyoto, Japan). SepPak C18 Cartridges were from
Waters (Boston, MA, USA). High-performance thin layer chromatography (HPTLC; SiO 60 F-254)
2
was from E. Merck (DE-Darmstadt). The GlcNAc primers were dissolved in sterilized DMSO to an
initial concentration of 50 mm.
Cell Culture. Mouse B16 melanoma cells were cultured using D-MEM/F-12 supplemented with 10%
FBS and 1% antibiotic-antimycotic solution in humidified atmosphere of 5% CO air at 378. Cells were
2
detached by application of 0.25% trypsin/ethylenediaminetetraacetate (EDTA) and passaged every 3 d.
6
Incubation of Cells with GlcNAc Primers. Cells (2.0ꢁ10 ) were seeded into 100-mm culture dishes
containing 7 ml of medium and incubated for 48 h. Then, the cells were washed with TI-DF (D-MEM/F-
1
2 containing 1% ITS-X without phenol red) to remove the serum, and were incubated with 50 mm of
each primer for 48 h at 378. After incubation, culture media were collected, and the cells were washed
with phosphate-buffered saline (PBS), harvested with 0.25% EDTA in PBS, and centrifuged at 1000 rpm
for 5 min.
3 3 2
/MeOH 2 :1, then with CHCl O
/iPrOH/H
The lipids were extracted from the cell pellet with CHCl
:11:2 in a sonicated bath. Lipids from the culture media were purified using a SepPak C18 column.
7
Lipids from the cell homogenate and culture medium fractions were analyzed by HPTLC with CHCl3/
MeOH/0.25% aq. KCl soln. 5 :4 :1 as developing solvent. HPTLC Plates were sprayed with resorcinol,
and then with orcinolꢀH SO reagent, and heated to detect the separated glycolipids, which were later
2
4
quantified using CS-9300PC dual-wavelength flying spot scanning densitometer (Shimadzu, Kyoto,
Japan).
Mass Spectrometry. The structure analyses of glycosylated products were carried out with an ESI
mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The primers and glycosylated products were
dissolved in MeOH and analyzed using 10% MeCN soln.
Digestion of Elongated Products by Enzymes. Enzymatic digestion of elongated products was carried
out in 100 ml of AcONa buffer (pH 5.5) containing 5 mU of a2,3-sialidase from salmonella typhimurium
LC2 (TAKARA BIO INC., Shiga, Japan). The reaction was carried out overnight at 378. The products
were extracted using SepPak C18 column and separated on an HPTLC plate with CHCl /MeOH/0.25%
3
aq. KCl soln. 5 :4 :1 as developing solvent. HPTLC Plates were sprayed with resorcinol, and with
orcinolꢀH SO reagent, and heated to detect the glycolipids.
2
4
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Received July 29, 2014