Synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches

Article abstract of DOI:10.1016/j.bmcl.2010.06.066

An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has been accomplished by means of chemical and enzymatic approaches. N-Acetyl-d-glucosamine (GlcNAc) clusters having O-glycosidic linkage or S-glycosidic linkage were chemically synthesized from known intermediates in high yields. The GlcNAc clusters were first used as substrates for β1,4 galactosyl transferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N-acetyllactosamine clusters. Further sugar elongation of the LacNAc clusters was demonstrated using α2,3 sialyl transferase and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.

Full text of DOI:10.1016/j.bmcl.2010.06.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4906–4910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of sialyllactosamine clusters using carbosilane as core scaffolds
by means of chemical and enzymatic approaches q
a
a
a
b
b
Koji Matsuoka a, , Reina Kaneko , Tetsuo Koyama , XiaoTao Ma , Yasuaki Esumi , Takemichi Nakamura ,
*
Ken Hatano a, Daiyo Terunuma a
a Area for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan
b The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has
Received 22 April 2010
Revised 9 June 2010
Accepted 11 June 2010
Available online 17 June 2010
been accomplished by means of chemical and enzymatic approaches. N-Acetyl-D-glucosamine (GlcNAc)
clusters having O-glycosidic linkage or S-glycosidic linkage were chemically synthesized from known
intermediates in high yields. The GlcNAc clusters were first used as substrates for b1,4 galactosyl trans-
ferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N-acetyllactosamine
clusters. Further sugar elongation of the LacNAc clusters was demonstrated using
and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.
Ó 2010 Elsevier Ltd. All rights reserved.
a2,3 sialyl transferase
Keywords:
N-Acetylglucosamine
N-Acetyllactosamine
Sialic acid
Sialyllactosamine
Glycosides
Oligosaccharides
Clusters
Chemo-enzymatic syntheses
Glycosyltransferases
Sialyl N-acetyllactosamine (SiaLacNAc; Neu5Ac
a
2?3Galb1?
preparation is possible because of the use of costly enzymes and su-
4GlcNAc) and N-acetyllactosamine (LacNAc; Galb1?4GlcNAc) are
known as extremely valuable structures of glycoconjugates such
as glycoproteins and glycolipids.1 Influenza virus recognizes the
gar nucleotides and that enzymatic synthesis is not suitable for
preparation of unnatural saccharide structures. Thus, an appropriate
combination of organic synthesis and biochemistry is a promising
convenient method for efficient construction of important oligosac-
charide structures.
Since monomeric oligosaccharides usually show low levels of
biological activity against sugar-binding proteins, the sugar cluster
effect7 is utilized in order to enhance the low binding affinities.8 In
our recent study on glyco-silicon functional materials, successful
enhancement of the interaction between carbohydrate chains
and proteins was also demonstrated.9 In this letter we report effi-
cient assembly of SiaLacNAc and LacNAc on carbosilane core scaf-
folds by means of a chemo-enzymatic approach.
Neu5Aca
2?3Gal structure of SiaLacNAc2 and adheres to the surface
of host cells, by which infection by the virus is established.3 LacNAc
is also known as a repeating sequence of the lactosaminoglycan,
which is a biologically important glycan chains on aging.4 Synthetic
assembly of the trisaccharidic and disaccharidic structures was suc-
cessfully accomplished by means of synthetic organic chemistry5
and by a combination of chemistry and biochemistry such as che-
mo-enzymatic methodology.6 The use of organic synthesis for con-
struction of an oligosaccharide structure has the merit of enabling
large-scale preparation and formation of unnatural saccharide se-
quences, but it also has disadvantages including the necessity of a
synthetic scheme involving multiple laborious steps and complica-
tion of purification. The use of enzymatic constructions using glyco-
syl transferases and sugar nucleotides has the merit on the basis of
natural pathway in biological systems. The disadvantages of enzy-
matic synthesis of the oligosaccharides are that only small-scale
Figure 1 summarizes our synthetic plan for the construction of
SiaLacNAc on a carbosilane scaffold. Known GlcNAc derivative and
carbosilane core frames bearing functional groups at
x-positions
are condensed to produce the corresponding multivalent-type
GlcNAc derivative. Sugar elongations are performed on dendritic
GlcNAc residues by using appropriate glycosyl transferases and
sugar nucleotides.
Scheme 1 summarizes chemical synthesis of glycoclusters.
The oxazoline derivative of GlcNAc 110 was allowed to glycosylate
with a known triol 211 in the presence of CSA to afford the
q
Glyco-silicon functional materials. Part 13. For part 12, see Ref. 9b.
* Corresponding author. Tel./fax: +81 48 858 3099.
E-mail address: koji@fms.saitama-u.ac.jp (K. Matsuoka).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.066

K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4906–4910
4907
OH
OH
O
HO
OH
O
OH
O
COOH
HO
O
X
HO
O
Si
AcHN
OH
NHAc
4
OH
Sia T
CMP-NANA
Enzymatic
elongations
OH
O
X
HO
HO
OH
O
O
HO
Si
OH
NHAc
4
UDP-Gal
Gal T
RO
RO
NHAc
X
O
OR
NHAc
X
RO
RO
O
Si
OR
NHAc
X
O
OR
NHAc
RO
RO
X
O
OR
Chemical
syntheses
RO
RO
Y
Y
Y
OAc
O
AcO
AcO
Si
OAc
NHAc
Y
Figure 1. Synthetic plan for construction of a carbohydrate chain on a multivalent-type core scaffold.
corresponding glycocluster 3 having three GlcNAc moieties in 69%
accomplished, preparation of similar glycoclusters having S-glyco-
sidic linkage was attempted. The target compound is of great interest
in comparison with a typical O-glycoside-type glycocluster for activ-
ity against glycosidase and glycosyl transferase. Thus, known 512
was treated with tris(3-bromopropyl)phenylsilane 613 in the pres-
17
yield, ½aꢀD ꢁ66° (c 1.1, CHCl3), 1H NMR (CDCl3) d 6.67 (d, 3H,
J2,NH = 8.8 Hz, N–H), 4.71 (d, 3H, J1,2 = 8.4 Hz, H-1), integral ratio of
the H atoms by 1H NMR: SiCH2/Ph/H-1 = 6:5:3, ESI MS calcd for
[M+Na]+: 1292.4881; found m/z: 1292.4914. Transesterification with
MeONa/MeOH followed by saponification with 0.1 M aqueous NaOH
for acetate 3 proceeded smoothly to give water-soluble glycocluster
4 in quantitative yield, 1H NMR (D2O) d 4.45 (d, 3H, J1,2 = 8.6 Hz, H-
1), integral ratio of the H atoms by 1H NMR: SiCH2/Ph/H-1 = 6:5:3,
13C NMR (D2O) d 100.82 (C-1). Since efficient introduction of GlcNAc
into the simple core scaffold through O-glycosidic linkage was
ence of NaOMe in MeOH–DMF14 to yield the corresponding thiogly-
17
coside 7 in 86% yield after reacetylation, ½aꢀD ꢁ27° (c 1.1, CHCl3), 1
H
NMR (CDCl3) d 6.09 (d, 3H, J2,NH = 9.6 Hz, N–H), 4.60 (d, 3H,
J1,2 = 10.2 Hz, H-1), integral ratio of the H atoms by 1H NMR:
SiCH2/Ph/H-1 = 6:5:3, ESI MS calcd for [M+Na]+: 1340.4196; found
m/z: 1340.4219. The chemical shift of C-1 in 7 was upfield shifted
by the results of 13C NMR, and a wider coupling constant between
H-1 and H-2 than that of O-glycoside 4 was also observed. Complete
removal of the protections in 7 was performed by a combination of
All new compounds with specific rotation data gave satisfactory results of
elemental analyses.

4908
K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4906–4910
RO
RO
OAc
NHAc
X
O
AcO
AcO
O
OR
NHAc
RO
RO
NHAc
X
O
N
X
O
RO
RO
Si
Si
Si
HO
Br
HO
OR
NHAc
O
CH3
Si
3
1
4
OR
(i)
(i)
(iii)
2
9
RO
RO
NHAc
Si
X
X
O
OR
NHAc
O
OR
(iii)
RO
RO
X
Si
X
NHAc
OAc
O
SAc
NHAc
Br
3
4
O
O
RO
RO
AcO
AcO
6
12
OR
OR
RO
RO
3: X = O, R = Ac
4: X = O, R = H
7: X = S, R = Ac
8: X = S, R = H
5
(ii)
(ii)
10: X = O, R = Ac
11: X = O, R = H
13: X = S, R = Ac
14: X = S, R = H
(ii)
(ii)
Scheme 1. Reagents and conditions: (i) CSA, CH2ClCH2Cl, 80 °C 2 h; (ii) NaOMe, MeOH, rt, then, 0.1 M aq NaOH; (iii) NaOMe, MeOH, 0 °C?rt, overnight, then, Ac2O–Pyr, rt.
transesterification and hydrolysis with 0.1 M aqueous NaOH to af-
ford trivalent-type thioglycosidic carbosilane 8 in 97% yield after
chromatographic purification on Sephadex LH-20 with MeOH as
the eluent, 1H NMR (D2O) d 4.54 (d, 3H, J1,2 = 10.2 Hz, H-1), integral
ratio of the H atoms by 1H NMR: SiCH2/Ph/H-1 = 6:5:3, 13C NMR
(D2O) d 82.84 (C-1). Similar chemical modifications were then per-
formed using known carbosilane 915 and 12.16 Thus, oxazoline 1
NMR (CDCl3) d 6.20 (br s, 4H, N–H), 4.66 (d, 4H, J1,2 = 9.8 Hz, H-1),
integral ratio of the H atoms by 1H NMR: SiCH2/H-1 = 8:4, which
was further treated under the same basic conditions as those de-
scribed for 3 to afford water-soluble 14 in 92% yield, 1H NMR
(D2O) d 4.48 (d, 4H, J1,2 = 10.2 Hz, H-1), integral ratio of the H atoms
by 1H NMR: SiCH2/H-1 = 8:4, 13C NMR (D2O) d 83.99 (C-1), ESI MS
calcd for [M+Na]+: 1163.3916; found m/z: 1163.3945.
26
was condensed with tetraol 9 to give 10 in 13% yield, ½aꢀD ꢁ14° (c
Since chemical synthesis of GlcNAc clusters was accomplished,
our attention was focused on enzymatic elongation of the sugar
chains on the dendritic scaffolds. Enzymatic elongation of saccha-
ride chains on a dendrimer was previously reported by Roy and
co-workers17a and Narvor and co-workers17b; however, carbosilane
dendrimers have not been used for such an objective. Carbosilanes
bearing an appropriate number of GlcNAc and glycosidic linkages
were treated with b1,4 galactosyl transferase (GalT) obtained from
bovine milk in the presence of UDP-galactose (UDP-Gal)18 followed
1.0, CHCl3), 1H NMR (CDCl3) d 6.76 (d, 4H, J2,NH = 6.8 Hz, N–H),
4.73 (d, 4H, J1,2 = 8.0 Hz, H-1), integral ratio of the H atoms by 1H
NMR: SiCH2/H-1 = 8:4, in which ester functional groups were re-
moved by the same method as that for 3 to give water-soluble 11
27
in 87% yield, ½aꢀD ꢁ33° (c 1.0, CHCl3), 1H NMR (D2O) d 4.49 (d, 4H,
J1,2 = 8.4 Hz, H-1), integral ratio of the H atoms by 1H NMR: SiCH2/
H-1 = 8:4, 13C NMR (D2O) d 100.73 (C-1), ESI MS calcd for [M+Na]+:
1099.4824; found m/z: 1099.4858. In comparison of the yield for
the preparation of 3, the yield for the preparation of 10 was low be-
cause of poor solubility of tetraol 9 in various organic solvents. Sul-
fide formation between 5 and 12 gave 13 having four GlcNAc
by treatment of
a
2,3 sialyl transferase (SiaT)19 obtained from rat
liver (recombinant) in the presence of CMP-sialic acid (CMP-
NANA). The schematic images are shown in Scheme 2. Trimeric
O-glycosides of GlcNAc 4 were allowed to react with UDP-Gal in
26
residues at each terminal in 94% yield, ½aꢀD ꢁ65° (c 1.1, CHCl3), 1H
UDP-Gal
OH
O
OH
O
HO
HO
OH
O
HO
HO
(i)
X
O
Si
X
HO
Si
NHAc
n
4-n
OH
NHAc
n
4-n
4: X = O, n = 3
8: X = S, n = 3
11: X = O, n = 4
14: X = S, n = 4
15: X = O, n = 3
16: X = S, n = 3
17: X = O, n = 4
18: X = S, n = 4
UDP-Gal
CMP-NANA
(iii)
(ii)
CMP-NANA
OH
OH
OH
O
HO
OH
O
COOH
HO
O
X
HO
O
O
Si
AcHN
OH
NHAc
n
4-n
OH
19: X = O, n = 3
20: X = S, n = 3
21: X = O, n = 4
22: X = S, n = 4
Scheme 2. Reagents and conditions: (i) UDP-Gal, GalT,
a-lactoalbumin, MgCl2, 50 mM HEPES buffer (pH 7.4), 37 °C, 3 d; (ii) CMP-NANA, SiaT, BSA, mercaptoethanol, CIAP,
MgCl2, 50 mM HEPES buffer (pH 7.4), 37 °C, 6 d; (iii) (i), then (ii) in one-pot.

K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4906–4910
4909
Table 1
the presence of GalT,
a
-lactoalbmin,20 and MgCl2 in 50 mM HEPES
Results of enzymatic sugar elongation on dendritic carbosilanes after isolation
buffer (pH 7.4) at 37 °C for 3 days to give 15 after the usual workup,
1H NMR (D2O) d 4.46 (br s, 4H, H-10), 4.41 (br s, 4H, H-1), integral
Primer compounds
Two step synthesis
One-pot
synthesis
Yield (%)
1
ratio of the H atoms by H NMR: SiCH2/Ph/H-1/H-10 = 6:5:3:3, ESI
MS calcd for [M+Na]+: 1400.55148; found m/z: 1400.3833. Galac-
tosylation of the other primer compounds was performed using
the same reaction conditions, and the reaction proceeded smoothly
to afford the corresponding glycoclusters having an appropriate
number of LacNAc residues. Further elongation of the sialic acid
residue was then carried out using CMP-NANA in the presence of
SiaT, bovine serum albumin (BSA), mercaptoethanol, calf intestinal
alkaline phosphatase (CIAP), and MgCl2 in 50 mM HEPES buffer (pH
7.4) at 37 °C for 6 days to give 19 after the usual workup, 1H NMR
(D2O) d 4.52 (br s, 3H, H-10), 4.44 (br s, 3H, H-1), 2.74 (br s, 3H,
Galactosylation
Sialylation
(%)
Total
yield (%)
(%)
4
8
15
97.2
16
19
65.3
20
63.5
88.1
NDa
64.4
96.8
63.7
NDa
93.5
92.4
17
95.3
21
11
14
NDa
18
NDa
22
87.4
73.7
a
ND means ‘not determined’ due to unsuccessful purification.
(a)
Acetone
NAc
SiCH2
CH2
HDO
H-1
SCH2
(b)
H-1
H-1
H-1
H-1’
(c)
SCH2
& H-3’’eq
H-3’’ax
5
4
3
2
1
0
Chemical shift (ppm)
Figure 2. Comparison of 1H NMR spectra on each reaction step. (a) Compound 14, (b) compound 18, (c) compound 22.

4910
K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4906–4910
H-300), integral ratio of the H atoms by 1H NMR: SiCH2/Ph/H-1/H-10/
H-300 = 6:5:3:3:3, MALDI-TOF MS calcd for [MꢁH]ꢁ: 2249.8; found
m/z: 2250.2. The results of the enzymatic elongation are shown in
Table 1. These reactions were conveniently monitored by TLC, and
the TLC indicated progress of the reaction step by step. After ultra-
filtration, chromatographic purification was performed by means
of a recycle-type SEC apparatus with water as the mobile phase.21
20 was isolated in 95% yield, ESI MS calcd for [Mꢁ3H]3ꢁ: 765.25;
found m/z: 765.26, and 22 was isolated in 74% yield, ESI MS calcd
for [Mꢁ4H+2Na]2ꢁ: 1497.47; found m/z: 1497.47. Unfortunately,
tetrameric compounds 17 and 21 were not isolated because of
the difficulty in removing impurities from the product mixtures.
Comparison of the results of 1H NMR of GlcNAc compound 14,
LacNAc compound 18, and SiaLacNAc compound 22 is shown in
Figure 2. These spectra clearly indicated that stepwise synthesis
is sufficiently accomplished.
Given the success of the stepwise elongation of carbohydrate
chains on the dendrimers, one-pot preparation was applied for this
reaction sequence. Thus, galactosylation for the dendrimer was
first carried out and the reaction was monitored by TLC. When
the reaction was completed, sialylation using the same protocol
as that for the preparation of 19 was carried out in the same reac-
tion vial. The results of the one-pot reaction are shown in Table 1.
Efficiency of the two-step synthesis was much higher than that of
the stepwise synthesis.
References and notes
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In conclusion, we have successfully demonstrated the prepara-
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efficient enzymatic elongation of carbohydrate moieties on multiva-
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preparation of carbosilane dendrimers with uniformly functional-
ized bioactive carbohydrate moieties. Further transformations
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Acknowledgments
We are grateful to Dr. T. Noguchi of Bioproducts Laboratory,
Biochemicals Division, Yamasa Co., Ltd for providing the CMP-
NANA used in this study. This work was partly supported by a
Health and Labour Sciences Research Grant for Research on
Advanced Medical Technology (14-N-O15) from the Ministry of
Health, Labor, and Welfare of Japan (D.T.).