A concise chemical synthesis of a fluorescent βgal-(1,4)-S-βGlc- Cer derivative and its enzymatic elongation by glycosyltransferases

Article abstract of DOI:10.1016/j.tetlet.2012.01.119

A straightforward chemical synthesis of lyso-lactosylceramide with the terminal galactose linked to glucose through a β-S-glycosidic bond is reported. The product is labeled on the amino-group with tetramethylrhodamine enabling its ultrasensitive detection in capillary electrophoresis using laser-induced fluorescence. The fluorescent product disaccharide is resistant to hydrolysis by glycosidases but is shown to remain as an acceptor substrate for glycosyltransferases for the conversion into the trisaccharides GM3 and Gb3.

Full text of DOI:10.1016/j.tetlet.2012.01.119

Tetrahedron Letters 53 (2012) 1812–1815
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Tetrahedron Letters
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A concise chemical synthesis of a fluorescent bGal-(1,4)-S-bGlc-Cer
derivative and its enzymatic elongation by glycosyltransferases
Hidenori Tanaka a, Yayoi Yoshimura a, Norman J. Dovichi b, Monica M. Palcic a, Ole Hindsgaul a,
⇑
a Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark
b Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A straightforward chemical synthesis of lyso-lactosylceramide with the terminal galactose linked to glu-
cose through a b-S-glycosidic bond is reported. The product is labeled on the amino-group with tetram-
ethylrhodamine enabling its ultrasensitive detection in capillary electrophoresis using laser-induced
fluorescence. The fluorescent product disaccharide is resistant to hydrolysis by glycosidases but is shown
to remain as an acceptor substrate for glycosyltransferases for the conversion into the trisaccharides GM3
and Gb3.
Received 24 November 2011
Revised 16 January 2012
Accepted 27 January 2012
Available online 3 February 2012
Keywords:
Glycolipid
Ó 2012 Elsevier Ltd. All rights reserved.
Lactosylceramide
Glycosyltransferase
S-glycosidic bond
Glycolipids are found on the surface of the cell membrane where
oligosaccharides are located outside the cell and can play an
important role in biological events such as cellular recognition, cell
adhesion, and infection.1 It is well-known that changes in the struc-
ture of such glycolipids accompany embryonic development, tumor
progression,2,3 and many inborn errors of metabolism, including
lysosomal storage diseases.4–6 There has been a long-standing inter-
est in understanding the metabolic pathways for both glycolipid
biosynthesis and catabolism.7 To study the biosynthetic (anabolic)
pathways, glycosidase-resistant glycolipid analogs can be a valu-
able tool.8–10
Labeling of natural glycolipids can be conveniently carried out
via the enzymatic cleavage of the long-chain fatty acids of the
ceramide and installation of a fluorescent tag on the resulting free
amine.13 This led us to select thiolactosylsphingolipid 3 as our
primary synthetic target, with a sulfur atom in the glycosidic link-
age: a class of compound known to be stable to glycosidase
enzymes.8–10 Our retrosynthetic analysis of 3 is shown in Scheme
1. To facilitate the final deprotection and retain the double bond,
acyl protecting groups are used in both thiolactosyl donor 4 and
sphingolipid acceptor 5.14 Formation of the thio-linkage in 4 is
achieved by coupling between galactosyl isothiouronium salt 6
and galactosyl triflate 7 via an SN2 reaction (Scheme 1).8
Lactosylceramide (1) is the common precursor for the biosyn-
thesis of both the ganglio- and globo-series of animal glycolipids
via the addition of sugars in the Golgi apparatus by the appropriate
The synthesis began with the formation of the thiolactoside.
Compounds 615 and 816 were prepared starting from galactose
pentaacetate 9. After conversion into triflate 7, condensation with
isothiouronium salt 6 in N,N0-dimethylformamide/dichlorometh-
ane using triethylamine was carried out to give thiolactoside 10
in a 44% yield.17 The new S-glycosidic bond was confirmed as a
Gal-b(1,4)-Glc linkage from coupling constants (JG1;a2l = 10 Hz,
glycosyltransferases (anabolism). It is also
a substrate for
lysosome-localized glycosidases resulting in its degradation to cer-
amide and beyond (catabolism). Wishing to selectively investigate
the anabolic pathways, we decided to prepare an analog of lacto-
sylceramide (1) that would be stable to glycosidases and that
was fluorescently tagged to enable the ultrasensitive detection
and quantitation of larger oligosaccharides at the single-cell level
(ca. 200 molecule detection).11,12 Herein, we report a facile route
to a tetramethylrhodamine (TMR)-labeled analog 2 of lactosylcer-
amide in which the terminal galactose is linked to glucose through
a b-S-glycosidic bond, and demonstrate its enzymatic elongation to
fluorescent-labeled trisaccharides.13
Glc
3;4
Glc
4;5
J
= 10 Hz and J = 10 Hz). Hydrolysis of phenyl thioglycoside
10 was effected using N-bromosuccinimide (NBS)/H2O. Surpris-
ingly, although an excess of NBS (5.0 equiv) was used, the inner
S-glycosidic linkage was not affected which was likely due to steric
hindrance. The resultant hemiacetal 11 was reacted with trichloro-
acetonitrile and 1,8-diazabicycloundec-7-ene (DBU) to give imi-
date donor 4 in a 58% yield over two steps from 10 (Scheme 2).
Next, the N-TFA protected sphingosine acceptor 5 was obtained
by olefin cross-metathesis. The addition of vinylmagnesium
⇑
Corresponding author. Tel.: +45 3327 5382; fax: +45 3327 4708.
E-mail address: Ole.Hindsgaul@carlsberglab.dk (O. Hindsgaul).
bromide to (S)-Garner’s aldehyde 12 afforded
a mixture of
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.01.119

H. Tanaka et al. / Tetrahedron Letters 53 (2012) 1812–1815
1813
O
diastereomerically pure form (11% from 12).20 Removal of the ace-
tal group gave the target acceptor 5 in a 67% yield (Scheme 3).
With imidate donor 4 and sphingosine acceptor 5 in hand, their
coupling could be effected in the presence of catalytic trimethyl-
silyl trifluoromethanesulfonate (TMSOTf) to give glycosphingolipid
16 in a 22% yield along with di-glycosylated compound 17 in a 23%
yield (Scheme 4). Even though the yield of 16 was low, this strategy
had the advantage that the final deprotection could be achieved in
one-step.
All the acyl groups in 16 were cleaved using sodium methoxide
in methanol to afford S-lactosylsphingolipid 3 in a quantitative
yield, which was easily converted into TMR-labeled glycolipid
2 as previously reported for the natural O-linked compound
(Scheme 5).13
HO
HO
OH
O
OH
O
HN
C15H31
O
O
C13H27
HO
HO
HO
OH
Lactosylceramide
1
Me2N
O
NMe2
Gb3
using GalT
COO
?
O
HN
HO
OH
O
OH
O
HN
O
HO
?
S
O
C13H27
Disaccharides with sulfur in the glycosidic linkage are known to
be resistant to glycosidases. For them to be useful as tools in the
study of glycolipid biosynthesis, however, they must remain as
substrates for glycosyltransferases which build up larger oligosac-
charides. In the biosynthesis of gangliosides in the common GM1
HO
HO
HO
OH
Synthetic target
GM3
using SiaT
2
pathway, this would require 2 to be a substrate for an
a(2,3)-
sialyltransferase, while for the production of glycosphingolipids
in the globoside pathway, glycolipid 2 must remain as a substrate
HO
HO
OH
O
OH
O
NH2
for an
Incubation of 2 with CMP-sialic acid in the presence of
sialyltransferase,21 or with UDP-galactose in the presence of
a(1,4) galactosyltransferase.
S
O
C13H27
a(2,3)-
HO
HO
HO
OH
3
a
(1,4)-galactosyltransferase22 resulted in the complete disappear-
ance of 2 and the production23 of the desired trisaccharides GM3
18 and Gb3 19, respectively (Scheme 5), as the sole products based
on the analysis by capillary electrophoresis with fluorescence
detection and MALDI-TOF mass-spectrometry (see Supplementary
data).
AcO
AcO
OAc
O
NHTFAc
OBz
NH
O
HO
C13H27
S
+
O
BzO
In summary, we have demonstrated a convergent synthesis of
S-lactosylceramide analog 2 and shown that, despite the incorpo-
ration of a sulfur atom in the glycosidic linkage and a large polyar-
omatic fluorescent tag, this compound remains as a substrate for
two key enzymes involved in the biosynthesis of larger common
glycolipids. This compound is being further evaluated as a sub-
strate in the investigation of glycolipid biosynthesis in single
cells.11,12
AcO
OH
BzO
CCl3
4
5
AcO
AcO
TfO
BzO
OAc
O
OBz
O
NH2Br
NH2
+
S
SPh
AcO
BzO
6
7
Acknowledgment
Scheme 1. Retrosynthesis of target 2 having a sulfur linkage between galactose and
glucose.
This work was supported by grant R33DK70317 from the
National Institute of Health.
trans- and syn-allylic alcohols 13 quantitatively with a dr = 5/1.18
The diastereomeric mixture was coupled with 1-pentadecene
using the 2nd generation Grubbs catalyst,19 and then protecting
group manipulations including benzylidene acetalation to separate
the minor isomer which led to the sphingosine derivative 15 in
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2012.01.119.
AcO
AcO
AcO
AcO
OAc
O
OAc
O
i
NH2Br
NH2
OAc
S
AcO
6(80%)
AcO
9
ref. 16
AcO
AcO
OAc
O
10: R = β-SPh (44%, 2 steps from 8)
HO
BzO
TfO
BzO
OBz
O
OBz
iv
ii
iii
O
11: R = α,β-OH
S
SPh
R
BzO
v
AcO
BzO
BzO
4: R = α,β-OC(=NH)CCl3
(58%, α/β = 6/1, 2 steps from 10)
8
7
Scheme 2. Synthesis of disaccharide donor 4. (i) (a) 33% wt. HBr in AcOH/CH2Cl2, rt, 2 h; (b) thiourea/acetone, reflux, 20 h; (ii) Tf2O, pyridine, CH2Cl2, 0 °C, 1 h; (iii) 6, Et3N,
DMF/CH2Cl2, rt, 24 h; (iv) NBS/wet acetone, rt, 2 h; (v) trichloroacetonitrile, DBU/CH2Cl2, rt, 1 h.

1814
H. Tanaka et al. / Tetrahedron Letters 53 (2012) 1812–1815
NBoc
NBoc
OH
NHTFA
O
H
3
i
5
iii
O
O
H
4
C13H27
NHTFA
C13H27
H
R
Ph
O
1
O
2
O
O
12
H
13: R = H
15 (11% from 12)
Ph
ii
14: R = C13H27
iv
NHTFA
HO
C13H27
5
OH
(67%)
Scheme 3. Synthesis of sphingosine acceptor 5. (i) vinylmagnesium bromide/THF, ꢀ78 °C, 2 h; (ii) 1-pentadecene, Grubbs 2nd generation catalyst/CH2Cl2, reflux, 4 h; (iii) (a)
aq. AcOH, 50 °C, 13 h; (b) TFA/CH2Cl2, rt, 2 h; (c) TFA2O, Et3N, MeOH/CH2Cl2, rt, 48 h; (d) benzaldehyde dimethyl acetal, TsOH monohydrate/MeCN, rt, 1 h; (iv) aq. AcOH, rt to
50 °C, 38 h.
TMSOTf
(0.2 equiv.)
3 Å MS
AcO
AcO
AcO
AcO
OAc
O
OAc
O
OBz
O
OBz
O
NHTFAc
OH
NHTFAc
O
S
S
O
C13H27
O
C13H27
4
+
5
BzO
BzO
AcO
AcO
CH2Cl2
-10 °C to RT
18 h
+
(1.2 equiv.) (1.0 equiv.)
BzO
BzO
16
17
O
OBz
(22%)
(23%)
BzO
OAc
O
AcO
AcO
OBz
S
AcO
Scheme 4. Coupling of disaccharide donor 4 with sphingosine acceptor 5.
HO
HO
O
OH
O
OH
O
HO OH
AcHN
OH
O
R
i
iii
HN
16
HO
S
O
HO
O
C13H27
HO
HO
HO
18
HO
OH
COOH
OH
3: R = H
2: R =
HO
OH
O
Me2N
O
NMe2
iv
HO
ii
HO
O
OH
O
COO
HO
HO
19
O
HN
O
Scheme 5. Deprotection, labeling with 6-TMR-b-Ala, and enzymatic reactions. (i) NaOMe/MeOH, rt, 4 days; (ii) 6-TMR-b-Ala NHS ester, DIEA/DMF, rt, 24 h; (iii) CMP-Neu5Ac,
(2,3)-sialyltransferase (MalE fusion protein from Campylobacter jejuni), MOPS buffer (pH 7.5) containing MnCl2 and 5% (v/v) DMSO; (iv) UDP-Gal, (1,4)-galactosyltrans-
ferase (Neisseria meningitidis), MOPS buffer (pH 7.0) containing MnCl2, BSA, DTT and 5% (v/v) DMSO.
a
a
Hindsgaul, O.; Palcic, M. M.; Prendergast, J.; Schnaar, R. L.; Dovichi, N. J. Anal.
Chem. 2011, 83, 2748–2753; (c) Krylov, S. N.; Zhang, Z.; Chan, N. W.; Arriaga, E.;
Palcic, M. M.; Dovichi, N. J. Cytometry 1999, 37, 14–20.
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coupling constants (J1ax,2 = J2,3 = approx. 10 Hz, J4,5 = 15.2 Hz).
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1815
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23. (a) Enzymatic synthesis of GM3 18. Compound 2 was dissolved in DMSO to
7.5) containing 60 mM MnCl2 and 5% (v/v) DMSO. After 5 h, CMP-Neu5Ac
(0.5 nmol) was added. The reaction was incubated at room temperature for a
total of 14 h. The formation of GM3 was confirmed by mass spectrometry and
capillary electrophoresis(CE). m/z (MALDI-TOF): found [MꢀH]ꢀ 1412.72,
C
69H99N5O24S calcd for [MꢀH]ꢀ 1412.63. (b) Enzymatic synthesis of Gb3 19.
The reaction mixture (40 ll) consisted of compound 2 (0.5 nmol), UDP-Gal
(1.5 nmol), and
a(1,4)-galactosyltransferase (1.6 lg) in 50 mM MOPS buffer
(pH 7.0) containing 20 mM MnCl2, 0.01% BSA, 0.5 mM DTT, and 5% (v/v) DMSO.
The reaction mixture was incubated at room temperature for 3 h. The
formation of Gb3 was confirmed by mass spectrometry and CE. m/z (MALDI-
TOF): found [M+H]+ 1285.83, C64H93N4O21S calcd for [M+H]+ 1285.64.
yield a 250
final concentration of DMSO was 5% of the reaction volume (40
reaction mixture (40 l) consisted of compound 2 (0.5 nmol), CMP-Neu5Ac
(0.6 nmol), and (2,3)-sialyltransferase (1.0 g) in 50 mM MOPS buffer (pH
lM solution; 2
l
l (0.5 nmol) of this solution was used so that the
ll). The
l
a
l