Acyl and silyl group effects in reactivity-based one-pot glycosylation: Synthesis of embryonic stem cell surface carbohydrates Lc4 and IV2Fuc-Lc4

Article abstract of DOI:10.1021/ja300284x

Relative reactivity evaluations showed the graded arming of toluenyl thioglucosides by variously positioned silyl groups but not by their acyl counterparts. These findings were applied in reactivity-based one-pot assembly of linker-attached Lc₄ and IV²Fuc-Lc₄, which are components of human embryonic stem cell surface. The sugar-galectin-1 binding was also examined.

Full text of DOI:10.1021/ja300284x

Communication
pubs.acs.org/JACS
Acyl and Silyl Group Effects in Reactivity-Based One-Pot
Glycosylation: Synthesis of Embryonic Stem Cell Surface
Carbohydrates Lc4 and IV2Fuc-Lc4
Yun Hsu,†,‡ Xin-An Lu,† Medel Manuel L. Zulueta,† Chih-Ming Tsai,† Kuo-I Lin,†
Shang-Cheng Hung,*,†,§ and Chi-Huey Wong*,†
†Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan
‡Department of Chemistry, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu 300, Taiwan
§Department of Applied Chemistry, National Chiao Tung University, 1001, Ta-Hsueh Road, Hsinchu 300, Taiwan
S
* Supporting Information
perbenzylated over peracylated donors, the armed/disarmed
concept7 was expanded into numerical values that define the
reactivities as imparted by protecting groups on glycosyl
donors.8,9 Currently, relative reactivity values (RRVs) have
been assigned to hundreds of thioglycosides, allowing the one-
pot assembly of many important oligosaccharides.10 While
unconventional for acyl groups, Demchenko’s group reported
that 2-O-benzoylated S-benzoxazoyl donors are considerably
more reactive than their 2-O-benzylated counterparts.11
Accordingly, it was proposed that cooperative arming arises
from the ability of a 2-O-acyl group, via an acyloxonium ion, to
stabilize the oxocarbenium ion intermediate formed during
glycosylation.
Another new finding is the arming effect of silyl-based
protection. Bols and co-workers12 showed that multiple large
silyl protecting groups, which are more inclined to orient
axially, increase donor reactivity by minimizing the electronic
interaction between the oxygen substituents and the developing
positive charge. They also asserted that silyl groups are devoid
of intrinsic arming electronic effects and that monosilylation,
because of its marginal effect on ring conformation, is
insufficient to provide a significant increase in reactivity.
Confirmations of reactivity enhancement by acyl and silyl
groups, however, have yet to be made using the existing RRV
database.
ABSTRACT: Relative reactivity evaluations showed the
graded arming of toluenyl thioglucosides by variously
positioned silyl groups but not by their acyl counterparts.
These findings were applied in reactivity-based one-pot
assembly of linker-attached Lc4 and IV2Fuc-Lc4, which are
components of human embryonic stem cell surface. The
sugar−galectin-1 binding was also examined.
arbohydrates are commonly found at the cell surface,
C
aiding recognition, adhesion, and signal transduction
events.1 Particularly abundant are glycosphingolipids (GSLs),
which have sugar components attached to ceramide. GSLs are
diverse and can be further subdivided into ganglio-, globo-,
isoglobo-, lacto-, and neolacto series on the basis of their core
sequence and connectivities.2 The variety and quantity of GSLs
differ among cell types at various developmental stages as well
as in cancer progression.3 For example, human embryonic stem
cells highly express globo- and lacto-series GSLs, but upon
differentiation to embryoid body outgrowth cells, these GSLs
are downregulated, and the expressions of gangliosides
increase.4 The lacto-series GSLs explicitly detected are
lactotetraosyl (Lc4) and 2‴-O-fucosyl-Lc4 (IV2Fuc-Lc4) ceram-
ide (Figure 1). Lc4 carries the core sequence common to all
Before moving to the oligosaccharide preparation, we
systematically investigated the positional effect of acyl and
bulky silyl groups on D-glucose-based thiotoluenyl donors by
RRV determination.13 Drawing on our regioselective one-pot
protection strategy,14 we prepared the full set of monoacety-
lated, monobenzoylated, monosilylated, and disilylated thio-
glucosides with benzyl groups masking the other hydroxy
positions. Thioglucosides carrying a free hydroxyl at different
locations were also synthesized. The 2-O position gave the
highest values for the acetyl (1; RRV = 983) and benzoyl (2;
RRV = 1265) groups (Figure 2). Nonetheless, we did not
observe a reactivity enhancement by the 2-O-acyl group
because the 2-alcohol 3 (RRV = 1900) and the tetrabenzylated
4 (RRV = 2656)9 are still more reactive. Consequently, the 4-
Figure 1. The structures of Lc4 and IV2Fuc-Lc4.
lacto-series GSLs, and IV2Fuc-Lc4 contains the H type 1
antigen. We report herein the chemical synthesis of these
carbohydrates through a reactivity-based one-pot strategy.
Their interactions with galectin-1, a prominent decoder of
cell-surface information,5 were also examined in solution.
The effect of protecting groups on glycosyl donor reactivity
is well-known.6 Initially deduced from the higher reactivity of
Received: January 11, 2012
Published: March 5, 2012
© 2012 American Chemical Society
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Communication
4, the reactivity increase ranged from 49-fold for the 2,3-di-O-
TIPS derivative 23 to 120-fold for the 3,4-di-O-TBS derivative
28.
For the Lc4 assembly, the 2-O-benzoylated thiogalactoside
3410c (RRV = 5200) could be used to affect the required β-
linkage upon glycosylation of alcohol 36 (RRV = 20)9 (Scheme
1). Like toluenyl thioglucoside, 34 was found to be less reactive
a
Scheme 1. Synthesis of Compound 43
Figure 2. RRVs of different silylated, benzylated, and acylated
thioglycosides (Ac, acetyl; Bz, benzoyl; Bn, benzyl; Tol, toluenyl).
O-acylated compounds 12 and 13 possessed the lowest RRVs,
with an approximately 5-fold lower reactivity than the
corresponding 2-O-acylated glucosides. As the 2-O and 4-O
positions are on opposite sides of the pyranosyl oxygen, the
presumed stabilization by the 2-O-acyl group on the transient
oxocarbenium ion is distinct from the destabilizing 4-O-acyl
electron-withdrawing influence. Notably, work on acid-
mediated hydrolysis of methyl glucosides in water detected a
minor rate increase attributed to 2-O participation.15 Recently,
the higher reaction rate manifested by 2-O-benzoylated relative
to 2-O-benzylated donors was extended from S-benzoxazoyl to
S-ethyl leaving groups, but differentiation was not significant for
O-pentenyl, S-phenyl, S-toluenyl, and S-thiazolinyl groups.16
The stereochemical orientation of the S-benzoxazoyl group was
also found to be vital for rate enhancement.17 These accounts
and our results imply that the 2-O-acyl arming tendency is
strongly modulated by the leaving group, which, although
without proof, may well be extended to the reaction solvent and
activator. Thus, a decrease in the leaving group’s propensity for
departure significantly dampens any rate effect caused by
formation of the acyloxonium ion.
Replacement of benzyl with a tert-butyldimethylsilyl (TBS)
or triisopropylsilyl (TIPS) group at different locations all
increased donor reactivity. Moreover, TIPS offered a slightly
better enhancement than TBS in all cases. The degree of
arming was greatest at 2-O, with approximately 14-fold (5) and
26-fold (6) increases in reactivity upon exchange of benzyl with
TBS and TIPS groups, respectively. As anticipated, the least
reactive of the monosilylated donors were found to be the ones
in which the silyloxy group is positioned at 6-C (20 and 21),
where it has a predictably minor influence on ring
conformation. Thus, we have shown here that monosilylation
certainly does provide substantial reactivity enhancement.
Further affirming the bulky group arming effect, disilylated
donors were generally more reactive than monosilylated ones.
Adjacent silyl groups gave more pronounced enhancements,
consistent with the torsional effect. Relative to tetrabenzylated
a
Reagents and conditions: (a) 3 Å molecular sieves, 1.2 equiv of NIS,
0.4 equiv of TMSOTf, CH2Cl2, −55 °C, 2 h; 37: 30%, 38: 78%. (b)
1.2 equiv of NIS, 1.0 equiv of AgOTf, 0 °C, 10 min, 40: 40% (one
pot). (c) (1) ethylenediamine, tBuOH, reflux, 20 h; (2) Ac2O, Et3N,
DMAP, CH2Cl2, rt, 18 h; 78% (two steps). (d) (1) TBAF, CH2Cl2, rt,
18 h; (2) NaOMe, MeOH, rt, 13 h; 70% (two steps). (e) Pd/C, H2,
MeOH with 5% formic acid; 93%. Phth: phthaloyl.
than toluenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactoside
(RRV = 17 000).9 Addition of N-iodosuccinimide (NIS) and
trimethylsilyl triflate (TMSOTf) to the CH2Cl2 solution of
building blocks 34 and 36 supplied the adduct 37 in a meager
30% yield. A significant amount of the unreacted alcohol 36
was also recovered. As an alternative to 34, the 3-O-silylated
thiogalactoside 35 was synthesized,14d and RRV measurements
revealed a higher reactivity comparable in magnitude to that for
the similar TBS group installation on the glucose core. Selective
activation of 35 in the presence of 36 fortunately gave the
disaccharide 38 in a satisfactory 78% yield, consistent with the
notion that raising the donor reactivity in the presence of
poorly reactive acceptors also increases the glycosylation
yield.18 Without quenching of the initial coupling step, further
assembly in one pot was attempted by adding the lactosyl diol
3910b followed by NIS and TMSOTf. Because the equatorial 3′-
hydroxyl of 39 is more reactive than the axial 4′-hydroxyl, the
β1→3 link should be formed preferentially. Unfortunately, the
desired tetrasaccharide 40 was not obtained. We figured that
strong acids negatively affect the outcome of the second
coupling. Thus, incorporation of tripropargylamine to
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Communication
neutralize the activator of the first coupling followed by
glycosylation of 39 promoted by the mild NIS/AgOTf
conditions delivered 40 in a one-pot yield of 40%. Here we
also isolated the disaccharide 38 in 18% yield. Conversion of
the phthalimido functionality to acetamido was carried out with
ethylenediamine to afford the free amine, which was
subsequently acetylated using Ac2O and N,N-dimethyl-4-
aminopyridine (DMAP). The 4′-hydroxyl was also acetylated
in this step, giving the product 41 in a two-step yield of 78%.
Stepwise cleavage of the silyl group by tetra-n-butylammonium
fluoride (TBAF) and the ester moiety by NaOMe/MeOH
delivered triol 42 (70%). Global hydrogenolysis led to the
linker-attached Lc4 (43) in 93% yield.
Table 1. Dissociation Constants (Kd) of Selected Sugars and
Galectin-1 Determined Using Isothermal Titration
a
Calorimetry
a
entry
sugar
Kd (μM)
1
2
3
4
compound 43
compound 47
lactose
31.5
33.8
71.4
b
galactose
−
a
The actual parameter measured by the technique is the association
b
constant Ka, which is the inverse of Kd. No binding was detected.
(33.8 μM) gave nearly identical Kd values upon interaction with
galectin-1. The synthetic sugars also bound the protein about 2
times more tightly than did lactose (71.4 μM). This implies
that the fucosylation of the terminal galactose in Lc4 does not
have any sizable effect on recognition of the sugar by the
protein. On the other hand, the higher affinity of 43 and 47
relative to lactose might be due to the lack of acetamido group
in the latter sugar.
To construct the IV2Fuc-Lc4 backbone, we started by
regioselective acetylation of compound 42 (Scheme 2).
a
Scheme 2. Synthesis of Compound 47
In summary, we systematically investigated the effect of acyl
and bulky silyloxy groups at different locations on donor
reactivity of the toluenyl thioglucoside by RRV analysis. It was
found that while silyl groups possess a strong arming influence
at all locations, this was not particularly demonstrated by 2-O-
acyl groups, as originally reported for donors with S-
benzoxazoyl leaving groups. A similar pattern was observed
for toluenyl thiogalactoside. This understanding was success-
fully implemented in the assembly of linker-attached Lc4
through a reactivity-based one-pot strategy. IV2Fuc-Lc4 was
also generated using an intermediate in the Lc4 transformation.
The synthesized sugars bound galectin-1 to the same extent.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and copies of H and 13C NMR
1
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
schung@gate.sinica.edu.tw, chwong@gate.sinica.edu.tw
a
Notes
Reagents and conditions: (a) 2.1 equiv of Ac2O, pyridine, CH2Cl2, rt,
The authors declare no competing financial interest.
18 h; 70%. (b) 3 Å molecular sieves, 1.2 equiv of NIS, 0.4 equiv of
TMSOTf, CH2Cl2, −60 to −40 °C, 1 h; 77%. (c) (1) NaOMe,
MeOH, rt, 6 h; (2) Pd/C, H2, MeOH with 5% formic acid; 90% (two
steps).
ACKNOWLEDGMENTS
■
This work was supported by the National Science Council
(NSC 97-2113-M-001-033-MY3 and NSC 98-2119-M-001-
008-MY2) and Academia Sinica.
Through the steric effect of the glycosidic linkage, regioselective
3‴,4′-di-O-acetylation of 42 with Ac2O was achieved, affording
2‴-alcohol 44 (70%). Glycosylation of 44 using fucosyl donor
4513 promoted by NIS/TMSOTf formed pentasaccharide 46
(77%). Subsequent deacetylation (NaOMe/MeOH) followed
by hydrogenolysis (Pd/C, H2) successfully furnished com-
pound 47 in 90% yield.
Using isothermal titration calorimetry, we investigated the
binding of compounds 43 and 47 with galectin-1, the first
mammalian galectin found among 15 members.5 Galectins are a
highly conserved family of β-galactoside-binding proteins that
play various roles in cancer progression, immune response,
inflammation, and development by interacting with cell-surface
carbohydrates.19 As shown in Table 1, 43 (31.5 μM) and 47
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