β-Mannosylation through O-Alkylation of Anomeric Cesium Alkoxides: Mechanistic Studies and Synthesis of the Hexasaccharide Core of Complex Fucosylated N-Linked Glycans

Article abstract of DOI:10.1002/ejoc.202000313

Several structurally diverse d-mannose-derived lactols, including various deoxy-d-mannoses and conformationally restricted bicyclic d-mannoses, have been synthesized and investigated in mechanistic studies of β-mannosylation through Cs₂CO₃-mediated anomeric O-alkylation. It was found that deoxy mannoses or conformationally restricted bicyclic d-mannoses are not as reactive as their corresponding parent mannose. This type of β-mannosylation proceeds efficiently when the C2-OH is left free, and protection of that leads to inferior results. NMR studies of d-mannose-derived anomeric cesium alkoxides indicated the predominance of the equatorial β-anomer after deprotonation. Reaction progress kinetic analysis suggested that monomeric cesium alkoxides be the key reactive species for alkylation with electrophiles. DFT calculations supported that oxygen atoms at C2, C3, and C6 of mannose promote the deprotonation of the anomeric hydroxyl group by Cs₂CO₃ and chelating interactions between Cs and these oxygen atoms favor the formation of equatorial anomeric alkoxides, leading to the highly β-selective anomeric O-alkylation. Based on experimental data and computational results, a revised mechanism for this β-mannosylation is proposed. The utilization of this β-mannosylation was demonstrated by an efficient synthesis of the hexasaccharide core of complex fucosylated N-linked glycans.

Full text of DOI:10.1002/ejoc.202000313

A Journal of
Accepted Article
Title: β-Mannosylation via O-Alkylation of Anomeric Cesium Alkoxides:
Mechanistic Studies and Synthesis of the Hexasaccharide Core
of Complex Fucosylated N-Linked Glycans
Authors: Shuai Meng, Bishwa Raj Bhetuwal, Hai Nguyen, Xiaotian
Qi, Cheng Fang, Kevin Saybolt, Xiaohua Li, Peng Liu, and
Jianglong Zhu
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000313
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
β-Mannosylation via O-Alkylation of Anomeric Cesium Alkoxides:
Mechanistic Studies and Synthesis of the Hexasaccharide Core of
Complex Fucosylated N-Linked Glycans
Shuai Meng,[a]† Bishwa Raj Bhetuwal,[a]† Hai Nguyen,[a]† Xiaotian Qi,[b] Cheng Fang,[b] Kevin Saybolt,[c]
Xiaohua Li,*[c] Peng Liu,*[b,d] and Jianglong Zhu*[a]
Abstract: A number of structurally diverse D-mannose-derived lactols, molecules have been developed as effective therapeutic agents
including various deoxy-D-mannoses and conformationally restricted
bicyclic D-mannoses, have been synthesized and investigated in
mechanistic studies of β-mannosylation via Cs2CO3-mediated
anomeric O-alkylation. It was found that deoxy mannoses or
conformationally restricted bicyclic D-mannoses are not as reactive
as their corresponding parent mannose. This type of β-mannosylation
proceeds efficiently when the C2-OH is left free, and protection of that
leads to inferior results. NMR studies of D-mannose-derived anomeric
cesium alkoxides indicated the predominance of the equatorial β-
anomer after deprotonation. Reaction progress kinetic analysis
suggested that monomeric cesium alkoxides be the key reactive
species for alkylation with electrophiles. DFT calculations supported
that oxygen atoms at C2, C3, and C6 of mannose promote the
deprotonation of the anomeric hydroxyl group by Cs2CO3 and
chelating interactions between Cs and these oxygen atoms favour the
formation of equatorial anomeric alkoxides, leading to the highly β-
selective anomeric O-alkylation. Based on experimental data and
computational results, a revised mechanism for this β-mannosylation
is proposed. The utilization of this β-mannosylation was demonstrated
by an efficient synthesis of the hexasaccharide core of complex
fucosylated N-linked glycans.
for treating various diseases. Sugar moieties, i.e. glycans, are
also known to influence physical, chemical, and biological
properties of their carrier molecules, including proteins, peptides,
and small molecules.2 In order to study their biological functions,
access to sufficient amounts of pure and structurally well-defined
carbohydrate molecules need to be available to researchers.
Isolation from natural sources sometimes has been successful,
but the highly heterogeneous nature of glycoforms limit the
practicality of this approach. In addition, the highly intrinsic
complexity of the diverse carbohydrate structures poses a great
challenge to the synthetic community, albeit great progress has
been achieved in their chemical3 and chemo-enzymatic
synthesis.4
Among various glycosidic linkages, β-mannopyranosides
(oftentimes referred to as β-mannosides) exist in a wide range of
biologically significant molecules including N-linked glycans,
bacterial capsular polysaccharides, fungal metabolites,
glycolipids, antimicrobial antibiotics, and lipopolysaccharides.5 As
a
class of 1,2-cis glycosides,6 β-mannopyranosides are
exceptionally difficult to construct due to the steric effect of the
axial C2-substituent as well as the absence of anomeric effect and
neighboring group participation. Despite the fact that remarkable
success has been achieved in stereoselective synthesis of β-
mannosides,7 development of a mild and easily operable β-
mannosylation method is desirable and of great interest to the
carbohydrate community.
Introduction
Tremendous glycobiological studies have demonstrated
oligosaccharides and glycoconjugates play essential roles in
numerous biological processes.1 In addition, carbohydrate
Initially developed by Schmidt8 and later by others,9 anomeric
O-alkylation has been demonstrated as a viable alternative to
traditional glycosylation for successful stereoselective synthesis
of oligosaccharides and glycoconjugates. With respect to β-
mannosides, Schmidt previously disclosed some studies on
anomeric O-alkylation of partially or fully protected D-
mannopyranoses with simple primary electrophiles in the
presence of NaH or KOtBu as the base.10 Oftentimes, poor to
moderate yields10c and moderate selectivity10d were observed. In
addition, over-alkylation was found to be a problem when 3,4,6-
tri-O-benzyl-D-mannopyranose (cf. 7, Table 1) was employed.10c
Based on our success in stereoselective synthesis of 2-deoxy
sugars,11,12 we recently reported a stereoselective synthesis of β-
mannopyranosides by cesium carbonate (Cs2CO3)-mediated
anomeric O-alkylation of D-mannose-derived 1,2-diols with
primary or secondary electrophiles.13,14 It is worth noting that this
mild and easily operable β-mannosylation method directly affords
the desired β-mannosides with a free C2-OH at the mannose
residue. The free C2-alcohol can be directly subjected to
subsequent chemical transformations as demonstrated in a
formal synthesis of potent calcium signal modulator
acremomannolipin A15 and a concise synthesis of a trisaccharide
[a]
Dr. S. Meng,† B. R. Bhetuwal,† H. Nguyen,† Prof. Dr. J. Zhu. †equal
contribution
Department of Chemistry and Biochemistry and School of Green
Chemistry and Engineering, The University of Toledo, 2801 W.
Bancroft Street, Toledo, Ohio 43606, United States
E-mail: Jianglong.Zhu@utoledo.edu
https://www.utoledo.edu/nsm/chemistry/people/Webpages/Zhu.html
Dr. X. Qi, C. Fang, Prof. Dr. P. Liu
Department of Chemistry, University of Pittsburgh, 219 Parkman
Avenue, Pittsburgh, Pennsylvania 15260, United States
E-mail: pengliu@pitt.edu
https://www.chem.pitt.edu/person/peng-liu
K. Saybolt, Prof. Dr. X. Li
Department of Natural Sciences, University of Michigan‒Dearborn,
4901 Evergreen Road, Dearborn, Michigan 48128, United States
E-mail: shannli@umich.edu
[b]
[c]
[d]
https://umdearborn.edu/users/shannli
Department of Chemical and Petroleum Engineering, University of
Pittsburgh, 3700 O’Hara Street, Pittsburgh, Pennsylvania 15261,
United States
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Scheme 1. Initially proposed β-mannosylation via anomeric O-alkylation under dual control of kinetic anomeric effect and metal chelation.
oligomer of the Hyriopsis schlegelii glycosphingolipid,
respectively.16
In our originally proposed hypothesis,13 after deprotonation of
D-mannose 1 with excess amounts of base, a mixture of dianions
Results and Discussion
2 and 4 may be produced and interconvert into each other via
open intermediate (Scheme 1). Due to the chelation
In order to gain more insight into this β-mannosylation, we
prepared additional deoxy D-mannose-derived lactol substrates
including 4,6-di-O-benzyl-3-deoxy-D-mannose 10, 4,6-di-O-
benzyl-2,3-dideoxy-D-mannose 12,18 4-O-benzyl-3,6-dideoxy-D-
3
effect,10equatorial anomeric alkoxide 4 would be preferentially
formed over the axial counterpart 2. In addition, equatorial
anomeric alkoxide 4 was believed to be more nucleophilic than
the axial counterpart 2 because of the double electron-electron
repulsion (also known as kinetic anomeric effect, colored in
purple).8,17 Subsequent SN2 reaction of equatorial anomeric
alkoxide 4 with suitable electrophiles may afford desired β-
mannopyranosides 5. It is believed that the synergy between
kinetic anomeric effect and the chelation of cesium ion with
oxygen atoms at C1 and C2 was the key for this β-selective
anomeric O-alkylation to succeed (cf. Newman projection,
Scheme 1).
19
mannose 14, and 4-O-benzyl-2,3,6-trideoxy-D-mannose 15
(Table 1).20 It was found that Cs2CO3-mediated anomeric O-
alkylation of 4,6-di-O-benzyl-3-deoxy-D-mannose 10, 4,6-di-O-
benzyl-2,3-dideoxy-D-mannose 12, and 4-O-benzyl-3,6-dideoxy-
D-mannose 14 with secondary triflate 6 (2.0 eq.) in the presence
of Cs2CO3 (2.5 eq.) under the same conditions afforded 4,6-di-O-
benzyl-3-deoxy-β-D-mannoside 25, 4,6-di-O-benzyl-2,3-dideoxy-
β-D-mannoside 27, 4-O-benzyl-3,6-dideoxy-β-D-mannoside 29 in
40%, 26%, and 3% yields (β only), respectively. However, no 4-
O-benzyl-2,3,6-trideoxy-β-D-mannoside 30 was detected when 4-
O-benzyl-2,3,6-trideoxy-D-mannose 15 reacted with triflate 6
under the same conditions.
Previously, in order to understand what structural features are
critical for the β-mannosylation to be effective, 3,4,6-tri-O-benzyl-
D-mannopyranose 7 and several D-mannose-derived lactol
substrates including 3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
D-mannose 8, 3,4,6-tri-O-benzyl-2-deoxy-D-mannose 9, 3,4-di-
O-benzyl-6-deoxy-D-mannose (D-rhamnose) 11, 3,4-di-O-
benzyl-2,6-dideoxy-D-mannose (D-olivose) 13, and a bicyclic 3-
O-benzyl-4,6-O-benzylidene-D-mannose 16 were prepared and
studied in the anomeric O-alkylation with D-galactose-derived
secondary triflate 6 in the presence of cesium carbonate (Table
1).13 It was found that replacing the C6 benzyl ether of D-mannose
7 with a TBDPS ether led to lower yield of corresponding
disaccharide 23 (40%, β only). Anomeric O-alkylation of 3,4,6-tri-
O-benzyl-2-deoxy-D-mannose 9 with secondary triflate 6 (2.0 eq.)
in the presence of Cs2CO3 (2.5 eq.) afforded the corresponding
desired disaccharide 24 in 64% yield (β only), which was
comparable to the reaction involving parent 3,4,6-tri-O-benzyl-D-
mannose 7 (67% yield, β only). In addition, 3,4-di-O-benzyl-6-
deoxy-D-mannose (D-rhamnose) 11 reacted with secondary
triflate 6 to give corresponding desired disaccharide 26 in only
30% yield (β only). Furthermore, anomeric O-alkylation of D-
olivose 13 and bicyclic D-mannose 16 with triflate 6 under the
same conditions afforded desired disaccharides 28 and 31 in 15%
and 0% yield, respectively.13 Those results suggested that our
originally proposed working model for this β-mannosylation (cf. 4
in Scheme 1) may need to be revisited.
Next, direct competition experiments were carried out for
comparative studies of the reactivity of the parent D-mannose 7
and its deoxy derivatives 9, 10, and 11. When a mixture of D-
mannose 7 (1.0 eq.), 2-deoxy-D-mannose 9 (1.0 eq.), and triflate
6 (2.0 eq.) in 1,2-dichloroethane was warmed at 40 oC for 24
hours in the presence of 2.5 equivalents of Cs2CO3, β-mannoside
22 and 2-deoxy disaccharide 24 were obtained in 48% and 13%,
respectively (β only) (a, Scheme 2). This interesting result
demonstrates that D-mannose 7 is much more reactive than 2-
deoxy-D-mannose 9 in this type of anomeric O-alkylation and
suggests the important role of C2-OH of the D-mannose, despite
the fact that previous experiments showed that anomeric O-
alkylation of parent D-mannose 7 or 2-deoxy-D-mannose 9 with
triflate 6 afforded 22 or 24 in similar yields and anomeric
selectivity (Table 1, vide supra). Under exactly the same
conditions, β-mannoside 22 and 3-deoxy disaccharide 25 were
obtained in 30% and 15%, respectively (β only) (b, Scheme 2).
Another competition experiment involving parent mannose 7 and
3,4-di-O-benzyl-6-deoxy-D-mannose 11 afforded β-mannoside
22 and 6-deoxy disaccharide 26 in 31% and 8%, respectively (β
only) (c, Scheme 2). These results indicate that deoxy mannoses
do not react as efficiently as their parent mannose in this type of
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Table 1. Anomeric O-alkylation of various deoxy-D-mannoses and bicyclic D-mannose-derived lactols with D-galactose-derived secondary triflate 6 in the presence
of Cs2CO3.a,b
o
[a] General conditions: D-mannose-derived lactols (1.0 eq.), triflate 6 (2.0 eq.), Cs2CO3 (2.5 eq.), ClCH2CH2Cl, 40 C, 24 h. [b]
Isolated yield. [c] See reference 13. [d] Disaccharide 22 was obtained in 75% yield when 2.5 eq. of triflate 6 and 3.0 eq. of Cs2CO3
were used, see reference 13.
Scheme 2. Competition anomeric O-alkylations between parent D-mannose and deoxy-D-mannoses.
Cs2CO3-mediated anomeric O-alkylation and suggest that all of
the oxygen atoms at C2, C3 and C6 of mannose contribute to the
reactivity of the mannose lactols.21
Electronically, anomeric alkoxides derived from deoxy sugars
should be more electron-rich than those derived from their
corresponding parent sugars due to the absence of electron-
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
withdrawing oxygen atom(s). In addition, deoxy sugars are less
sterically hindered in comparison with their parent sugar
molecules. Therefore, deoxy sugar-derived anomeric alkoxides
should be more nucleophilic than those derived from their parent
sugar molecules and afford the corresponding deoxy glycosides
in higher yields via anomeric O-alkylation, which is consistent with
our previous observations.11 However, our experimental results
demonstrated that parent 3,4,6-tri-O-benzyl-D-mannose 7 was
superior to those deoxy-D-mannoses (9-15) in this cesium
carbonate-promoted anomeric O-alkylation (Table 1), which
seemed somewhat contradictory to what we had observed
previously for the stereoselective synthesis of 2-deoxy-β-
glycosides.11 Such findings encouraged us to further investigate
the mechanistic aspects of this β-selective mannosylation.
glucopyranose-dervived anomeric alkoxide 41. Anomeric O-
alkylation of this alkoxide 41 gave rise to β-glucoside 40 under
kinetic anomeric effect.8,17 This result indicated that 2,3,4,6-tetra-
O-benzyl-D-mannopyranose 38 is less reactive than 3,4,6-tri-O-
benzyl-D-mannopyranose 7, probably due to more severe steric
effects. Despite that the presence of a free hydroxyl group at C2
of the mannose is not essential for this β-mannosylation, leaving
this C2 hydroxyl group unprotected helps achieve superior yields
and excellent β-selectivity.
Scheme 3. Anomeric O-alkylation of 2,3,4,6-tetra-O-benzyl-D-mannopyranose.
Previous studies also indicated that conformationally restricted
bicyclic 3-O-benzyl-4,6-O-benzylidene-D-mannose 16 did not
react with triflate 6 in the presence of cesium carbonate to afford
corresponding disaccharide 31,13 which suggests that
a
conformationally flexible sugar ring may be critical for this type of
anomeric O-alkylation. In order to further study the impact of the
conformation and ring strain to the reactivity of the D-mannose-
derived anomeric alkoxide in the β-mannosylation reaction, we
prepared additional bicyclic D-mannose-derived lactol substrates.
They include 3,4-O-bisketal-protected mannose 17, 4,6-di-O-
benzyl-2,3-O-isopropylidene-D-mannose 18,22 3,6-anhydro-4-O-
benzyl-D-mannose 19, 2,6-anhydro-3,4-di-O-benzyl-D-mannose
20, and 4-O-benzyl-3,6-O-(o-xylylene)-D-mannose 21.20,23 As
shown in Table 1, Cs2CO3-mediated anomeric O-alkylation of 3,4-
O-bisketal-protected mannose 17 and 4,6-di-O-benzyl-2,3-O-
isopropylidene-D-mannose 18 bearing conformationally flexible
C6-oxygen atom with secondary triflate 6 under standard
conditions afforded 3,4-O-bisketal-protected mannoside 32 and
4,6-di-O-benzyl-2,3-O-isopropylidene-D-mannoside 33 in 23%
and 34% yields (β only), respectively. However, none of 3,6-
We also carried out NMR studies for studying lactol 7 and its
corresponding anomeric cesium alkoxide (42). In the event, to a
0.1 M solution of 3,4,6-tri-O-benzyl-D-mannose (7, α/β = 3.0/1) in
Scheme 4. Preparation of anomeric cesium alkoxides in CDCl3 and subsequent
alkylation with allyl bromide.
anhydro-4-O-benzyl-D-mannose
benzyl-D-mannose 20,
19,
and
2,6-anhydro-3,4-di-O-
4-O-benzyl-3,6-O-(o-
xylylene)mannose 21 reacted with triflate 6 to afford detectable
amounts of corresponding disaccharides 34, 35 or 36,
respectively.24 These results suggested the relatively poor
nucleophilicity of these bicyclic mannose-derived anomeric
alkoxides. After careful analysis of the reaction mixtures, none of
the bicyclic lactol donors (16, 17, 19-21) was detected except that
29% of the relatively more flexible 6,5-cis-fused lactol 18 was
recovered. The decomposition of those strained bicyclic lactols
may indicate their instabililty in addition to their low reactivity.25
Furthermore,
we
prepared
2,3,4,6-tetra-O-benzyl-D-
mannopyranose 38 and studied this substrate in this Cs2CO3-
mediated anomeric O-alkylation. As shown in Scheme 3, 2,3,4,6-
tetra-O-benzyl-D-mannopyranose 38 was able to react with C4-
triflate 6 (2.0 eq.) in the presence of cesium carbonate (2.5 eq.)
to afford desired β-mannopyranoside 39β in 46% isolated yield
and α-mannopyranoside 39α in 3% yield, respectively (β/α =
15.3/1). In addition, a small amount (5%) of β-glucoside 40 was
also isolated from the reaction mixture. Presumably,
deprotonation of the anomeric hydroxyl group of 38 followed by a
sequential ring opening, epimerization of the C2 stereocenter
through enolization of the aldehyde and re-protonation, and
cyclization afforded a small amount of 2,3,4,6-tetra-O-benzyl-D-
Figure 1. Comparison of 1H NMR spectra of 3,4,6-tri-O-CD3-D-mannose (44,
α/β = 5.2/1, colored in blue, bottom spectrum) and its corresponding cesium
alkoxide 45 (α/β = 1/5.0, colored in red, top spectrum).
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Figure 2. Kinetic studies of anomeric O-alkylation of mannose 7 with allyl bromide (2.5 eq.) in the presence of Cs2CO3 (3.0 eq.) in 1,2-dichloroethane at 40 oC.
CDCl326 was added 3 eq. of Cs2CO3 and the reaction mixture was
stirred at 40 C for 1 hour. The resulting mixture was allowed to
as H-4 and H-5.20 These data indicate that both anomeric cesium
alkoxides (45α and 45β) mainly adopt a stable chair conformation
at room temperature or 40 oC.27 In addition, proton signal
broadening (except H4) may indicate the possible chelation of
cesium with various oxygen atoms. The predominance of the
equatorial β-anomer after deprotonation (α/β = 1/5.0) was
probably due to the chelation of the cesium ion to C2-oxygen at
room temperature, while in the axial α-anomer such a chelation
would not be possible due to the 1,2-trans relationship. In addition,
this solution of anomeric cesium alkoxide 45 in CDCl3 was treated
o
stand for 0.5 hour and the supernatant, presumably containing
cesium alkoxide (cf. 42, Scheme 4) in CDCl3, was taken out for
NMR studies. It was found that cesium alkoxide 42 exists as a
mixture of α and β anomers (α/β = 1/3.0).20 Next, 1.5 eq. of allyl
bromide was added to this supernatant containing cesium
alkoxide 42 in CDCl3 and the resulting mixture was stirred at 40
oC for 24 hours to afford desired allyl 3,4,6-tri-O-benzyl-β-D-
mannoside (cf. 43,15 Scheme 4) in 23% yield. This result indicated
that cesium alkoxide 42β is the reactive species in the anomeric
O-alkylation.
o
with 1.5 eq. of allyl bromide and stirred at 40 C for 24 hours to
afford the desired allyl 3,4,6-tri-O-CD3-β-D-mannoside (46) in
22% yield (β only, Scheme 5). This result was comparable to that
involving 3,4,6-tri-O-benzyl-D-mannose (7), which indicates that
the benzyl protecting group is not required for this reaction. In
addition, we also prepared 3,4,6-tri-O-(2-naphthylmethyl(NAP))-
D-mannopyranose (47) and the corresponding cesium alkoxide
48 following the same procedure.20 Unfortunately, various
attempts to crystallize D-mannose-derived anomeric alkoxides 42,
45, and 48 suitable for X-ray crystallographic analysis were
unsuccessful.28
We also prepared 3,4,6-tri-O-CD3-D-mannose (44) and its
corresponding anomeric cesium alkoxide (45) for extensive NMR
studies. The use of CD3 ether instead of benzyl ether protecting
group (cf. 7) was to simplify the NMR spectra. As shown in Figure
1, 1H NMR spectrum of 3,4,6-tri-O-CD3-D-mannose (44) (0.1 M in
CDCl3) showed that the more thermodynamically stable axial α-
anomer was predominant before deprotonation due to the
anomeric effect (α/β = 5.2/1).20 Next, 3 eq. of Cs2CO3 was added
to this 0.1 M solution of 44 in CDCl3 and the reaction mixture was
o
stirred at 40 C for 1 hour. The resulting mixture was allowed to
Previously, anomeric O-alkylation of 3,4,6-tri-O-benzyl-D-
mannose 7 with allyl bromide in the presence of Cs2CO3 was
found to afford allyl 3,4,6-tri-O-benzyl-β-D-mannoside 43 in 94%
yield (β only).15 Based on that, we conducted reaction progress
kinetic analysis involving two different initial concentrations of
mannose 7, in order to establish whether monomeric cesium
alkoxide 42β or its dimeric form is the key reactive intermediate
for this anomeric O-alkylation (Figure 2).20 The measured
concentration of mannose 7 and allyl β-D-mannoside 43
dependence on time is depicted in Figure 2 (a, 0.1 M of initial
stand for 0.5 hour and the supernatant containing cesium alkoxide
45 was taken out and subjected to extensive NMR experiments
(Scheme 5) at room temperature.20,27 The 1H NMR spectrum
indicated that deprotonation of anomeric hydroxyl groups of 44
with Cs2CO3 cleanly afforded the corresponding anomeric cesium
alkoxides (45) as a mixture of anomers (α/β = 1/5.0, Figure 1).
Both signals of H-4 from cesium alkoxides 45β (major) and 45α
(minor) were shown as triplets (J(H-3,H-4) = J(H-4,H-5) = 9.6 Hz)
indicating a trans-diaxial relationship between H-3 and H-4 as well
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Bn
B− = 7-Cs
HB = 7
O
B−
HB
BnO
BnO
Cs
OH
O
HO
O
BnO
BnO
O
OH
OH
BnO
DG = 0.0
7
7-Cs
Bn
B−
HB
O
BnO
Cs
O
BnO
BnO
O
BnO
BnO
O
DG = 3.2
7-Cs
9-Cs
9
9-Cs
(most stable)
B−
HB
Bn
O
BnO
BnO
OH
O
Cs
HO
O
BnO
BnO
DG = 2.8
O
OH
OH
10
10-Cs
B−
HB
OH
O
Cs
HO
O
BnO
BnO
O
BnO
DG = 6.6
10-Cs
11-Cs
11
11-Cs
Figure 3. Relative acidity of anomeric OH of D-mannose 7 and deoxy-D-mannoses 9, 10, and 11. Cesium alkoxide 7-Cs is used as the base to calculate the
reaction energies of all deprotonation reactions. All energies are in kcal/mol. Bond lengths are in angstrom.
Figure 4. Calculated activation free energies of the O-allylation of anomeric cesium alkoxides 7-Cs, 9-Cs, 10-Cs, and 11-Cs with allyl bromide. All energies are
in kcal/mol. Bond lengths are in angstrom.
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Scheme 5. Revised mechanism for stereoselective β-mannosylation via Cs2CO3-mediated anomeric O-alkylation.
alkoxide, and thus make the anomeric OH of 7 more acidic than
that of 10.
concentration of mannose 7), while kinetic data corresponding to
0.2 M of initial concentration of mannose 7 are provided in the
Supporting Information.20 Next, kinetic analysis proved that this
reaction exhibits a strict first-order dependence on mannose 7 at
both 0.1 M and 0.2 M initial concentrations (b, Figure 2). As the
deprotonation is complete in a relatively short time period
(Scheme 4) and the subsequent SN2 reaction is believed to be
the rate-determining step, our kinetic studies indicate that
monomeric cesium alkoxide 42β should be the real reactive
Next, we investigated the relative nucleophilic reactivities of the
anomeric cesium alkoxides using allyl bromide as the electrophile.
The computed activation energies of the SN2 transition states with
respect to each of the cesium alkoxide intermediates are shown
in Figure 4. The allylation of all four cesium aloxides require
comparable activation energies (∆G‡ = 19.4 – 21.6 kcal/mol),
suggesting once these alkoxides are formed, their reaction rates
in the subsequent O-alkylation would be similar.
species for this anomeric O-alkylation.29
Taken together, the above computational analyses indicate the
Cs ion forms strong chelating interactions with multiple oxygen
atoms in anomeric alkoxides. The possible chelation of cesium
ion with the oxygen atoms on the sugar is also supported by 133Cs
NMR studies as well as 18-crown-6 titration studies.20 This
stabilizing chelation effect promotes the deprotonation of D-
mannose. The importance of the Cs−O chelation is evident in the
experimentally observed low reactivities of deoxy-D-mannoses 9,
10, and 11. Removal of oxygen substituents at either C2, C3, or
C6 positions destabilizes the chelated cesium alkoxides, and thus
makes the deprotonation of anomeric hydroxyl group in deoxy-
manoses more difficult.
Based on aforementioned discussions, we revised the
originally proposed mechanistic hypothesis as shown in Scheme
5. After deprotonation of anomeric hydroxyl group of D-mannose
7 with cesium carbonate, a mixture of axial alkoxide 52 and
equatorial alkoxide 54 may be produced and interconvert into
each other via open aldehyde intermediate 53. Due to chelation,
Equatorial anomeric cesium alkoxide 5433 (cf. 7-Cs, Figure 3),
would be preferentially formed. In addition, the effective chelation
may also alleviate the steric effect of the C2-hydroxyl group.
Subsequent anomeric O-alkylation of the cesium alkoxide 54 with
the assistance of kinetic anomeric effect, i.e. double electron-
electron repulsion, with suitable electrophiles via SN2 substitution
then affords the desired β-mannopyranosides 55 (Scheme 5).34
This β-mannosylation via anomeric O-alkylation was next
utilized in the synthesis of the hexasaccharide core35 of complex
fucosylated N-linked glycans.36 For instance, bi-antennary α-
(2,3)-sialylated core-fucosylated N-glycan dodecasaccharide was
found on alpha fetoprotein (AFP) isolated from patients with
hepatocellular carcinoma. The structure of this N-glycan was
believed to be a much more reliable marker than AFP to
differentiate benign and malignant liver diseases.37 This
dodecasaccharide was also detected on the surface of
erythropoietin38 and is believed to be important for the in vivo
With all of the experimental results in hand, we next conducted
computational studies to explore the role of Cs2CO3 in the
anomeric O-alkylation and the origin of the decreased reactivities
of deoxy-D-mannoses 9, 10, and 11.30 Based on the experimental
studies discussed above, we surmised that the cesium salt may
either promote the deprotonation of the anomeric OH group to
form anomeric cesium alkoxides (e.g. 44) or enhance the
nucleophilicity of the anomeric cesium alkoxides in the
subsequent alkylation step. These two potential effects were
computationally analyzed using density functional theory (DFT)
calculations. All calculations were performed at the M06-2X/def2-
QZVP/SMD(dichloroethane)//M06-2X/SDD-6-31G(d) level of
theory (see SI for computational details).
We first investigated the relative acidities of the anomeric OH
groups in tri-O-benzyl-D-mannose 7 and deoxy-D-mannoses 9,
10, and 11. We computed the reaction energies of the
deprotonation of these substrates using anomeric cesium
alkoxide 7-Cs as the base.31 All three deoxy-D-mannoses require
much higher energy to deprotonate than D-mannose 7, indicating
cesium alkoxide 7-Cs is much more stable than cesium alkoxides
derived from the deoxy-D-mannoses. Examination of the lowest
energy conformers of the deprotonated cesium alkoxides (Figure
3) indicated that the pyranose rings in 7-Cs adopts a 4C1
conformation. The 1C4 conformer of 7-Cs is 6.4 kcal/mol less
stable (see SI for details). A strong chelation of the cesium ion
with four oxygen atoms, O1, O2, O6, and O5 (the endocyclic
oxygen), was observed.32 The lower stability of anomeric cesium
alkoxides 9-Cs and 11-Cs is attributed to the lack of O2 and O6
substituents, respectively, which diminished the chelating Cs–O
interactions. On the other hand, 10-Cs is stabilized by chelation
with O1, O2, O6, and O5 atoms, which is similar to that in 7-Cs.
Nonetheless, inductive effects of the C3-OBn group in 7-Cs
provide additional stabilization of the negative charge in the
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Scheme 6. Synthesis of the hexasaccharide core of fucosylated N-linked glycans.
activity of erythropoietin.39 As shown in Scheme 6, known
the formation of predominant equatorial anomeric cesium
alkoxides after deprotonation, due to the chelation of the cesium
ion to the oxygen atoms. In addition, the chelating interactions of
cesium ion with the oxygen atoms on the sugar are also supported
by 133Cs NMR experiments as well as 18-crown-6 titration
studies.20 Kinetic studies supported that the anomeric O-
alkylation involves monomeric cesium alkoxides as the key
reactive species. DFT calculations suggested that that the oxygen
atoms at C2, C3 and C6 of the mannose enhances the acidity of
the anomeric hydroxyl group to facilitate the deprotonation by
Cs2CO3. In particular, the C2-oxygen atom is believed to play a
major role in the chelation with the cesium ion. Such chelation
preferentially favors the formation of equatorial anomeric
alkoxides, leading to the highly stereoselective alkylation to form
the β-anomer of the products. Based on experimental data and
protected
β-D-galactosamine-derived
thioglycoside
5640
underwent standard deacetylation (94%) followed by triflation of
the resulting C4-alcohol to afford triflate 57 (85%). Next, cesium
carbonate-mediated anomeric O-alkylation of known 3,6-di-O-(4-
methoxybenzyl)-4-O-benzyl-D-mannopyranose
5813
with
aforementioned triflate acceptor 57 (2.5 eq.) under the optimized
conditions gave the desired β-disaccharide 59 in 80% yield (β
only). Standard benzylation of the C2’-OH of disaccharide 59
afforded 60 which was subjected to the traditional glycosylation
with known disaccharide acceptor 6136c (NIS, cat. TfOH) to
produce the corresponding tetrasaccharide 62 in 58% yield. After
DDQ-mediated deprotection of bis-PMB ethers of 62 under
neutral conditions, 63 was obtained in 74% yield whose
spectroscopic data were found to be identical to those previously
reported.36c
Previously,
double
α-mannosylation
of
computational results, a revised mechanism for this β-
tetrasaccharide 63 with thioglycoside donor 65 (AgOTf, p-
toluenesulfenyl chloride; then catalytic TMSOTf) afforded
hexasaccharide 67 in 77% yield.36c In our hands, double α-
mannosylation of tetrasaccharide 63 with trichloroacetimidate
donor 64 in the presence of catalytic amount of TMSOTf afforded
complex hexasaccharide 66 in 95% yield.
mannosylation is proposed. We also isolated and characterized a
side product, 3,4,6-tri-O-benzyl-D-fructose, which was
presumably formed via an α-keto rearrangement of the open
aldehyde intermediate.20 This umpolung-type β-mannosylation
has also been demonstrated in efficient synthesis of the
hexasaccharide core of complex fucosylated N-linked glycans.
Application of this method to the stereoselective construction of
other types of challenging glycosidic linkages, such as β-2-amino-
2-deoxy-D-mannosides, and synthesis of complex biologically
significant carbohydrate molecules are currently underway.
complex hexasaccharide 66 in 95% yield.
Conclusions
In conclusion, various structurally diverse deoxy-D-mannoses
and conformationally restricted bicyclic D-mannoses have been
prepared and subjected to the mechanistic studies of this
umpolung-type β-mannosylation via anomeric O-alkylation.
Deoxy mannoses were found to be not as reactive as parent
mannose, and conformationally restricted bicyclic D-mannoses
afforded no product or lower yields than conformationally flexible
D-mannoses. When the hydroxyl group at C2 was masked, this
type of β-mannosylation did not proceed as efficiently, albeit the
corresponding β-mannoside was still detected as the major
product. Studies of various alkali metal bases indiated that cesium
carbonate (Cs2CO3) was the optimal base for this β-selective
anomeric O-alkylation.20 Extensive NMR studies demonstrated
Experimental Section
General procedure for Cs2CO3 mediated O-alkylation.
To a mixture of sugar lactol donor (0.1 mmol, 1.0 eq.), sugar
derived triflate acceptor 6 (2.0 eq.), and Cs2CO3 (2.5 eq.) was
added 1,2-dichloroethane (1.0 mL). The reaction mixture was
stirred at 40 ℃ for 24 hours. The crude reaction mixture was
purified by preparative TLC. The β-configuration of the new
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
7.37 – 7.27 (m, 11H, HAr), 7.24 – 7.13 (m, 9H, HAr), 6.89 – 6.81
(m, 4H, HAr), 6.78 – 6.69 (m, 5H, HAr), 5.52 (d, J = 9.9 Hz, 1H, H-
1), 4.96 (d, J = 13.0 Hz, 1H, -OCH2Ar), 4.91 (s, 2H, -OCH2Ar),
4.86 (d, J = 10.9 Hz, 1H, -OCH2Ar), 4.60 (d, J = 12.0 Hz, 1H, -
OCH2Ar), 4.56 (s, 1H, H-1′), 4.55 – 4.49 (m, 2H, -OCH2Ar), 4.49
– 4.41 (m, 4H, -OCH2Ar), 4.38 (d, J = 11.6 Hz, 1H, -OCH2Ar), 4.32
– 4.23 (m, 2H, H-3, H-2), 4.02 (dd, J = 9.8, 8.0 Hz, 1H, H-4), 3.90
(t, J = 9.6 Hz, 1H, H-4′), 3.81 – 3.78 (m, 4H, H-2′, -OCH3), 3.77 –
3.70 (m, 5H, -OCH3, H-6a, H-6′a), 3.66 – 3.59 (m, 3H, H-6b, H-
6′b, H-5), 3.42 – 3.34 (m, 2H, H-3′, H-5′); 13C NMR (150 MHz,
CDCl3) δ 168.08, 167.31, 159.25, 158.98, 139.03, 138.91, 138.63,
138.06, 133.86, 133.69, 132.82, 132.06, 131.80, 131.69, 130.74,
130.48, 129.33, 129.28, 128.87, 128.58, 128.38, 128.25, 128.06,
128.00, 127.94, 127.85, 127.78, 127.72, 127.66, 127.45, 126.82,
123.44, 123.33, 113.86, 113.73, 101.79, 83.37, 82.50, 79.26,
78.57, 76.04, 75.21, 75.11, 74.99, 74.81, 74.18, 73.59, 73.07,
71.61, 69.22, 68.91, 55.36, 55.31, 54.86; LRMS (ESI) calculated
for C70H69NNaO13S [M+Na]+ 1187.44, found 1187.30.
formed mannosidic linkage was unambiguously assigned by
measuring the 1JC-H for the anomeric carbon.
Synthesis of the hexasaccharide core of the fucosylated N-
linked glycans.
Phenyl-3,6-di-O-benzyl-4-O-trifluoromethylsulfonyl-2-deoxy-
2-phthalimido-1-thio-β-D-galactopyranoside (57): To
a
solution of known compound 56[40] (2.67 g, 4.6 mmol) in MeOH
(25 mL) and THF (25 mL) was added 0.5 M NaOMe solution in
MeOH (6.4 mL, 3.22 mmol). The mixture was stirred at room
temperature for 7 hours. The reaction mixture was neutralized by
the addition of Amberlyst IR-120 (H+), filtered, and concentrated.
The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 5/1 to 3/1) to give the corresponding alcohol
(2.52 g, 94 %). To a solution of this alcohol (0.67 g, 1.2 mmol) and
pyridine (1.0 mL, 12 mmol) in CH2Cl2 (3.0 mL) cooled at 0 ℃ was
added Tf2O (0.35 mL, 2.0 mmol) dropwise. The resulting mixture
was stirred at 0 ℃ for 2 h before being quenched with ice water.
The organic layer was separated and the aqueous layer was
extracted with CH2Cl2 (50 mL × 3). The combined organic layer
was washed sequentially with saturated CuSO4 (50 mL × 3) and
water (50 mL × 3), dried over anhydrous Na2SO4, filtered and
concentrated. The residue was purified by silica gel column
chromatography with CH2Cl2 to afford the sugar derived triflate 57
Benzyl-O-2,4-di-O-benzyl-3,6-di-O-(para-methoxybenzyl)-β-
D-mannopyranosyl-(1→4)-O-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-β-D-gluopyranosyl-(1→4)-O-[2,3,4-tri-O-
benzyl-α-L-fucopyranosyl-(1→6)]-3-O-benzyl-2-deoxy-2-
phthalimido-β-D-gluopyranoside (62): To a mixture of donor 60
(87 mg, 0.075 mmol), acceptor 61[36c] (45 mg, 0.05 mmol),
activated 4 Å molecular sieves (300 mg), and NIS (84 mg) was
added CH2Cl2 (2.5 mL). The solution was cooled to -40 ℃ and
TfOH (2.0 µL) was added. The resulting mixture was stirred at this
temperature overnight and then filtered through celite. The filtrate
was quenched and washed with saturated Na2S2O3 aqueous
solution. The organic layer was dried over Na2SO4, filtered, and
concentrated. The crude reaction mixture was purified by
preparative TLC (EtOAc/ CH2Cl2/Toluene = 1/5/5) to furnish the
title tetra-saccharide 62 (57 mg, 58%). [휶]ퟐ퐃ퟑ = -3.3 (c 1.0, CHCl3);
FT-IR (thin film): 3065, 3033, 2934, 2867, 1777, 1716, 1614,
1
(725 mg, 85%). H NMR (600 MHz, CDCl3) δ 7.85 (m, 1H, HAr),
7.74 (m, 1H, HAr), 7.70 (m, 1H, HAr), 7.58 (dd, J = 7.3, 1.1 Hz, 1H,
HAr), 7.42 – 7.32 (m, 7H, HAr), 7.25 – 7.18 (m, 3H, HAr), 6.98 –
6.93 (m, 3H, HAr), 6.90 – 6.83 (m, 2H, HAr), 5.54 (d, J = 2.9 Hz,
1H, H-4), 5.52 (d, J = 10.5 Hz, 1H, H-1), 4.70 (d, J = 12.6 Hz, 1H,
-OCH2Ar), 4.65 (d, J = 11.2 Hz, 1H, -OCH2Ar), 4.52 – 4.45 (m, 2H,
-OCH2Ar, H-2), 4.38 (dd, J = 10.5, 2.9 Hz, 1H, H-3), 4.21 (d, J =
12.6 Hz, 1H, -OCH2Ar), 4.01 (dd, J = 8.3, 5.6 Hz, 1H, H-5), 3.79
(dd, J = 9.2, 5.6 Hz, 1H, H-6a), 3.69 (dd, J = 9.2, 8.4 Hz, 1H, H-
6b); 13C NMR (150 MHz, CDCl3) δ 168.05, 166.87, 137.36, 136.51, 1514, 1454, 1388, 1249, 1091, 1038, 749, 724, 700 cm-1;1H NMR
134.26, 133.98, 132.81, 131.67, 131.62, 131.61, 129.01, 128.69,
128.44, 128.36, 128.34, 128.28, 128.24, 128.01, 123.80, 123.45,
118.68 (q, JC-F = 319.7 Hz), 84.21, 80.57, 75.14, 73.91, 72.97,
(600 MHz, CDCl3) δ 7.88 (d, J = 7.3 Hz, 1H, HAr), 7.82 (d, J = 7.2
Hz, 1H, HAr), 7.76 – 7.56 (m, 7H, HAr), 7.50 (d, J = 7.3 Hz, 1H, HAr),
7.46 – 7.11 (m, 32H, HAr), 7.07 (m, 1H, HAr), 7.04 – 6.91 (m, 6H,
HAr), 6.87 – 6.69 (m, 12H, HAr), 5.59 (d, J = 8.4 Hz, 1H, H-1), 5.01
– 4.91 (m, 5H), 4.91 – 4.81 (m, 4H), 4.80 – 4.72 (m, 2H), 4.66 –
4.47 (m, 9H), 4.45 – 4.26 (m, 8H), 4.21 – 4.16 (m, 2H), 4.12 (dd,
J = 10.7, 8.5 Hz, 1H), 4.07 – 4.01 (m, 2H), 3.99 (dd, J = 10.2, 2.8
Hz, 1H), 3.90 – 3.85 (m, 2H), 3.83 – 3.77 (m, 4H), 3.77 – 3.72 (m,
3H), 3.68 (s, 3H), 3.65 – 3.57 (m, 3H), 3.41 (dd, J = 10.8, 3.0 Hz,
1H), 3.38 (dd, J = 9.4, 3.0 Hz, 1H), 3.35 (ddd, J = 9.8, 5.1, 1.7 Hz,
1H), 3.28 (ddd, J = 9.9, 3.0, 1.6 Hz, 1H), 1.01 (d, J = 6.5 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 168.27, 167.89, 167.78, 167.67,
159.19, 158.91, 139.21, 139.17, 139.05, 138.96, 138.86, 138.73,
138.07, 137.16, 133.91, 133.73, 133.67, 133.58, 132.01, 131.79,
131.66, 130.87, 130.59, 129.28, 129.19, 128.62, 128.60, 128.51,
128.42, 128.34, 128.23, 128.19, 128.10, 128.07, 128.05, 127.91,
127.89, 127.74, 127.72, 127.62, 127.60, 127.57, 127.55, 127.47,
127.44, 127.37, 127.08, 126.95, 126.72, 123.55, 123.31, 123.20,
113.81, 113.68, 101.45, 96.90, 96.86, 96.70, 82.46, 79.57, 79.24,
77.71, 77.33, 76.09, 76.05, 75.82, 75.30, 75.09, 75.06, 74.84,
74.82, 74.69, 74.63, 74.43, 74.36, 73.79, 73.39, 73.20, 73.09,
72.52, 71.30, 69.96, 69.27, 68.51, 66.07, 63.94, 56.68, 55.95,
55.35, 55.25, 16.51; LRMS (ESI) calculated for C119H118N2Na2O24
[M+2Na]2+ 1002.89, found 1002.80.
1
71.89, 67.20, 50.99.
Phenyl-O-2,4-di-O-benzyl-3,6-di-O-(para-methoxybenzyl)-β-
D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-β-D-gluopyranoside (60): To a mixture of
mannopyranosyl donor 58[13] (1.02 g, 2.0 mmol), sugar-derived
triflate acceptor 57 (3.7 g, 5.0 mmol), and Cs2CO3 (2.0 g, 6.0
mmol) was added 1,2-dichloroethane (20 mL). The reaction
mixture was stirred at 40 ℃ for 24 hours. The crude reaction
mixture was purified by silica gel column chromatography
(Hexanes/EtOAc = 5/1 to 1/1) to give disaccharide 59 (1.73 g,
80%). To a solution of 59 (1.0 g, 0.93 mmol) in DMF (4.0 mL)
cooled at 0 ℃ was added NaH (75 mg, 1.86 mmol, 60% in mineral
oil) portion wise. The resulting mixture was stirred at 0 ℃ for 1 h
before BnBr (0.17 mL, 1.4 mmol) was added. The reaction mixture
was warmed up and stirred at ambient temperature for 3 h before
being quenched with water. The resulting mixture was extracted
with EtOAc three times, and combined organic extracts were
washed with water and brine, dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by silica gel
column chromatography (Hexanes/EtOAc = 5/1 to 3/1) to give the
1
title compound 60 (1.01 g, 94%). The JC-H of mannosidic
anomeric carbon for 60 was determined to be 159.0 Hz. [휶]퐃ퟐퟑ
=
Benzyl-O-2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl-(1→3)-O-[2-O-benzoyl-3,4,6-tri-O-benzyl-α-
D-mannopyranosyl-(1→6)]-O-2,4-di-O-benzyl-β-D-
mannopyranosyl-(1→4)-O-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-β-D-gluopyranosyl-(1→4)-O-[2,3,4-tri-O-
benzyl-α-L-fucopyranosyl-(1→6)]-3-O-benzyl-2-deoxy-2-
+5.9 (c 1.0, CHCl3); FT-IR (thin film): 3064, 3032, 2938, 2863,
1777, 1714, 1678, 1512, 1455, 1388, 1250, 1174, 1102, 1070,
917, 807, 701 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.80 (d, J = 7.3
Hz, 1H, HAr), 7.70 (m, 1H, HAr), 7.65 (m, 1H, HAr), 7.58 (d, J = 7.3
Hz, 1H, HAr), 7.50 – 7.44 (m, 2H, HAr), 7.42 – 7.38 (m, 2H, HAr),
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
phthalimido-β-D-gluopyranoside (66): To a solution of tetra-
saccharide 62 (57 mg, 0.029 mmol) in CH2Cl2 (3.0 mL) was added
a solution of pH 7 (1.0 mL) buffer and 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) (18 mg, 0.079 mmol). The resulting
mixture was stirred at room temperature for 3 h before additional
amount of DDQ (13 mg, 0.057 mmol) was added. The mixture
was stirred for another 2 hours and another portion of DDQ (13
mg, 0.057 mmol) was added. After 2 hours, the mixture was
quenched with saturated NaHCO3 solution and extracted with
ethyl acetate (10 mL × 3). The organic layer was dried over
Na2SO4, filtered, and concentrated. The crude reaction mixture
was purified by preparative TLC (EtOAc/CH2Cl2 = 1/10) to furnish
the corresponding diol 63 (37 mg, 74%). The spectroscopic data
was consistent with the data reported in the literature.[36c] To a
mixture of diol acceptor 63 (74 mg, 0.043 mmol), donor 64 (95 mg,
0.13 mmol), activated 4 Å molecular sieves (100 mg) was added
CH2Cl2 (1.0 mL). The solution was cooled to -20 ℃ and TMSOTf
(1.0 µL) was added. The resulting mixture was slowly warmed up
to room temperature over 2 hours. The reaction was quenched
with Et3N (200 µL) and filtered through celite. The filtrate was
washed with saturated NaHCO3 aqueous solution and brine. The
organic layer was dried over Na2SO4, filtered, and concentrated.
The residue was purified by preparative TLC (EtOAc/Toluene =
1/10) to afford hexasaccharide 66 (115 mg, 95%). [훂]ퟐ퐃ퟑ = -7.0 (c
1.0, CHCl3); FT-IR (thin film): 3091, 3061, 3030, 2933, 2872,
1777, 1717, 1496, 1455, 1390, 1266, 1099, 1077, 737, 696 cm-1;
1H NMR (600 MHz, CDCl3) δ 8.07 (dd, J = 8.2, 1.4 Hz, 2H), 7.83
– 7.78 (m, 2H), 7.76 (d, J = 7.4 Hz, 1H), 7.67 – 7.63 (m, 2H), 7.61
– 7.55 (m, 2H), 7.49 – 7.43 (m, 2H), 7.43 (m, 1H), 7.40 – 7.34 (m,
6H), 7.33 – 7.08 (m, 60H), 7.06 – 7.00 (m, 2H), 6.97 (t, J = 7.6 Hz,
2H), 6.95 – 6.92 (m, 2H), 6.90 – 6.87 (m, 2H), 6.76 – 6.68 (m, 4H),
6.63 – 6.55 (m, 1H), 6.50 (t, J = 7.5 Hz, 2H), 5.72 (t, J = 2.5 Hz,
1H), 5.54 (t, J = 2.3 Hz, 1H), 5.46 (d, J = 8.3 Hz, 1H), 5.20 (d, J =
2.0 Hz, 1H), 4.95 – 4.78 (m, 12H), 4.74 – 4.56 (m, 8H), 4.55 –
4.37 (m, 11H), 4.34 (d, J = 11.3 Hz, 1H), 4.29 (d, J = 12.3 Hz, 1H),
4.27 – 4.18 (m, 4H), 4.17 – 4.10 (m, 3H), 4.09 – 4.04 (m, 2H),
4.00 – 3.91 (m, 7H), 3.90 – 3.83 (m, 2H), 3.80 (m, 1H), 3.76 –
3.69 (m, 5H), 3.64 (dd, J = 10.8, 1.5 Hz, 1H), 3.61 – 3.53 (m, 5H),
3.29 (dd, J = 10.8, 3.4 Hz, 1H), 3.24 – 3.19 (m, 2H), 0.95 (d, J =
6.5 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 168.05, 167.87, 167.64,
165.56, 165.07, 139.01, 138.91, 138.89, 138.78, 138.76, 138.70,
138.41, 138.20, 138.04, 138.01, 137.94, 137.18, 133.75, 133.55,
133.24, 132.76, 131.95, 131.80, 131.63, 130.10, 130.00, 129.97,
129.95, 128.74, 128.58, 128.57, 128.54, 128.52, 128.49, 128.46,
128.37, 128.33, 128.30, 128.24, 128.21, 128.18, 128.02, 128.01,
127.98, 127.89, 127.87, 127.85, 127.77, 127.67, 127.63, 127.61,
127.59, 127.57, 127.53, 127.46, 127.43, 127.38, 127.15, 126.91,
126.84, 126.78, 123.52, 123.25, 102.06, 99.60, 98.34, 96.91,
96.69, 82.21, 79.77, 79.46, 78.35, 78.23, 77.80, 76.51, 76.31,
75.54, 75.24, 75.20, 75.17, 75.06, 74.81, 74.78, 74.58, 74.54,
74.47, 74.43, 74.16, 74.13, 73.85, 73.55, 73.41, 73.36, 72.66,
72.48, 72.10, 71.60, 71.03, 69.94, 69.08, 69.04, 69.00, 68.48,
67.99, 66.55, 66.04, 63.93, 56.64, 55.92, 16.51. LRMS (ESI)
calculated for C171H166N2Na2O34 [M+2Na]2+ 1419.56, found
1419.80.
University of Pittsburgh and the Extreme Science and
Engineering Discovery Environment (XSEDE) supported by the
NSF. We thank Dr. Yong Wah Kim for assistance in the 133Cs
NMR studies, Mr. James K. Dunaway and Rodney A. Park from
University of Toledo, and Mr. Luke Harding and Justin Woodward
from University of University of Michigan‒Dearborn for
experimental assistance.
Keywords: β-mannosylation • anomeric O-alkylation •
carbohydrates • glycosylation • N-linked glycans
[1] (a) A. Varki, Glycobiology 1993, 3, 97-130. (b) C. R. Bertozzi, L. L.
Kiessling, Science 2001, 291, 2357-2364. (c) A. Varki, Glycobiology 2017,
27, 3-49.
[2] (a) D. F. Wyss, J. S. Choi, J. Li, M. H. Knoppers, K. J. Willis, A. R. N.
Arulanandam, A. Smolyar, E. L. Reinherz, G. Wagner, Science 1995, 269,
1273-1278. (b) A. C. Weymouth-Wilson, Nat. Prod. Rep. 1997, 14, 99-
110; (c) V. Kren, L. Martinkova, Curr. Med. Chem. 2001, 8, 1303-1328.
[3] For select reviews, see: (a) P. O. Adero, H. Amarasekara, P. Wen, L. Bohé,
D. Crich, Chem. Rev. 2018, 118, 8242-8284. (b) C. S. Bennett, M. C.
Galan, Chem. Rev. 2018, 118, 7931-7985. (c) J. Zeng, Y. Xu, H. Wang,
L. Meng, Q. Wan, Sci. China Chem. 2017, 60, 1162-1179. (d) X. Li, J.
Zhu, Eur. J. Org. Chem. 2016, 4724-4767. (e) S. C. Ranade, A. V.
Demchenko, J. Carbohydr. Chem. 2013, 32, 1-43. (f) M. J. McKay, H. M.
Nguyen, ACS Catal. 2012, 2, 1563–1595. (g) X. Li, J. Zhu, J. Carbohydr.
Chem. 2012, 31, 284–324. (h) X. Zhu, R. R. Schmidt, Angew. Chem. Int.
Ed. 2009, 48, 1900-1934; Angew. Chem. 2009, 121, 1932-1967. (i) A. V.
Demchenko, Editor. Handbook of chemical glycosylation; Advances in
stereoselectivity and therapeutic relevance. 2008, 501 pp; (j) P. Fuegedi,
The Organic Chemistry of Sugars. Levy, D. E., Fuegedi, P., Eds.; CRC
Press, 2006, pp 89–179; (k) D. P. Galonić, D. Y. Gin, Nature 2007, 446,
1000-1007; (l) K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93, 1503–1531.
[4] (a) W. Li, J. B. McArthur, X. Chen, Carbohydr. Res. 2019, 472, 86−97. (b)
L.-X. Wang, M. N. Amin, Chem. Biol. 2014, 21, 51-66.
[5] J. J. Gridley, H. M. I. Osborn, J. Chem. Soc., Perkin Trans. 1, 2000, 1471–
1491.
[6] (a) S. S. Nigudkar, A. V. Demchenko, Chem. Sci. 2015, 6, 2687-2704. (b)
A. V. Demchenko, Curr. Org. Chem. 2003, 7, 35–79. (c) A. V. Demchenko,
Synlett 2003, 1225-1240. (d) M. Tanaka, A. Nakagawa, N. Nishi, K. Iijima,
R. Sawa, D. Takahashi, K. Toshima, J. Am. Chem. Soc. 2018, 140, 3644–
3651.
[7] (a) K. Sasaki, K. Tohda, Tetrahedron Lett. 2018, 59, 496–503. (b) T. J.
Boltje, T. Buskas, G.-J. Boons, Nat. Chem. 2009, 1, 611-622. (c) A.
Ishiwata, Y. J. Lee, Y. Ito, Org. Biomol. Chem. 2010, 8, 3596-3608. (d) Y.
Zeng, F. Kong, Prog. Chem. 2006, 18, 907-926. (e) F. Barresi, O.
Hindsgaul, Modern Methods in Carbohydrate Synthesis; S. H. Khan, R. A.
Eds. O'Neill, Harwood Academic Publishers: Amsterdam, 1996; 251-276.
(f) K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93, 1503–1531. (g) H.
Paulsen, Angew. Chem. Int. Ed. 1982, 21, 155-173; Angew. Chem. 1982,
94, 184-201.
[8] (a) R. R. Schmidt, J. Michel, Tetrahedron Lett. 1984, 25, 821−824. (b) R.
R. Schmidt, Angew. Chem. Int. Ed. 1986, 25, 212−235; Angew. Chem.
1986, 98, 213−236. (c) R. R. Schmidt, Pure Appl. Chem. 1989, 61,
1257−1270. (d) R. R. Schmidt, W. Klotz, Synlett 1991, 168−170. (e) Y. E.
Tsvetkov, W. Klotz, R. R. Schmidt, Liebigs Ann. Chem. 1992, 371−375. (f)
R. R. Schmidt, Front. Nat. Prod. Res. 1996, 1, 20−54.
Acknowledgements
[9] (a) S. S. Pertel, O. A. Gorkunenko, E. S. Kakayan, V. J. Chirva, Carbohydr.
Res. 2011, 346, 685−688. (b) D. A. Ryan, D. Y. Gin, J. Am. Chem. Soc.
2008, 130, 15228−15229. (c) W. J. Morris, M. Shair, D. Org. Lett. 2009,
11, 9-12. (d) G. Trewartha, J. N. Burrows, A. G. M. Barrett, Tetrahedron
Lett. 2005, 46, 3553–3556. (e) B. Vauzeilles, B. Dausse, S. Palmier, J.-
M. Beau, Tetrahedron Lett. 2001, 42, 7567–7570. (f) S. Izumi, Y.
Kobayashi, Takemoto, Y. Org. Lett. 2019, 21, 665–670.
We are grateful to National Science Foundation (CHE-1464787 to
X.L. and J.Z.), National Institutes of Health Common Fund
Glycosciences Program (1U01GM125290 to P.L. and
1U01GM125289 to J.Z.), The University of Toledo, and University
of Michigan‒Dearborn for supporting this research. Calculations
were performed at the Center for Research Computing at the
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
[10] (a) R. R. Schmidt, M. Reichrath, Angew. Chem., Int. Ed. 1979, 18, 466–
467. (b) R. R. Schmidt, M. Reichrath, U. Moering, Tetrahedron Lett. 1980,
21, 3561–3564. (c) J. Tamura, R. R. Schmidt, J. Carbohydr. Chem. 1995,
14, 895–911. (d) R. R. Schmidt, U. Moering, M. Reichrath, Chem. Ber.
1982, 115, 39–49.
H. Y. Cheong, J. Org. Chem. 2017, 82, 7183-7189. (c) M. Anand, R. B.
Sunoj, H. F. Schaefer, III, J. Am. Chem. Soc. 2014, 136, 5535-5538.
[31] Computationally it is not feasible to accurately model the deprotonation
using the experimentally used base Cs2CO3, which does not completely
dissolve under the experimental conditions. Here, using 7-Cs as the
model base will not affect the relative acidity trend.
[11] D. Zhu, K. N. Baryal, S. Adhikari, J. Zhu, J. Am. Chem. Soc. 2014, 136,
3172–3175.
[32] The chelating Cs−O distances in the optimized structures are typically
shorter than 3 Å, which indicates relatively strong interactions, see: O. C.
Gagné, F. C. Hawthorne, Acta Crystallogr. B Struct. Sci. 2018, 74, 63-78.
[33] Whether the C2-OH of intermediates 52 and 54 is deprotonated or not can
not be concluded from the 1H NMR data; however, it is believed that it
remains mainly as undeprotonated form in consideration of its pKa value.
In addition, our experimental analysis of the weight of cesium alkoxide 42
is consistent with the presence of one cesium ion in the majority
component.
[12] D. Zhu, S. Adhikari, K. N. Baryal, B. N. Abdullah, J. Zhu, J. Carbohydr.
Chem. 2014, 33, 438-451.
[13] H. Nguyen, D. Zhu, X. Li, J. Zhu, Angew. Chem. Int. Ed. 2016, 55, 4767-
4771; Angew. Chem. 2016, 128, 4845–4849.
[14] D. Takahashi, Trends. Glycosci. Glyc. 2016, 28, E119-E120.
[15] X. Li, N. Berry, K. Saybolt, U. Ahmed, Y. Yuan, Tetrahedron Lett. 2017,
58, 2069–2072.
[16] B. R. Bhetuwal, J. Woodward, X. Li, J. Zhu, J. Carbohydr. Chem. 2017,
36, 162-172.
[34] Deprotonation of C2-OH is not required for this anomeric O-alkylation to
occur, as anomeric O-alkylation of 2,3,4,6-tetra-O-benzyl-D-
mannopyranose also afford the desired β-mannoside in good yield and
anomeric selectivity. However, we can not rule out the possibility that the
C2-OH of mannose is deprotonated in a thermodynamically unfavorable,
rate-limiting step to generate a bis-alkoxide which is rapidly alkylated at
the anomeric position. We sincerely thank one of the reviewers for
pointing this out.
[17] Previous studies also indicated that due to electron-electron repulsion
anomeric C1-alkoxide is more nucleophilic than non-anomeric alkoxide,
see: refs 9c, 11 and 12.
[18] M. T. Yang, K. A. Woerpel, J. Org. Chem. 2009, 74, 545–553.
[19] I. S. Aidhen, N. Satyamurthi, Indian J. Chem., Sect B 2008, 47B, 1851-
1857.
[20] See Supporting Information for details.
[21] See Supporting Information for additional direct competition experiments
that produce similar results when allyl bromide is used as the electrophile.
[22] V. S. Borodkin, M. A. J. Ferguson, A. V Nikolaev, Tetrahedron Lett. 2001,
42, 5305-5308.
[35] T. Katoh, T. Katayama, Y. Tomabechi, Y. Nishikawa, J. Kumada, Y.
Matsuzaki, K. Yamamoto, J. Biol. Chem. 2016, 291, 23305–23317.
[36] (a) B. Wu, Z. Hua, J. D. Warren, K. Ranganathan, Q. Wan, G. Chen, Z.
Tan, J. Chen, A. E ndo, S. J. Danishefsky, Tetrahedron Lett. 2006, 47,
5577– 5579. (b) B. Wu, Z. Tan, G. Chen, J. Chen, Z. Hua, Q. Wan, K.
Ranganathan, S. J. Danishefsky, Tetrahedron Lett. 2006, 47, 8009– 8011.
(c) B. Sun, B. Srinivasan, X. Huang, Chem. Eur. J. 2008, 14, 7072–7081.
(d) P. Nagorny, B. Fasching, X. Li, G. Chen, B. Aussedat, S. J.
Danishefsky, J. Am. Chem. Soc. 2009, 131, 5792–5799.
[23] For examples of using conformationally restricted o-xylylene protecting
group in carbohydrate synthesis, see: (a) A. J. Poss, M. S. Smyth, Synth.
Commun. 1989, 19, 3363-3366. (b) P. Balbuena, E. M. Rubio, C. O. Mellet,
J. M. G. Fernández, Chem. Commun. 2006, 2610-2612. (c) A. Imamura,
T. L. Lowary, Org. Lett. 2010, 12, 3686–3689. (d) Y. Okada, N. Asakura,
M. Bando, Y. Ashikaga, H. Yamada, J. Am. Chem. Soc. 2012, 134,
6940−6943. (e) N. Asakura, A. Motoyama, T. Uchino, K. Tanigawa, H.
Yamada, J. Org. Chem. 2013, 78, 9482−9487. (f) T. Uchino, Y.Tomabechi,
A. Fukumoto, H. Yamada, Carbohydr. Res. 2015, 402, 118-123. (g) L.
Zhang, K. Shen, H. A. Taha, T. L. Lowary, J. Org. Chem. 2018, 83, 7659-
7671.
[37] (a) P. J. Johnson, T. C. W. Poon, N. M. Hjelm, C. S. Ho, S. K. W. Ho, C.
Welby, D. Stevenson, T. Patel, R. Parekh, R. R. Townsend, Br. J. Can.
1999, 81, 1188–1195. (b) K. Taketa, Electrophoresis 1998, 19, 2595–
2602.
[38] F.-T. A. Chen, R. A. Evangelista, Electrophoresis 1998, 19, 2639 –2644.
[39] E. Watson, A. Bhide, H. van Halbeek, Glycobiology 1994, 4, 227–237.
[40] T. Sawada, S. Fujii, H. Nakano, S. Ohtake, K. Kimata, O. Habuchi,
Carbohydr. Res. 2005, 340, 1983-1996.
[24] When 2,6-anhydro-3,4-di-O-benzyl-D-mannose 20 was subjected to
anomeric O-alkylation, the formation of corresponding disaccharide 35
was not observed, instead an enal (ref. S19, Supporting Information) was
isolated in 50% yield. Presumably, enal was formed via deprotonation of
anomeric hydroxyl of 20 followed by ring opening, enolization, and
elimination of C3-benzyloxy group.
[25] A small amount of side product, 3,4,6-tri-O-benzyl-D-fructose 37 (Table
1), was always detected in all of the previous anomeric O-alkylation
reactions involving 3,4,6-tri-O-benzyl-D-mannose 7 as the lactol donor.
Presumably, 37 was obtained via an α-keto rearrangement of the open
aldehyde intermediate derived from lactol 7.
[26] This cesium carbonate-mediated β-mannosylation via anomeric O-
alkylation gives comparable results in 1,2-dichloroethane (DCE) and
chloroform (CHCl3).
[27] Extensive NMR studies of anomeric cesium alkoxide (45) at room
temperature or 40 ℃ did not show much difference.
[28] Attempted crystallization of anomeric alkoxides 42, 45, and 48 using
various solvents or mixed solvents, e.g. dichloromethane, 1,2-
dichloroethane, chloroform, and dichloromethane/n-pentane, was
unsuccessful.
[29] Although the data prove that monomeric cesium alkoxide 42β should be
the real reactive species for this anomeric O-alkylation at 0.1~0.2 M of
initial concentration of mannose 7, other forms of cesium alkoxide 42β,
e.g., dimeric form, can not be ruled out at higher concentration,
[30] (a) H. Xu, K. Muto, J. Yamaguchi, C. Zhao, K. Itami, D. G. Musaev, J. Am.
Chem. Soc. 2014, 136, 14834-14844. (b) D. M. Walden, A. A. Jaworski,
R. C. Johnston, M. T. Hovey, H. V. Baker, M. P. Meyer, K. A. Scheidt, P.
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10.1002/ejoc.202000313
European Journal of Organic Chemistry
RESEARCH ARTICLE
Entry for the Table of Contents (Please choose one layout)
RESEARCH ARTICLE
Shuai Meng,[a]† Bishwa Raj Bhetuwal,[a]†
Hai Nguyen,[a]† Xiaotian Qi,[b] Cheng
Fang,[b] Kevin Saybolt,[c] Xiaohua Li,*[c]
Peng Liu,*[b,d] and Jianglong Zhu*[a]
Page No. – Page No.
β-Mannosylation via O-Alkylation of
Anomeric Cesium Alkoxides:
Mechanistic Studies and Synthesis of
the Hexasaccharide Core of Complex
Fucosylated N-Linked Glycans
Extensive mechanistic studies of β-mannosylation via Cs2CO3-mediated anomeric O-alkylation have been described. Experimental
results from various NMR studies and reaction progress kinetic analysis as well as DFT calculations suggested that equatorial
monomeric cesium alkoxides be the key reactive species for alkylation with electrophiles. A revised mechanism for this β-
mannosylation is proposed. This β-mannosylation was utilized in the synthesis of the hexasaccharide core of complex fucosylated N-
linked glycans.
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