Synthesis of Arabinoxylan Oligosaccharides by Preactivation-Based Iterative Glycosylations

Article abstract of DOI:10.1021/acs.joc.9b02529

A concise synthetic strategy has been developed for assembling densely substituted arabinoxylan oligosaccharides, which are valuable substrates for characterizing hemicellulose-degrading enzymes. The xylan backbone has been prepared by an iterative preact

Full text of DOI:10.1021/acs.joc.9b02529

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Article
Synthesis of Arabinoxylan Oligosaccharides by
Pre-Activation-Based Iterative Glycosylations
Emilie N. Underlin, Maximilian Böhm, and Robert Madsen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02529 • Publication Date (Web): 25 Nov 2019
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Synthesis of Arabinoxylan Oligosaccharides by Pre-Activation-Based
Iterative Glycosylations
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Emilie N. Underlin, Maximilian Böhm, and Robert Madsen*
Department of Chemistry, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
E-mail: rm@kemi.dtu.dk
_
____________________________________________________________________________
O
O
BzO
BzO
HO
R'O
SPh
+
SPh
BzO
R"O
Iterative pre-activation-based assembly
O
BzO
BzO
O
O
O
R'O
O
BzO
O
O
R'O
R"O
CF3
SPh
R'O
R"O
R"O
BzO
O
O
NPh
BzO
OBz
Ara1
Ara1
2
2
Xyl1-4Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl1-4Xyl
3
3
3
3
3
3
3
Ara1 Ara1 Ara1
Ara1 Ara1
Ara1 Ara1
Abstract
A concise synthetic strategy has been developed for assembling densely substituted
arabinoxylan oligosaccharides, which are valuable substrates for characterizing hemicellulose-
degrading enzymes. The xylan backbone has been prepared by an iterative pre-activation-based
glycosylation approach with phenyl thioglycosides. The pre-activation has been performed with
in situ generated p-nitrobenzenesulfenyl triflate prior to addition of the acceptor. The
glycosylation temperature was shown to have an important impact on the yield of the coupling.
The arabinose substituents have been introduced in one high-yielding glycosylation with a N-
phenyl trifluoroacetimidate donor. The strategy has been successfully employed for the
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synthesis of three heptasaccharides in seven steps and overall yields of 24 – 36% from the
corresponding monosaccharide building blocks.
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INTRODUCTION
The hemicellulose polysaccharides comprise 15 – 35% by weight of the plant cell
1
wall which is the main source of lignocellulosic biomass. The largest group of
polysaccharides in hemicellulose are the xylans which are characterized by a common
1
backbone of (1→4)-linked xylopyranosides. The xylan structures are highly complex
and heterogeneous due to the differences in the substitution patterns. One of the most
abundant xylan subclasses are the arabinoxylans where some of the xylose units are
substituted with L-arabinofuranosyl residues through either single substitution by

(1→2) or (1→3) linkages or double substitution by both linkages.1,2
The arabinoxylans are dietary fibers that are not hydrolyzed by the digestive
3
enzymes. Instead they are fermented by microbes in the gut and in this way influences
3
the composition and abundance of the colonic microbiota. The result is a prebiotic
effect where the health of the host is benefitted. Arabinoxylans have been shown to
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strengthen the immune system and to lower the risk for obesity, type 2 diabetes, colon
3
cancer and cardiovascular diseases. The physiological functions, however, are
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strongly connected to the structure of the arabinoxylans, i.e. the length of the xylan
3
backbone and the distribution of the arabinose units. Besides food applications
arabinoxylans are also used as a renewable source to prepare biodegradable materials
such as films and hydrogels.1,4
Arabinoxylans are cleaved by glycosyl hydrolases (GH) to form smaller saccharides.
The hydrolysis of the xylose linkages can be achieved with GH10 and GH11 xylanases,
which prefer several unsubstituted xylose units, and with GH5 xylanases where an
5
arabinose substituent is allowed close to the cleavage site. Hydrolysis of the arabinose
residues can be performed with GH43, GH51 and GH62 arabinofuranosidases where
some of the enzymes exclusively cleave arabinoses linked to the 2 or the 3 position of
single substituted xylose residues, whereas others are able to remove the pentose from
disubstituted xylose moieties.5,6 All the enzyme classes, however, suffer from poor
access in the densely substituted regions of arabinoxylans and the difficulties are
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further complicated by the lack of well-defined substrates with multiple arabinose
substituents.
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Arabinoxylan-degrading enzymes are usually investigated with rather heterogeneous
polysaccharides isolated from natural sources by suitable pretreatment procedures. A
few saccharides containing 2 – 4 xylose units and 1 – 2 arabinose residues can be
7
isolated from enzymatic degradation of arabinoxylans, but these simple substrates do
not resemble the more densely substituted regions. Thus, well-defined xylans with
several arabinose substituents would be valuable tools for mapping the active site of
enzymes implicated in arabinoxylan degradation and to understand the interplay
between different enzymes in order to achieve the most efficient deconstruction.
Chemical synthesis offers the possibility to prepare pure and well-defined
oligosaccharides, but the arabinoxylans have so far received relatively little attention.
Xylobiose has been glycosylated with one or two equiv. of an arabinofuranosyl donor to
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afford tri- and tetrasaccharides. Recently, arabinoxylans with up to 6 xylose residues
and 1 – 2 arabinose units were prepared by solid-phase synthesis and used for
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determining the epitopes for monoclonal antibodies and the substrate specificity for
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xylanases and arabinofuranosidases. In one case, two arabinoses were glycosylated
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onto the 2 and the 3 position of the same xylose residue although a very low yield was
obtained illustrating the challenge of producing more densely substituted
arabinoxylans. Linear xylans, on the other hand, where no additional substituents are
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present, are more readily assembled by either stepwise or blockwise approaches and
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so far linear xylans up to decaxylans have been prepared, which includes an
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approach by cleaving the hydroxymethyl group of glucans and the assembly of S-
linked oligoxylans.13
Herein, we report a novel pre-activation-based synthetic strategy for assembling
arabinoxylans, which has led to the preparation of heptasaccharides 1 – 3 with three
arabinose substituents (Scheme 1). A tetraxylan is chosen as the backbone since this
constitutes a sufficient length to give the necessary enzyme activities.5,9a The three
arabinose substituents are connected to either a xylotriose unit (as in 1) or a xylobiose
moiety (as in 2 and 3) in order to resemble the densely substituted region. The
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preparation of 1 – 3 extends our work on the synthesis of plant cell wall components
where we have previously assembled oligomers of rhamnogalacturonan I and
homogalacturonan by using n-pentenyl glycosides as glycosyl donors.14
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Scheme 1. Structures of Arabinoxylans 1 – 3 and Retrosynthetic Strategy
O
HO
HO
O
O
O
O
O
HO
HO
O
O
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HO
HO
O
O
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HO
O
HO OH
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OH
HO
OH
HO
OH
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OH
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HO OH
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HO
OH
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OH
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OH
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BzO
BzO
O
BzO
O
CF3
O
RO
O
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BzO
O
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BzO
RO
+
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SPh
RO
NPh
BzO
OBz
4
Pre-activation-based assembly
O
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BzO
BzO
HO
RO
HO
RO
SPh
SPh
SPh
+
+
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RO
BzO
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RESULTS AND DISCUSSION
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The recent solid-phase synthesis of arabinoxylans was achieved by a stepwise approach
where one xylose or arabinose unit was glycosylated at a time onto the growing oligosaccharide
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chain followed by cleavage from the resin and global deprotection. Up to ten equiv. of a
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xylopyranosyl phosphate (prepared from the corresponding tolyl thioglycoside) or an ethyl 1-
thioarabinofuranoside was used as the donor for each glycosylation reaction. We opted for a
solution-phase strategy involving fewer steps where all the arabinose units are introduced in one
coupling reaction (Scheme 1). The linear xylan backbone will be prepared by a pre-activation-
1
5
based synthesis from the appropriate phenyl 1-thioxylopyranosides where deprotection steps
1
6
will not be necessary after each glycosylation reaction. The pre-activation-based thioglycoside
protocol has previously been used for coupling of a variety of monosaccharides15,17 including
1
8
arabinofuranosides, but it has so far not been applied to the coupling of xylose units. The
benzoyl group will serve as a permanent protecting group for the xylose residues since it allows
for neighboring group participation from the 2 position to afford the -linkage in the
glycosylations. The arabinose units will also be protected with benzoyl groups and the
corresponding N-phenyl trifluoroacetimidate 4 will serve as the donor since it has previously
given high yields in the coupling with different secondary alcohols.19
Scheme 2. Synthesis of Building Blocks 5 and 6
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O
HO
HO
HO
OH
D-xylose
1) Ac O, DMAP, Et N
2
3
2
) PhSH, BF ·OEt2
3
3) NaOMe, MeOH, 40% (3 steps)
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) 2-methoxypropene, CSA, 70%
) PMBCl, NaH
3) CSA, MeOH, 91% (2 steps)
HO
HO
O
PMBO
HO
O
SPh
SPh
HO
HO
8
7
1
) BzCl, Et N, DMAP
then DMAPA
2) DDQ, 94% (2 steps)
3
BzCl, pyridine
84%
O
O
BzO
BzO
HO
BzO
SPh
SPh
BzO
BzO
5
6
Several phenyl 1-thioxylopyranoside building blocks were required for the xylan assembly
by the pre-activation-based approach. This involves fully benzoylated glycoside 5, 2,3-di-O-
benzoylated glycoside 6 and two glycosides with different protecting groups at position 2 and 3.
The building blocks were all prepared from phenyl 1-thio--D-xylopyranoside (7), which is
available from xylose in three simple steps involving peracetylation in the presence of 4-(N,N-
2
0
dimethylamino)pyridine (DMAP), thiolation and deacetylation with no purification of the
intermediates (Scheme 2). Perbenzoylation of 7 then afforded tribenzoate 5 while the
preparation of dibenzoate 6 required selective transformations of the hydroxy groups. In
xylopyranosides the three hydroxy groups are all equatorial and similar in reactivity making
2
1
regioselective reactions a particular challenge. One of the most effective procedures is to block
the 2 and the 3 position with an isopropylidene group, which affords only minor amounts of the
2
2
corresponding 3,4-acetal. Thus, treatment of triol 7 with 2-methoxyprop-1-ene and
camphorsulfonic acid (CSA) led to the acetonide at position 2 and 3 as the major product.
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Subsequently, a para-methoxybenzyl group (PMB) was installed at position 4 and the acetonide
2
3
was removed under acidic conditions to provide diol 8. The hydroxy groups were benzoylated
and excess benzoyl chloride removed with 3-(dimethylamino)-1-propylamine (DMAPA).24
Without further purification the crude product was subjected to PMB deprotection with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to afford dibenzoate 6.
1
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Scheme 3. Synthesis of Building Blocks 8 and 10
1
) LevOH, DMAP, DCC
PMBO
HO
O
2) DDQ
HO
LevO
O
SPh
SPh
HO
74%, 2 steps
LevO
8
9
+
-
BzCl, nBu N HSO
4
4
1
M NaOH, -5 °C
68%
1
2
) LevOH, DMAP, DCC
) DDQ
PMBO
HO
O
HO
LevO
O
SPh
SPh
78% (2 steps)
BzO
10
BzO
11
The two remaining building blocks required a different protecting group to be installed at
position 3 as well as at position 2 and 3 since these sites will be linked to the arabinose units.
The temporary protecting group should be an ester to ensure neighboring group participation in
the glycosylation, but should at the same time be removable in the presence of the benzoates. A
chloroacetate was first considered and installed selectively at position 3, but the following
glycosylation with 5 under the pre-activation protocol only furnished a moderate yield of the
disaccharide. This was attributed to the use of a silver salt in the glycosylation and a levulinyl
(Lev) group was therefore chosen instead. N,N’-Dicyclohexylcarbodiimide (DCC) coupling of
diol 8 with levulinic acid followed by direct treatment with DDQ furnished dilevulinate 9
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(
Scheme 3). For the selective introduction of the temporary protecting group, diol 8 was first
subjected to regioselective benzoylation at position 2. With benzoyl chloride in pyridine the
esterification was slow and actually produced the dibenzoate as the main product. However,
when diol 8 was submitted to 1.3 equiv. of benzoyl chloride in a mixture of CH Cl and 1 M
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NaOH under phase transfer catalysis, 2-benzoate 10 was obtained in 68% yield with only
minor amounts of the corresponding 3-benzoate as a byproduct. Subsequent esterification with
levulinic acid and deprotection of the PMB group gave the desired 3-levulinate 11.
The stage was now set for assembling the xylan backbone by the pre-activation-based
protocol. The original procedure with tolylthio glycosides called for activation of the donor with
p-toluenesulfenyl chloride and silver triflate at a temperature of –60 °C to generate the reactive
1
6
electrophile. The acceptor was then added followed by stirring for 15 min and warming to rt.
The procedure was later on modified with the formation of the reactive electrophile at –78 °C
1
7
followed by adding the acceptor and raising the temperature to rt. Using this latest protocol,
26
donor 5 was coupled to acceptor 6 with phenylsulfenyl chloride and silver triflate to give
disaccharide 12 in 64% yield (Scheme 4, Method A). The coupling could also be carried out in a
1
6
one-pot iterative manner by adding more promoter and acceptor to furnish trisaccharide 13
although the yield in this case was only 34% since disaccharide 12 was also formed in 59%
2
7
yield. Attempts to improve the glycosylations by using Me S /Tf O or 1-benzenesulfinyl
2
2
2
2
8
piperidine/Tf O as the promoter were unsuccessful and only led to a mixture of products.
2
29
However, when p-nitrobenzenesulfenyl chloride and silver triflate was employed as the
promoter, the yield of 12 and 13 in the two reactions increased to 79 and 69%, respectively. p-
Nitrobenzenesulfenyl chloride is commercially available as opposed to phenylsulfenyl chloride
and p-toluenesulfenyl chloride which are prepared from the corresponding thiols with sulfuryl
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chloride. In addition, p-nitrobenzenesulfenyl chloride is a solid making this reagent procedurally
more convenient to deliver while the other two sulfenyl chlorides are liquids. The quality of
commercial p-nitrobenzenesulfenyl chloride, however, varied and we obtained lower
glycosylation yields when using light brown samples with a mp of ~43 °C. Attempts to
recrystallize the compound failed, but sublimation yielded material with a light yellow color and
a mp of 47 – 51 °C which could be stored at 5 °C for months. When p-nitrobenzenesulfenyl
chloride purified by sublimation was used, the yields in the glycosylations were consistent.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
During the couplings, the disulfide byproduct from the activation (i.e. pNO PhSSPh)
2
precipitated and did therefore not take part in any additional reactions.
Scheme 4. Synthesis of Saccharides 12 and 13
Method A:
1
) AgOTf, RSCl, CH Cl2
-
2
78 oC, 10 min
2) 6 (0.9 equiv)
-
78 oC, 15 min
O
o
O
BzO
BzO
-78 C  rt, 1 h
BzO
BzO
SPh
O
O
BzO
SPh
BzO
Method B:
BzO
BzO
5
1) AgOTf, RSCl, CH2Cl2
12
o
-
65 C, 10 min
Method A:
2
) 6 (0.9 equiv)
o
R = Ph: 64%
R = pNO Ph: 79%
1) AgOTf, RSCl
CH Cl , -78 oC
-55 C, 15 min
-55
oC  -15 oC, 25 min
2
2
2
1
0 min
Method B:
2
) 6 (0.9 equiv)
R = pNO Ph: 90%
o
2
-78 C, 15 min
o
-78 C  rt, 15 min
) RSCl, -78 C, 10 min
o
3
4
) 6 (0.8 equiv)
-
-
78 oC, 15 min
78 C  rt, 15 min
BzO
BzO
O
O
o
O
BzO
O
O
BzO
BzO
SPh
BzO
BzO
13
R = Ph: 34%
R = pNO Ph: 69%
2
To further improve the glycosylation outcome the influence of the temperature was also
investigated. These experiments were performed in a two-neck Schlenk flask that allowed for
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2
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2
2
2
2
2
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3
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5
5
6
measuring the internal temperature in the reaction mixture at any time during the glycosylation.
The activation of donor 5 was observed to be quick and goes to completion in less than 10 min
at –78 °C according to TLC analysis. This is in accordance with investigations on the activation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30
of similar thioglycosides under these conditions. However, in the ensuing glycosylation
reaction after adding acceptor 6 the reaction temperature becomes important. If the temperature
3
1
is kept at –78 °C the glycosylation is sluggish and an intermolecular aglycon transfer becomes
32
a competing reaction leading to the regeneration of donor 5. Under these conditions
disaccharide 12 was only obtained in 54% yield together with 10% of donor 5 although full
activation of the donor had been achieved. Thus, at very low temperature the hydroxy group and
the sulfur in acceptor 6 are both able to serve as a nucleophile in the reaction with the donor.
Further experiments revealed that the aglycon transfer was mainly observed when the
temperature was kept below –60 °C. Therefore, three separate experiments were set up where
acceptor 6 was added at –55 °C (Figure 1). In the first experiment, the temperature was
immediately raised to rt over 1 h while in the two other experiments the temperature was first
maintained at –55 °C for 10 – 15 min before being raised. The first reaction gave 73% yield of
disaccharide 12 whereas 85 and 90% yield were obtained in the two other experiments (Figure 1
and Scheme 4, Method B). This is a notable difference and illustrates the significance of the
temperature in securing an optimal coupling reaction. If the temperature is too low the aglycon
transfer becomes a side reaction while if the temperature is too high the activated intermediate
33
of donor 5 (which is mostly the corresponding 1,2-benzoxonium ion) presumably decomposes.
Thus, a coupling temperature of –55 °C was included in the protocol in the following
experiments. The reactions were moinitored by TLC at –55 °C and the temperature was only
raised when the coupling had gone to completion.
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1
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9
0
1
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6
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9
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2
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5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Reaction Temperatures and Yields for Coupling of 5 and 6 to afford 12
Using this optimized procedure donor 5 was then coupled to Lev-protected acceptor 11 to
afford disaccharide 14 in 86% yield (Scheme 5). Unfortunately, when the same coupling was
performed in a one-pot fashion with the further addition of acceptor 6, trisaccharide 15 was only
obtained in 28% yield. The addition of 2,4,6-tri-tert-butylpyrimidine15,34 to mitigate the possible
influence of the increasing amount of acid lowered the yield even further to 17%. Instead, large
amounts of unreacted acceptor 6 were recovered in both cases. The low yield was not caused by
the inability of Lev-protected thioglycosides to serve as donors since the coupling between
disaccharide donor 14 and acceptor 6 gave rise to trisaccharide 15 in 69% yield. Possibly, the
mediocre result is caused by the Lev group undergoing further transformations due to the longer
reaction time in the one-pot protocol. Therefore, the xylan backbones in targets 1 – 3 will be
assembled by an iterative glycosylation approach with pre-activation where the glycosylation
products once isolated will be immediately used as donors for the next glycosylation step
without any further modifications.
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1
1
1
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Synthesis of Saccharides 14 and 15
1) AgOTf, pNO PhSCl
2
O
CH Cl , -78 oC, 10 min
O
BzO
BzO
BzO
BzO
2
2
SPh
O
O
LevO
SPh
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BzO
2) 11 (0.9 equiv)
BzO
o
BzO
-55 C, 15 min
5
14
86%
1
) AgOTf, pNO PhSCl
2
1
) AgOTf, pNO PhSCl
2
CH2Cl2, -65 oC, 10 min
CH Cl , -65 oC, 10 min
2
2
69%
) 11 (0.9 equiv), -55 oC, 15 min
28%
2) 6 (0.9 equiv)
2
o
-55 C  -15 oC, 15 min
o
-55 C, 15 min
3
4
) pNO PhSCl, -65 oC, 10 min
2
) 6 (0.8 equiv), -55 oC, 15 min
o
55 C  -15 oC, 15 min
-
BzO
BzO
O
O
O
LevO
O
O
BzO
BzO
BzO
SPh
BzO
15
Accordingly, to prepare arabinoxylan 1 disaccharide 14 was then coupled twice with Lev-
protected acceptor 11 to afford trisaccharide 16 and tetrasaccharide 17 in 88 and 73% yields,
respectively (Scheme 6). The three Lev groups in the 3 positions were removed with hydrazine
to give triol 18 in 96% yield. Subsequent glycosylation with four equiv. of imidate donor 4
afforded protected arabinoxylan 19 in 90% yield. The arabinofuranose donor 4 was prepared in
four straightforward steps from the parent pentose.35,19b Thus, heptasaccharide 19 has been
assembled from monosaccharides 4, 5 and 11 in a total of five steps and 48% overall yield,
which illustrates the advantage of the iterative pre-activation-based strategy.
Scheme 6. Synthesis of Heptasaccharide 19
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9
1
) AgOTf, pNO PhSCl
2
CH Cl , -65 oC, 10 min
O
BzO
BzO
2
2
O
O
1
4
O
LevO
O
2
) 11 (0.9 equiv)
BzO
SPh
LevO
o
BzO
16
-55 C, 15 min
BzO
88%
1) AgOTf, pNO PhSCl
2
CH Cl , -65 oC, 10 min
) 11 (0.9 equiv)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
73%
2
o
-55 C, 60 min
O
BzO
BzO
O
O
O
RO
O
RO
BzO
O
O
BzO
SPh
RO
BzO
BzO
1
7: R = Lev
8: R = H
H NNH , 96%
2
2
1
1
) AcCl, MeOH
) BzCl, pyridine
(63%, 2 steps)
HO
HO
2
BzO
O
CF3
NPh
O
O
TMSOTf
3) HBr, AcOH, H O (77%)
HO
OH
2
BzO
OBz
90%
4) 2,2,2-Trifluoro-N-phenyl-
L-arabinose
4
acetimidoyl chloride
Cs CO3 (84%)
2
D
O
BzO
BzO
C
O
O
B
O
O
O
A
BzO
BzO
O
O
O
BzO
SPh
O
E
O
BzO
BzO
O
BzO
BzO
G
O
F
BzO
OBz
BzO
OBz
BzO
OBz
19
The same sequence of reactions were then employed to prepare the protected forms of
arabinoxylans 2 and 3. To assemble the former, disaccharide 14 was used again and now
coupled with 2,3-Lev-protected acceptor 9 to give trisaccharide 20 in 71% yield (Scheme 7).
Further glycosylation with acceptor 6 produced 61% yield of tetrasaccharide 21, which was
subjected to Lev deprotection to afford triol 22. The arabinoses were then installed with donor 4
to afford heptasaccharide 23 in 95% yield. Likewise, the route to arabinoxylan 3 began with the
coupling between donor 5 and 2,3-Lev-protected acceptor 9 to furnish disaccharide 24 in 95%
yield (Scheme 8). The following glycosylation with acceptor 11 produced trisaccharide 25 in
5
2% yield. The moderate yield in this coupling as well as between 20 and 6 in Scheme 7 may
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
indicate a lower stability of the activated species with a Lev group at the 2 position. Next,
trisaccharide 25 was reacted with acceptor 6 to give tetrasaccharide 26 in 75% yield, which was
followed by Lev deprotection to provide triol 27. The final triple glycosylation with arabinose
donor 4 gave heptasaccharide 28 in 91% yield. Thus, protected heptasaccharides 23 and 28 have
each been prepared in five steps from monosaccharides 4, 5, 9 and 11 and with overall yields of
34 and 33%, respectively. In all cases, the three arabinoses were installed in very high yields
with imidate donor 4 proving the strength of this method even for forming densely substituted
substrates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7. Synthesis of Heptasaccharide 23
1
) AgOTf, pNO PhSCl
2
CH Cl , -65 oC, 10 min
O
BzO
BzO
2
2
O
O
LevO
14
O
O
LevO
2
) 9 (0.9 equiv)
-55 C, 50 min
BzO
SPh
o
BzO
LevO
20
71%
1) AgOTf, pNO PhSCl
2
CH Cl , -65 oC, 10 min
2
2
61%
2
) 6 (0.9 equiv)
o
-
55 C, 40 min
4
O
TMSOTf
BzO
BzO
O
O
RO
O
O
O
RO
95%
BzO
O
BzO
SPh
BzO
RO
BzO
2
1: R = Lev
2: R = H
H NNH , 97%
2
2
2
D
O
BzO
BzO
C
O
O
B
O
O
O
O
A
BzO
BzO
O
BzO
O
O
BzO
BzO
SPh
O
O
BzO
G
BzO
F
O
BzO
OBz
E
BzO
OBz
BzO
OBz
23
The -linkages of the xylose backbones were confirmed by measuring the JCH coupling
constants for the anomeric carbon atoms, which were found to be between 155 and 165 Hz
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where a coupling around 160 Hz is characteristic of a -glycosidic bond. The anomeric carbon
atoms for the -glycosidic linkages were located in the  = 99.0 – 102.5 ppm range, which is in
11
accordance with earlier observations. Xylopyranosides are more conformationally flexible
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
than e.g. glucopyranosides and the J
coupling constants can therefore not always be used
H-1,H-2
to determine their anomeric configuration. On the contrary, for the arabinofuranose substituents
the JH-1,H-2 coupling constants could be used to verify the -linkages since small values of 0 – 2
Hz are characteristic for the  anomers.37
Scheme 8. Synthesis of Heptasaccharide 28
1
) AgOTf, pNO PhSCl
2
CH Cl , -65 oC, 10 min
O
BzO
BzO
2
2
O
5
O
SPh
LevO
2
) 9 (0.9 equiv)
BzO
o
LevO
-55 C, 25 min
24
1) AgOTf, pNO PhSCl
95%
2
CH Cl , -65 oC, 10 min
2
2
5
2% 2) 11 (0.9 equiv)
o
-55 C, 25 min
O
BzO
BzO
O
O
LevO
O
O
LevO
BzO
SPh
LevO
BzO
1
) AgOTf, pNO PhSCl
2
25
CH Cl , -65 oC, 10 min
2
2
2
) 6 (0.9 equiv)
-55 C, 65 min
75%
o
O
BzO
BzO
O
O
RO
4
O
O
RO
BzO
O
BzO
O
TMSOTf
RO
SPh
BzO
BzO
91%
26: R = Lev
H NNH , 99%
2
2
27: R = H
D
O
BzO
BzO
C
O
O
O
O
B
O
O
A
BzO
G
O
BzO
O
O
BzO
SPh
O
BzO
BzO
BzO
O
BzO
O
BzO
OBz
BzO
E
F OBz
BzO
OBz
28
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Deprotection of the three heptasaccharides is a two-step process since the thiophenyl moiety
and the benzoates are removed under different conditions. In the first approach, the thiophenyl
3
8
group was cleaved in the first step with NBS in a mixture of acetone and water. This
transformation proceeded uneventfully for all three heptasaccharides and gave rise to reducing
sugars 29 – 31 in 92 – 93% yield (Scheme 9). The products were isolated as / mixtures with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the  anomers as the major components according to NMR. The following Zemplén reaction to
3
9
remove the benzoates was carried out with approx. 20 equiv. of NaOMe in a 1:1 methanol –
dichloromethane solution for up to 7 days to achieve complete deprotection of all benzoyl
groups. Unfortunately, a mixture of saccharides of different length were obtained and it became
apparent that the alkaline deprotection procedure had been accompanied by a so-called peeling
4
0
reaction (endwise degradation). In this process reducing saccharides are degraded with one
monosaccharide at the time from the reducing end through aldose – ketose isomerizations and -
4
0
alkoxy eliminations. To circumvent this side reaction, the order of the deprotections were
reversed. First, the protected heptasaccharides were subjected to cleavage of the benzoyl groups,
which proceeded cleanly to give compounds 32 – 34 in 77 – 91% yield. Then, attempts were
made to remove the thiophenyl moiety under the same conditions as applied to the
perbenzoylated substrates. However, these conditions now resulted in partial hydrolysis of the
arabinofuranose residues, which is presumably due to the higher acid lability of these
substituents in the absence of the benzoyl groups. Hence, 2,6-lutidine was added during the
4
1
deprotections to ensure a pH of the reaction media around 6, which led to clean removal of the
thiophenyl groups to afford the completely deprotected target structures 1 – 3 in 84 – 97% yield.
Heptasaccharides 1 – 3 were fully characterized by 800 MHz NMR spectroscopy and although
1
13
they were obtained as / mixtures, the differences in the H and the C chemical shifs between
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the  and the  anomers were only observed for the reducing end xylose unit and when an
arabinose residue was attached to this moiety (as in 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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Scheme 9. Deprotection of Heptasaccharides 19, 23 and 28
19
23
28
O
NBS
BzO
BzO
O
O
RO
O
O
acetone
O
BzO
2
O
1
R O
R O
4
H O
3
R O
2
R O
OH
BzO
9: R, R , R = 2,3,5-tri-O-benzoyl--L-arabinofuranosyl, R , R3 = Bz (92%)
2
4
1
2
3
3
0: R, R , R = 2,3,5-tri-O-benzoyl--L-arabinofuranosyl, R , R4 = Bz (93%)
2
3
1
1: R, R , R = 2,3,5-tri-O-benzoyl--L-arabinofuranosyl, R , R4 = Bz (92%)
1
2
3
NaOMe
MeOH
CH Cl
2
2
NaOMe, MeOH, CH Cl2
2
Peeling
O
HO
HO
O
O
RO
O
O
2
HO
O
O
1
R O
R O
4
X
3
R O
R O
HO
2
4
1
3
3
3
3
2: R, R , R = -L-arabinofuranosyl, R , R = H, X = SPh (77%)
2
3
1
4
3: R, R , R = -L-arabinofuranosyl, R , R = H, X = SPh (82%)
1
2
3
4
4: R, R , R = -L-arabinofuranosyl, R , R = H, X = SPh (91%)
NBS
2,6-lutidine
2
4
1
3
acetone
1
: R, R , R = -L-arabinofuranosyl, R , R = H, X = OH (97%)
H O
2
3
1
4
2
2
3
: R, R , R = -L-arabinofuranosyl, R , R = H, X = OH (84%)
1
2
3
4
: R, R , R = -L-arabinofuranosyl, R , R = H, X = OH (88%)
In conclusion, three arabinoxylan heptasaccharides have been assembled in seven steps from
monosaccharide building blocks in overall yields of 36 (1), 24 (2) and 27% (3). The key
transformations are the pre-activation-based thioglycoside glycosylation to prepare the
tetraxylan backbones and the triple glycosylation with imidate donor 4 to install the arabinose
substituents. p-Nitrobenzenesulfenyl chloride was employed as a potent and convenient source
of the sulfenium electrophile for the pre-activation of the phenyl thioglycoside donors. In the
ensuing glycosylation reaction, temperature monitoring revealed an optimum temperature for
the coupling where side reactions are diminished. The strategy allows for the expeditious
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preparation of a diverse set of densely substituted arabinoxylans in good overall yields as
valuable substrates for characterization of hemicellulose-degrading enzymes.
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EXPERIMENTAL SECTION
General Information. Starting materials, reagents and solvents were purchased from
commercial suppliers and used without further purification unless otherwise noted. All solvents
were of analytical HPLC grade. Anhyd solvents were obtained from an Innovative Technology
PS-MD-7 PureSolv solvent purification system except for CH Cl and toluene for
2
2
glycosylations, which were dried over 3Å mol sieves prior to use. All reactions were performed
under inert atm (N ) in oven-dried glassware. TLC was carried out using Merck aluminium
2
sheets pre-coated with 0.25 mm silica gel, C-60 F254 plates. TLC plates were inspected under
UV light or visualized by charring after dipping in a cerium ammonium sulfate solution (1%
cerium(IV)sulfate and 2.5% ammonium heptamolybdate in a 10% sulfuric acid solution). Flash
column chromatography was performed using Merck Geduran silica gel 60 Å (40‒63 m) while
4
2
dry column chromatography was accomplished using Merck silica gel 60 Å (15‒40 m).
NMR spectra were recorded with a Bruker AscendTM 400 or a Bruker AscendTM 800
spectrometer. The chemical shift are reported in ppm relative to the residual solvent peak from
CDCl ( = 7.26 ppm,  = 77.16 ppm), CD OD ( = 4.87 ppm,  = 49.00 ppm) or D O (
3
H
C
3
H
C
2
H
1
13
=
4.79 ppm). Assignment of H and C resonances were based on APT, DQF-COSY, HSQC,
H2BC, HMBC, TOCSY and HSQC-TOSCY experiments. Xylose residues are numbered A – D
from the reducing end while arabinose substituents are labelled E – G as shown in Scheme 6 –
8
. Optical rotation was measured on a Perkin Elmer Model 341 polarimeter. HRMS analysis
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was performed on either a UHPLC-QTOF system (Dionex UltiMate 3000 and Bruker MaXis)
with an electrospray ionization (ESI) source or a MALDI-TOF system (Bruker Solarix XR 7T).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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8
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0
Phenyl 1-thio--D-xylopyranoside (7). D-Xylose (29.9 g, 199 mmol), Et N (223 mL, 160 mmol)
3
and DMAP (4.90 g, 40.1 mmol) were suspended in CH Cl (300 mL) at 0 °C. Upon dropwise
2
2
addition of Ac O (94.0 mL, 996 mmol) the solution turned yellow. After stirring for 2 h at rt
2
TLC analysis (hexane/EtOAc 1:1) showed the consumption of the starting material and
formation of the product. The mixture was washed with 1 M HCl and brine followed by
concentration in vacuo. The crude xylose tetraacetate was dissolved in dry CH Cl (160 mL)
2
2
followed by addition of PhSH (25.0 mL, 243 mmol) and BF ·OEt (76.0 mL, 600 mmol) at 0 °C
3
2
under N atm. The reaction was stirred overnight at rt where it turned purple. TLC analysis
2
(hexane/EtOAc 2:1) showed full consumption of the starting material and formation of the
product. The solution was diluted with CH Cl and washed with saturated aq NaHCO and
2
2
3
brine, dried over Na SO , filtered and concd in vacuo. The residue was dissolved in methanol
2
4
(160 mL) and solid sodium was added until a basic medium was obtained. After 1 h TLC
analysis (hexane/EtOAc 2:1) showed the consumption of the starting material and formation of
+
the product. The solution was neutralized with Amberlite IR-120 (H ), filtered and concd in
vacuo. The residue was purified by dry column chromatography (10% acetone in toluene, 5%
gradient) followed by crystallization from hexane and acetone to afford a white crystalline
20
1
product (19.2 g, 40%). R 0.27 (toluene/acetone 1:1). Mp 142.3‒143.8 °C (lit. 143‒145 °C). H
f
NMR (400 MHz, CD OD)  7.54‒7.51 (m, 2H), 7.34‒7.26 (m, 3H), 4.56 (d, J = 9.3 Hz, 1H,
3
1,2
H1), 3.95 (dd, J5,5’ = 11.3 Hz, J = 5.2 Hz, 1H, H5), 3.51‒3.45 (m, 1H, H4), 3.38‒3.31 (m, 3H,
4,5
1
3
1
H3, OH), 3.26‒3.19 (m, 2H, H2, H5’). C{ H} NMR (101 MHz, CD OD)  134.9, 133.1 (×2),
3
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29.9 (×2), 128.5, 90.1 (C1), 79.2 (C3), 73.7 (C2), 70.9 (C4), 70.4 (C5). NMR data are in
accordance with literature values.43
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Phenyl 2,3,4-tri-O-benzoyl-1-thio--D-xylopyranoside (5). Triol 7 (5.12 g, 21.1 mmol) was
dissolved in pyridine (45 mL) and BzCl (7.4 mL, 63.4 mmol) was added. The reaction was
stirred at rt for 1 h and excess of BzCl was quenched by the addition of methanol (10 mL)
followed by stirring for an additional 10 min. The mixture was diluted with CH Cl and washed
2
2
with 1 M HCl and water, dried over Na SO , filtered, and concd in vacuo. The residue was
2
4
purified by flash column chromatography to afford 5 as write crystals (5.1 g, 84%). R 0.28
f
1
(heptane/EtOAc 7:3). Mp 102.9‒104.3 °C (EtOH). H NMR (400 MHz, CDCl )  8.05 (dd, J =
3
8.2, 1.1 Hz, 2H), 8.01‒7.98 (m, 4H), 7.56‒7.51 (m, 5H), 7.42‒7.32 (m, 9H), 5.78 (t, J = 6.6 Hz,
H, H3), 5.46 (t, J = 6.3 Hz, 1H, H2), 5.33‒5.26 (m, 2H, H1, H4), 4.71 (dd, J5eq,5ax = 12.3 Hz,
1
1
3
1
J5eq,4 = 4.0 Hz, 1H, H5 ), 3.83 (dd, J5ax,5eq = 12.3 Hz, J5ax,4 = 6.5 Hz, 1H, H5 ). C{ H} NMR
eq
ax
(101 MHz, CDCl )  165.6, 165.3, 165.3, 133.6, 133.5, 133.5, 133.2, 132.8 (×2), 130.2 (×2),
3
1
8
30.1 (×2), 130.1 (×2), 129.3 (×2), 129.2 (×2), 129.1, 128.6 (×2), 128.6 (×2), 128.5 (×2), 128.3,
6.5 (C1), 70.6 (C3), 70.1 (C2), 68.8 (C4), 63.7 (C5). Anal. calcd for C H O S: C, 69.30; H,
3
2
26
7
4.73; S, 5.78. Found: C, 69.26; H, 4.70; S, 5.66. NMR data are in accordance with literature
values.44
Phenyl 4-O-(p-methoxy)benzyl-1-thio--D-xylopyranoside (8). To a solution of triol 7 (29.5 g,
1
22 mmol) in dry DMF (200 mL) was added CSA (2.83 g, 12.0 mmol) and the mixture was
heated to 60 °C. 2-Methoxy prop-1-ene (37.5 mL, 366 mmol) was added slowly to the solution.
The reaction was stirred for 1 h and then cooled to rt and quenched with Et N (30 mL).
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Concentration in vacuo followed by purification of the residue by flash column chromatography
(heptane/EtOAc/CH Cl 4:1:1) afforded phenyl 2,3-O-isopropylidene-1-thio--D-
2
2
1
xylopyranoside as a colorless oil (24.1 g, 70%). R 0.62 (toluene/acetone 3:1). H NMR (400
f
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CDCl )  7.58‒7.55 (m, 2H), 7.34‒7.30 (m, 3H), 4.80 (d, J = 9.6 Hz, 1H, H1), 4.13 (dd,
3
1,2
J5,5’ = 11.6 Hz, J = 5.3 Hz, 1H, H5 ), 4.03‒3.96 (m, 1H, H4), 3.54 (t, J = 9.1 Hz, 1H, H3),
4,5
eq
3
.27‒3.22 (m, 2H, H2, H5 ), 2.17 (d, J
= 4.0 Hz, 1H, OH), 1.49 (s, 3H), 1.45 (s, 3H).
ax
OH,4
1
3
1
C{ H} NMR (101 MHz, CDCl ) δ 133.0 (×2), 132.0, 129.0 (×2), 128.3, 111.5, 85.6 (C1), 83.0
3
(C3), 75.3 (C2), 70.0 (C5), 69.2 (C4), 26.8, 26.7. NMR data are in accordance with literature
45
values. The intermediate (17.5 g, 62.0 mmol) was dissolved in dry DMF (120 mL) and NaH
(3.0 g, 74.4 mmol, 60% in mineral oil) was added at 0 °C. After 10 min PMBCl (10.9 mL, 80.6
mmol, 1.3 equiv.) was added. The mixture was stirred at rt for 16 h and then quenched with 10%
HCl (28 mL) and diluted with CH Cl . The solution was washed with saturated aq NaHCO ,
2
2
3
dried over Na SO , filtered, and concd in vacuo. The crude residue was dissolved in
2
4
CH Cl /MeOH 1:1 (200 mL), CSA (14.4 g, 62.0 mmol) was added and the mixture was allowed
2
2
to stir at rt overnight. The reaction was quenched by addition of Et N and concd in vacuo. The
3
residue was purified by flash column chromatography (heptane/EtOAc 3:2) to furnish
compound 8 as a white crystalline solid (20.5 g, 91% over 2 steps). R 0.47 (hexane/EtOAc 1:1).
f
Mp 72.7‒75.6 °C (EtOH). []25 ‒55.9 (c 1, CHCl ). H NMR (400 MHz, CDCl )  7.53‒7.50
1
D
3
3
(m, 2H), 7.33‒7.29 (m, 3H), 7.27‒7.23 (m, 2H), 6.90‒6.86 (m, 2H), 4.61 (d, Jgem = 11.4 Hz, 1H,
PMB), 4.58‒4.54 (m, 2H, H1 (J = 8.9 Hz) PMB), 4.07 (dd, J = 11.5 Hz, J5eq,4 = 4.8 Hz,
1
,2
5eq,5ax
1H, H5 ), 3.80 (s, 3H, OMe), 3.66 (t, J = 8.4 Hz, 1H, H3), 3.49‒3.43 (m, 1H, H4), 3.40 (t, J =
eq
8
.6 Hz, 1H, H2), 3.27 (dd, J5ax,5eq = 11.5 Hz, J5ax,4 = 9.7 Hz, 1H, H5 ), 2.52 (bs, 2H, OH).
ax
1
3
1
C{ H} NMR (101 MHz, CDCl )  159.7, 132.8 (×2), 132.3, 130.1, 129.7 (×2), 129.2 (×2),
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28.3, 114.2 (×2), 88.8 (C1), 76.6 (C4), 76.5 (C3), 72.8 (PMB), 72.1 (C2), 67.1 (C5), 55.4
+
(OMe). HRMS: m/z calcd for C H O SNa [M + Na] 385.1080, found 385.1090.
19 22
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Phenyl 2,3-di-O-benzoyl-1-thio--D-xylopyranoside (6). Compound 8 (1.97 g, 5.43 mmol) was
dissolved in CH Cl (32 mL) and Et N (2.4 mL, 17.2 mmol), DMAP (0.069 g, 0.56 mmol) and
2
2
3
BzCl (1.9 mL, 16.4 mmol) were added at 0°C. After 5 min the reaction was left to stir at rt
overnight. 3-(Dimethylamino)-1-propylamine (2.2 mL, 17.5 mmol) was added and the mixture
was stirred for 1.5 h followed by washing with 1 M HCl and brine, drying over MgSO4,
filtration, and concentration in vacuo. The product phenyl 2,3-di-O-benzoyl-4-O-(p-
methoxy)benzyl-1-thio--D-xylopyranoside (3.12 g, 100%) was obtained as a light yellow syrup
2
5
and used without further purification. R 0.28 (hexane/EtOAc 4:1). []
+62.0 (c 1.0, CHCl3).
D
f
1
H NMR (400 MHz, CDCl )  7.99‒7.95 (m, 3H), 7.56‒7.27 (m, 12H), 7.15‒7.12 (m, 2H),
3
6
.74‒6.71 (m, 2H), 5.60 (t, J = 8.0 Hz, 1H, H3), 5.34 (t, J = 8.0 Hz, 1H, H2), 5.04 (d, J = 8.1
1,2
Hz, 1H, H1), 4.56 (d, Jgem = 11.9 Hz, 1H, PMB), 4.52 (d, Jgem = 11.8 Hz, 1H, PMB), 4.28 (dd,
J5eq,5ax = 11.9 Hz, J5eq,4 = 4.6 Hz, 1H, H5 ), 3.80‒3.76 (m, 1H, H4), 3.75 (s, 3H, OMe), 3.57
eq
1
3
1
(dd, J5ax,5eq = 11.9 Hz, J5ax,4 = 8.5 Hz, 1H, H5 ). C{ H} NMR (101 MHz, CDCl )  165.7,
ax
3
165.4, 159.5, 133.4 (×2), 133.2, 132.5 (×2), 130.1 (×2), 130.0 (×2), 129.7, 129.7 (×2), 129.5,
1
7
29.4, 129.1 (×2), 128.5 (×2), 128.5 (×2), 128.1, 113.9 (×2), 87.0 (C1), 73.9 (C4), 73.6 (C3),
+
2.5 (PMB), 70.6 (C2), 66.4 (C5), 55.4 (OMe). HRMS: m/z calcd for C H O SNa [M + Na]
3
3
30
7
1
3
593.1604, found 593.1617. NMR data are in accordance with literature values. To a well
stirred emulsion of the above PMB ether (0.696 g, 1.22 mmol) in CH Cl (10 mL) and H O (1.0
2
2
2
mL), DDQ (0.415 g, 1.83 mmol) was added. The reaction was stirred in the dark for 7 h and
then filtered through a large pad of Celite. The filtrate was washed with saturated aq NaHCO3
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and brine, filtered and concd in vacuo. Purification by flash column chromatography
(
pentane/EtOAc 4:1) yielded 6 as a white crystalline compound (0.514 g, 94%). R 0.53
f
hexane/EtOAc 2:1). Mp 133.1‒134.0 °C (EtOH). []25D +66.1 (c 1.0, CHCl ). 1H NMR (400
(
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
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5
5
5
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1
2
3
4
5
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7
8
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1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
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2
3
4
5
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7
8
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2
3
4
5
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7
8
9
0
MHz, CDCl )  8.05‒7.99 (m, 4H), 7.57‒7.49 (m, 4H), 7.43‒7.39 (m, 4H), 7.35‒7.29 (m, 3H),
3
5.43 (t, J = 7.4 Hz, 1H, H2), 5.33 (t, J = 7.4 Hz, 1H, H3), 5.09 (d, J = 7.3 Hz, 1H, H1), 4.45
1
,2
(
dd, J5eq,5ax = 12.0 Hz, J5eq,4 = 4.4 Hz, 1H, H5 ), 4.00 (q, J = 6.9 Hz, 1H, H4), 3.61 (dd, J
=
5ax,5eq
eq
1
3
1
1
2.0 Hz, J5ax,4 = 7.9 Hz, 1H, H5 ), 3.03 (s, 1H, OH). C{ H} NMR (101 MHz, CDCl )  167.1,
ax
3
165.2, 133.8, 133.6, 133.0, 132.8 (×2), 130.2 (×2), 130.0 (×2), 129.3, 129.2 (×2), 128.9, 128.7
(×2), 128.7 (×2), 128.3, 86.8 (C1), 76.0 (C3), 70.2 (C2), 68.4 (C4), 67.6 (C5). HRMS: m/z calcd
+
for C H O SNa [M + Na] 473.1029, found 473.1048.
2
5
22
6
Phenyl 2,3-di-O-levulinoyl-1-thio--D-xylopyranoside (9). A solution of diol 8 (1.98 g, 5.47
mmol) and 4-oxopentanoic acid (1.61 g, 13.8 mmol) in CH Cl (8.5 mL) was cooled to 0 °C.
2
2
Then a solution of DMAP (0.070 g, 0.58 mmol) and DDC (3.42 g, 16.8 mmol) in CH Cl (1.0
2
2
mL) was added. The reaction stirred at rt overnight and then filtered and concd. The residue was
dissolved in CH Cl (20 mL) and H O (2.0 mL) and stirred vigorously. DDQ (1.86 g, 8.21
2
2
2
mmol) was added and the mixture was stirred at rt for 4.5 h. Then the solution was filtered
through a large pad of Celite and washed with saturated aq NaHCO and brine, dried over
3
Na SO , filtered, and concd in vacuo. The residue was purified by flash column chromatography
2
4
(
hexane/EtOAc 1:1) to provide 9 as a light yellow wax (1.78 g, 74% over 2 steps). R 0.58
f
1
(
EtOAc). []25 ‒23.4 (c 1.0, CHCl ). H NMR (400 MHz, CDCl )  7.48‒7.46 (m, 2H),
D
3
3
7
.33‒7.29 (m, 3H), 5.02 (t, J = 8.4 Hz, 1H, H3), 4.94 (t, J = 8.7 Hz, 1H, H2), 4.73 (d, J = 8.8
1,2
Hz, 1H, H1), 4.19 (dd, J5eq,5ax = 11.7 Hz, J5eq,4 = 5.1 Hz, 1H, H5 ), 3.84‒3.79 (m, 1H, H4), 3.39
eq
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(
dd, J5ax,5eq = 11.7 Hz, J5ax,4 = 9.5 Hz, 1H, H5 ), 3.16 (bs, 1H, OH), 2.89‒2.51 (m, 8H), 2.17 (2
ax
1
3
1
× s, 6H). C{ H} NMR (101 MHz, CDCl )  208.0, 206.4, 173.0, 171.4, 132.7, 132.6 (×2),
3
1
2
29.1 (×2), 128.2, 86.8 (C1), 76.5 (C3), 69.7 (C2), 68.6 (C5), 68.5 (C4), 38.4, 37.9, 29.9, 29.9,
0
1
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0
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0
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
8.3, 28.1. HRMS: m/z calcd for C H O SNa [M + Na] 461.1241, found 461.1242.
2
1
26
8
Phenyl 2-O-benzoyl-4-O-(p-methoxy)benzyl-1-thio-β-D-xylopyranoside (10). To a vigorously
stirred solution of diol 8 (1.0 g, 2.76 mmol), tetrabutylammonium hydrogen sulfate (0.187 g,
0
.55 mmol) and BzCl (0.43 mL, 3.72 mmol) in CH Cl (50 mL) at ‒5 °C was added a 1 M aq
2 2
NaOH solution (7.0 mL, 7.0 mmol). The mixture was stirred for 30 min (TLC indicated traces
of 8 and the dibenzoylated product). The organic layer was separated, washed with H O, dried
2
over Na SO , filtered, and concd in vacuo. The residue was purified by flash column
2
4
chromatography (hexane/EtOAc 8.5:1.5) affording compound 10 as white crystals (0.877 g,
6
8%). R 0.35 (hexane/EtOAc 3:1). Mp 101.7‒102.4 °C (EtOH). []25D ‒13.2 (c 1.0, CHCl3).
f
1
H NMR (400 MHz, CDCl )  8.09‒8.07 (m, 2H), 7.61‒7.57 (m, 1H), 7.48‒7.41 (m, 4H),
3
7
.29‒7.24 (m, 5H), 6.89‒6.86 (m, 2H), 5.04 (dd, J = 9.5 Hz, J = 9.0 Hz, 1H, H2), 4.78 (d,
1,2 2,3
J = 9.6 Hz, 1H, H1), 4.65 (d, J = 11.5 Hz, 1H, PMB), 4.60 (d, Jgem = 11.5 Hz, 1H, PMB),
1
,2
gem
4
.10 (dd, J5eq,5ax = 11.5 Hz, J5eq,4 = 5.1 Hz, 1H, H5 ), 3.85 (t, J = 8.8 Hz, 1H, H3), 3.81 (s, 3H,
eq
OMe), 3.61‒3.55 (m, 1H, H4), 3.30 (dd, J5ax,5eq = 11.5 Hz, J5ax,4 = 10.1 Hz, 1H, H5 ), 2.62 (bs,
ax
1
3
1
1
H, OH). C{ H} NMR (101 MHz, CDCl )  166.1, 159.7, 133.5, 132.8 (×2), 132.7, 130.1
3
(×2), 130.1, 129.8, 129.7 (×2), 129.1 (×2), 128.6 (×2), 128.2, 114.2 (×2), 86.8 (C1), 77.4 (C4),
76.1 (C3), 73.1 (PMB), 73.0 (C2), 67.6 (C5), 55.4 (OMe). HRMS: m/z calcd for C H O SNa
2
6
26
6
+
[M + Na] 489.1342, found 489.1343.
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Phenyl 2-O-benzoyl-3-O-levulinoyl-1-thio--D-xylopyranoside (11). To a solution of 10 (3.94 g,
8.45 mmol) and 4-oxopentanoic acid (1.22 g, 10.5 mmol) in CH Cl (14 mL) cooled to 0 °C was
2
2
added DMAP (0.104 g, 0.85 mmol) and DCC (2.61 g, 12.6 mmol). The solution was left to stir
at rt for 3.5 h, then filtered and concd in vacuo. The residue was dissolved in CH Cl (60 mL)
1
1
1
1
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1
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0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
and H O (6.0 mL) and stirred vigorously. DDQ (2.88 g, 12.7 mmol) was added and the mixture
2
was left to stir at rt overnight in the dark. Then the solution was filtered through a large pad of
Celite and washed with saturated aq NaHCO and brine, dried over Na SO , filtered, and concd
3
2
4
in vacuo. The residue was purified by flash column chromatography (hexane/EtOAc 2:1)
affording 11 as a white crystalline solid (2.935 g, 78% over 2 steps). R 0.28 (hexane/EtOAc
f
1
1
8
:1). Mp 88.7‒90.3 °C (EtOH). []25 ‒1.8 (c 1.0, CHCl ). H NMR (400 MHz, CDCl ) 
D
3
3
.03‒8.01 (m, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.47‒7.44 (m, 4H), 7.29‒7.28 (m, 3H), 5.23‒5.16
(m, 2H, H2, H3), 4.94‒4.91 (m, 1H, H1), 4.29 (dd, J5eq,5ax = 11.8 Hz, J5eq,4 = 4.9 Hz, 1H, H5eq),
.94‒3.89 (m, 1H, H4), 3.49 (dd, J5ax,5eq = 11.7 Hz, J5ax,4 = 9.1 Hz, 1H, H5 ), 3.14 (bs, 1H,
3
ax
1
3
1
OH), 2.81‒2.65 (m, 2H), 2.58‒2.51 (m, 1H), 2.44‒2.37 (m, 1H), 2.11 (s, 3H). C{ H} NMR
(101 MHz, CDCl )  207.7, 172.8, 165.3, 133.6, 132.9, 132.7 (×2), 130.0 (×2), 129.4, 129.1
3
(×2), 128.7 (×2), 128.2, 87.0 (C1), 76.4 (C3), 70.3 (C2), 68.5 (C4), 68.3 (C5), 38.5, 29.8, 28.3.
+
HRMS: m/z calcd for C H O SNa [M + Na] 467.1135, found 467.1136.
23 24
7
Temperature Screening (General Procedure A). Crushed mol sieves (3Å, 1.0 g) were added a
-neck Schlenk flask, where the middle neck was fitted with a glass stopper and the other with a
2
septum. The flask was placed under vacuum, heated with a heatgun and then subjected to an atm
of N and cooled to rt. Donor 5 (0.200 g, 0.36 mmol) dissolved in dry CH Cl (3.0 mL) was
2
2
2
added to the flask together with AgOTf (0.185 g, 0.72 mmol) dissolved in dry toluene (2.0 mL).
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Stirring of the reaction was initiated and the glass stopper was exchanged for a thermometer,
and the solution was cooled to ‒65 °C. pNO PhSCl (0.068 g, 0.36 mmol) was dissolved in dry
2
CH Cl (0.5 mL) and slowly added followed by stirring for an additional 10 min. Acceptor 6
2
2
0
1
2
3
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8
9
0
(
0.146 g, 0.33 mmol) was dissolved in dry CH Cl (0.5 mL) and added quickly. The temperature
2 2
was raised as indicated in Figure 1, where the reaction was allowed to warm to ‒15 °C at which
point Et N (0.15 mL, 1.08 mmol) was added. The solution was filtered through a pad of Celite
3
and concd in vacuo. The residue was purified by flash column chromatography to afford the
product 12.
Phenyl 2,3,4-tri-O-benzoyl--D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-1-thio--D-
xylopyranoside (12). To a Schlenk flask containing 3Å mol sieves (1.20 g) was added donor 5
(0.200 g, 0.36 mmol) dissolved in dry CH Cl (6.0 mL) and AgOTf (0.185 g, 0.72 mmol)
2
2
dissolved in dry Et O (5.0 mL). The mixture was stirred for 1 h at rt and then cooled to ‒78 °C
2
at which point PhSCl (0.062 g, 0.43 mmol) or pNO PhSCl (0.082 g, 0.43 mmol) dissolved in
2
dry CH Cl (1.0 mL) was added dropwise followed by stirring for 10 min. Acceptor 6 (0.146 g,
2
2
0
.33 mmol) dissolved in dry CH Cl (0.5 mL) was then added slowly. The reaction was stirred
2 2
at ‒78 °C for 15 min, then for 15 min at rt after which Et N (0.21 mL, 2.88 mmol) was added.
3
The mixture was filtered through a pad of Celite, concd in vacuo, and purified by flash column
chromatography (pentane/EtOAc 3:2) to give 12 as a white amorphous solid (0.186 g, 64% with
PhSCl and 0.229 g, 79% with pNO PhSCl). Alternatively, general procedure A was employed
2
2
5
1
to afford 0.260 g (90%) of 12. R 0.18 (heptane/EtOAc 7:3). []
‒22 (c 1.0, CHCl ). H NMR
3
f
D
(
400 MHz, CDCl )  8.02‒7.92 (m, 10H), 7.56‒7.50 (m, 5H), 7.45‒7.27 (m, 15H), 5.69‒5.62
3
A
B
A
(
m, 2H, H3 , H3 ), 5.34 (t, J = 8.0 Hz, 1H, H2 ), 5.24 (dd, J
= 6.5 Hz, J1B,2B = 5.0 Hz, 1H,
B,3B
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A
B
B
H2 ), 5.06‒5.01 (m, 2H, H1 , H4 ), 4.95 (d, J
= 4.8 Hz, 1H, H1 ), 4.27 (dd, J
=
1
B,2B
5Aeq,5Aax
A
A
1
2.2 Hz, J5Aeq,4A = 4.7 Hz, 1H, H5 ), 4.09 (td, J = 8.3 Hz, J
= 4.9 Hz, 2H, H4 ), 4.03
eq
5Aeq,4A
B
(dd, J5Beq,5Bax = 12.4 Hz, J5Beq,4B = 3.9 Hz, 1H, H5 ), 3.55 (dd, J
= 12.1 Hz, J5Aax,4A =
eq
5Aax,5Aeq
1
1
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1
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1
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0
1
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4
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7
8
9
0
1
2
3
4
5
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9
0
1
2
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4
5
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8
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0
1
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9
0
A
B
13
1
8
.6 Hz, 1H, H5 ), 3.43 (dd, J
= 12.4 Hz, J5Bax,4B = 6.3 Hz, 1H, H5 ). C{ H} NMR
ax
5Bax,5Beq ax
(
101 MHz, CDCl )  165.6, 165.5, 165.4, 165.4, 165.2, 133.5 (×2), 133.5, 133.4 (×2), 132.8,
3
1
1
32.7 (×2), 130.1 (×2), 130.0 (×2), 130.0 (×2), 129.9 (×2), 129.9 (×2), 129.5, 129.4, 129.3,
B
A
A
29.2, 129.1 (×2), 129.0, 128.6 (×6), 128.5 (×4), 128.2, 99.8 (C1 ), 86.8 (C1 ), 75.2 (C4 ), 73.1
A
A
B
B
B
A
B
(
C3 ), 70.6 (C2 ), 70.2 (C2 ), 69.7 (C3 ), 68.6 (C4 ), 65.6 (C5 ), 60.9 (C5 ). HRMS: m/z
+
calcd for C H O SNa [M + Na] 917.2238, found 917.2258.
5
1
42 13
Phenyl 2,3,4-tri-O-benzoyl--D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl--D-xylopyranosyl-
1→4)-2,3-di-O-benzoyl-1-thio--D-xylopyranoside (13). To a Schlenk flask containing 3Å mol
sieves (1.20 g) was added donor 5 (0.200 g, 0.36 mmol) dissolved in dry CH Cl (6.0 mL) and
(
2
2
AgOTf (0.185 g, 0.72 mmol) dissolved in dry Et O (5.0 mL). The mixture was stirred for 1 h at
2
rt and then cooled to ‒78 °C at which point PhSCl (0.062 g, 0.43 mmol) or pNO PhSCl (0.082
2
g, 0.43 mmol) dissolved in dry CH Cl (1.0 mL) was added dropwise followed by stirring for 10
2
2
min. Acceptor 6 (0.146 g, 0.33 mmol) dissolved in dry CH Cl (0.5 mL) was then added slowly.
2
2
The reaction was stirred at ‒78 °C for 15 min and then for 15 min at rt. AgOTf (0.093 g, 0.36
mmol) dissolved in dry Et O (2.0 mL) was added and the mixture was cooled to ‒78 °C over 20
2
min. PhSCl (0.058 g, 0.40 mmol) or pNO PhSCl (0.075 g, 0.40 mmol) dissolved in dry CH Cl
2
2
2
(1.0 mL) was added dropwise followed by stirring for 10 min. Acceptor 6 (0.132 g, 0.29 mmol)
dissolved in dry CH Cl (0.4 mL) was added slowly. The reaction was stirred at ‒78 °C for 15
2
2
min, then for 15 min at rt after which Et N (0.4 mL, 5.44 mmol) was added. The mixture was
3
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