Regioselective silyl/acetate exchange of disaccharides yields advanced glycosyl donor and acceptor precursors

Article abstract of DOI:10.1021/jo4013805

Glycoconjugates are composed of carbohydrate building blocks linked together in a multitude of ways giving rise to diverse biological functions. Carbohydrates are especially difficult to synthetically manipulate because of the similar reactivity of their numerous and largely equivalent hydroxyl groups. Hence, methodologies for both the efficient protection and selective modification of carbohydrate alcohols are considered important synthetic tools in organic chemistry. When per-O-TMS protected mono- or disaccharides in a mixture of pyridine and acetic anhydride are treated with acetic acid, regioselective exchange of silicon for acetate protecting groups occurs. Acid concentration, thermal conditions, and microwave assistance mediate the silyl/acetate exchange reaction. Regiocontrol is achieved by limiting the equivalents of acetic acid, and microwave irradiation hastens the process. We coined the term Regioselective Silyl Exchange Technology (ReSET) to describe this process, which essentially sets the protecting groups anew. To demonstrate the scope of the reaction, the conditions were applied to lactose, melibiose, cellobiose, and trehalose. ReSET provided rapid access to a wide range of orthogonally protected disaccharides that would otherwise require multiple synthetic steps to acquire. The resulting bifunctional molecules are poised to serve as modular building blocks for more complex glycoconjugates.

Full text of DOI:10.1021/jo4013805

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Article
Regioselective Silyl/Acetate Exchange of Disaccharides
Yields Advanced Glycosyl Donor and Acceptor Precursors
Hsiao-Wu Hsieh, Matthew W. Schombs, Mark A. Witschi, and Jacquelyn Gervay-Hague
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo4013805 • Publication Date (Web): 27 Aug 2013
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Regioselective Silyl/Acetate Exchange of Disaccharides Yields Advanced
Glycosyl Donor and Acceptor Precursors
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HsiaoꢀWu Hsieh,a,b Matthew W. Schombs,a,b Mark A. Witschia and
Jacquelyn GervayꢀHague*a
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jgervayhague@ucdavis.edu
a Department of Chemistry, University of California at Davis, One Shields Avenue,
Davis, CA 95616, USA
b These authors contributed equally to this work
Abstract
Glycoconjugates are comprised of carbohydrate building blocks linked together
in a multitude of ways giving rise to diverse biological functions. Carbohydrates are
especially difficult to synthetically manipulate due to the similar reactivity of their
numerous and largely equivalent hydroxyl groups. Hence, methodologies for both
the efficient protection and selective modification of carbohydrate alcohols are
considered important synthetic tools in organic chemistry. When perꢀOꢀTMS
protected monoꢀ or disaccharides in a mixture of pyridine and acetic anhydride are
treated with acetic acid, regioselective exchange of silicon for acetate protecting
groups occurs. Acid concentration, thermal conditions and microwave assistance
mediate the silyl/acetate exchange reaction. Regiocontrol is achieved by limiting the
equivalents of acetic acid and microwave irradiation hastens the process. We coined
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the term Regioselective Silyl Exchange Technology (ReSET) to describe this process,
which essentially sets the protecting groups anew. To demonstrate the scope of the
reaction, the conditions were applied to lactose, melibiose, cellobiose and trehalose.
ReSET provided rapid access to a wide range of orthogonally protected disaccharides
that would otherwise require multiple synthetic steps to acquire. The resulting
biꢀfunctional molecules are poised to serve as modular building blocks for more
complex glycoconjugates.
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Introduction
In humans, carbohydrates are important constituents of secreted and cellꢀsurface
glycoproteins, membrane components in the form of glycolipids and gangliosides as
well as various types of extracellular matrix molecules.1 As such, they mediate a
wide range of biological processes from embryonic development to differentiation,
cellꢀcell recognition, signaling, hostꢀpathogen interactions, cancer immunology,
intracellular trafficking and localization.2, 3 The nine common monosaccharides
found in mammalian cells can be linked in an astonishing number of ways, resulting
in much higher complexity than is possible from amino acids or nucleotides. The
development of synthetic methods to generate such complex structures in an efficient
and controlled fashion is a cornerstone of glycobiology and glycochemistry research.1
A major challenge in developing methods for oligosaccharide synthesis is the ability
to differentiate between seemingly equivalent hydroxyl groups in order to achieve
specific chemical linkages. Often, several protection and deprotection sequences are
required, which increases production time and decreases step economy.4 Methods
that streamline the pathway to selectively protected carbohydrate hydroxyl groups are
continually under development and the topic of this report.5
Acetyl protecting groups are among the most versatile and widely used because
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they are easily introduced, robust and can be removed without affecting glycosidic
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linkages.
And because of the numerous methods available for selective
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saponification of the anomeric acetate,6ꢀ9 it can be considered orthogonal to other
acetates. Accordingly, significant resources have been dedicated to research focused
on the selective acetylation of glycosides both enzymatically and chemically.10
Enzymatic acylation11 has enabled the preparation of selectively acetylated
pyranosides,12ꢀ15 furanosides,16 sialosides,17 oligosaccharides18ꢀ20 and natural
glycosides.21, 22 However, enzyme specificity and substrate solubility often limit the
diversity of substrates amenable to modification.23, 24 Chemical methods for the
selective acylation of sugars have also had limited success.10, 25 The main factor
hindering progress in this area can be attributed to the insolubility of free sugars in
organic solvents. Further complicating matters is anomalous behavior resulting from
the hydrogen bonding network present in unprotected carbohydrates.26 In these
instances, alkyl glycosides have proved useful and, not surprisingly, selective
acetylation of the primary hydroxyl at Cꢀ6 can be achieved preferentially.27ꢀ29
However, hydrolysis of the anomeric acetal (acetolysis) typically requires harsh acidic
and thermal conditions.30 Since disaccharides can also be readily hydrolysed under
these conditions, this strategy is largely limited to monosaccharides.31, 32
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As opposed to using either alkyl glycosides or unprotected sugars, we recently
reported a complimentary protocol allowing for the direct exchange of trimethylsilyl
ethers with acetyl protecting groups (Scheme 1).33 The methodology begins with a
free monosaccharide, which is perꢀOꢀsilylated to confer maximal solubility in organic
solvent. Silylation can be carried out on multiꢀgram quantities in nearly quantitative
yield. Subsequent treatment of the perꢀOꢀsilylated sugar with acetic acid in a
pyridine acetic anhydride mixture results in regioselective silyl acetate exchange
giving rise to selectively acetylated monosaccharides. We refer to this process by
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the acronym ReSET (Regioselective Silyl Exchange Technology) because silyl
protecting groups are being set anew to acetates during the reaction. As illustrated in
Scheme 1, when ReSET was applied to galactose four uniquely protected analogs
were prepared via a single transformation.33 Careful monitoring of the reaction
revealed that the Cꢀ6 trimethylsilyl group was the first to exchange followed by the
anomeric silyl acetal. This order of reactivity held true for all the monosaccharides
studied. 33 Upon increasing the concentration of acetic acid from 2 equiv. to 4 equiv.,
the secondary trimethyl silyl groups began to exchange and the order and rate of
exchange varied depending upon the structure of the monosaccahride. In the case of
galactose, the Cꢀ2 silyl protecting group was more labile than the Cꢀ3, which in turn
was more reactive than the Cꢀ4 position.
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Scheme 1. Selective acetylation of perꢀOꢀtrimethylsilyl galactose
The orthogonally protected compounds generated by ReSET can be readily
converted to a wide range of glycosyl donors through activation of the anomeric
acetate or silyl ether. Alternatively, protodesilylation of the silicon protecting groups
reveals selectively acetylated glycosyl acceptors (Scheme 2). In this manner, these
biꢀfunctional intermediates are poised to serve as modular building blocks en route to
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more complex glycoconjugates.
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OAc
O
1. Dowex H+ resin
OR
HO
AcO
A glycosyl acceptor
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OR
TMSO
AcO
OAc
O
1. Anomeric activation
O
TMSO
AcO
OAc
2. Acceptor
OH
or
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AcO
O
O
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TMSO
AcO
AcO
Scheme 2. Concept of a selectively acetylated bifunctional intermediate
As part of a program targeting the synthesis of complex oligosaccharides, we
sought to extend ReSET to disaccharides. We were especially interested in lactose,
since a similar reactivity profile to galactose could potentially provide highly
diversified building blocks in a single step. One might expect that disaccharides
would be bound by the reactivity of the monosaccharides from which they are
constructed, however it is not uncommon for disaccharides varying only in
connectivity to exhibit remarkably different behaviors. Moreover, unprotected
disaccharides are far less soluble in organic solvents than the monosaccharides from
which they are composed. Further complicating matters is the susceptibility of the
internal glycosidic linkages to hydrolysis under a variety of conditions. For example,
Bhat et al. observed rapid cleavage of the internal glycosidic linkages of
perꢀOꢀTMSꢀmelibiose, perꢀOꢀTMSꢀcellobiose and perꢀOꢀTMSꢀlactose upon addition
of TMSI, even at 0 °C.34 In fact microwave conditions, similar to those used for
ReSET, have been employed for the degradation of oligosaccharides and
polysaccharides.35ꢀ38
Collectively, these challenges made disaccharides ideal
substrates for developing the technology.
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We reasoned that in order to apply the silyl exchange reaction to disaccharides,
conditions would need to be identified under which acetate exchange occured more
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rapidly than glycoside cleavage. It had been shown that the cleavage process can be
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attenuated through careful selection of protecting groups.
Indeed, a significant
decrease in the rate of internal glycosidic bond scission has been observed upon
replacement of electron donating protecting groups (such as TMS) with electron
withdrawing acetates. Herein, is described the results of extending ReSET to both
reducing and nonreducing disaccharides including the biologically relevant lactose,
melibiose, cellobiose and trehalose substrates.
Results and Discussion
Lactose (Dꢀgalpꢀβ (1 → 4)ꢀDꢀglcp) is a key component of several tumor
associated carbohydrate antigens (TACA).42ꢀ46 Thus, it is not surprising that this
important structural motif has been incorporated into several carbohydrate based
cancer vaccines.47ꢀ49 Moreover, lactosides have been extensively used to target and
image asialoglycoprotein receptors, which are overexpressed on cancer cells.50 In
2000, Bruehl and Bertozzi synthesized a series of monoꢀ and disulfated lactose
derivatives in order to determine the optimal configuration for L
ꢀselectin binding.51, 52
Structurally defined multivalent lactoseꢀcontaining clusters have also been designed
for optimal galectin binding.48, 53 As such, there is a need for ready access to
differentially protected lactosides to further investigate these biological processes.
Our studies began with the preparation of perꢀOꢀTMS lactose, which was
accomplished in one step from commercially available lactose.34 The conditions
developed for the regioselective acetylation of monosaccharides served as the starting
point for the disaccharide system (Table 1). As such, perꢀOꢀsilylated lactose was
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diluted into a mixture of anhydrous pyridine and acetic anhydride followed by
addition of 3 equiv. of glacial acetic acid (Table 1, Entry 1).54 The homogeneous
solution was stirred under argon at rt for 5 d, after which time the solvent was
removed and the mixture was purified using flash column chromatography affording
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the
6,6’ꢀdiꢀOꢀacetylꢀlactoside
(1)
in
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55%
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the
1,6,6’ꢀtriꢀOꢀacetylꢀlactoside (2) and 12% of the 1,2,6,6’ꢀtetraꢀOꢀacetylꢀlactoside (3).
The initial exchange followed the same trend as the monosaccharides in that the
primary hydroxyls reacted first followed by anomeric exchange. The formation of 1
was consistent with a recently reported exchange of a perꢀOꢀTMS protected betaꢀthio
maltoside.55
Table 1. Selective acetylation of perꢀOꢀTMS lactose
It is worthy of note that the results shown in Table 1 constitute the first example
of a silyl exchange reaction on a disaccharide bearing an anomeric silyl acetal.
Moreover, both the silyl acetal (1) and the anomeric acetate (2) can be activated with
trimethylsilyl iodide (TMSI) affording the same glycosyl iodide (2I, Scheme 3).34, 54
The in situ convergence of 1 and 2 to the same glycosyl donor (2I) can be considered
additive. As such, 2I was obtained in high yield (73% from commercially available
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lactose, Table 1, Entry 5).
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Scheme 3. Compounds 1 and 2 converge to the same glycosyl iodide (2I)
Next, a thermal study was conducted in order to probe the effects of temperature
on the reaction rate and selectivity. Similar product mixtures were obtained by
increasing the reaction temperature from rt to 60 °C. However, the increased
temperature was accompanied by a significant decrease in reaction time (Table 1,
Entries 2 and 3). Under thermal conditions, compound 4 containing a single TMS
ether at the 4’ position was also observed. Recognizing the potential of 4 to serve as
a modular building block for many of the more complex tumor associated
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carbohydrate antigens,
we sought to increase its production thermally. After
heating at 80 °C for 1 d, the main products recovered were 4 and perꢀOꢀacetylated
lactose (5, Table 1, Entry 4). Hence, heating the reaction to 80 °C reduced the
reaction time to 1 d and resulted in approximately a fiveꢀfold increase in the yield of 4.
Ready access to compounds 1ꢀ4 opens the door to a wide range of glycosides with
minimal synthetic manipulation. Importantly, all compounds were isolated using
column chromatography illustrating the enhanced stability conferred upon the
remaining TMS ethers by the newly installed acetates. All structures were
unambiguously characterized, with key assignments being identified via NMR (1H,
13C, DEPT, HSQC, HMBC and COSY experiments; see Supporting Information).
The previously results from the microwave studies on silylated monosaccharides
suggested a similar strategy with disaccharides may serve to further increase the
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reaction rate.33 At the same time, we were mindful of the susceptibility of the
glycosidic linkage. Accordingly, perꢀOꢀTMS lactose was irradiated at 125 °C for 3
cycles of 25 min (Table 1, Entry 5). After each cycle, the reaction progress was
monitored via thin layer chromatography (TLC). These conditions afforded a
similar product distribution of 1 and 2 as that obtained under thermal conditions,
albeit in a much shorter time. No cleavage or hydrolysis products were observed,
which can be attributed to the rapid incorporation of the Cꢀ6,6’ acetates which confer
stability to the glycosidic linkage. Next, the amount of acetic acid was varied to
determine its effect on the product distribution and reaction time. Increasing the
amount of acetic acid accelerated the reaction and resulted in the exclusive formation
of compounds 4 and 5, (Table 1, Entries 6 and 7). Thus, the orthogonally protected
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species (4) was prepared in 2 steps from commercially available
under 4 h from perꢀOꢀTMS lactose.
Dꢀlactose and in just
In order to reveal the selectively protected lactosyl acceptor (6), compound 4 was
simply treated with Dowex® acidic resin in methanol. After 10 h at rt, TLC showed
complete consumption of the starting material and subsequent formation of the key
intermediate (6) in 90% yield (Scheme 4). Interestingly, the removal of a single
TMS group required stirring with acidic resin in methanol for 10 h. Further
investigation revealed that as the number of acetate protecting groups increased, the
time required to remove the remaining TMS groups also lengthened providing further
evidence of the inductively stabilizing effects of the acetate groups during
protodesilylation. As such, the exchange process not only adds stability to the
glycosidic linkages, but to the remaining silyl ethers as well.
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Scheme 4. Unmasking of acceptor 6 upon acidic hydrolysis of the TMS ether
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TMS groups are often used as transient protecting groups due to their labile
nature.59 When stability is a concern and the orthogonality associated with the use
of silyl protection strategies is required, bulky silyl protecting groups such as
tertꢀbutyldimethylsilyl (TBDMS) or tertꢀbutyldiphenylsilyl (TBDPS) are frequently
employed. However, conformational changes associated with the steric bulk of these
groups are often observed when incorporated into carbohydrates.60 The technology
disclosed here allows for the extended use of TMS protecting groups in multistep
sequences, thereby providing access to silicon bearing carbohydrates without
affecting sugar conformation. As far as we are aware, this is the first reported
synthesis of compounds 1ꢀ4. Moreover, perꢀOꢀTMS lactose could be regenerated
via a salvage pathway. Simple deacetylation and subsequent reꢀsilylation allows any
unwanted derivatives to be recycled in a nearly quantitative manner (Scheme 5).
Scheme 5. Substrate recycling
Having established the optimal conditions for ReSET of perꢀOꢀTMS lactose, we
next explored the effects of shifting the internal glycosidic linkage from the 4ꢀposition
to the 6ꢀposition. Melibiose (Dꢀgalpꢀα(1→6)ꢀDꢀglcp) is composed of the same
monomeric units as lactose, however in melibiose the glycosidic linkage is
α(1→6)
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to glucose; so unlike lactose, there is only one Cꢀ6 hydroxyl, which is one sugar
removed from the anomeric center. There are several natural product based
glycolipids containing a macrolactone linkage between the anomeric position of the
glucosyl moiety and the Cꢀ6’ position of the galactosyl residue. These compounds
have been isolated from plants and yeast and are of growing commercial interest as
biodegradable emulsifiers.61, 62 The application of ReSET to melibiose not only
provides the opportunity to investigate the reactivity differences between
constitutional isomers, but also grants access to a series of orthogonally protected
derivatives that can be useful for structure activity relationship studies (Table 2).
Table 2. Selective acetylation of perꢀOꢀTMS melibiose
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As described earlier, perꢀOꢀTMS melibiose was prepared in one step from
commercially available melibiose.34 After coꢀevaporation with anhydrous benzene,
perꢀOꢀTMS melibiose was subjected to the optimized selective acetylation conditions
starting with 2.0 equiv. of glacial acetic acid. The reaction mixture was irradiated at
125 °C for 6 x 25 min cycles. Once TLC revealed the starting material had been
consumed, the solvent was removed and the residue was purified using flash column
chromatography. The reaction afforded a mixture containing the 6’ꢀmonoꢀOꢀacetate
(7) in 39% yield and 1,6’ꢀdiꢀOꢀacetate (8) in 20% yield as a mixture of α/β anomers
(Table 3, Entry 1). This result closely mirrors the lactose case, in that the first
groups to exchange are the primary and anomeric silyl ethers.
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Next, access to melibiose derivatives with a higher degree of exchange was
explored. Based on the results of lactose, further exchange can be achieved either
through prolonged microwave irradiation or by increasing the concentration of acetic
acid. As microwave irradiation has been used to degrade oligosaccharides, we opted
to increase the acetic acid content for our studies (Table 3, Entry 2). Under these
conditions, instead of obtaining the monoꢀ and diꢀOꢀacetylated analogs; the pentaꢀ,
hexaꢀ, heptaꢀ and perꢀOꢀacetylated compounds (9ꢀ12) were isolated in moderate
yields. Each analog was found to exist as a mixture of α/β anomers. It is also
worthy of note that for lactose a mixture containing approximately 10ꢀ15% of
unresolved partially acetylated compounds was always obtained, in addition to the
major products. However, for melibiose the partially acetylated intermediates were
wellꢀresolved via TLC and column chromatography. As such, isolation and
characterization of each compound was possible. Assignment of the structures to the
corresponding TLC spots observed during the course of the reaction led to the
conclusion that acetylation proceeds in a pseudoꢀstepwise fashion. After the primary
and anomeric positions are acetylated, the remaining glucose silyl ethers (2, 3 and 4
positions) appear to exchange nonꢀspecifically, followed somewhat sequentially by
the 2’, 3’ and 4’ positions of the galactosyl moiety. This twoꢀstep process enabled
the first reported synthesis of these selectively acetylated melibiose building blocks
(9ꢀ12) that would otherwise require multiple synthetic steps.
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Based upon the results with lactose and melibiose, which indicated that the rate of
exchange for the glucose silyl ethers is relatively rapid after the primary, C6 and C6’
positions exchange, we predicted that cellobiose might pose challenges in terms of
achieving regioselective control. Cellobiose (Dꢀglcp- β (1 → 4)ꢀDꢀglcp) is the
disaccharide repeat unit of cellulose, which is the most abundant organic material on
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the planet and a major renewable resource. Research efforts have been dedicated to
achieving the selective chemical modification of cellobiose for over twentyꢀfive
years.63 It has been used as a model for the development of carbohydrate based
sensors.64 Cellobiose has also served as the core for synthetic glycolipid based
adjuvants and was found to induce a strong immunogenic response when coupled to
short peptide antigens.65 With applications as a food preservative, conjugation of
cellobiose to the aminopolysaccharide chitosan resulted in higher water solubility at
neutral pH and conferred increased antibacterial activity against E. coli.66
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With all of its hydroxyls in an equatorial orientation, cellobiose was deemed an
interesting substrate for the ReSET reaction. Subsequent studies revealed that as
with the other reducing sugars evaluated, selectivity was achieved for the primary and
anomeric positions of cellobiose yielding compounds 13 and 14 (Table 3, Entries 1
and 2). Beyond this initial exchange, both extended reaction times and higher
concentrations of acetic acid resulted in inseparable mixtures that quickly converged
to perꢀOꢀacetylated cellobiose. Thus, under the current conditions, selectivity
beyond the primary and anomeric positions is not observed for cellobiose.
Table 3. Selective acetylation of perꢀOꢀTMS cellobiose
While all of the substrates described up to this point have been reducing
disaccharides, only melibiose contained an internal αꢀlinkage. The silyl exchange
reaction on melibiose also proceeded in the most stepꢀwise manner. In order to
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determine if there is a correlation between the orientation of the internal glycosidic
linkage and the order of exchange, we turned our attention to a nonꢀreducing
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disaccharide of particular biological relevance. Trehalose (Dꢀglcp-α(1→1)ꢀDꢀglcp)
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contains two glucosyl moieties connected by an α,αꢀ1,1 glycosidic bond. Trehalose
is utilized by plants, bacteria and all invertebrates67,68 for the protection it confers
against oxidative stress, desiccation and freezing.69 Accordingly, trehalose variants are
widely considered potentially powerful therapeutic agents for the treatment of various
diseases involving oxidative stress and chronic inflammation. Analogues of
trehalose have shown potential as antibiotics and fungicides,70 while other derivatives
are able to disrupt cell wall biosynthesis and may act as novel agents in the fight
against Mycobacterium tuberculosis.67 For these reasons, ready access to
nonꢀsymmetrical trehalose analogues is particularly desirable. As such, trehalose
was selected as the capstone example for ReSET, as applied to disaccharides.
Being a nonꢀreducing symmetric disaccharide, we expected product isolation and
characterization to be simplified by elimination of α/β product mixtures. However,
the propensity for glycosidic bond cleavage remained a significant concern.
Beginning with the azeotropic distillation of perꢀOꢀTMS trehalose with anhydrous
benzene, subsequent acetylation was evaluated with 2.0 equiv. of glacial acetic under
microwave conditions (Table 4, Entry 1). The reaction afforded the monoꢀacetylated
(15) and diꢀacetylated (16) compounds after 1.5 h. When the amount of acetic acid
was increased to 4.0 equiv. only 30 mins was required for the complete consumption
of starting material. After purification, monoꢀacetylated (15) and diꢀacetylated (16)
compounds were isolated in 22% and 56% yields respectively (Table 4, Entry 2). In
order to access a higher degree of acetylation, the acetic acid was increased to 8 equiv.
under the same microwave conditions and after 1.5 h, the reaction afforded a product
distribution of pentaꢀacetate 17 in 14% yield, hexaꢀacetate 18 in 9% yield, and
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perꢀacetate 19 in 44% yield (Table 4, Entry 2). Interestingly, NMR analysis revealed
that pentaꢀacetate 17 and hexaꢀacetate 18 were intermediates in which one glucose
ring had fully exchanged (acetylated). A similar proximal activation was observed
with melibiose suggesting that exchange of one secondary TMS group activates
neighboring ethers towards exchange; but further studies are needed to understand the
underlying stereoelectronic effects of neighboring activation. Compound 18 has two
secondary silyl ethers remaining (2’ꢀ and 3’ꢀ positions) indicating that the 4’ꢀ position
is slightly more reactive than the 2’ꢀ and 3’ꢀ positions, a fact that may be of use for
further regioselective reactions. Most importantly, ReSET offers an efficient
methodology for breaking the symmetry of trehalose as evidenced by the formation of
15, 17 and 18.
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Table 4. Selective acetylation of perꢀOꢀTMS trehalose
As a basis for expanding ReSET in the future, we are interested in
understanding the mechanism of this reaction. Two important experimental
outcomes emerged from the studies thus far: silyl exchange is facilitated by
proximal acetate groups, while at the same time protodesilylation becomes more
difficult with increasing numbers of acetate protecting groups. To help explain these
results we looked to the recent work of Wu and coꢀworkers. In 2011, they published
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a proposed mechanism for the direct esterification of TBDMS ethers under strongly
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acidic conditions.71
Inspired by this report, we propose a mechanism for
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regioselective silyl exchange (Figure 1). The reaction first involves the formation of
pyridiniumꢀacetate salt upon addition of a stoichiometric amount of AcOH to a
mixture containing Ac2O and pyridine. Recall that AcOH plays a crucial role in the
reaction sequence and is necessary for the exchange to proceed. Once a perꢀOꢀTMS
protected disaccharide is introduced, the acetate ion is poised to attack silicon leading
to SiꢀO bond cleavage and subsequent acetylation. Which silicon is attacked is
likely a consequence of both its steric and electronic environments. Studies indicate
thus far that the primary hydroxyls are always the first to exchange suggesting that
sterics are a primary factor. A similar selectivity was recently reported for the
stepꢀwise selective desilylation of the primary TMS ethers on a panel of
monosaccharides and subsequent acetylation of the resulting free hydroxyls.72, 73
Among the remaining TMS ethers, the anomeric position would be most stabilized
upon removal of the silicon group putting it next in line for exchange. Incorporation
of the acetate at the anomeric center places an inductively withdrawing group in close
proximity to the Cꢀ2 position making this trimethylsilyl group most susceptible; and
for the most part the trend continues around the ring i.e. Cꢀ3 is next to exchange and
so on. Although at times sterics may interfere. For example, in the formation of 3
there is no discrimination between the Cꢀ3 position and the Cꢀ2’ and Cꢀ3’ positions.
The data for both melibiose and trehalose follow this trend quite well. For trehalose,
one entire ring is fully acetylated before any of the secondary TMS ethers are
exchanged on the second ring. In general, the secondary OTMS groups in a glucosyl
moiety are less selective than the secondary OTMS groups in a galactosyl moiety,
presumably due to steric differences between axial and equatorial substituents.
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O
-O
Me
O
Me
Si
Me
Me
Si
O
H
N
AcO
Me
Me
O
O
Me
Me
AcOH (2-8 equiv)
Me
pyridine
per-OTMS-Lactose
AcO
N
+
Me
N
O
O
Me
O
TMSO
O
O
TMSO
O
OTMS
OTMS
TMSO
TMSO
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Acetate attacks most accessible Si atom
O
OAc
TMSO
TMSO
OAc
O
Me
N
O
OAc
O
O
O
TMSO
O
CH3
O
TMSO
TMSO
OAc
O
TMSO
-O
Me
Me
Si
O
O
O
O
TMSO
O
TMSO
OTMS
N
Me
Me
Me
Me
Si
-O
Me
Me
Me
Acetate attacks most electrophilic Si atom
Inductive effect of neighboring acetates
increases Si electrophilicity
Figure 1. Plausible mechanism of regioselective silyl/acetate exchange
An alternative mechanism worthy of consideration could involve pyridinium
acetate catalysing protodesilylation giving selectively deprotected alcohols, which
then react with acetic anyhydride. To test this possibility, the ReSET reaction was
performed in the absence of acetic anyhydride. Under these conditions, the reaction
was orders of magnitude slower and selectivity was not observed. This finding lends
support to the hypothesis that the acylpyridinium rather than pyridinium acetate is
attacked by the TMS ether oxygen and also demonstrates the need to incorporate
acetate protecting groups to confer selectivity.
On the matter of protodesilylation, the mechanism begins with protonation of the
silyl ether oxygen atom (Figure 2). An increased number of acetate groups around
the sugar ring would concommitently decrease the basicity of the ether oxygen atoms
thus slowing the reaction. The basicity of acetal oxygen would be similarly affected
which explains the increased stability of the glycosidic linkage with incorporation of
acetate protecting groups. The addition of a single acetate group confers remarkable
stability as observed in the formation of 15.
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Me
Si
Me
Me
Me
HOMe
O
H+
Me
Me
O
Si
H
5
OAc
O
O
OAc
O
OAc
O
OAc
O
6
7
8
9
6
AcO
O
AcO
OAc
AcO
OAc
AcO
AcO
AcO
AcO
AcO
4
Electron withdrawing acetates decrease basicity of ether and acetal oxygen lone pairs
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Figure 2. Protodesilylation of TMS protecting group by acidic resin
Conclusion
ReSET provides a robust platform for the synthesis of orthogonally protected
monoꢀ and disaccharides in a rapid and efficient manner. PerꢀOꢀTMS lactose was
selected as the model system for the systematic evaluation of the effects of acetic acid
equivalents as well as identification of optimal thermal and microwave conditions.
Acetic acid was found to be an essential component of the reaction mixture, resulting
in increased exchange rates and the formation of more highly substituted derivatives.
A mechanism for the silyl/acetate exchange reaction is also proposed. The
remaining TMS ethers appended to the reaction products were found to be remarkably
stabile. As such, ReSET allows these TMS groups to be used in multiꢀstep reaction
sequences, which would otherwise preclude the use of transient TMS groups. This
stability also allows for their extended storage and subsequent unmasking without
concern for transꢀacetylation or transꢀsilylation. These orthogonally protected
advanced intermediates are poised for selective deprotection and subsequent
conversion to a wide range of biologically relevant modifications including sulfation
and phosphorylation. The products generated by this technology can be readily
converted to a wide range of glycosyl donors. Alternatively, selective deprotection
reveals their potential as glycosyl acceptors. In this way, these biꢀfunctional
intermediates are groomed to serve as modular building blocks en route to more
complex glycoconjugates. ReSET also allows one to tune the reactivity of the
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resulting glycosides based on the degree of silyl/acetate exchange.74 The optimal
conditions established for lactose translated seamlessly to other biologically relevant
disaccharides such as melibiose, cellobiose and trehalose. Importantly, when
applied to trehalose ReSET led to facile desymmetrizaton. This process not only
occurs with respect to the primary hydroxyls, but also by virtue of the fact that one
ring becomes completely acetylated before the secondary hydroxyls of the other ring
exchange. In addition to ReSET being timeꢀ and stepꢀeconomical, high value
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starting materials can be easily recycled in a nearly quantitative manner.
A
motivation of this work is the application of these selectively acetylated disaccharides
to the assembly and functionalization of a wide variety of complex glycoconjugates.
As such, applications to the synthesis of biologically relevant complex glycosides are
onꢀgoing and will be reported in due course.
Experimental Section
General procedure for regioselective silyl exchange of perꢀOꢀTMS disaccharides
(Table 1 ꢀ Table 4): In a 10 mL microwave reactor vessel or 25 mL round bottom
flask, perꢀOꢀTMS disaccharide (lactose, melibiose, cellobiose or trehalose) (500 mg,
0.54 mmol or 230 mg, 0.25 mmol) was dissolved in anhydrous benzene (3 mL). The
solvent was removed under rotary evaporation with argon backfilling. The
azeotropic distillation was repeated two additional times to dryness affording viscous
syrup. To the reaction flask was added a dry stirring bar, anhydrous pyridine (2.0
mL/per mmol TMS sugar), acetic anhydride (1.5 mL/per mmol TMS sugar), and
glacial acetic acid (2ꢀ8 equiv.). The reaction vessel was either placed under room
temperature or was heated by conventional oil bath or was subjected to microwave
irradiation (standard mode, 100 W, 125 °C or 110 °C, ~40 psi) for various time
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intervals. Once TLC showed the reaction was complete, the reaction mixture was
transferred into a 50 mL round bottom flask where it was azeotroped with copious
amounts of anhydrous benzene to dryness. The crude foam was immediately
purified by flash column chromatography (hexanes/ethyl acetate/5% triethhylamine)
to afford the partially acetylated products.
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General procedure for perꢀOTMSꢀsubstrare recycling (Scheme 5): Compund 5
(1.00g, 1.46 mmol) was dissolved in dry MeOH (5 mL) and treated with wt 25%
NaOMe in MeOH solution (0.3 mL) at 0 ˚C ice bath. After 10 min, H2O (~3 mL)
was added to dissolve the forming white precipitate and continued stirring at rt.
After 1 h, the reaction mixture was added Dowex H+ resin until pH 7, filtered and
concentrated under reduced pressure. The resulting residue was then dissolved in
dry pyridine (6 mL), follow by addition of TMSCl (1.8 mL, 14.1 mmol) and catalytic
amount of DMAP (18 mg, 0.15 mmol). After 5 h, the reaction mixture was diluted
with EA/Hex (v/v=1/1) solution and washed with water and brine. The oganic layer
was dried over anhydrous Na2SO4 and concertated under reduced pressure to afford
perꢀOTMSꢀlactose as (1.25 g, 92%) a light yellow oil.
(6ꢀOꢀAcetylꢀ2,3,4ꢀtriꢀOꢀtrimethylsilylꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(6ꢀOꢀacetylꢀ
1,2,3ꢀtriꢀOꢀtrimethylsilyl)ꢀ ꢀglucopyranoside (1): Following the general procedure
D
described above, perꢀOꢀTMS lactose (500 mg, 0.54 mmol) was acetylated using
Ac2O/pyridine and glacial acetic acid (93 ꢀL, 1.62 mmol, 3.0 equiv.). The reaction
mixture was stirred under rt for 5d until the starting material was completely
consumed. The reaction mixture was purified using flash column chromatography
(EA/Hex/NEt3 = 5:85:10 – 14:85:1 – 29:70:1) to afford compounds 1, 2 and 3 (Table
1, Entry 1). Diꢀacetylated compound 1 (94 mg, 19%) was obtained as a white
amorphous foam. TLC (EA/Hex = 30:70) Rf 0.70. 1H NMR (800 MHz, CDCl3) δ
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4.99 (d, J = 3.1 Hz, 1H, Hꢀ1), 4.37 – 4.33 (m, 2H, Hꢀ6ab), 4.24 (dd, J = 5.8, 10.7 Hz,
1H, Hꢀ6’a), 4.08 (dd, J = 7.8, 10.7 Hz, 1H, Hꢀ6’b), 4.04 (d, J = 7.5 Hz, 1H, Hꢀ1’),
4.02 – 3.97 (m, 1H, Hꢀ5), 3.74 (t, J = 8.8 Hz, 1H, Hꢀ3), 3.71 (d, J = 2.3 Hz, 1H, Hꢀ4’),
3.63 (app t, J = 7.5, 9.2 Hz, 1H, Hꢀ2’), 3.50 – 3.45 (m, 2H, Hꢀ4, Hꢀ5’), 3.41 (dd, J =
3.2, 9.0 Hz, 1H, Hꢀ2), 3.32 (dd, J = 2.5, 9.2 Hz, 1H, Hꢀ3’), 2.09 (s, 3H), 2.05 (s, 3H),
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0.13 (m, 54H). 13C NMR (200 MHz, CDCl3)
δ 170.90, 170.62, 103.0, 93.8, 77.0,
75.1, 74.0, 72.3, 71.93, 71.79, 71.51, 68.9, 63.28, 62.73, 21.1, 20.95, 0.91, 0.87, 0.60,
0.53, 0.49, 0.16. ESIꢀHRMS (m/z) calcd. for C34H74O13Si6 [M+NH4]+: 876.4089;
found: 876.4090.
(6ꢀOꢀAcetylꢀ2,3,4ꢀtriꢀOꢀtrimethylsilylꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(1,6ꢀdiꢀOꢀac
etylꢀ2,3ꢀdiꢀOꢀtrimethylsilyl)ꢀ ꢀglucopyranoside (2): Compound 2 (228 mg, 55%)
D
was obtained as a white amorphous foam (Table 1, Entry 1; see compound 1 for
reaction and purification condition). Compound 2 can be separated into two single
anomers 2α and 2β by gradient flash column chromatography (EA/Hex/NEt3 = 5:90:5
to EA/Hex = 10:90 – 20:80). 2α: TLC (EA/Hex = 2:8) Rf 0.36. 1H NMR (800 MHz,
CDCl3) δ 6.07 (d, J = 3.7, 1H, Hꢀ1), 4.41 (dd, J = 12.1, 2.0 Hz, 1H, Hꢀ6a), 4.30 (dd, J
= 12.1, 5.0 Hz, 1H, Hꢀ6b), 4.25 (dd, J = 10.7, 5.8 Hz, 1H, Hꢀ6’a), 4.08 (dd, J = 10.7,
7.8 Hz, 1H, Hꢀ6’b), 4.06 (d, J = 7.5 Hz, 1H, Hꢀ1’), 3.86 (ddd, J = 10.1, 4.9, 1.9 Hz,
1H, Hꢀ5), 3.72 (d, J = 2.1 Hz, 1H, Hꢀ4’), 3.69 (t, J = 8.9 Hz, 1H, Hꢀ2’), 3.64 (dd, J =
9.1, 7.5 Hz, 1H, Hꢀ2), 3.60 (dd, J = 9.0, 3.7 Hz, 1H, Hꢀ4), 3.55 (dd, J = 10.1, 8.8 Hz,
1H, Hꢀ5’), 3.49 (dd, J = 7.3, 6.2 Hz, 1H, Hꢀ5’), 3.32 (dd, J = 9.2, 2.5 Hz, 1H, Hꢀ3’),
2.13 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H), 0.15 (s, 9H), 0.13 (s, 9H), 0.11 (s, 9H), 0.103
(s, 9H), 0.102 (s, 9H), 0.10. 13C NMR (200 MHz, CDCl3) δ 170.6, 170.5, 169.4,
102.8, 91.8, 75.9, 74.9, 72.5, 72.0, 71.8, 71.5, 71.2, 71.1, 62.4, 62.3, 20.94, 20.91,
20.8, 0.7, 0.6, 0.4, 0.3, ꢀ0.03. 2β: TLC (EA/Hex = 2:8) Rf 0.32. 1H NMR (800
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MHz, CDCl3) δ 5.42 (d, J = 7.7 Hz, 1H, Hꢀ1), 4.42 (dd, J = 2.0, 11.9 Hz, 1H, Hꢀ6a),
4.29 (dd, J = 5.6, 12.0 Hz, 1H, Hꢀ6b), 4.24 (dd, J = 6.0, 10.8 Hz, 1H, Hꢀ6’a), 4.07 (dd,
J = 7.4, 10.8 Hz, 1H, Hꢀ6’b), 4.03 (d, J = 7.5, 1H, Hꢀ1’), 3.71 (d, J = 2.3 Hz, 1H,
Hꢀ4’), 3.65 (app t, J = 7.5, 9.1 Hz, 1H, Hꢀ2’), 3.62 – 3.57 (m, 1H, Hꢀ5), 3.57 – 3.44
(m, 4H, Hꢀ2, Hꢀ3, Hꢀ4, Hꢀ5’), 3.30 (dd, J = 2.5, 9.2 Hz, 1H, Hꢀ3’), 2.12 (s, 3H), 2.08
(s, 3H), 2.06 (s, 3H), 0.17 – 0.13 (m, 27H), 0.098 (s, 9H), 0.089 (s, 9H). 13C NMR
(200 MHz, CDCl3) δ 170.71, 170.65, 169.4, 103.0, 94.3, 76.16, 76.12, 75.2, 74.23,
74.14, 72.1, 71.51, 71.50, 62.75, 62.68, 21.41, 21.06, 20.94, 1.1, 0.96, 0.94, 0.60, 0.52.
ESIꢀHRMS (m/z) calcd. for C33H68O14Si5 [M+NH4]+: 846.3799; found: 846.3828.
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(6ꢀOꢀAcetylꢀ2,3,4ꢀtriꢀOꢀtrimethylsilylꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(6ꢀOꢀacetylꢀ
2,3ꢀdiꢀOꢀtrimethylsilyl)ꢀαꢀ ꢀglucopyranosyl iodide (2I): In an ovenꢀdried NMR
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tube, compound 1, 2α or 2β (0.04 mmol) was added and dissolved in dry benzeneꢀd6
(0.4 mL). After introducing TMSI (7 ꢀL, 0.048 mmol) to the reaction vessel, the
reaction was kept at 0 ˚C and gradually warmed to 25 ˚C over 4ꢀ5 h. The reaction
was monitored by NMR until its corresponding iodide formed. The iodide is highly
reactive and moisture sensitive. Column chromatography or aqueous workꢀup
should be avoided. After azotroped the iodide with anhydrous benzene (5 mL x 3)
and concentrated under reduced pressure, a light yellow amorphous foam of 2I could
be obtained. In situ 1H NMR (800 MHz, C6D6)
δ 6.69 (d, J = 4.0 Hz, 1H, Hꢀ1), 4.72
– 4.67 (m, 2H, Hꢀ6ab), 4.38 – 4.28 (m, 4H, Hꢀ1’, Hꢀ6’ab, Hꢀ5), 4.05 (t, J = 8.7 Hz,
1H, Hꢀ3), 3.94 (dd, J = 8.9, 10.1 Hz, 1H, Hꢀ4), 3.89 (ddd, J = 10.1, 7.6, 2.5 Hz, 1H,
Hꢀ5’), 3.75 (d, J = 2.4 Hz, 1H, Hꢀ4’), 3.53 (dd, J = 12.1, 5.3 Hz, 1H, Hꢀ2’), 3.36 (dd,
J = 2.6, 9.3 Hz, 1H, Hꢀ3’), 2.56 (dd, J = 3.9, 8.6 Hz, 1H, Hꢀ2), 1.74 (s, 3H), 1.64 (s,
3H), 0.36 (s, 9H), 0.31 (s, 9H), 0.23 (s, 9H), 0.22 (s, 9H), 0.14 (s, 9H), 0.11 (s, 9H),
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0.02 (s, 9H). In situ C NMR (200 MHz, C6D6)
δ 169.4, 169.3, 103.0, 83.6, 76.8,
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75.2, 75.1, 75.0, 72.8, 72.1, 71.7, 71.6, 62.6, 61.5, 22.0, 19.9, 1.7, 0.7, 0.6, 0.4, 0.4,
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acetylꢀ3ꢀOꢀtrimethylsilyl)ꢀ ꢀglucopyranoside (3): Compund 3 (50 mg, 12%) was
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obtained as a white amorphous foam (Table 1, Entry 1; see compound 1 for reaction
and purification condition). TLC (EA/Hex = 3:7) Rf 0.28. 1H NMR (600 MHz,
CDCl3) δ 5.59 (d, J = 8.4 Hz, 1H, Hꢀ1), 4.99 – 4.93 (m, 1H, Hꢀ2), 4.47 (dd, J = 2.0,
12.0 Hz, 1H, Hꢀ6a), 4.28 (dd, J = 5.3, 12.0 Hz, 1H, Hꢀ6b), 4.17 (dd, J = 6.2, 10.9 Hz,
1H, Hꢀ6’a), 4.11 (dd, J = 6.9, 10.9 Hz, 1H, Hꢀ6’b), 4.06 (d, J = 7.5 Hz, 1H, Hꢀ1’),
3.71ꢀ3.67 (m, 3H, Hꢀ3, Hꢀ5, Hꢀ4’), 3.63ꢀ3.60 (m, 2H, Hꢀ2’, Hꢀ4), 3.49 (t, J = 6.5 Hz,
1H, Hꢀ5’), 3.31 (dd, J = 2.5, 9.2 Hz, 1H, Hꢀ3’), 2.09 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H),
2.06 (m, 3H), 0.15 (s, 9H), 0.11 (s, 9H), 0.10 (s, 18H). 13C NMR (150 MHz, CDCl3)
δ 170.69, 170.62, 169.50, 169.42, 102.8, 92.3, 75.68, 75.12, 74.44, 73.76, 72.96,
72.37, 71.70, 71.62, 62.97, 62.37, 21.14, 21.07, 20.99, 20.98, 0.96, 0.62, 0.59, 0.53.
ESIꢀHRMS (m/z) calcd. for C32H62O15Si4 [M+NH4]+: 816.3510; found: 816.3488.
(2,3,6ꢀTriꢀOꢀacetylꢀ4ꢀOꢀtrimethylsilylꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(1,2,3,6ꢀtetr
aꢀOꢀacetyl)ꢀ ꢀglucopyranoside (4): Following the general procedure described
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above, perꢀOꢀTMS lactose (500 mg, 0.54 mmol) was acetylated using Ac2O/pyridine
and glacial acetic acid (216 ꢀL, 3.78 mmol, 7.0 equiv.) followed by nine microwave
irradiation cycles (standard mode, 100 W, 125 °C, ~40 psi, 25 min). The reaction
mixture was purified using flash column chromatography (EA/Hex = 20:80 – 50:50)
to afford compounds 4 and 5 (Table 1, Entry 7). Monoꢀsilylated compound 4 (69
mg, 18%) was obtained as a white amorphous foam. TLC (EA/Hex = 5:5) Rf 0.34.
1H NMR (600 MHz, CDCl3) δ 6.24 (d, J = 3.7 Hz, 2H, Hꢀ1α), 5.66 (d, J = 8.3 Hz, 1H,
Hꢀ1β), 5.45 (app t, J = 9.6, 10.2 Hz, 1H, Hꢀ3α), 5.22 (t, J = 9.2 Hz, 1H, Hꢀ3β),
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5.16ꢀ5.12 (m, 2H, Hꢀ2’α, Hꢀ2’β), 5.03 (dd, J = 8.4, 9.3 Hz, 1H, Hꢀ2β), 4.99 (dd, J =
3.7, 10.3 Hz, 1H, Hꢀ2α), 4.85 – 4.80 (m, 2H, Hꢀ3’α, Hꢀ3’β), 4.47 – 4.41 (m, 4H, Hꢀ1’α,
Hꢀ6α, Hꢀ1’β, , Hꢀ6β), 4.26ꢀ4.21 (m, 2H, Hꢀ6α, Hꢀ6β ), 4.10ꢀ4.07 (m, 2H, Hꢀ6’α, Hꢀ6’β),
4.05 – 3.97 (m, 5H, Hꢀ6’α, Hꢀ4’α, Hꢀ5α, Hꢀ6’β,Hꢀ4’β), 3.83ꢀ3.78 (m, 2H, Hꢀ4α, Hꢀ4β),
3.74 (ddd, J = 2.0, 5.0, 9.9 Hz, 1H, Hꢀ5β), 3.66ꢀ3.63 (m, 2H, Hꢀ5’α, Hꢀ5’β), 2.16 (s,
3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H),
2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.015 (s, 3H), 2.00 (s, 3H), 0.12 (s, 9H), 0.117
(s, 9H). 13C NMR (150 MHz, CDCl3) δ 170.50, 170.48, 170.47, 170.46, 170.32,
170.10, 169.85, 169.72, 169.33, 169.24, 169.09, 169.00, 101.3, 101.1, 91.7, 89.2,
75.58, 75.49, 73.67, 73.53, 73.50, 72.81, 72.34, 72.33, 71.0, 70.7, 69.8, 69.7, 69.64,
69.60, 68.1, 62.00, 61.82, 61.77, 61.71, 21.08, 20.98, 20.90, 20.85, 20.7, 0.32, 0.32.
ESIꢀHRMS (m/z) calcd. for C29H44O18Si [M+NH4]+: 726.2641; found: 726.2639.
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(2,3,4,6ꢀTetraꢀOꢀacetylꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(1,2,3,6ꢀtetraꢀOꢀacetylꢀ)ꢀDꢀ
glucopyranoside (5): Compund 5 (224 mg, 61%) was obtained as a white
amorphous foam (Table 1, Entry 7; see compound 4 for reaction and purification
condition). TLC (EA/Hex = 5:5) Rf 0.18. 1H NMR (600 MHz, CDCl3) δ 6.25 (d, J
= 3.7 Hz, 1H), 5.66 (d, J = 8.3 Hz, 1H), 5.47 (app t, J = 9.6, 10.2 Hz, 1H), 5.38 – 5.31
(m, 2H), 5.24 (t, J = 9.2 Hz, 1H), 5.11 (ddd, J = 8.0, 9.1, 10.3 Hz, 2H), 5.04 (dd, J =
8.3, 9.4 Hz, 1H), 5.00 (dd, J = 3.7, 10.3 Hz, 1H), 4.95 (ddd, J = 3.5, 9.1, 10.4 Hz, 2H),
4.51 – 4.39 (m, 5H), 4.18 – 4.04 (m, 7H), 4.00 (ddd, J = 2.0, 4.1, 10.1 Hz, 1H), 3.87
(app dd, J = 6.6, 12.5 Hz, 3H), 3.85 – 3.78 (m, 2H), 3.75 (ddd, J = 2.0, 4.8, 9.9 Hz,
1H), 2.07 (m, 61H). 13C NMR (150 MHz, CDCl3) δ 170.50, 170.47, 170.45, 170.42,
170.27, 170.26, 170.20, 170.19, 170.06, 169.75, 169.73, 169.70, 169.26, 169.14,
169.06, 168.99, 101.4, 101.1, 91.6, 89.1, 75.94, 75.80, 73.6, 72.7, 71.12, 71.08, 70.85,
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70.61, 69.71, 69.23, 69.09, 66.70, 66.69, 61.85, 61.56, 60.95, 60.89, 21.00, 20.99,
20.82, 20.80, 20.80, 20.78, 20.76, 20.75, 20.65. ESIꢀHRMS (m/z) calcd. for
C28H38O19 [M+NH4]+: 696.2351; found: 696.2346.
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(2,3,6ꢀTriꢀOꢀacetylꢀ4ꢀolꢀβꢀDꢀgalactopyranosyl)ꢀ(1ꢀ4)ꢀOꢀ(1,2,3,6ꢀtetraꢀOꢀacetyl)ꢀDꢀ
glucopyranoside (6): To a MeOH (2 mL) solution of compound 4 (63 mg, 0.09 mmol)
was added Dowex H+ resin (~250 mg). The suspension was allowed to stir at rt until
TLC showed the starting material was completely consumed (12 h). Next, the resin
was removed via filtration and the solution was concentrated to afford a viscous oil.
The crude mixture was immediately purified using flash column chromatography
(EA/Hex = 70:30) to obtain compounds 6 (50 mg, 90%) as a white amorphous foam.
TLC (EA/Hex = 2:1) Rf 0.27. 1H NMR (800 MHz, CDCl3) δ 6.25 (d, J = 3.7 Hz, 1H,
Hꢀ1α), 5.66 (d, J = 8.3 Hz, 1H, Hꢀ1β), 5.44 (t, J = 9.8 Hz, 1H, Hꢀ3α), 5.23 (t, J = 9.2
Hz, 1H, Hꢀ3β), 5.18ꢀ5.14 (m, 2H, Hꢀ2’α, Hꢀ2’β), 5.05 (app t, J = 8.4, 9.0 Hz, 1H, Hꢀ2β),
5.01 (dd, J = 3.7, 10.3 Hz, 1H, Hꢀ2α), 4.92 – 4.84 (m, 2H, Hꢀ3’α, Hꢀ3’β), 4.48 – 4.41
(m, 4H, Hꢀ1’α, Hꢀ6aα, Hꢀ1’β,Hꢀ6aβ), 4.33 – 4.23 (m, 4H, Hꢀ6’abα, Hꢀ6’abβ ), 4.13ꢀ4.11
(m, 2H, Hꢀ6bα, Hꢀ6bβ), 4.01ꢀ3.98 (m, 3H, Hꢀ4’α, Hꢀ5α, Hꢀ4’β,), 3.83ꢀ3.79 (m, 2H,
Hꢀ4α, Hꢀ4β), 3.77ꢀ3.75 (m, 1H, Hꢀ5β), 3.71ꢀ3.69 (m, 2H, Hꢀ5’α, Hꢀ5’β), 2.46 (s, 2H),
2.17 (s, 3H), 2.14 – 1.98 (m, 43H). 13C NMR (200 MHz, CDCl3) δ 170.91, 170.88,
170.49, 170.45, 170.18, 170.07, 169.98, 169.95, 169.68, 169.44, 169.36, 169.08,
169.00, 101.2, 101.0, 91.7, 89.1, 75.9, 75.7, 73.7, 73.42, 73.37, 72.7, 72.23, 72.18,
70.9, 70.6, 69.7, 69.6, 69.5, 66.9, 66.8, 62.03, 62.00, 61.97, 61.7, 21.08, 21.00, 20.98,
20.96, 20.94, 20.90, 20.83, 20.77, 20.74, 20.65. ESIꢀHRMS (m/z) calcd. for
C26H36O18 [M+NH4]+: 654.2245; found: 654.2243.
(2,3,4ꢀTriꢀOꢀtrimethylsilylꢀ6ꢀOꢀacetylꢀαꢀDꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(1,2,3,4ꢀtetr
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aꢀOꢀtrimethylsilyl)ꢀαꢀDꢀglucopyranoside (7): Following the general procedure
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described above, perꢀOꢀTMS melibiose (500 mg, 0.54 mmol) was acetylated using
Ac2O/pyridine and glacial acetic acid (62 ꢀL, 1.08 mmol, 2 equiv.) followed by six
irradiation cycles (standard mode, 100 W, 125 °C, ~40 psi, 25 min). The reaction
mixture was purified using flash column chromatography (EA/Hex = 5:95 – 12:88) to
afford compounds 7 and 8 (Table 2, Entry 1). Monoꢀacetylated analogue 10 (190
mg, 39%) was obtained as a white foam. TLC (EA/Hex = 15:85) Rf 0.59. 1H NMR
(800 MHz, CDCl3) δ 5.07 (d, J = 3.4 Hz, 1H, Hꢀ1’), 4.92 (d, J = 3.0, 1H, Hꢀ1), 4.17 –
4.10 (m, 2H, Hꢀ6’ab), 3.88 – 3.64 (m, 9H, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6ab, Hꢀ2’, Hꢀ3’, Hꢀ4’,
Hꢀ5’), 3.33 (dd, J = 3.1, 9.1 Hz, 1H, Hꢀ2), 2.06 (s, 3H), 0.20 (s, 9H), 0.14 (s, 9H),
0.13 (s, 9H), 0.127 (s, 9H), 0.122 (s, 9H), 0.121 (s, 9H), 0.11 (s, 9H). 13C NMR (200
MHz, CDCl3) δ 170.8, 99.6, 93.8, 74.3, 74.2, 72.9, 72.82, 71.82, 70.6, 69.2, 68.6,
63.7, 21.0, 1.4, 1.1, 0.69, 0.63, 0.53, 0.33, 0.30. ESIꢀHRMS (m/z) calcd. for
C35H80O12Si7 [M+NH4]+: 906.4378; found: 906.4390.
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(2,3,4ꢀTriꢀOꢀtrimethylsilylꢀ6ꢀOꢀacetylꢀαꢀDꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(2,3,4ꢀtriꢀOꢀ
trimethylsilylꢀ1ꢀOꢀacetyl)ꢀ ꢀglucopyranoside (8): Compound 8 (90 mg, 20%) was
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obtained as a white foam (Table 2, Entry 1; see compound 7 for reaction and
purification condition). TLC (EA/Hex = 15:85) Rf 0.40. 1H NMR (800 MHz,
CDCl3) δ 6.02 (d, J = 3.5 Hz, 4H, Hꢀ1α), 5.33 (d, J = 8.0 Hz, 1H, Hꢀ1β), 5.06 (d, J =
3.4 Hz, 1H, Hꢀ1’β), 5.00 (d, J = 3.4 Hz, 4H, Hꢀ1’α), 4.15 – 4.07 (m, 12H, Hꢀ6ab), 3.90
– 3.63 (m, 50H, Hꢀ5, Hꢀ2’, Hꢀ6’, Hꢀ3’, Hꢀ5’, Hꢀ4’, Hꢀ4, Hꢀ3, Hꢀ6’), 3.53 (dd, J = 3.6,
9.0 Hz, 4H, Hꢀ3α), 3.45 (t, J = 8.8 Hz, 1H), 3.42 – 3.39 (m, 1H), 3.36 (d, J = 9.2 Hz,
1H), 2.08 (s, 12H), 2.07 (s, 4H), 2.07 (s, 13H), 2.05 (s, 3H), 0.21 – 0.11 (m, 313H).
13C NMR (200 MHz, CDCl3) δ 170.9, 170.8, 169.7, 169.4, 99.7, 99.6, 94.5, 92.1, 78.1,
77.9, 75.4, 74.9, 74.2, 73.0, 72.9, 72.5, 70.9, 70.6, 70.5, 69.1, 69.1, 68.8, 68.7, 63.8,
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63.7, 62.7, 62.1, 21.3, 21.2, 21.08, 21.06, 1.5, 1.4, 1.10, 1.05, 1.01, 0.7, 0.63, 0.60, 0.4,
0.31, 0.25, 0.2. ESIꢀHRMS (m/z) calcd. for C34H74O13Si6 [M+NH4]+: 876.4089;
found: 876.4105.
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(2,3,4ꢀTriꢀOꢀtrimethylsilylꢀ6ꢀOꢀacetylꢀαꢀ
Dꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(1,2,3,4ꢀtetr
aꢀOꢀacetyl)ꢀ ꢀglucopyranoside (9): Following the general procedure described
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above, perꢀOꢀTMS melibiose (500 mg, 0.54 mmol) was acetylated using
Ac2O/pyridine and glacial acetic acid (216 ꢀL, 3.78 mmol, 7.0 equiv.) followed by six
cycles of microwave irradiation (standard mode, 100 W, 125 °C, ~40 psi, 25 min).
The reaction mixture was purified using flash column chromatography (EA/Hex =
40:60 – 60:40) to afford compounds 9, 10, 11 and 12 (Table 2, Entry 2).
Pentaꢀacetylated analogue 9 (21 mg, 7%) was obtained as a white foam. TLC
(EA/Hex = 1/1) Rf 0.81. 1H NMR (800 MHz, CDCl3) δ 6.27 (d, J = 3.7 Hz, 1H,
Hꢀ1α), 5.64 (d, J = 8.4 Hz, 1H, Hꢀ1β), 5.46 (t, J = 9.8 Hz, 1H, Hꢀ3α), 5.23 (t, J = 9.4
Hz, 1H, Hꢀ3β), 5.16 (td, J = 6.2, 9.8, 2H, Hꢀ4α, Hꢀ4β), 5.09 (app t, J = 8.0, 9.6 Hz, 1H,
Hꢀ2β), 5.03 (dd, J = 3.7, 10.3 Hz, 1H, Hꢀ2α), 4.75 (d, J = 3.2 Hz, 1H, Hꢀ1’β), 4.73 (d, J
= 4.0 Hz, 1H, Hꢀ1’α), 4.13 (dd, J = 5.6, 11.2 Hz, 1H, Hꢀ6’β), 4.15 – 4.08 (m, 3H, Hꢀ6’α,
Hꢀ6’α, Hꢀ5β), 4.05 (dd, J = 7.3, 11.2 Hz, 1H, Hꢀ6’β), 3.91 (app t, J = 6.4 Hz, 1H,
Hꢀ5’β), 3.90 – 3.85 (m, 3H, Hꢀ2’α, Hꢀ5’α, Hꢀ2’β), 3.83 (m, 3H, Hꢀ5α, Hꢀ3’α, Hꢀ3’β),
3.79 (d, J = 1.2 Hz, 2H, Hꢀ4’α, Hꢀ4’β), 3.70 (dd, J = 5.3, 11.6 Hz, 2H, Hꢀ6α, Hꢀ6β),
3.59 (dd, J = 1.7, 11.6 Hz, 1H, Hꢀ6β), 3.52 (dd, J = 1.8, 11.5 Hz, 1H, Hꢀ6α), 2.16 (s,
3H), 2.07 (app d, J = 3.0 Hz, 9H), 2.04 (s, 3H), 2.03 (app dd, J = 3.3, 7.7 Hz, 12H),
2.01 (s, 3H), 0.17 (s, 9H), 0.16 (s, 9H), 0.14 (s, 9H), 0.13 (app d, J = 1.1 Hz, 27H).
13C NMR (200 MHz, CDCl3) δ 170.8, 170.5, 170.4, 169.9, 169.5, 169.4, 169.3, 169.1,
169.0, 99.9, 91.9, 89.1, 74.2, 73.3, 73.1, 72.9, 71.1, 70.5, 70.4, 70.3, 69.5, 69.3, 69.2,
69.0, 68.7, 68.5, 65.8, 65.5, 63.9, 63.7, 21.0, 20.9, 20.8, 20.8, 20.7, 0.72, 0.72, 0.60,
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0.58, 0.43, 0.37. ESIꢀHRMS (m/z) calcd. for C31H56O16Si3 [M+NH4]+: 786.3220;
found: 786.3183.
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(3,4ꢀDiꢀOꢀtrimethylsilylꢀ2,6ꢀdiꢀOꢀacetylꢀαꢀ
Dꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(1,2,3,4ꢀte
traꢀOꢀacetyl)ꢀ ꢀglucopyranoside (10): Compound 10 (52 mg, 26%) was obtained as
D
a white foam (Table 2, Entry 2; see compound 9 for reaction and purification
condition). TLC (EA/Hex = 1/1) Rf 0.70. 1H NMR (800 MHz, CDCl3) δ 6.26 (d, J
= 3.7 Hz, 3H, Hꢀ1α), 5.64 (d, J = 8.3 Hz, 1H, Hꢀ1β), 5.45 (t, J = 9.9 Hz, 3H, Hꢀ3α),
5.22 (t, J = 9.4 Hz, 1H, Hꢀ3β), 5.17 (td, J = 5.0, 9.8 Hz, 4H, Hꢀ4α, Hꢀ4β), 5.11 (d, J =
3.5, 4H, Hꢀ1’α, Hꢀ1’β), 5.05 (app t, J = 8.8, 9.6 Hz, 1H, Hꢀ2β) 5.01 (dd, J = 3.7, 10.3
Hz, 3H, Hꢀ2α), 4.90 – 4.85 (m, 4H, Hꢀ2’α, Hꢀ2’β), 4.14 – 4.05 (m, 9H, Hꢀ6’α, Hꢀ6’β,
Hꢀ6’α, Hꢀ6’β), 4.03 (m, 7H, Hꢀ3’α, Hꢀ5α, Hꢀ3’β), 3.85 (d, J = 2.6 Hz, 3H, Hꢀ4’α), 3.84
– 3.79 (m, 6H, Hꢀ5’α, Hꢀ5’β, Hꢀ4’β), 3.73 (dd, J = 2.6, 9.8 Hz, 1H, Hꢀ5β), 3.66 (dt, J =
5.4, 10.8 Hz, 4H, Hꢀ6α, Hꢀ6β), 3.54 (dd, J = 1.7, 11.8 Hz, 1H, Hꢀ6’β), 3.49 (dd, J = 1.7,
11.7 Hz, 3H, Hꢀ6’α), 2.17 (s, 8H), 2.14 (s, 3H), 2.10 (s, 9H), 2.09 (s, 4H), 2.06 (s,
12H), 2.04 (s, 9H), 2.03 (app d, J = 3.1 Hz, 7H), 2.02 (app d, J = 3.3 Hz, 18H), 2.00 (s,
4H), 0.18 (s, 10H), 0.17 – 0.16 (m, 25H), 0.11 (app d, J = 3.4 Hz, 37H). 13C NMR
(200 MHz, CDCl3) δ 170.8, 170.74, 170.72, 170.69, 170.4, 170.3, 169.9, 169.5, 169.4,
169.3, 169.1, 168.96, 96.6, 96.2, 91.7, 89.1, 77.3, 77.2, 77.0, 73.8, 73.2, 72.5, 72.4,
71.42, 71.35, 70.5, 70.3, 70.2, 69.4, 69.0, 68.8, 68.5, 68.4, 68.2, 67.9, 64.9, 63.7, 63.5,
21.23, 21.22, 21.00, 20.90, 20.83, 20.75, 20.7, 20.6, 0.6, 0.50, 0.49. ESIꢀHRMS
(m/z) calcd. for C30H50O17Si2 [M+NH4]+: 756.2930; found: 756.2903.
(2,3,6ꢀTriꢀOꢀacetylꢀ4ꢀOꢀtrimethylsilylꢀαꢀDꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(1,2,3,4ꢀtetr
aꢀOꢀacetyl)ꢀDꢀglucopyranoside (11): Compound 11 (43 mg, 22%) was obtained as a
white foam (Table 2, Entry 2; see compound 9 for reaction and purification condition).
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The Journal of Organic Chemistry
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TLC (EA/Hex = 1/1) Rf 0.55. 1H NMR (800 MHz, CDCl3) δ 6.27 (d, J = 3.6 Hz, 1H,
Hꢀ1α), 5.66 (d, J = 8.3 Hz, 1H, Hꢀ1β), 5.46 (t, J = 9.9 Hz, 1H, Hꢀ3α), 5.25 – 5.20 (m,
3H, Hꢀ3’α, Hꢀ3’β, Hꢀ3β), 5.18 – 5.09 (m, 6H, Hꢀ4α, Hꢀ2’α, Hꢀ1’α, Hꢀ4β, Hꢀ2’β, Hꢀ1’β),
5.06 (app t, J = 8.8, 9.6 Hz, 1H, Hꢀ2β), 5.03 (dd, J = 4.0, 10.4 Hz, 1H, Hꢀ2α), 4.17 –
4.10 (m, 4H, Hꢀ4’α, Hꢀ6’α, Hꢀ4’β, Hꢀ6’β), 4.04 – 4.06 (m, 1H, Hꢀ5α), 4.03 – 3.96 (m,
3H, Hꢀ6’α, Hꢀ5’α, Hꢀ6’β), 3.94 (app t, J = 6.5 Hz, 1H, Hꢀ5’β), 3.78 – 3.74 (m, 1H,
Hꢀ5β), 3.70 (dd, J = 4.5, 11.7 Hz, 2H, Hꢀ6α, Hꢀ6β), 3.59 (dd, J = 2.4, 11.7 Hz, 1H,
Hꢀ6β), 3.54 (dd, J = 2.5, 11.5 Hz, 1H, Hꢀ6α), 2.18 (s, 3H), 2.11 (s, 3H), 2.09 – 2.08 (m,
12H), 2.06 (app d, 6H), 2.04 – 2.03 (m, 6H), 2.02 (app d, 6H), 2.00 (app d, 6H), 0.11
(app d, 18H). 13C NMR (200 MHz, CDCl3) δ 170.76, 170.67, 170.6, 170.5, 170.4,
170.3, 170.2, 169.8, 169.5, 169.4, 169.09, 169.05, 96.5, 96.1, 91.7, 89.0, 73.5, 73.0,
70.5, 70.3, 70.0, 69.91, 69.4, 69.3, 69.2, 68.6, 68.4, 68.34, 68.26, 68.1, 65.7, 65.6,
64.5, 62.8, 62.6, 21.29, 21.27, 21.03, 20.95, 20.94, 20.90, 20.83, 20.75, 20.74, 20.73,
20.71, 20.6, 0.4. ESIꢀHRMS (m/z) calcd. for C29H44O18Si [M+NH4]+: 726.2641;
found: 726.2616.
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(2,3,4,6ꢀTetraꢀOꢀacetylꢀαꢀDꢀgalactopyranosyl)ꢀ(1ꢀ6)ꢀOꢀ(1,2,3,4ꢀtetraꢀOꢀacetyl)ꢀDꢀ
glucopyranoside (12): Compound 12 (81 mg, 44%) was obtained as a white foam
(Table 2, Entry 2; see compound 9 for reaction and purification condition). TLC
(EA/Hex = 1/1) Rf 0.33. 1H NMR (800 MHz, CDCl3) δ 6.25 (d, J = 3.4 Hz, 1H),
5.64 (d, J = 8.3 Hz, 1H), 5.46 – 5.39 (m, 4H), 5.30 (ddd, J = 3.3, 6.7, 10.2 Hz, 3H),
5.21 (t, J = 9.5 Hz, 1H), 5.14 – 5.09 (m, 5H), 5.08 – 4.97 (m, 5H), 4.16 (dt, J = 6.5,
21.6 Hz, 3H), 4.06 – 4.00 (m, 7H), 3.75 (d, J = 10.0 Hz, 1H), 3.68 (dd, J = 4.2, 11.8
Hz, 3H), 3.59 (d, J = 11.7 Hz, 1H), 3.55 (d, J = 11.5 Hz, 1H), 2.16 (s, 3H), 2.10 (dd, J
= 3.5, 5.7 Hz, 13H), 2.08 (d, J = 6.5 Hz, 8H), 2.01 (dd, J = 3.3, 7.5 Hz, 17H), 2.00 (d,
J = 2.3 Hz, 7H), 1.98 (d, J = 4.9 Hz, 8H), 1.95 (s, 7H). 13C NMR (200 MHz, CDCl3)
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